本發(fā)明涉及一套植物基因組堿基定點(diǎn)突變的人工系統(tǒng)及定點(diǎn)突變方法，特別是用于水稻基因組單堿基置換的人工系統(tǒng)。

背景技術(shù)：

水稻(oryzasatival.)是世界主要糧食作物之一，世界上近一半人口，包括幾乎整個(gè)東亞和東南亞的人口，都以稻米為食。在中國(guó)，水稻播種面積占全國(guó)糧食作物的1/4，而產(chǎn)量則占一半以上。提高產(chǎn)量，改善稻米品質(zhì)，增加水稻植株抗病、抗逆性等研究以保證糧食的穩(wěn)定供給是人類社會(huì)可持續(xù)性發(fā)展的重大課題。水稻也是單子葉植物的模式系統(tǒng)，其研究技術(shù)、方法、理論和成果對(duì)其它禾本科植物，如小麥、玉米、高粱等具有重要指導(dǎo)作用。

進(jìn)入二十一世紀(jì)以來，全基因組測(cè)序，基因挖掘和基因編輯等技術(shù)手段的不斷涌現(xiàn)讓現(xiàn)代生物技術(shù)在農(nóng)業(yè)上發(fā)揮了前所未有的作用。其中，基因功能的鑒定以及對(duì)有重大應(yīng)用價(jià)值的基因的轉(zhuǎn)化應(yīng)用是最為關(guān)鍵的步驟。隨著基因組大數(shù)據(jù)工程的快速發(fā)展，海量的植物基因組序列等待著解讀，基因的挖掘和應(yīng)用嚴(yán)重滯后，由此顯得更加迫切。值得注意的是，利用現(xiàn)代生物技術(shù)所獲得的轉(zhuǎn)基因材料，在應(yīng)用上涉及田間釋放的轉(zhuǎn)基因生物安全問題，近些年來引發(fā)了公眾的普遍顧慮。

目前在植物研究領(lǐng)域，crispr/cas9技術(shù)已在擬南芥、煙草、水稻、大豆、番茄、玉米、大麥、小麥、馬鈴薯、蘑菇等模式植物和農(nóng)作物上展現(xiàn)出驕人的成績(jī)，獲得了大量基因突變材料和基因編輯產(chǎn)品。但是這些編輯事件基本上都是利用植物細(xì)胞非同源末端連接修復(fù)機(jī)制獲得的，屬于基因功能喪失性突變體。

獲得基因功能獲得性突變體，對(duì)于基礎(chǔ)研究和應(yīng)用研究的意義更為重大。然而，傳統(tǒng)的基因功能獲得性突變體是通過同源重組機(jī)制而獲得的。在大部分復(fù)雜生物體內(nèi)，非同源末端連接修復(fù)機(jī)制在外源dna片段的插入過程中占主導(dǎo)地位，同源重組發(fā)生的概率非常低，從而定制突變體的效率只有千分之一到百分之二。最近出現(xiàn)的利用胞嘧啶去氨酶介導(dǎo)的單堿基替換技術(shù)，為人們高效獲得基因功能獲得性突變體提供了全新思路。然而，初步結(jié)果發(fā)現(xiàn)，現(xiàn)有的單堿基替換技術(shù)，如rbe3系統(tǒng)，是基于來源于小鼠的apobec1蛋白，其對(duì)靶位點(diǎn)tc和cc具有偏好性，而對(duì)靶位點(diǎn)gc和ac編輯效率較低。

技術(shù)實(shí)現(xiàn)要素：

由于水稻基因組中g(shù)c和ac位點(diǎn)含量極高，因此，為了克服現(xiàn)有技術(shù)對(duì)gc和ac位點(diǎn)不敏感的缺陷，本申請(qǐng)?zhí)峁┝艘惶兹斯は到y(tǒng)，所述人工系統(tǒng)包括

第i調(diào)節(jié)元件，其包括能夠編碼如氨基酸序列i的核苷酸序列；其中所述氨基酸序列i選自seqidno.1、seqidno.2、seqidno.3、seqidno.4、seqidno.5和seqidno.6中的一種；

第ii調(diào)節(jié)元件，其包括依次從5’端到3’端的第ii-1核苷酸序列和第ii-2核苷酸序列；所述第ii-1核苷酸序列包括靶核苷酸序列；所述第ii-2核苷酸序列包括來源于化膿鏈球菌(streptococcuspyogenes)的sgrna核酸序列；所述第ii-1核苷酸序列和所述第ii-2核苷酸序列轉(zhuǎn)錄融合，其產(chǎn)物能引導(dǎo)第i調(diào)控元件編碼的蛋白至目標(biāo)生物基因組中待突變的靶位點(diǎn)處，并將所述靶位點(diǎn)處的c突變?yōu)閠、a和g中的一種，或?qū)⑺霭形稽c(diǎn)處的g突變?yōu)閍、t和c中的一種；

當(dāng)所述第ii調(diào)節(jié)元件為多個(gè)時(shí)，包含在其中的多個(gè)第ii-1核苷酸序列兩兩不相同。另外，當(dāng)所述第ii調(diào)節(jié)元件為多個(gè)時(shí)，它們可以串聯(lián)的形成連接在一起。

通過利用本申請(qǐng)的人工系統(tǒng)，可以將水稻基因組內(nèi)源位點(diǎn)的特定的c定點(diǎn)突變?yōu)閠、a和g中的一種，或g定點(diǎn)突變?yōu)閍、t和c中的一種，篩選得到水稻基因功能獲得性突變體。但是，使用的靶核苷酸序列為靶位點(diǎn)處為c那條鏈上的核苷酸序列。通過本申請(qǐng)seqidno.2的驗(yàn)證，加之生物信息學(xué)的分析，以及結(jié)合本領(lǐng)域的常規(guī)技術(shù)知識(shí)，可以合理的得出seqidno.1、seqidno.3、seqidno.4、seqidno.5和seqidno.6也均適用于本申請(qǐng)的人工系統(tǒng)。

在一個(gè)具體實(shí)施方式中，所述第i調(diào)節(jié)元件的核苷酸序列為能夠適于在水稻中表達(dá)的核苷酸序列，所述第ii調(diào)節(jié)元件的核苷酸序列為能夠適于在水稻中發(fā)生轉(zhuǎn)錄的核苷酸序列。

在一個(gè)具體實(shí)施方式中，能夠編碼如seqidno.1所示蛋白的核苷酸編碼序列如seqidno.7所示；能夠編碼如seqidno.2所示蛋白的核苷酸編碼序列如seqidno.8所示；能夠編碼如seqidno.3所示蛋白的核苷酸編碼序列如seqidno.9所示；能夠編碼如seqidno.4所示蛋白的核苷酸編碼序列如seqidno.10所示；能夠編碼如seqidno.5所示蛋白的核苷酸編碼序列如seqidno.11所示；能夠編碼如seqidno.6所示蛋白的核苷酸編碼序列如seqidno.12所示。經(jīng)過密碼子優(yōu)化分析，在本申請(qǐng)中隨機(jī)挑選出適合在水稻中表達(dá)如seqidno.2所示氨基酸的核苷酸序列其中之一如seqidno.8所示。相應(yīng)地，根據(jù)如seqidno.8所示的核苷酸序列信息，根據(jù)生物信息學(xué)的分析，以及結(jié)合本領(lǐng)域的常規(guī)技術(shù)知識(shí)，可以合理的得出適合在水稻中表達(dá)如seqidno.1、seqidno.3、seqidno.4、seqidno.5和seqidno.6所示氨基酸的核苷酸序列。

在一個(gè)具體實(shí)施方式中，所述第ii-2核苷酸序列如seqidno.13所示。

在一個(gè)具體實(shí)施方式中，所述第ii-1核苷酸序列包括iis型限制性內(nèi)切酶的酶切位點(diǎn)，所述靶核苷酸序列通過所述iis型限制性內(nèi)切酶的酶切位點(diǎn)的切割被克隆，以使所述第ii-1核苷酸序列與第ii-2序列轉(zhuǎn)錄融合；當(dāng)所述第ii調(diào)節(jié)元件為多個(gè)時(shí)，用于克隆不同靶核苷酸序列的所述iis型限制性內(nèi)切酶的酶切位點(diǎn)兩兩不相同。

其中，由于所述靶核苷酸序列是根據(jù)基因編輯位點(diǎn)而變化的，因此可以將其他元件構(gòu)建好，包括事先克隆到相關(guān)位置的限制性內(nèi)切酶的酶切位點(diǎn)。在使用的之前，再根據(jù)基因編輯目的將所述靶核苷酸序列通過限制性內(nèi)切酶的酶切位點(diǎn)的切割而被克隆。當(dāng)所述第ii調(diào)節(jié)元件為多個(gè)時(shí)，包含在其中的多個(gè)第ii-1核苷酸序列中的限制性內(nèi)切酶的酶切位點(diǎn)兩兩不相同，如此，可以有效的保障不同的靶核苷酸順利的被克隆到目標(biāo)位置。多個(gè)靶核苷酸序列可用于目標(biāo)生物基因組上的多個(gè)待突變的靶位點(diǎn)的堿基替換。

在一個(gè)具體實(shí)施方式中，優(yōu)選所述克隆位點(diǎn)的核苷酸序列包括seqidno.14和/或seqidno.15。

在一個(gè)具體實(shí)施方式中，通過如下方式確定所述靶核苷酸序列：

1)確定水稻基因組上需要被改造的核苷酸序列；

2)判斷步驟1)中確定的需要被改造的核苷酸序列或其反向互補(bǔ)序列中是否攜帶有待突變的核苷酸c，并判斷所述待突變的核苷酸c突變?yōu)閠、a和g中的一種、或所述待突變的核苷酸g突變?yōu)閍、t和c中的一種后引起的改變是否符合預(yù)期；

3)在需要被改造的核苷酸序列或其反向互補(bǔ)序列中篩選靶標(biāo)序列：向所述待突變的核苷酸c的3′端方向搜索以確認(rèn)存在能夠被氨基酸序列i識(shí)別的識(shí)別模序。且所述待突變的核苷酸c處在所述識(shí)別模序5′端上游的-19至-13的位置，由此確定的所述識(shí)別模序5′端上游17至21個(gè)核苷酸序列(不含識(shí)別模序)為所述靶核苷酸序列。

在一個(gè)具體實(shí)施方式中，當(dāng)?shù)趇調(diào)節(jié)元件中含有能夠編碼seqidno.1、seqidno.2和seqidno.3所示中的一種的氨基酸序列時(shí)，所識(shí)別模序?yàn)?′-ngg-3′、5′-nga-3′、5′-gagn-3′、5′-aagn-3′中的一種，所述靶核苷酸序列上游的17至21個(gè)核苷酸序列，淘汰含有連續(xù)五個(gè)t的核苷酸序列；其中，所述n為a、g、c和t中的一種。

在一個(gè)具體實(shí)施方式中，當(dāng)?shù)趇調(diào)節(jié)元件中含有能夠編碼seqidno.4、seqidno.5和seqidno.6所示中的一種氨基酸序列時(shí)，所述模序?yàn)?′-nga-3′、5′-tgcg-3′、5′-tgtg-3′、5′-gaag-3′、5′-cgcg-3′中的一種，所述靶核苷酸序列為所述識(shí)別模序5′端上游的17至21個(gè)核苷酸序列，淘汰含有連續(xù)五個(gè)t的核苷酸序列；其中，所述n為a、g、c和t中的一種。

在一個(gè)具體實(shí)施方式中，所述靶核苷酸序列如seqidno.16所示。

在一個(gè)具體實(shí)施方式中，所述人工系統(tǒng)還包括在所述第i調(diào)節(jié)元件的5’端的能夠用于水稻中的，且能夠啟動(dòng)所述第i調(diào)節(jié)元件轉(zhuǎn)錄的第一啟動(dòng)子；和/或所述人工系統(tǒng)還包括在所述第ii調(diào)節(jié)元件的5’端的能夠用于水稻中的，且能夠啟動(dòng)所述第ii調(diào)節(jié)元件轉(zhuǎn)錄的第二啟動(dòng)子。

在一個(gè)具體實(shí)施方式中，所述第一啟動(dòng)子為rna聚合酶ii型啟動(dòng)子；和/或第二啟動(dòng)子為rna聚合酶iii型啟動(dòng)子。

在一個(gè)具體實(shí)施方式中，第一啟動(dòng)子為seqidno.17；和/或第二啟動(dòng)子為seqidno.18和/或seqidno.19。

在一個(gè)具體實(shí)施方式中，所述人工系統(tǒng)還包括在所述第i調(diào)節(jié)元件的3’端的能夠終止所述第i調(diào)節(jié)元件轉(zhuǎn)錄的第一終止子；和/或所述人工系統(tǒng)還包括在所述第ii調(diào)節(jié)元件的3’端的能夠終止所述第ii調(diào)節(jié)元件轉(zhuǎn)錄的第二終止子。

在一個(gè)具體實(shí)施方式中，第一終止子為seqidno.20；和/或第二終止子為seqidno.21。

在一個(gè)具體實(shí)施方式中，所述第i調(diào)節(jié)元件和所述第ii元件能夠被克隆到至少一個(gè)載體上。例如，所述第i調(diào)節(jié)元件表達(dá)框架和所述第ii調(diào)節(jié)元件表達(dá)框架能夠被克隆或整合到同一個(gè)載體上，或第i調(diào)節(jié)元件表達(dá)框架和第ii調(diào)節(jié)元件表達(dá)框架混合或分別位于不同的載體上時(shí)，可以采用基因槍法、農(nóng)桿菌侵染法或peg介導(dǎo)轉(zhuǎn)化法將兩個(gè)表達(dá)框架或含有兩個(gè)表達(dá)框架的載體導(dǎo)入到水稻的愈傷組織或水稻原生質(zhì)體中。

在一個(gè)具體實(shí)施方式中，所述第i調(diào)節(jié)元件能夠被克隆到pubi-ccdb上；所述第ii調(diào)節(jié)元件被克隆到入門載體pentr4上。pubi-ccdb為基于gateway反應(yīng)并用于水稻遺傳轉(zhuǎn)化的雙元載體。

在一個(gè)具體實(shí)施方式中，所述第一啟動(dòng)子、第i調(diào)節(jié)元件和第一終止子能夠被克隆到pubi-ccdb載體上。

在一個(gè)具體實(shí)施方式中，第二啟動(dòng)子、第ii調(diào)節(jié)元件和第二終止子被克隆到pentr4載體上。

本申請(qǐng)之二提供了一種如本申請(qǐng)之一中任意一人工系統(tǒng)在用于將水稻基因組中的c定點(diǎn)突變?yōu)閠、a和g中的一種，或?qū)⑺净蚪M中的g定點(diǎn)突變?yōu)閍、t和c中的一種的應(yīng)用。

本申請(qǐng)之三提供了一種將水稻基因組上的c定點(diǎn)突變?yōu)閠、a或g的方法，其包括如下步驟：

1)將本申請(qǐng)之一中任意一人工系統(tǒng)通過農(nóng)桿菌介導(dǎo)的方法導(dǎo)入到水稻愈傷組織，然后培養(yǎng)以獲得水稻植株；

2)篩選獲得含有定點(diǎn)突變的水稻植株。

進(jìn)一步地，所述水稻植株能夠產(chǎn)生含有定點(diǎn)替換堿基的種子，水稻獲得了新的經(jīng)濟(jì)性狀。

在進(jìn)行所述的人工系統(tǒng)導(dǎo)入時(shí)，可以采用農(nóng)桿菌侵染法，也可以采用基因槍法或peg介導(dǎo)轉(zhuǎn)化的方法中的一種將所述的人工系統(tǒng)導(dǎo)入到水稻愈傷組織或水稻原生質(zhì)體中，這是本領(lǐng)域技術(shù)人員容易理解的。

本領(lǐng)域的技術(shù)人員公知，水稻基因組dna由兩條鏈組成，因此，所述靶核苷酸序列可以在其中的任意一條鏈上，并且靶位點(diǎn)處為c。例如，當(dāng)所述靶核苷酸序列位于某一功能基因的一條鏈中時(shí)，該功能基因的特定位點(diǎn)上的c被定點(diǎn)突變?yōu)閠、a和g中的一種后，如果其中的一種突變能夠獲得預(yù)期的其對(duì)應(yīng)的功能蛋白中的氨基酸，則可以采用此系統(tǒng)來實(shí)現(xiàn)，即可以通過直接的堿基替換三聯(lián)體密碼子中的c為t、a和g中的一種，或通過間接的堿基替換三聯(lián)體密碼子中的g為a、t和c中的一種來改變所述三聯(lián)體密碼子編碼氨基酸為所需氨基酸的核苷酸序列，得到水稻基因功能獲得性突變體；或當(dāng)所述靶核苷酸序列位于某一功能基因的另一條鏈中時(shí)，該功能基因的特定位點(diǎn)上的g被定點(diǎn)突變?yōu)閍、t和c中的一種后，如果其中的一種突變能夠獲得預(yù)期的其對(duì)應(yīng)的功能蛋白中的氨基酸，也可以采用此系統(tǒng)來實(shí)現(xiàn)，即可以通過將該鏈中的g替換為a、t和c中的一種來改變?cè)撴溨械乃鋈?lián)體密碼子編碼氨基酸，或通過間接的堿基替換三聯(lián)體密碼子中的g為a、t和c中的一種，得到水稻基因功能獲得性突變體。

本申請(qǐng)的有益效果在于：

a)第ii調(diào)節(jié)元件可以為多個(gè)，這樣可以同時(shí)編輯水稻細(xì)胞內(nèi)多個(gè)基因靶位點(diǎn)。

b)本申請(qǐng)的人工系統(tǒng)的堿基編輯效率高，特別是gc和ac處的靶位點(diǎn)堿基編輯效率高，可達(dá)26.9％，而現(xiàn)有的rbe3系統(tǒng)不能實(shí)現(xiàn)此位點(diǎn)相應(yīng)的編輯。因此，本申請(qǐng)的人工系統(tǒng)在水稻基因功能研究和分子育種中具有廣泛的應(yīng)用價(jià)值。

附圖說明

圖1顯示了本申請(qǐng)實(shí)施例帶有pi-d2基因的靶向序列相對(duì)應(yīng)pi-d2基因的位置，以及利用rbe5系統(tǒng)獲得的pi-d2基因功能獲得性突變體核苷酸突變信息。其中，識(shí)別模序序列由下劃實(shí)線表示；靶核苷酸序列為加粗顯示部分，氨基酸替換位點(diǎn)由下劃虛線表示。

具體實(shí)施方式

pubi-ccdb載體的來源：pubi-ccdb載體由本實(shí)驗(yàn)室通過改造pcambia1300而來，其中插入了attr1-ccdb-attr2模塊，用于gateway反應(yīng)接受來自于入門載體的attl1-靶向序列轉(zhuǎn)錄模塊-attl2模塊。

pentr4載體的來源：購(gòu)自美國(guó)invitrogen公司。

實(shí)施例1

1.載體的構(gòu)建

通過dna克隆的常規(guī)操作，將玉米的組成型啟動(dòng)子ubi-p(seqidno.17)、seqidno.8、nos終止子(seqidno.20)按照從5’到3’的順序克隆到pubi-ccdb載體上，命名為pubi:rbe5，用于水稻的轉(zhuǎn)基因植株研究。

將osu6-p啟動(dòng)子(seqidno.18)、兩個(gè)bsai酶切位點(diǎn)(seqidno.14)、sgrna序列(seqidno.13)、(t)8終止子(seqidno.21)、粳稻u6snrna啟動(dòng)子(seqidno.19)、兩個(gè)btgzi酶切位點(diǎn)(seqidno.15)、sgrna序列(seqidno.13)、(t)8終止子(seqidno.21)按照從5’到3’的順序克隆到pentr4載體的多克隆位點(diǎn)上，命名為pentr4:sgrna。其中的兩個(gè)btgzi或兩個(gè)bsai酶切位點(diǎn)可以用于分別克隆以下實(shí)施例2中特定基因的靶向序列。

2.針對(duì)pi-d2基因的識(shí)別序列設(shè)計(jì)和克隆。

每個(gè)基因的轉(zhuǎn)錄本序列和基因組序列從msu水稻基因組數(shù)據(jù)庫中獲得(http://rice.plantbiology.msu.edu/)。1)判斷編輯位點(diǎn)的正鏈或負(fù)鏈?zhǔn)欠駭y帶有核酸堿基c，相應(yīng)堿基c突變?yōu)閠、a或g后引起的氨基酸改變是否符合預(yù)期；2)在3′端方向搜索ngg、nga、gagn、aagn等模序，所述n是a或g或c或t，使突變堿基c處在所述模序上游的的-19至-13位置；3)合成19-20bp的靶向序列，進(jìn)行載體構(gòu)建。

針對(duì)pi-d2基因的靶核苷酸序列為5′-gagcataatgacaataataa-3′(seqidno.16)。分別合成gpi-d2-f1(5′-gtgtgagcataatgacaataataa-3′，seqidno.22)和gpi-d2-r1(5′-aaacttattattgtcattatgctc-3′，seqidno.23)引物后，使用t4多聚核苷酸激酶進(jìn)行磷酸化處理，退火形成雙鏈，克隆到pentr4:sgrna載體的bsai酶切位點(diǎn)中，測(cè)序確認(rèn)插入片段(靶核苷酸序列)完全正確，獲得pentr4:gpi-d2。將pentr4:gpi-d2用aatii酶切進(jìn)行線性化，再通過gateway反應(yīng)將第ii核苷酸序列轉(zhuǎn)錄元件轉(zhuǎn)移到pubi:rbe5中，獲得為pubi:rbe5-gpi-d2最終載體，其對(duì)于pi-d2基因上的改造位點(diǎn)序列為5′-ttattattgtcattatgctc-3′(seqidno.24)，該序列與seqidno.16互補(bǔ)；基因編輯后序列為5′-ttattattgtcattatactc-3′(seqidno.25)。

3.rbe系統(tǒng)轉(zhuǎn)化粳稻品種kitaake。

1)水稻愈傷誘導(dǎo)：

去殼的未成熟的水稻種子用50％的市售消毒液處理25分鐘；無菌水清洗3-5次，然后將種子轉(zhuǎn)移至無菌的培養(yǎng)皿中，吸出多余的水份；將種子放置于msd平板上(4.43g/lms粉；30g/l蔗糖；2ml/l2,4-d；8g/l植物凝膠；ph5.7)，于光照培養(yǎng)室培養(yǎng)10天，誘導(dǎo)愈傷組織形成；去除種子的胚和芽，將愈傷組織轉(zhuǎn)移至一個(gè)新的msd培養(yǎng)皿上，培養(yǎng)5天，直至能夠用于農(nóng)桿菌的轉(zhuǎn)化。

2)農(nóng)桿菌轉(zhuǎn)化：

將pubi:rbe5-gpi-d2通過電擊法轉(zhuǎn)入到農(nóng)桿菌菌株eha105中，在lb培養(yǎng)基中過夜培養(yǎng)12小時(shí)；收集農(nóng)桿菌，用msd溶液重懸，使其od600＝0.1，待用。

3)水稻愈傷的農(nóng)桿菌侵染：

將愈傷組織置于上述農(nóng)桿菌懸浮液中30分鐘；除去農(nóng)桿菌懸浮液，將愈傷組織轉(zhuǎn)移至無菌的吸水紙上；將愈傷組織轉(zhuǎn)移至新的含有100μm乙酰丁香酮的msd培養(yǎng)基上，室溫避光培養(yǎng)3天。

4)水稻抗性愈傷篩選：

將暗培養(yǎng)后的愈傷組織轉(zhuǎn)移至msd培養(yǎng)基(200mg/l特美汀；50mg/l潮霉素b)上，光照培養(yǎng)2周至1個(gè)月，直至愈傷組織的表面出現(xiàn)抗性愈傷，轉(zhuǎn)移抗性愈傷到新的msd培養(yǎng)基(200mg/l特美汀，50mg/l潮霉素b)上，每2周換一次培養(yǎng)基。

5)抗性愈傷分化與生根

將抗性愈傷組織轉(zhuǎn)移至再生培養(yǎng)基上(4.43g/lms粉；30g/l蔗糖；25g/l山梨醇；0.5mg/lnaa；3mg/lba；100mg/l特美?。?0mg/l潮霉素b；12g/l瓊脂粉；ph＝5.7)，直至長(zhǎng)成植株幼苗，每7-10天轉(zhuǎn)移一次；轉(zhuǎn)移幼苗至1/2ms培養(yǎng)基(2.21g/lms粉；15g/l蔗糖；8g/l植物凝膠；ph5.7)中生根。

4.對(duì)t0代轉(zhuǎn)基因水稻中pi-d2基因靶位點(diǎn)進(jìn)行鑒定。

用ctab法提取抗性愈傷和轉(zhuǎn)基因植株的基因組dna。根據(jù)pi-d2基因的靶位點(diǎn)dna序列設(shè)計(jì)特異性的pcr引物：pi-d2-f1：5′-cgggttgtaagagtgcctgt-3′(seqidno.26)，pi-d2-r1：5′-ctccagcttcttcacagcaa-3′(seqidno.27)，利用i-5高保真酶混合液(maclab)進(jìn)行pcr擴(kuò)增目的片段491bp，pcr產(chǎn)物直接測(cè)序或連接pgem-t載體后測(cè)序，基因編輯效果如圖1所示，pubi:rbe5系統(tǒng)對(duì)pi-d2的靶位點(diǎn)編輯效率高達(dá)26.9％。

實(shí)施例2

通過dna克隆的常規(guī)操作，將玉米的組成型啟動(dòng)子ubi-p(seqidno.17)、seqidno.28、nos終止子(seqidno.20)按照從5’到3’的順序克隆到pubi-ccdb載體上，命名為pubi:rbe3。將pentr4:gpi-d2用aatii酶切進(jìn)行線性化，再通過gateway反應(yīng)將靶向序列轉(zhuǎn)錄元件轉(zhuǎn)移到pubi:rbe3中，獲得為pubi:rbe3-gpi-d2最終載體，用于水稻的轉(zhuǎn)基因植株研究。其對(duì)于pi-d2在基因組靶位點(diǎn)序列與實(shí)施例1相同，為5′-ttattattgtcattatgctc-3′(seqidno.24)。seqidno.28所示的核苷酸序列編碼如seqidno.29所示的氨基酸序列。

其他操作同實(shí)施例1。

轉(zhuǎn)基因植株群體中未篩選獲得到pi-d2的編輯植株。

本申請(qǐng)通過轉(zhuǎn)化水稻愈傷，獲得含有rbe3(pubi:rbe3-gpi-d2)和rre5(pubi:rbe5-gpi-d2)系統(tǒng)的轉(zhuǎn)基因水稻，通過靶位點(diǎn)測(cè)序發(fā)現(xiàn)，對(duì)于gc靶位點(diǎn)，rbe5系統(tǒng)成功地在轉(zhuǎn)基因水稻植株中對(duì)pi-d2位點(diǎn)進(jìn)行了單堿基定點(diǎn)替換。我們從26株獨(dú)立的轉(zhuǎn)基因水稻株系中篩選獲得到7株目標(biāo)突變體，基因編輯效率為26.9％，而已報(bào)道的單堿基替換載體rbe3系統(tǒng)對(duì)其的編輯效率為零。綜上所述，我們構(gòu)建的這套新載體進(jìn)行針對(duì)gc和ac靶位點(diǎn)的定點(diǎn)堿基突變方法，對(duì)于用基因編輯技術(shù)獲得指定基因功能獲得性水稻突變體具有重要價(jià)值。

lha1760261核苷酸序和氨基酸列表

<110>中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所

<120>一種定點(diǎn)突變的人工載體系統(tǒng)及定點(diǎn)突變方法

<130>lha1760261

<160>29

<170>patentinversion3.5

<210>1

<211>1572

<212>prt

<213>人工序列

<223>seqidno.1

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<210>2

<211>1558

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<213>人工序列

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<210>3

<211>1676

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<223>seqidno.3

<400>3

mdsllmnrreflyqfknvrwakgrretylcyvvkrrdsatsfsldfgylrnkngchvellflryisdwdldpgrcyrvtwfiswspcydcarhvadflrgnpnlslriftarlyfcedrkaepeglrrlhragvqiaimtfkdyfycwntfvenhgrtfkaweglhensvrlsrqlrrillplyevddlrdafrtsgsetpgtsesatpesdkkysiglaigtnsvgwavitdeykvpskkfkvlgntdrhsikknligallfdsgetaeatrlkrtarrrytrrknricylqeifsnemakvddsffhrleesflveedkkherhpifgnivdevayhekyptiyhlrkklvdstdkadlrliylalahmikfrghfliegdlnpdnsdvdklfiqlvqtynqlfeenpinasgvdanailsarlsksrrlenliaqlpgekknglfgnlialslgltpnfksnfdlaedaklqlskdtydddldnllaqigdqyadlflaaknlsdaillsdilrvnteitkaplsasmikrydehhqdltllkalvrqqlpekykeiffdqskngyagyidggasqeefykfikpilekmdgteellvklnredllrkqrtfdngsiphqihlgelhailrrqedfypflkdnrekiekiltfripyyvgplargnsrfawmtrkseetitpwnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyftvyneltkvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyfkkiecfdsveisgvedrfnaslgtyhdllkiikdkdfldneenediledivltltlfedremieerlktyahlfddkvmkqlkrrrytgwgrlsrklingirdkqsgktildflksdgfanrnfmqlihddsltfkediqkaqvsgqgdslhehianlagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqkgqknsrermkrieegikelgsqilkehpventqlqneklylyylqngrdmyvdqeldinrlsdydvdhivpqsflkddsidnkvltrsdknrgksdnvpseevvkkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlvetrqitkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqfykvreinnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmiakseqeigkatakyffysnimnffkteitlangeirkrplietngetgeivwdkgrdfatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliarkkdwdpkkyggfdsptvaysvlvvakvekgkskklksvkellgitimerssfeknpidfleakgykevkkdliiklpkyslfelengrkrmlasagelqkgnelalpskyvnflylashyeklkgspedneqkqlfveqhkhyldeiieqisefskrviladanldkvlsaynkhrdkpireqaeniihlftltnlgapaafkyfdttidrkrytstkevldatlihqsitglyetridlsqlggdsggstnlsdiieketgkqlviqesilmlpeeveevignkpesdilvhtaydestdenvmlltsdapeykpwalviqdsngenkikmlsggspkkkrkv；

<210>4

<211>1572

<212>prt

<213>人工序列

<223>seqidno.4

<400>4

mdsllmnrreflyqfknvrwakgrretylcyvvkrrdsatsfsldfgylrnkngchvellflryisdwdldpgrcyrvtwfiswspcydcarhvadflrgnpnlslriftarlyfcedrkaepeglrrlhragvqiaimtfkdyfycwntfvenhgrtfkaweglhensvrlsrqlrrillplyevddlrdafrtdkkysiglaigtnsvgwavitdeykvpskkfkvlgntdrhsikknligallfdsgetaeatrlkrtarrrytrrknricylqeifsnemakvddsffhrleesflveedkkherhpifgnivdevayhekyptiyhlrkklvdstdkadlrliylalahmikfrghfliegdlnpdnsdvdklfiqlvqtynqlfeenpinasgvdanailsarlsksrrlenliaqlpgekknglfgnlialslgltpnfksnfdlaedaklqlskdtydddldnllaqigdqyadlflaaknlsdaillsdilrvnteitkaplsasmikrydehhqdltllkalvrqqlpekykeiffdqskngyagyidggasqeefykfikpilekmdgteellvklnredllrkqrtfdngsiphqihlgelhailrrqedfypflkdnrekiekiltfripyyvgplargnsrfawmtrkseetitpwnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyftvyneltkvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyfkkiecfdsveisgvedrfnaslgtyhdllkiikdkdfldneenediledivltltlfedremieerlktyahlfddkvmkqlkrrrytgwgrlsrklingirdkqsgktildflksdgfanrnfmqlihddsltfkediqkaqvsgqgdslhehianlagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqkgqknsrermkrieegikelgsqilkehpventqlqneklylyylqngrdmyvdqeldinrlsdydvdhivpqsflkddsidnkvltrsdknrgksdnvpseevvkkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlvetrqitkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqfykvreinnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmiakseqeigkatakyffysnimnffkteitlangeirkrplietngetgeivwdkgrdfatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliarkkdwdpkkyggfvsptvaysvlvvakvekgkskklksvkellgitimerssfeknpidfleakgykevkkdliiklpkyslfelengrkrmlasagelqkgnelalpskyvnflylashyeklkgspedneqkqlfveqhkhyldeiieqisefskrviladanldkvlsaynkhrdkpireqaeniihlftltnlgapaafkyfdttidrkrytstkevldatlihqsitglyetridlsqlggdrpkkkrkvgg；

<210>5

<211>1588

<212>prt

<213>人工序列

<223>seqidno.5

<400>5

mdsllmnrreflyqfknvrwakgrretylcyvvkrrdsatsfsldfgylrnkngchvellflryisdwdldpgrcyrvtwfiswspcydcarhvadflrgnpnlslriftarlyfcedrkaepeglrrlhragvqiaimtfkdyfycwntfvenhgrtfkaweglhensvrlsrqlrrillplyevddlrdafrtsgsetpgtsesatpesdkkysiglaigtnsvgwavitdeykvpskkfkvlgntdrhsikknligallfdsgetaeatrlkrtarrrytrrknricylqeifsnemakvddsffhrleesflveedkkherhpifgnivdevayhekyptiyhlrkklvdstdkadlrliylalahmikfrghfliegdlnpdnsdvdklfiqlvqtynqlfeenpinasgvdanailsarlsksrrlenliaqlpgekknglfgnlialslgltpnfksnfdlaedaklqlskdtydddldnllaqigdqyadlflaaknlsdaillsdilrvnteitkaplsasmikrydehhqdltllkalvrqqlpekykeiffdqskngyagyidggasqeefykfikpilekmdgteellvklnredllrkqrtfdngsiphqihlgelhailrrqedfypflkdnrekiekiltfripyyvgplargnsrfawmtrkseetitpwnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyftvyneltkvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyfkkiecfdsveisgvedrfnaslgtyhdllkiikdkdfldneenediledivltltlfedremieerlktyahlfddkvmkqlkrrrytgwgrlsrklingirdkqsgktildflksdgfanrnfmqlihddsltfkediqkaqvsgqgdslhehianlagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqkgqknsrermkrieegikelgsqilkehpventqlqneklylyylqngrdmyvdqeldinrlsdydvdhivpqsflkddsidnkvltrsdknrgksdnvpseevvkkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlvetrqitkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqfykvreinnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmiakseqeigkatakyffysnimnffkteitlangeirkrplietngetgeivwdkgrdfatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliarkkdwdpkkyggfvsptvaysvlvvakvekgkskklksvkellgitimerssfeknpidfleakgykevkkdliiklpkyslfelengrkrmlasagelqkgnelalpskyvnflylashyeklkgspedneqkqlfveqhkhyldeiieqisefskrviladanldkvlsaynkhrdkpireqaeniihlftltnlgapaafkyfdttidrkrytstkevldatlihqsitglyetridlsqlggdrpkkkrkvgg；

<210>6

<211>1676

<212>prt

<213>人工序列

<223>seqidno.6

<400>6

mdsllmnrreflyqfknvrwakgrretylcyvvkrrdsatsfsldfgylrnkngchvellflryisdwdldpgrcyrvtwfiswspcydcarhvadflrgnpnlslriftarlyfcedrkaepeglrrlhragvqiaimtfkdyfycwntfvenhgrtfkaweglhensvrlsrqlrrillplyevddlrdafrtsgsetpgtsesatpesdkkysiglaigtnsvgwavitdeykvpskkfkvlgntdrhsikknligallfdsgetaeatrlkrtarrrytrrknricylqeifsnemakvddsffhrleesflveedkkherhpifgnivdevayhekyptiyhlrkklvdstdkadlrliylalahmikfrghfliegdlnpdnsdvdklfiqlvqtynqlfeenpinasgvdanailsarlsksrrlenliaqlpgekknglfgnlialslgltpnfksnfdlaedaklqlskdtydddldnllaqigdqyadlflaaknlsdaillsdilrvnteitkaplsasmikrydehhqdltllkalvrqqlpekykeiffdqskngyagyidggasqeefykfikpilekmdgteellvklnredllrkqrtfdngsiphqihlgelhailrrqedfypflkdnrekiekiltfripyyvgplargnsrfawmtrkseetitpwnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyftvyneltkvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyfkkiecfdsveisgvedrfnaslgtyhdllkiikdkdfldneenediledivltltlfedremieerlktyahlfddkvmkqlkrrrytgwgrlsrklingirdkqsgktildflksdgfanrnfmqlihddsltfkediqkaqvsgqgdslhehianlagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqkgqknsrermkrieegikelgsqilkehpventqlqneklylyylqngrdmyvdqeldinrlsdydvdhivpqsflkddsidnkvltrsdknrgksdnvpseevvkkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlvetrqitkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqfykvreinnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmiakseqeigkatakyffysnimnffkteitlangeirkrplietngetgeivwdkgrdfatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliarkkdwdpkkyggfvsptvaysvlvvakvekgkskklksvkellgitimerssfeknpidfleakgykevkkdliiklpkyslfelengrkrmlasagelqkgnelalpskyvnflylashyeklkgspedneqkqlfveqhkhyldeiieqisefskrviladanldkvlsaynkhrdkpireqaeniihlftltnlgapaafkyfdttidrkrytstkevldatlihqsitglyetridlsqlggdsggstnlsdiieketgkqlviqesilmlpeeveevignkpesdilvhtaydestdenvmlltsdapeykpwalviqdsngenkikmlsggspkkkrkv；

<210>7

<211>4719

<212>dna

<213>人工序列

<223>seqidno.7

<400>7

atggatagccttctcatgaacagaagagagtttctctatcagtttaaaaatgttcggtgggcgaaggggaggagagagacatatctctgctatgttgttaagcggagagattctgcgacctcattctcactcgattttggttatttgaggaacaagaatggatgtcatgtcgaattgttgtttctccggtatatttccgactgggatttggacccagggcggtgttaccgggtcacatggtttatttcctggagtccatgttacgactgtgcgcgccatgtcgccgacttcctcaggggtaatcctaacttgtccttgcggatttttacagccagactctatttctgtgaggatcggaaggcggaacccgaggggctgagaagactgcaccgcgctggcgtccaaatcgccatcatgacttttaaggattatttctactgttggaacacgttcgtcgagaaccacggtcggaccttcaaagcctgggaagggctgcatgaaaattccgtgaggttgtcccggcaactccgcagaatactcctgcccctttatgaggtcgacgatctcagagacgcctttagaactgataaaaagtattcaatcggacttgctattgggacaaactctgtgggctgggcggtaattaccgacgagtacaaggtgcctagtaagaaatttaaagtgctcggaaacactgacaggcactctataaagaagaacctgatcggggcactgcttttcgactccggagagacggcggaggcgacgcgtctcaagcgtaccgcgcgccgcaggtacacaagaaggaagaataggatctgctacttgcaggaaatcttcagtaacgagatggcgaaggtcgacgatagtttctttcatcggttggaagaatcgttcctcgtagaggaggacaaaaagcacgagcgtcacccaatattcgggaatattgttgacgaggttgcctaccatgagaaatatcctacaatatatcacctccgtaagaagcttgtcgattcaactgataaggctgatctcagactcatctatcttgccctcgcacatatgattaagtttcgtggccacttcttgattgaaggcgacctcaacccggacaactcagatgttgacaagctttttatacagctcgtccagacatataaccagctgtttgaagagaatcccatcaatgcgagtggggttgatgctaacgccattttgtccgccaggttgtccaaatctcgcagactggaaaacctgatcgcacagcttcccggtgaaaagaaaaacgggctcttcggcaatctcatcgcactgtccctcggcctcaccccaaacttcaagtctaacttcgacctggccgaggatgcgaagctccagctgtcaaaagatacatacgacgacgatttggacaatctgcttgcgcaaataggcgaccagtatgcggacctgttcctggctgccaaaaatctgtcagatgcaatcctcctgtccgatatattgcgtgtgaacaccgaaatcacgaaggcaccgcttagcgcatccatgatcaagagatacgacgagcaccatcaggacctcacactcctcaaggcgcttgttcgtcagcagcttcccgagaaatataaggaaatttttttcgatcaaagcaagaatggatatgctggctatattgacggtggcgcttcgcaggaggagttctataaattcattaagccgattctggagaagatggacggaacggaggagctcctcgtcaagcttaaccgggaagacctgttgcggaagcagaggacttttgataacggctctattccgcaccaaatccatctgggtgagttgcacgcaatcttgagaagacaagaggatttctacccgttccttaaggataacagagagaagatagaaaaaatactgaccttcaggataccatactatgtgggcccactggcgcgcggaaatagtcgtttcgcatggatgactagaaagtccgaagaaacgatcacgccatggaattttgaggaagtggtcgacaagggcgcctctgcccagagcttcatcgaaaggatgaccaattttgacaaaaatctgcctaacgaaaaggtgcttccgaagcacagcctgttgtatgaatacttcacagtttataacgagctcactaaggtcaagtacgtcacggagggcatgcgtaagcctgctttcctgtctggtgaacaaaaaaaggcgattgtggacctccttttcaagacgaaccgtaaagttactgtgaagcaactgaaagaggattactttaagaaaattgagtgcttcgacagtgtggagatttccggtgtcgaggaccggtttaacgccagcctgggtacgtatcatgacctgcttaaaattatcaaggataaagatttcctggataatgaagagaacgaagatatactggaggacattgtgttgactttgaccctcttcgaggacagagagatgattgaggaaagactgaagacctacgcacacctttttgatgacaaggtcatgaaacaactcaagcgccggcgctatactggctggggccggctttctcgcaagctcatcaatgggattcgggataagcaatcaggcaagacaattttggacttcctcaaatccgacggattcgcaaataggaattttatgcagctgatacatgacgactctttgacattcaaagaagacatacagaaggctcaggtctccggccaaggagattctttgcacgagcatatcgctaacttggcaggtagccccgccataaaaaagggcattcttcaaacggtaaaagttgttgacgaactcgtgaaggttatgggccgtcataagccggaaaacattgttattgaaatggctagggaaaatcagacgacccagaagggacagaaaaatagcagggagcggatgaagagaattgaagagggaattaaggagcttggatctcagattcttaaggagcaccctgtggagaacacccaacttcagaatgaaaagctctacctttactaccttcaaaacggccgggatatgtacgtcgatcaggaacttgacattaaccggttgagcgattatgacgttgaccatattgtgccccaatctttccttaaagacgactctatcgacaataaagtgctgacgcgcagcgataaaaatcgcggtaagtcggataatgtcccgtcggaagaggtggttaaaaaaatgaagaactattggaggcaactcctgaatgccaagctgatcactcagaggaaattcgacaatctcaccaaggcagaaaggggtggacttagcgagctcgacaaggccggttttatcaaaagacagctggtggagacacgccaaatcaccaaacacgttgcccagatcctggattcgaggatgaacacgaagtatgacgagaacgacaagttgattagggaagtcaaggtcatcactttgaagtccaagctggtgagcgactttcgcaaagacttccagttttacaaagtcagggaaattaataactaccaccacgcccacgacgcctaccttaacgccgtggttggcacagcactcatcaagaaataccctaagctcgaatctgagttcgtctatggcgactataaggtctacgacgttagaaaaatgatcgcgaaatctgagcaggaaataggcaaggcaactgccaagtacttcttctattccaatatcatgaacttttttaagacggagattaccctggcgaatggtgagatccgcaagcgccctttgattgagacaaacggagaaacaggagagatcgtatgggacaaagggcgggactttgctactgttaggaaggtgctctctatgccacaagttaacattgtcaaaaaaactgaagtgcagacaggtgggtttagcaaggaatctatcctgccgaagaggaactctgacaagctgatcgcccgcaagaaagattgggatccgaaaaagtacggaggattcgactcccccacagttgcgtactccgtgcttgtcgtggccaaagtggagaagggcaagtctaagaagctcaagagcgtcaaagagttgttggggatcacgattatggagcggtcgtctttcgaaaagaatccgatagattttctcgaggccaagggttataaagaagtcaagaaggatcttatcatcaagctccctaagtactccctctttgagcttgaaaacggacggaaaagaatgctggcttcagcgggtgaacttcagaagggtaatgaactcgctctgccctcaaaatatgtgaatttcctttacctggcatcacactatgagaagcttaaggggtctccagaggacaacgagcagaagcaactgttcgttgaacaacacaagcactaccttgacgagattatcgagcaaatcagcgagtttagcaagcgcgttatactggcagacgcaaatcttgataaggtccttagcgcctacaacaagcatagagacaaacccatccgggagcaggccgagaacattattcatctcttcaccttgacgaatcttggggccccggccgcgttcaagtacttcgatactaccatagacagaaagcgctatacatcgacaaaggaagttcttgacgccacgctgatccaccaaagtataacaggcctctatgagacacgcatcgacctttcgcagttgggcggtgaccgccccaaaaagaagaggaaagttggcgggtga；

<210>8

<211>4767

<212>dna

<213>人工序列

<223>seqidno.8

<400>8

atggatagccttctcatgaacagaagagagtttctctatcagtttaaaaatgttcggtgggcgaaggggaggagagagacatatctctgctatgttgttaagcggagagattctgcgacctcattctcactcgattttggttatttgaggaacaagaatggatgtcatgtcgaattgttgtttctccggtatatttccgactgggatttggacccagggcggtgttaccgggtcacatggtttatttcctggagtccatgttacgactgtgcgcgccatgtcgccgacttcctcaggggtaatcctaacttgtccttgcggatttttacagccagactctatttctgtgaggatcggaaggcggaacccgaggggctgagaagactgcaccgcgctggcgtccaaatcgccatcatgacttttaaggattatttctactgttggaacacgttcgtcgagaaccacggtcggaccttcaaagcctgggaagggctgcatgaaaattccgtgaggttgtcccggcaactccgcagaatactcctgcccctttatgaggtcgacgatctcagagacgcctttagaactagcggaagcgagacgccagggacttctgaatcggccacccccgagagcgataaaaagtattcaatcggacttgctattgggacaaactctgtgggctgggcggtaattaccgacgagtacaaggtgcctagtaagaaatttaaagtgctcggaaacactgacaggcactctataaagaagaacctgatcggggcactgcttttcgactccggagagacggcggaggcgacgcgtctcaagcgtaccgcgcgccgcaggtacacaagaaggaagaataggatctgctacttgcaggaaatcttcagtaacgagatggcgaaggtcgacgatagtttctttcatcggttggaagaatcgttcctcgtagaggaggacaaaaagcacgagcgtcacccaatattcgggaatattgttgacgaggttgcctaccatgagaaatatcctacaatatatcacctccgtaagaagcttgtcgattcaactgataaggctgatctcagactcatctatcttgccctcgcacatatgattaagtttcgtggccacttcttgattgaaggcgacctcaacccggacaactcagatgttgacaagctttttatacagctcgtccagacatataaccagctgtttgaagagaatcccatcaatgcgagtggggttgatgctaacgccattttgtccgccaggttgtccaaatctcgcagactggaaaacctgatcgcacagcttcccggtgaaaagaaaaacgggctcttcggcaatctcatcgcactgtccctcggcctcaccccaaacttcaagtctaacttcgacctggccgaggatgcgaagctccagctgtcaaaagatacatacgacgacgatttggacaatctgcttgcgcaaataggcgaccagtatgcggacctgttcctggctgccaaaaatctgtcagatgcaatcctcctgtccgatatattgcgtgtgaacaccgaaatcacgaaggcaccgcttagcgcatccatgatcaagagatacgacgagcaccatcaggacctcacactcctcaaggcgcttgttcgtcagcagcttcccgagaaatataaggaaatttttttcgatcaaagcaagaatggatatgctggctatattgacggtggcgcttcgcaggaggagttctataaattcattaagccgattctggagaagatggacggaacggaggagctcctcgtcaagcttaaccgggaagacctgttgcggaagcagaggacttttgataacggctctattccgcaccaaatccatctgggtgagttgcacgcaatcttgagaagacaagaggatttctacccgttccttaaggataacagagagaagatagaaaaaatactgaccttcaggataccatactatgtgggcccactggcgcgcggaaatagtcgtttcgcatggatgactagaaagtccgaagaaacgatcacgccatggaattttgaggaagtggtcgacaagggcgcctctgcccagagcttcatcgaaaggatgaccaattttgacaaaaatctgcctaacgaaaaggtgcttccgaagcacagcctgttgtatgaatacttcacagtttataacgagctcactaaggtcaagtacgtcacggagggcatgcgtaagcctgctttcctgtctggtgaacaaaaaaaggcgattgtggacctccttttcaagacgaaccgtaaagttactgtgaagcaactgaaagaggattactttaagaaaattgagtgcttcgacagtgtggagatttccggtgtcgaggaccggtttaacgccagcctgggtacgtatcatgacctgcttaaaattatcaaggataaagatttcctggataatgaagagaacgaagatatactggaggacattgtgttgactttgaccctcttcgaggacagagagatgattgaggaaagactgaagacctacgcacacctttttgatgacaaggtcatgaaacaactcaagcgccggcgctatactggctggggccggctttctcgcaagctcatcaatgggattcgggataagcaatcaggcaagacaattttggacttcctcaaatccgacggattcgcaaataggaattttatgcagctgatacatgacgactctttgacattcaaagaagacatacagaaggctcaggtctccggccaaggagattctttgcacgagcatatcgctaacttggcaggtagccccgccataaaaaagggcattcttcaaacggtaaaagttgttgacgaactcgtgaaggttatgggccgtcataagccggaaaacattgttattgaaatggctagggaaaatcagacgacccagaagggacagaaaaatagcagggagcggatgaagagaattgaagagggaattaaggagcttggatctcagattcttaaggagcaccctgtggagaacacccaacttcagaatgaaaagctctacctttactaccttcaaaacggccgggatatgtacgtcgatcaggaacttgacattaaccggttgagcgattatgacgttgaccatattgtgccccaatctttccttaaagacgactctatcgacaataaagtgctgacgcgcagcgataaaaatcgcggtaagtcggataatgtcccgtcggaagaggtggttaaaaaaatgaagaactattggaggcaactcctgaatgccaagctgatcactcagaggaaattcgacaatctcaccaaggcagaaaggggtggacttagcgagctcgacaaggccggttttatcaaaagacagctggtggagacacgccaaatcaccaaacacgttgcccagatcctggattcgaggatgaacacgaagtatgacgagaacgacaagttgattagggaagtcaaggtcatcactttgaagtccaagctggtgagcgactttcgcaaagacttccagttttacaaagtcagggaaattaataactaccaccacgcccacgacgcctaccttaacgccgtggttggcacagcactcatcaagaaataccctaagctcgaatctgagttcgtctatggcgactataaggtctacgacgttagaaaaatgatcgcgaaatctgagcaggaaataggcaaggcaactgccaagtacttcttctattccaatatcatgaacttttttaagacggagattaccctggcgaatggtgagatccgcaagcgccctttgattgagacaaacggagaaacaggagagatcgtatgggacaaagggcgggactttgctactgttaggaaggtgctctctatgccacaagttaacattgtcaaaaaaactgaagtgcagacaggtgggtttagcaaggaatctatcctgccgaagaggaactctgacaagctgatcgcccgcaagaaagattgggatccgaaaaagtacggaggattcgactcccccacagttgcgtactccgtgcttgtcgtggccaaagtggagaagggcaagtctaagaagctcaagagcgtcaaagagttgttggggatcacgattatggagcggtcgtctttcgaaaagaatccgatagattttctcgaggccaagggttataaagaagtcaagaaggatcttatcatcaagctccctaagtactccctctttgagcttgaaaacggacggaaaagaatgctggcttcagcgggtgaacttcagaagggtaatgaactcgctctgccctcaaaatatgtgaatttcctttacctggcatcacactatgagaagcttaaggggtctccagaggacaacgagcagaagcaactgttcgttgaacaacacaagcactaccttgacgagattatcgagcaaatcagcgagtttagcaagcgcgttatactggcagacgcaaatcttgataaggtccttagcgcctacaacaagcatagagacaaacccatccgggagcaggccgagaacattattcatctcttcaccttgacgaatcttggggccccggccgcgttcaagtacttcgatactaccatagacagaaagcgctatacatcgacaaaggaagttcttgacgccacgctgatccaccaaagtataacaggcctctatgagacacgcatcgacctttcgcagttgggcggtgaccgccccaaaaagaagaggaaagttggcgggtga；

<210>9

<211>5031

<212>dna

<213>人工序列

<223>seqidno.9

<400>9

atggatagccttctcatgaacagaagagagtttctctatcagtttaaaaatgttcggtgggcgaaggggaggagagagacatatctctgctatgttgttaagcggagagattctgcgacctcattctcactcgattttggttatttgaggaacaagaatggatgtcatgtcgaattgttgtttctccggtatatttccgactgggatttggacccagggcggtgttaccgggtcacatggtttatttcctggagtccatgttacgactgtgcgcgccatgtcgccgacttcctcaggggtaatcctaacttgtccttgcggatttttacagccagactctatttctgtgaggatcggaaggcggaacccgaggggctgagaagactgcaccgcgctggcgtccaaatcgccatcatgacttttaaggattatttctactgttggaacacgttcgtcgagaaccacggtcggaccttcaaagcctgggaagggctgcatgaaaattccgtgaggttgtcccggcaactccgcagaatactcctgcccctttatgaggtcgacgatctcagagacgcctttagaactagcggaagcgagacgccagggacttctgaatcggccacccccgagagcgataaaaagtattcaatcggacttgctattgggacaaactctgtgggctgggcggtaattaccgacgagtacaaggtgcctagtaagaaatttaaagtgctcggaaacactgacaggcactctataaagaagaacctgatcggggcactgcttttcgactccggagagacggcggaggcgacgcgtctcaagcgtaccgcgcgccgcaggtacacaagaaggaagaataggatctgctacttgcaggaaatcttcagtaacgagatggcgaaggtcgacgatagtttctttcatcggttggaagaatcgttcctcgtagaggaggacaaaaagcacgagcgtcacccaatattcgggaatattgttgacgaggttgcctaccatgagaaatatcctacaatatatcacctccgtaagaagcttgtcgattcaactgataaggctgatctcagactcatctatcttgccctcgcacatatgattaagtttcgtggccacttcttgattgaaggcgacctcaacccggacaactcagatgttgacaagctttttatacagctcgtccagacatataaccagctgtttgaagagaatcccatcaatgcgagtggggttgatgctaacgccattttgtccgccaggttgtccaaatctcgcagactggaaaacctgatcgcacagcttcccggtgaaaagaaaaacgggctcttcggcaatctcatcgcactgtccctcggcctcaccccaaacttcaagtctaacttcgacctggccgaggatgcgaagctccagctgtcaaaagatacatacgacgacgatttggacaatctgcttgcgcaaataggcgaccagtatgcggacctgttcctggctgccaaaaatctgtcagatgcaatcctcctgtccgatatattgcgtgtgaacaccgaaatcacgaaggcaccgcttagcgcatccatgatcaagagatacgacgagcaccatcaggacctcacactcctcaaggcgcttgttcgtcagcagcttcccgagaaatataaggaaatttttttcgatcaaagcaagaatggatatgctggctatattgacggtggcgcttcgcaggaggagttctataaattcattaagccgattctggagaagatggacggaacggaggagctcctcgtcaagcttaaccgggaagacctgttgcggaagcagaggacttttgataacggctctattccgcaccaaatccatctgggtgagttgcacgcaatcttgagaagacaagaggatttctacccgttccttaaggataacagagagaagatagaaaaaatactgaccttcaggataccatactatgtgggcccactggcgcgcggaaatagtcgtttcgcatggatgactagaaagtccgaagaaacgatcacgccatggaattttgaggaagtggtcgacaagggcgcctctgcccagagcttcatcgaaaggatgaccaattttgacaaaaatctgcctaacgaaaaggtgcttccgaagcacagcctgttgtatgaatacttcacagtttataacgagctcactaaggtcaagtacgtcacggagggcatgcgtaagcctgctttcctgtctggtgaacaaaaaaaggcgattgtggacctccttttcaagacgaaccgtaaagttactgtgaagcaactgaaagaggattactttaagaaaattgagtgcttcgacagtgtggagatttccggtgtcgaggaccggtttaacgccagcctgggtacgtatcatgacctgcttaaaattatcaaggataaagatttcctggataatgaagagaacgaagatatactggaggacattgtgttgactttgaccctcttcgaggacagagagatgattgaggaaagactgaagacctacgcacacctttttgatgacaaggtcatgaaacaactcaagcgccggcgctatactggctggggccggctttctcgcaagctcatcaatgggattcgggataagcaatcaggcaagacaattttggacttcctcaaatccgacggattcgcaaataggaattttatgcagctgatacatgacgactctttgacattcaaagaagacatacagaaggctcaggtctccggccaaggagattctttgcacgagcatatcgctaacttggcaggtagccccgccataaaaaagggcattcttcaaacggtaaaagttgttgacgaactcgtgaaggttatgggccgtcataagccggaaaacattgttattgaaatggctagggaaaatcagacgacccagaagggacagaaaaatagcagggagcggatgaagagaattgaagagggaattaaggagcttggatctcagattcttaaggagcaccctgtggagaacacccaacttcagaatgaaaagctctacctttactaccttcaaaacggccgggatatgtacgtcgatcaggaacttgacattaaccggttgagcgattatgacgttgaccatattgtgccccaatctttccttaaagacgactctatcgacaataaagtgctgacgcgcagcgataaaaatcgcggtaagtcggataatgtcccgtcggaagaggtggttaaaaaaatgaagaactattggaggcaactcctgaatgccaagctgatcactcagaggaaattcgacaatctcaccaaggcagaaaggggtggacttagcgagctcgacaaggccggttttatcaaaagacagctggtggagacacgccaaatcaccaaacacgttgcccagatcctggattcgaggatgaacacgaagtatgacgagaacgacaagttgattagggaagtcaaggtcatcactttgaagtccaagctggtgagcgactttcgcaaagacttccagttttacaaagtcagggaaattaataactaccaccacgcccacgacgcctaccttaacgccgtggttggcacagcactcatcaagaaataccctaagctcgaatctgagttcgtctatggcgactataaggtctacgacgttagaaaaatgatcgcgaaatctgagcaggaaataggcaaggcaactgccaagtacttcttctattccaatatcatgaacttttttaagacggagattaccctggcgaatggtgagatccgcaagcgccctttgattgagacaaacggagaaacaggagagatcgtatgggacaaagggcgggactttgctactgttaggaaggtgctctctatgccacaagttaacattgtcaaaaaaactgaagtgcagacaggtgggtttagcaaggaatctatcctgccgaagaggaactctgacaagctgatcgcccgcaagaaagattgggatccgaaaaagtacggaggattcgactcccccacagttgcgtactccgtgcttgtcgtggccaaagtggagaagggcaagtctaagaagctcaagagcgtcaaagagttgttggggatcacgattatggagcggtcgtctttcgaaaagaatccgatagattttctcgaggccaagggttataaagaagtcaagaaggatcttatcatcaagctccctaagtactccctctttgagcttgaaaacggacggaaaagaatgctggcttcagcgggtgaacttcagaagggtaatgaactcgctctgccctcaaaatatgtgaatttcctttacctggcatcacactatgagaagcttaaggggtctccagaggacaacgagcagaagcaactgttcgttgaacaacacaagcactaccttgacgagattatcgagcaaatcagcgagtttagcaagcgcgttatactggcagacgcaaatcttgataaggtccttagcgcctacaacaagcatagagacaaacccatccgggagcaggccgagaacattattcatctcttcaccttgacgaatcttggggccccggccgcgttcaagtacttcgatactaccatagacagaaagcgctatacatcgacaaaggaagttcttgacgccacgctgatccaccaaagtataacaggcctctatgagacacgcatcgacctttcgcagttgggcggtgactccggcggaagtacaaacctttcagacattatagaaaaggaaaccggcaagcaactcgtcatccaggaatccatacttatgctccctgaagaggtggaagaagtgatcggtaataaaccagagagcgacatacttgtccacaccgcttatgacgaaagtacagacgaaaacgtcatgcttctgacgagtgatgcccccgaatacaaaccttgggcgctcgtcatccaggattccaatggggagaataaaataaagatgctctctggaggcagcccaaagaagaagagaaaggtctga；

<210>10

<211>4719

<212>dna

<213>人工序列

<223>seqidno.10

<400>10

atggatagccttctcatgaacagaagagagtttctctatcagtttaaaaatgttcggtgggcgaaggggaggagagagacatatctctgctatgttgttaagcggagagattctgcgacctcattctcactcgattttggttatttgaggaacaagaatggatgtcatgtcgaattgttgtttctccggtatatttccgactgggatttggacccagggcggtgttaccgggtcacatggtttatttcctggagtccatgttacgactgtgcgcgccatgtcgccgacttcctcaggggtaatcctaacttgtccttgcggatttttacagccagactctatttctgtgaggatcggaaggcggaacccgaggggctgagaagactgcaccgcgctggcgtccaaatcgccatcatgacttttaaggattatttctactgttggaacacgttcgtcgagaaccacggtcggaccttcaaagcctgggaagggctgcatgaaaattccgtgaggttgtcccggcaactccgcagaatactcctgcccctttatgaggtcgacgatctcagagacgcctttagaactgataaaaagtattcaatcggacttgctattgggacaaactctgtgggctgggcggtaattaccgacgagtacaaggtgcctagtaagaaatttaaagtgctcggaaacactgacaggcactctataaagaagaacctgatcggggcactgcttttcgactccggagagacggcggaggcgacgcgtctcaagcgtaccgcgcgccgcaggtacacaagaaggaagaataggatctgctacttgcaggaaatcttcagtaacgagatggcgaaggtcgacgatagtttctttcatcggttggaagaatcgttcctcgtagaggaggacaaaaagcacgagcgtcacccaatattcgggaatattgttgacgaggttgcctaccatgagaaatatcctacaatatatcacctccgtaagaagcttgtcgattcaactgataaggctgatctcagactcatctatcttgccctcgcacatatgattaagtttcgtggccacttcttgattgaaggcgacctcaacccggacaactcagatgttgacaagctttttatacagctcgtccagacatataaccagctgtttgaagagaatcccatcaatgcgagtggggttgatgctaacgccattttgtccgccaggttgtccaaatctcgcagactggaaaacctgatcgcacagcttcccggtgaaaagaaaaacgggctcttcggcaatctcatcgcactgtccctcggcctcaccccaaacttcaagtctaacttcgacctggccgaggatgcgaagctccagctgtcaaaagatacatacgacgacgatttggacaatctgcttgcgcaaataggcgaccagtatgcggacctgttcctggctgccaaaaatctgtcagatgcaatcctcctgtccgatatattgcgtgtgaacaccgaaatcacgaaggcaccgcttagcgcatccatgatcaagagatacgacgagcaccatcaggacctcacactcctcaaggcgcttgttcgtcagcagcttcccgagaaatataaggaaatttttttcgatcaaagcaagaatggatatgctggctatattgacggtggcgcttcgcaggaggagttctataaattcattaagccgattctggagaagatggacggaacggaggagctcctcgtcaagcttaaccgggaagacctgttgcggaagcagaggacttttgataacggctctattccgcaccaaatccatctgggtgagttgcacgcaatcttgagaagacaagaggatttctacccgttccttaaggataacagagagaagatagaaaaaatactgaccttcaggataccatactatgtgggcccactggcgcgcggaaatagtcgtttcgcatggatgactagaaagtccgaagaaacgatcacgccatggaattttgaggaagtggtcgacaagggcgcctctgcccagagcttcatcgaaaggatgaccaattttgacaaaaatctgcctaacgaaaaggtgcttccgaagcacagcctgttgtatgaatacttcacagtttataacgagctcactaaggtcaagtacgtcacggagggcatgcgtaagcctgctttcctgtctggtgaacaaaaaaaggcgattgtggacctccttttcaagacgaaccgtaaagttactgtgaagcaactgaaagaggattactttaagaaaattgagtgcttcgacagtgtggagatttccggtgtcgaggaccggtttaacgccagcctgggtacgtatcatgacctgcttaaaattatcaaggataaagatttcctggataatgaagagaacgaagatatactggaggacattgtgttgactttgaccctcttcgaggacagagagatgattgaggaaagactgaagacctacgcacacctttttgatgacaaggtcatgaaacaactcaagcgccggcgctatactggctggggccggctttctcgcaagctcatcaatgggattcgggataagcaatcaggcaagacaattttggacttcctcaaatccgacggattcgcaaataggaattttatgcagctgatacatgacgactctttgacattcaaagaagacatacagaaggctcaggtctccggccaaggagattctttgcacgagcatatcgctaacttggcaggtagccccgccataaaaaagggcattcttcaaacggtaaaagttgttgacgaactcgtgaaggttatgggccgtcataagccggaaaacattgttattgaaatggctagggaaaatcagacgacccagaagggacagaaaaatagcagggagcggatgaagagaattgaagagggaattaaggagcttggatctcagattcttaaggagcaccctgtggagaacacccaacttcagaatgaaaagctctacctttactaccttcaaaacggccgggatatgtacgtcgatcaggaacttgacattaaccggttgagcgattatgacgttgaccatattgtgccccaatctttccttaaagacgactctatcgacaataaagtgctgacgcgcagcgataaaaatcgcggtaagtcggataatgtcccgtcggaagaggtggttaaaaaaatgaagaactattggaggcaactcctgaatgccaagctgatcactcagaggaaattcgacaatctcaccaaggcagaaaggggtggacttagcgagctcgacaaggccggttttatcaaaagacagctggtggagacacgccaaatcaccaaacacgttgcccagatcctggattcgaggatgaacacgaagtatgacgagaacgacaagttgattagggaagtcaaggtcatcactttgaagtccaagctggtgagcgactttcgcaaagacttccagttttacaaagtcagggaaattaataactaccaccacgcccacgacgcctaccttaacgccgtggttggcacagcactcatcaagaaataccctaagctcgaatctgagttcgtctatggcgactataaggtctacgacgttagaaaaatgatcgcgaaatctgagcaggaaataggcaaggcaactgccaagtacttcttctattccaatatcatgaacttttttaagacggagattaccctggcgaatggtgagatccgcaagcgccctttgattgagacaaacggagaaacaggagagatcgtatgggacaaagggcgggactttgctactgttaggaaggtgctctctatgccacaagttaacattgtcaaaaaaactgaagtgcagacaggtgggtttagcaaggaatctatcctgccgaagaggaactctgacaagctgatcgcccgcaagaaagattgggatccgaaaaagtacggaggattcgtctcccccacagttgcgtactccgtgcttgtcgtggccaaagtggagaagggcaagtctaagaagctcaagagcgtcaaagagttgttggggatcacgattatggagcggtcgtctttcgaaaagaatccgatagattttctcgaggccaagggttataaagaagtcaagaaggatcttatcatcaagctccctaagtactccctctttgagcttgaaaacggacggaaaagaatgctggcttcagcgggtgaacttcagaagggtaatgaactcgctctgccctcaaaatatgtgaatttcctttacctggcatcacactatgagaagcttaaggggtctccagaggacaacgagcagaagcaactgttcgttgaacaacacaagcactaccttgacgagattatcgagcaaatcagcgagtttagcaagcgcgttatactggcagacgcaaatcttgataaggtccttagcgcctacaacaagcatagagacaaacccatccgggagcaggccgagaacattattcatctcttcaccttgacgaatcttggggccccggccgcgttcaagtacttcgatactaccatagacagaaagcgctatacatcgacaaaggaagttcttgacgccacgctgatccaccaaagtataacaggcctctatgagacacgcatcgacctttcgcagttgggcggtgaccgccccaaaaagaagaggaaagttggcgggtga；

<210>11

<211>4767

<212>dna

<213>人工序列

<223>seqidno.11

<400>11

atggatagccttctcatgaacagaagagagtttctctatcagtttaaaaatgttcggtgggcgaaggggaggagagagacatatctctgctatgttgttaagcggagagattctgcgacctcattctcactcgattttggttatttgaggaacaagaatggatgtcatgtcgaattgttgtttctccggtatatttccgactgggatttggacccagggcggtgttaccgggtcacatggtttatttcctggagtccatgttacgactgtgcgcgccatgtcgccgacttcctcaggggtaatcctaacttgtccttgcggatttttacagccagactctatttctgtgaggatcggaaggcggaacccgaggggctgagaagactgcaccgcgctggcgtccaaatcgccatcatgacttttaaggattatttctactgttggaacacgttcgtcgagaaccacggtcggaccttcaaagcctgggaagggctgcatgaaaattccgtgaggttgtcccggcaactccgcagaatactcctgcccctttatgaggtcgacgatctcagagacgcctttagaactagcggaagcgagacgccagggacttctgaatcggccacccccgagagcgataaaaagtattcaatcggacttgctattgggacaaactctgtgggctgggcggtaattaccgacgagtacaaggtgcctagtaagaaatttaaagtgctcggaaacactgacaggcactctataaagaagaacctgatcggggcactgcttttcgactccggagagacggcggaggcgacgcgtctcaagcgtaccgcgcgccgcaggtacacaagaaggaagaataggatctgctacttgcaggaaatcttcagtaacgagatggcgaaggtcgacgatagtttctttcatcggttggaagaatcgttcctcgtagaggaggacaaaaagcacgagcgtcacccaatattcgggaatattgttgacgaggttgcctaccatgagaaatatcctacaatatatcacctccgtaagaagcttgtcgattcaactgataaggctgatctcagactcatctatcttgccctcgcacatatgattaagtttcgtggccacttcttgattgaaggcgacctcaacccggacaactcagatgttgacaagctttttatacagctcgtccagacatataaccagctgtttgaagagaatcccatcaatgcgagtggggttgatgctaacgccattttgtccgccaggttgtccaaatctcgcagactggaaaacctgatcgcacagcttcccggtgaaaagaaaaacgggctcttcggcaatctcatcgcactgtccctcggcctcaccccaaacttcaagtctaacttcgacctggccgaggatgcgaagctccagctgtcaaaagatacatacgacgacgatttggacaatctgcttgcgcaaataggcgaccagtatgcggacctgttcctggctgccaaaaatctgtcagatgcaatcctcctgtccgatatattgcgtgtgaacaccgaaatcacgaaggcaccgcttagcgcatccatgatcaagagatacgacgagcaccatcaggacctcacactcctcaaggcgcttgttcgtcagcagcttcccgagaaatataaggaaatttttttcgatcaaagcaagaatggatatgctggctatattgacggtggcgcttcgcaggaggagttctataaattcattaagccgattctggagaagatggacggaacggaggagctcctcgtcaagcttaaccgggaagacctgttgcggaagcagaggacttttgataacggctctattccgcaccaaatccatctgggtgagttgcacgcaatcttgagaagacaagaggatttctacccgttccttaaggataacagagagaagatagaaaaaatactgaccttcaggataccatactatgtgggcccactggcgcgcggaaatagtcgtttcgcatggatgactagaaagtccgaagaaacgatcacgccatggaattttgaggaagtggtcgacaagggcgcctctgcccagagcttcatcgaaaggatgaccaattttgacaaaaatctgcctaacgaaaaggtgcttccgaagcacagcctgttgtatgaatacttcacagtttataacgagctcactaaggtcaagtacgtcacggagggcatgcgtaagcctgctttcctgtctggtgaacaaaaaaaggcgattgtggacctccttttcaagacgaaccgtaaagttactgtgaagcaactgaaagaggattactttaagaaaattgagtgcttcgacagtgtggagatttccggtgtcgaggaccggtttaacgccagcctgggtacgtatcatgacctgcttaaaattatcaaggataaagatttcctggataatgaagagaacgaagatatactggaggacattgtgttgactttgaccctcttcgaggacagagagatgattgaggaaagactgaagacctacgcacacctttttgatgacaaggtcatgaaacaactcaagcgccggcgctatactggctggggccggctttctcgcaagctcatcaatgggattcgggataagcaatcaggcaagacaattttggacttcctcaaatccgacggattcgcaaataggaattttatgcagctgatacatgacgactctttgacattcaaagaagacatacagaaggctcaggtctccggccaaggagattctttgcacgagcatatcgctaacttggcaggtagccccgccataaaaaagggcattcttcaaacggtaaaagttgttgacgaactcgtgaaggttatgggccgtcataagccggaaaacattgttattgaaatggctagggaaaatcagacgacccagaagggacagaaaaatagcagggagcggatgaagagaattgaagagggaattaaggagcttggatctcagattcttaaggagcaccctgtggagaacacccaacttcagaatgaaaagctctacctttactaccttcaaaacggccgggatatgtacgtcgatcaggaacttgacattaaccggttgagcgattatgacgttgaccatattgtgccccaatctttccttaaagacgactctatcgacaataaagtgctgacgcgcagcgataaaaatcgcggtaagtcggataatgtcccgtcggaagaggtggttaaaaaaatgaagaactattggaggcaactcctgaatgccaagctgatcactcagaggaaattcgacaatctcaccaaggcagaaaggggtggacttagcgagctcgacaaggccggttttatcaaaagacagctggtggagacacgccaaatcaccaaacacgttgcccagatcctggattcgaggatgaacacgaagtatgacgagaacgacaagttgattagggaagtcaaggtcatcactttgaagtccaagctggtgagcgactttcgcaaagacttccagttttacaaagtcagggaaattaataactaccaccacgcccacgacgcctaccttaacgccgtggttggcacagcactcatcaagaaataccctaagctcgaatctgagttcgtctatggcgactataaggtctacgacgttagaaaaatgatcgcgaaatctgagcaggaaataggcaaggcaactgccaagtacttcttctattccaatatcatgaacttttttaagacggagattaccctggcgaatggtgagatccgcaagcgccctttgattgagacaaacggagaaacaggagagatcgtatgggacaaagggcgggactttgctactgttaggaaggtgctctctatgccacaagttaacattgtcaaaaaaactgaagtgcagacaggtgggtttagcaaggaatctatcctgccgaagaggaactctgacaagctgatcgcccgcaagaaagattgggatccgaaaaagtacggaggattcgtctcccccacagttgcgtactccgtgcttgtcgtggccaaagtggagaagggcaagtctaagaagctcaagagcgtcaaagagttgttggggatcacgattatggagcggtcgtctttcgaaaagaatccgatagattttctcgaggccaagggttataaagaagtcaagaaggatcttatcatcaagctccctaagtactccctctttgagcttgaaaacggacggaaaagaatgctggcttcagcgggtgaacttcagaagggtaatgaactcgctctgccctcaaaatatgtgaatttcctttacctggcatcacactatgagaagcttaaggggtctccagaggacaacgagcagaagcaactgttcgttgaacaacacaagcactaccttgacgagattatcgagcaaatcagcgagtttagcaagcgcgttatactggcagacgcaaatcttgataaggtccttagcgcctacaacaagcatagagacaaacccatccgggagcaggccgagaacattattcatctcttcaccttgacgaatcttggggccccggccgcgttcaagtacttcgatactaccatagacagaaagcgctatacatcgacaaaggaagttcttgacgccacgctgatccaccaaagtataacaggcctctatgagacacgcatcgacctttcgcagttgggcggtgaccgccccaaaaagaagaggaaagttggcgggtga；

<210>12

<211>5031

<212>dna

<213>人工序列

<223>seqidno.12

<400>12

atggatagccttctcatgaacagaagagagtttctctatcagtttaaaaatgttcggtgggcgaaggggaggagagagacatatctctgctatgttgttaagcggagagattctgcgacctcattctcactcgattttggttatttgaggaacaagaatggatgtcatgtcgaattgttgtttctccggtatatttccgactgggatttggacccagggcggtgttaccgggtcacatggtttatttcctggagtccatgttacgactgtgcgcgccatgtcgccgacttcctcaggggtaatcctaacttgtccttgcggatttttacagccagactctatttctgtgaggatcggaaggcggaacccgaggggctgagaagactgcaccgcgctggcgtccaaatcgccatcatgacttttaaggattatttctactgttggaacacgttcgtcgagaaccacggtcggaccttcaaagcctgggaagggctgcatgaaaattccgtgaggttgtcccggcaactccgcagaatactcctgcccctttatgaggtcgacgatctcagagacgcctttagaactagcggaagcgagacgccagggacttctgaatcggccacccccgagagcgataaaaagtattcaatcggacttgctattgggacaaactctgtgggctgggcggtaattaccgacgagtacaaggtgcctagtaagaaatttaaagtgctcggaaacactgacaggcactctataaagaagaacctgatcggggcactgcttttcgactccggagagacggcggaggcgacgcgtctcaagcgtaccgcgcgccgcaggtacacaagaaggaagaataggatctgctacttgcaggaaatcttcagtaacgagatggcgaaggtcgacgatagtttctttcatcggttggaagaatcgttcctcgtagaggaggacaaaaagcacgagcgtcacccaatattcgggaatattgttgacgaggttgcctaccatgagaaatatcctacaatatatcacctccgtaagaagcttgtcgattcaactgataaggctgatctcagactcatctatcttgccctcgcacatatgattaagtttcgtggccacttcttgattgaaggcgacctcaacccggacaactcagatgttgacaagctttttatacagctcgtccagacatataaccagctgtttgaagagaatcccatcaatgcgagtggggttgatgctaacgccattttgtccgccaggttgtccaaatctcgcagactggaaaacctgatcgcacagcttcccggtgaaaagaaaaacgggctcttcggcaatctcatcgcactgtccctcggcctcaccccaaacttcaagtctaacttcgacctggccgaggatgcgaagctccagctgtcaaaagatacatacgacgacgatttggacaatctgcttgcgcaaataggcgaccagtatgcggacctgttcctggctgccaaaaatctgtcagatgcaatcctcctgtccgatatattgcgtgtgaacaccgaaatcacgaaggcaccgcttagcgcatccatgatcaagagatacgacgagcaccatcaggacctcacactcctcaaggcgcttgttcgtcagcagcttcccgagaaatataaggaaatttttttcgatcaaagcaagaatggatatgctggctatattgacggtggcgcttcgcaggaggagttctataaattcattaagccgattctggagaagatggacggaacggaggagctcctcgtcaagcttaaccgggaagacctgttgcggaagcagaggacttttgataacggctctattccgcaccaaatccatctgggtgagttgcacgcaatcttgagaagacaagaggatttctacccgttccttaaggataacagagagaagatagaaaaaatactgaccttcaggataccatactatgtgggcccactggcgcgcggaaatagtcgtttcgcatggatgactagaaagtccgaagaaacgatcacgccatggaattttgaggaagtggtcgacaagggcgcctctgcccagagcttcatcgaaaggatgaccaattttgacaaaaatctgcctaacgaaaaggtgcttccgaagcacagcctgttgtatgaatacttcacagtttataacgagctcactaaggtcaagtacgtcacggagggcatgcgtaagcctgctttcctgtctggtgaacaaaaaaaggcgattgtggacctccttttcaagacgaaccgtaaagttactgtgaagcaactgaaagaggattactttaagaaaattgagtgcttcgacagtgtggagatttccggtgtcgaggaccggtttaacgccagcctgggtacgtatcatgacctgcttaaaattatcaaggataaagatttcctggataatgaagagaacgaagatatactggaggacattgtgttgactttgaccctcttcgaggacagagagatgattgaggaaagactgaagacctacgcacacctttttgatgacaaggtcatgaaacaactcaagcgccggcgctatactggctggggccggctttctcgcaagctcatcaatgggattcgggataagcaatcaggcaagacaattttggacttcctcaaatccgacggattcgcaaataggaattttatgcagctgatacatgacgactctttgacattcaaagaagacatacagaaggctcaggtctccggccaaggagattctttgcacgagcatatcgctaacttggcaggtagccccgccataaaaaagggcattcttcaaacggtaaaagttgttgacgaactcgtgaaggttatgggccgtcataagccggaaaacattgttattgaaatggctagggaaaatcagacgacccagaagggacagaaaaatagcagggagcggatgaagagaattgaagagggaattaaggagcttggatctcagattcttaaggagcaccctgtggagaacacccaacttcagaatgaaaagctctacctttactaccttcaaaacggccgggatatgtacgtcgatcaggaacttgacattaaccggttgagcgattatgacgttgaccatattgtgccccaatctttccttaaagacgactctatcgacaataaagtgctgacgcgcagcgataaaaatcgcggtaagtcggataatgtcccgtcggaagaggtggttaaaaaaatgaagaactattggaggcaactcctgaatgccaagctgatcactcagaggaaattcgacaatctcaccaaggcagaaaggggtggacttagcgagctcgacaaggccggttttatcaaaagacagctggtggagacacgccaaatcaccaaacacgttgcccagatcctggattcgaggatgaacacgaagtatgacgagaacgacaagttgattagggaagtcaaggtcatcactttgaagtccaagctggtgagcgactttcgcaaagacttccagttttacaaagtcagggaaattaataactaccaccacgcccacgacgcctaccttaacgccgtggttggcacagcactcatcaagaaataccctaagctcgaatctgagttcgtctatggcgactataaggtctacgacgttagaaaaatgatcgcgaaatctgagcaggaaataggcaaggcaactgccaagtacttcttctattccaatatcatgaacttttttaagacggagattaccctggcgaatggtgagatccgcaagcgccctttgattgagacaaacggagaaacaggagagatcgtatgggacaaagggcgggactttgctactgttaggaaggtgctctctatgccacaagttaacattgtcaaaaaaactgaagtgcagacaggtgggtttagcaaggaatctatcctgccgaagaggaactctgacaagctgatcgcccgcaagaaagattgggatccgaaaaagtacggaggattcgtctcccccacagttgcgtactccgtgcttgtcgtggccaaagtggagaagggcaagtctaagaagctcaagagcgtcaaagagttgttggggatcacgattatggagcggtcgtctttcgaaaagaatccgatagattttctcgaggccaagggttataaagaagtcaagaaggatcttatcatcaagctccctaagtactccctctttgagcttgaaaacggacggaaaagaatgctggcttcagcgggtgaacttcagaagggtaatgaactcgctctgccctcaaaatatgtgaatttcctttacctggcatcacactatgagaagcttaaggggtctccagaggacaacgagcagaagcaactgttcgttgaacaacacaagcactaccttgacgagattatcgagcaaatcagcgagtttagcaagcgcgttatactggcagacgcaaatcttgataaggtccttagcgcctacaacaagcatagagacaaacccatccgggagcaggccgagaacattattcatctcttcaccttgacgaatcttggggccccggccgcgttcaagtacttcgatactaccatagacagaaagcgctatacatcgacaaaggaagttcttgacgccacgctgatccaccaaagtataacaggcctctatgagacacgcatcgacctttcgcagttgggcggtgactccggcggaagtacaaacctttcagacattatagaaaaggaaaccggcaagcaactcgtcatccaggaatccatacttatgctccctgaagaggtggaagaagtgatcggtaataaaccagagagcgacatacttgtccacaccgcttatgacgaaagtacagacgaaaacgtcatgcttctgacgagtgatgcccccgaatacaaaccttgggcgctcgtcatccaggattccaatggggagaataaaataaagatgctctctggaggcagcccaaagaagaagagaaaggtctga；

<210>13

<211>76

<212>dna

<213>化膿鏈球菌（streptococcuspyogenes）

<223>seqidno.13

<400>13

gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc；

<210>14

<211>20

<212>dna

<213>人工序列

<223>seqidno.14

<400>14

agagaccaaaggaggtctca；

<210>15

<211>37

<212>dna

<213>人工序列

<223>seqidno.15

<400>15

ggctaggatccatcgcagtcagcgatgagtacagcaa；

<210>16

<211>20

<212>dna

<213>水稻（oryzasatival）

<223>針對(duì)pi-d2的靶核苷酸序列

<400>16

gagcataatgacaataataa；

<210>17

<211>1765

<212>dna

<213>玉米（zeamaysl.）

<223>seqidno.17

<400>17

gcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcacggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgctttttttttgtcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgca；

<210>18

<211>326

<212>dna

<213>水稻（oryzasatival）

<223>seqidno.18

<400>18

aagaacgaactaagccggacaaaaaaaggagcacatatacaaaccggttttattcatgaatggtcacgatggatgatggggctcagacttgagctacgaggccgcaggcgagagaagcctagtgtgctctctgcttgtttgggccgtaacggaggatacggccgacgagcgtgtactaccgcgcgggatgccgctgggcgctgcgggggccgttggatggggatcggtgggtcgcgggagcgttgaggggagacaggtttagtaccacctcgcctaccgaacaatgaagaacccaccttataaccccgcgcgctgccgcttgtgtt；

<210>19

<211>245

<212>dna

<213>水稻（oryzasatival）

<223>seqidno.19

<400>19

ggatcatgaaccaacggcctggctgtatttggtggttgtgtagggagatggggagaagaaaagcccgattctcttcgctgtgatgggctggatgcatgcgggggagcgggaggcccaagtacgtgcacggtgagcggcccacagggcgagtgtgagcgcgagaggcgggaggaacagtttagtaccacattgcccagctaactcgaacgcgaccaacttataaacccgcgcgctgtcgcttgtgt；

<210>20

<211>253

<212>dna

<213>camv(califlowermosaicvirus)

<223>seqidno.20

<400>20

gatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatc；

<210>21

<211>8

<212>dna

<213>人工序列

<223>seqidno.21

<400>21

tttttttt；

<210>22

<211>24

<212>dna

<213>人工序列

<223>gpi-d2-f1

<400>22

gtgtgagcataatgacaataataa；

<210>23

<211>24

<212>dna

<213>人工序列

<223>gpi-d2-r1

<400>23

aaacttattattgtcattatgctc；

<210>24

<211>20

<212>dna

<213>人工序列

<223>pi-d2基因上的編輯位點(diǎn)序列

<400>24

ttattattgtcattatgctc；

<210>25

<211>20

<212>dna

<213>人工序列

<223>pi-d2基因被編輯后的部分核苷酸序列

<400>25

ttattattgtcattatactc；

<210>26

<211>20

<212>dna

<213>人工序列

<223>pi-d2-f1

<400>26

cgggttgtaagagtgcctgt；

<210>27

<211>20

<212>dna

<213>人工序列

<223>pi-d2-r1

<400>27

ctccagcttcttcacagcaa；

<210>28

<211>5133

<212>dna

<213>人工序列

<223>seqidno.28

<400>28

atgagtagcgagacaggtcctgttgcagttgacccgacccttcggagaaggatagagccacacgaatttgaagtgtttttcgaccctagagaactgaggaaggagacgtgccttctgtacgagataaactggggtggtcgccactctatttggaggcacacttcgcaaaacacgaacaagcatgtggaggtgaactttatagaaaaatttacgactgagagatacttctgccctaatacccggtgctccatcacctggttccttagctggagcccttgtggcgaatgctcgagggcaatcaccgagtttctgtccagatacccacatgtgacgctttttatatatattgcccgcttgtatcaccacgctgaccctagaaaccgccagggtcttcgcgatctgatatcttcaggagttaccatccaaataatgacggaacaagaatccggttactgttggcgcaatttcgtcaactatagcccttccaatgaagctcattggcctagatatccgcacctctgggtccggctgtatgttctcgagctttactgcattatacttggacttcccccctgcttgaatattctccgcagaaagcagcctcagcttacgttttttacgattgcactccaaagttgtcattatcagagactgccaccccatatcttgtgggctacgggactgaagagcggaagcgagacgccagggacttctgaatcggccacccccgagagcgataaaaagtattcaatcggacttgctattgggacaaactctgtgggctgggcggtaattaccgacgagtacaaggtgcctagtaagaaatttaaagtgctcggaaacactgacaggcactctataaagaagaacctgatcggggcactgcttttcgactccggagagacggcggaggcgacgcgtctcaagcgtaccgcgcgccgcaggtacacaagaaggaagaataggatctgctacttgcaggaaatcttcagtaacgagatggcgaaggtcgacgatagtttctttcatcggttggaagaatcgttcctcgtagaggaggacaaaaagcacgagcgtcacccaatattcgggaatattgttgacgaggttgcctaccatgagaaatatcctacaatatatcacctccgtaagaagcttgtcgattcaactgataaggctgatctcagactcatctatcttgccctcgcacatatgattaagtttcgtggccacttcttgattgaaggcgacctcaacccggacaactcagatgttgacaagctttttatacagctcgtccagacatataaccagctgtttgaagagaatcccatcaatgcgagtggggttgatgctaaggccattttgtccgccaggttgtccaaatctcgcagactggaaaacctgatcgcacagcttcccggtgaaaagaaaaacgggctcttcggcaatctcatcgcactgtccctcggcctcaccccaaacttcaagtctaacttcgacctggccgaggatgcgaagctccagctgtcaaaagatacatacgacgacgatttggacaatctgcttgcgcaaataggcgaccagtatgcggacctgttcctggctgccaaaaatctgtcagatgcaatcctcctgtccgatatattgcgtgtgaacaccgaaatcacgaaggcaccgcttagcgcatccatgatcaagagatacgacgagcaccatcaggacctcacactcctcaaggcgcttgttcgtcagcagcttcccgagaaatataaggaaatttttttcgatcaaagcaagaatggatatgctggctatattgacggtggcgcttcgcaggaggagttctataaattcattaagccgattctggagaagatggacggaacggaggagctcctcgtcaagcttaaccgggaagacctgttgcggaagcagaggacttttgataacggctctattccgcaccaaatccatctgggtgagttgcacgcaatcttgagaagacaagaggatttctacccgttccttaaggataacagagagaagatagaaaaaatactgaccttcaggataccatactatgtgggcccactggcgcgcggaaatagtcgtttcgcatggatgactagaaagtccgaagaaacgatcacgccatggaattttgaggaagtggtcgacaagggcgcctctgcccagagcttcatcgaaaggatgaccaattttgacaaaaatctgcctaacgaaaaggtgcttccgaagcacagcctgttgtatgaatacttcacagtttataacgagctcactaaggtcaagtacgtcacggagggcatgcgtaagcctgctttcctgtctggtgaacaaaaaaaggcgattgtggacctccttttcaagacgaaccgtaaagttactgtgaagcaactgaaagaggattactttaagaaaattgagtgcttcgacagtgtggagatttccggtgtcgaggaccggtttaacgccagcctgggtacgtatcatgacctgcttaaaattatcaaggataaagatttcctggataatgaagagaacgaagatatactggaggacattgtgttgactttgaccctcttcgaggacagagagatgattgaggaaagactgaagacctacgcacacctttttgatgacaaggtcatgaaacaactcaagcgccggcgctatactggctggggccggctttctcgcaagctcatcaatgggattcgggataagcaatcaggcaagacaattttggacttcctcaaatccgacggattcgcaaataggaattttatgcagctgatacatgacgactctttgacattcaaagaagacatacagaaggctcaggtctccggccaaggagattctttgcacgagcatatcgctaacttggcaggtagccccgccataaaaaagggcattcttcaaacggtaaaagttgttgacgaactcgtgaaggttatgggccgtcataagccggaaaacattgttattgaaatggctagggaaaatcagacgacccagaagggacagaaaaatagcagggagcggatgaagagaattgaagagggaattaaggagcttggatctcagattcttaaggagcaccctgtggagaacacccaacttcagaatgaaaagctctacctttactaccttcaaaacggccgggatatgtacgtcgatcaggaacttgacattaaccggttgagcgattatgacgttgaccatattgtgccccaatctttccttaaagacgactctatcgacaataaagtgctgacgcgcagcgataaaaatcgcggtaagtcggataatgtcccgtcggaagaggtggttaaaaaaatgaagaactattggaggcaactcctgaatgccaagctgatcactcagaggaaattcgacaatctcaccaaggcagaaaggggtggacttagcgagctcgacaaggccggttttatcaaaagacagctggtggagacacgccaaatcaccaaacacgttgcccagatcctggattcgaggatgaacacgaagtatgacgagaacgacaagttgattagggaagtcaaggtcatcactttgaagtccaagctggtgagcgactttcgcaaagacttccagttttacaaagtcagggaaattaataactaccaccacgcccacgacgcctaccttaacgccgtggttggcacagcactcatcaagaaataccctaagctcgaatctgagttcgtctatggcgactataaggtctacgacgttagaaaaatgatcgcgaaatctgagcaggaaataggcaaggcaactgccaagtacttcttctattccaatatcatgaacttttttaagacggagattaccctggcgaatggtgagatccgcaagcgccctttgattgagacaaacggagaaacaggagagatcgtatgggacaaagggcgggactttgctactgttaggaaggtgctctctatgccacaagttaacattgtcaaaaaaactgaagtgcagacaggtgggtttagcaaggaatctatcctgccgaagaggaactctgacaagctgatcgcccgcaagaaagattgggatccgaaaaagtacggaggattcgactcccccacagttgcgtactccgtgcttgtcgtggccaaagtggagaagggcaagtctaagaagctcaagagcgtcaaagagttgttggggatcacgattatggagcggtcgtctttcgaaaagaatccgatagattttctcgaggccaagggttataaagaagtcaagaaggatcttatcatcaagctccctaagtactccctctttgagcttgaaaacggacggaaaagaatgctggcttcagcgggtgaacttcagaagggtaatgaactcgctctgccctcaaaatatgtgaatttcctttacctggcatcacactatgagaagcttaaggggtctccagaggacaacgagcagaagcaactgttcgttgaacaacacaagcactaccttgacgagattatcgagcaaatcagcgagtttagcaagcgcgttatactggcagacgcaaatcttgataaggtccttagcgcctacaacaagcatagagacaaacccatccgggagcaggccgagaacattattcatctcttcaccttgacgaatcttggggccccggccgcgttcaagtacttcgatactaccatagacagaaagcgctatacatcgacaaaggaagttcttgacgccacgctgatccaccaaagtataacaggcctctatgagacacgcatcgacctttcgcagttgggcggtgactccggcggaagtacaaacctttcagacattatagaaaaggaaaccggcaagcaactcgtcatccaggaatccatacttatgctccctgaagaggtggaagaagtgatcggtaataaaccagagagcgacatacttgtccacaccgcttatgacgaaagtacagacgaaaacgtcatgcttctgacgagtgatgcccccgaatacaaaccttgggcgctcgtcatccaggattccaatggggagaataaaataaagatgctctctggaggcagcccaaagaagaagagaaaggtctga；

<210>29

<211>1710

<212>prt

<213>人工序列

<223>seqidno.29

<400>29

mssetgpvavdptlrrriephefevffdprelrketcllyeinwggrhsiwrhtsqntnkhvevnfiekftteryfcpntrcsitwflswspcgecsraiteflsryphvtlfiyiarlyhhadprnrqglrdlissgvtiqimteqesgycwrnfvnyspsneahwpryphlwvrlyvlelyciilglppclnilrrkqpqltfftialqschyqrlpphilwatglksgsetpgtsesatpesdkkysiglaigtnsvgwavitdeykvpskkfkvlgntdrhsikknligallfdsgetaeatrlkrtarrrytrrknricylqeifsnemakvddsffhrleesflveedkkherhpifgnivdevayhekyptiyhlrkklvdstdkadlrliylalahmikfrghfliegdlnpdnsdvdklfiqlvqtynqlfeenpinasgvdakailsarlsksrrlenliaqlpgekknglfgnlialslgltpnfksnfdlaedaklqlskdtydddldnllaqigdqyadlflaaknlsdaillsdilrvnteitkaplsasmikrydehhqdltllkalvrqqlpekykeiffdqskngyagyidggasqeefykfikpilekmdgteellvklnredllrkqrtfdngsiphqihlgelhailrrqedfypflkdnrekiekiltfripyyvgplargnsrfawmtrkseetitpwnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyftvyneltkvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyfkkiecfdsveisgvedrfnaslgtyhdllkiikdkdfldneenediledivltltlfedremieerlktyahlfddkvmkqlkrrrytgwgrlsrklingirdkqsgktildflksdgfanrnfmqlihddsltfkediqkaqvsgqgdslhehianlagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqkgqknsrermkrieegikelgsqilkehpventqlqneklylyylqngrdmyvdqeldinrlsdydvdhivpqsflkddsidnkvltrsdknrgksdnvpseevvkkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlvetrqitkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqfykvreinnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmiakseqeigkatakyffysnimnffkteitlangeirkrplietngetgeivwdkgrdfatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliarkkdwdpkkyggfdsptvaysvlvvakvekgkskklksvkellgitimerssfeknpidfleakgykevkkdliiklpkyslfelengrkrmlasagelqkgnelalpskyvnflylashyeklkgspedneqkqlfveqhkhyldeiieqisefskrviladanldkvlsaynkhrdkpireqaeniihlftltnlgapaafkyfdttidrkrytstkevldatlihqsitglyetridlsqlggdsggstnlsdiieketgkqlviqesilmlpeeveevignkpesdilvhtaydestdenvmlltsdapeykpwalviqdsngenkikmlsggspkkkrkv。