本文選題：CRISPR/Cas9　切入點(diǎn)：雙元載體　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：對(duì)于生命規(guī)律的探究很大程度上依賴于基因功能的闡釋,而全基因組測(cè)序及基因組學(xué)的發(fā)展使得對(duì)強(qiáng)大的基因組編輯工具的需求更為迫切。CRISPR/Cas9基因組編輯技術(shù)以其設(shè)計(jì)原理簡(jiǎn)單、技術(shù)門檻低、操作便捷、通用性強(qiáng)等特點(diǎn)在2013年初迅速成為基因組編輯研究領(lǐng)域的關(guān)注焦點(diǎn)。為了測(cè)試CRISPR/Cas9在植物中的應(yīng)用效果,建立全面的CRISPR/Cas9工具系統(tǒng),助力植物基因功能的研究,加快對(duì)植物功能基因的挖掘,本研究工作首先基于CRISPR/Cas9技術(shù)創(chuàng)制了植物的基因組編輯工具箱,涵蓋各種雙元載體和sgRNA模板載體以及相應(yīng)的sgRNA組裝方法。工具箱包括適用于雙子葉植物的雙元載體pHSE401、pBSE401、pKSE401和適用于單子葉植物的雙元載體pHUE411、pBUE411。通過在玉米和擬南芥中對(duì)CRISPR/Cas9工具箱進(jìn)行功能驗(yàn)證,證明該CRISPR/Cas9工具箱在雙子葉和單子葉植物中均能高效誘導(dǎo)基因組編輯,因此可為相關(guān)研究人員提供更為簡(jiǎn)單、便捷的工具平臺(tái),實(shí)現(xiàn)高效的突變體創(chuàng)制目的。在應(yīng)用CRISPR/Cas9工具時(shí),我們發(fā)現(xiàn)以胚性愈傷為轉(zhuǎn)化受體的單子葉植物玉米可以在T0代高效產(chǎn)生純合或雙等位突變體,但轉(zhuǎn)化方法最簡(jiǎn)單高效的模式植物擬南芥的T1代突變體卻存在嚴(yán)重的嵌合現(xiàn)象,影響了突變傳遞到下一代的效率。我們猜測(cè)轉(zhuǎn)基因T1代嵌合體的形成可能是與使用的CaMV 35S等組成型啟動(dòng)子在卵細(xì)胞或者單細(xì)胞期胚中的活性較低有關(guān)。為此,本研究采用卵細(xì)胞特異性啟動(dòng)子驅(qū)動(dòng)Cas9,創(chuàng)制了卵細(xì)胞特異性表達(dá)的CRISPR/Cas9系統(tǒng),即EPC(egg cell-specific promoter-controlled)CRISPR/Cas9 系統(tǒng),接著對(duì)EPC CRISPR/Cas9 進(jìn)行了功能測(cè)試,結(jié)果表明使用該系統(tǒng)在擬南芥的轉(zhuǎn)基因T1代成功地實(shí)現(xiàn)了兩基因(CHLI1 CHLI2)和三基因(TRY CPC ETC2)的純合突變,解決了CRISPR/Cas9技術(shù)在擬南芥中應(yīng)用時(shí)遇到的T1代突變體嵌合嚴(yán)重的問題。上述三基因和兩基因同時(shí)發(fā)生純合或雙等位突變的的株系占相應(yīng)的全部T1代轉(zhuǎn)基因植株的比例可以分別達(dá)到8.3%和13.1%。通過對(duì)三種啟動(dòng)子(EC1.2p/35Sp/EC1.1p)和兩種終止子(E9t/Nost)進(jìn)行組合,比較相應(yīng)載體的打靶效率發(fā)現(xiàn),Cas9終止子的選擇對(duì)于EPC CRISPR/Cas9發(fā)揮作用至關(guān)重要。此外,為了進(jìn)一步提高EPCCRISPR/Cas9系統(tǒng)的基因打靶效率,采用啟動(dòng)子融合策略對(duì)EPC CRISPR/Cas9系統(tǒng)進(jìn)行了優(yōu)化嘗試。通過嘗試不同的增強(qiáng)子與EC1.2或Ec1.1的啟動(dòng)子進(jìn)行融合,發(fā)現(xiàn)將EC1.2的增強(qiáng)子與EC1.1的啟動(dòng)子融合后顯著提高了EPC CRISPR/Cas9系統(tǒng)對(duì)靶基因的純合突變效率(17.0%),達(dá)到了優(yōu)化的目的。CRISPR/Cas9和胞嘧啶脫氨酶介導(dǎo)的堿基編輯系統(tǒng),最先在動(dòng)物中得到成功應(yīng)用。為了測(cè)試堿基編輯系統(tǒng)在植物中的應(yīng)用效果,本研究創(chuàng)制了一個(gè)適用于雙子葉植物(pHSE901、pBSE901、pKSE901、pHEE901)和單子葉植物(pHUE911、pBUE911)的堿基編輯工具箱并在擬南芥中進(jìn)行了測(cè)試。通過對(duì)T1代和T2代靶基因AtALS的編輯效果分析發(fā)現(xiàn),在T1代得到靶基因編輯的株系占全部株系的1.7%(4/240);在T1代得到編輯的靶點(diǎn)可以高效地傳遞到T2代,其中三個(gè)T1株系的T2代植株中,獲得除草劑抗性的堿基編輯植株的平均比例達(dá)84.1%。此外,研究結(jié)果提示在植物中,PAM區(qū)可能是一個(gè)新型的"脫氨酶作用窗口"�？傊�,本研究中建立的植物基因組編輯和堿基編輯系統(tǒng)無論在基礎(chǔ)理論研究還是作物改良中都有很好的應(yīng)用前景。
[Abstract]:For exploring the law of life depends largely on the interpretation of gene function, and the development of whole genome sequencing and genomics makes powerful genome editing tools is more urgent than.CRISPR/Cas9 genome editing technology with its simple design principle, low technical threshold, convenient operation, strong versatility and so quickly become the focus of research field genome editing in early 2013. In order to apply the test results of CRISPR/Cas9 in plant, the establishment of CRISPR/Cas9 tool system comprehensively, research on power plant gene function, accelerate the mining of plant gene function, this paper firstly based on the CRISPR/Cas9 technology to create a plant genome editing toolbox, covering a variety of binary vector and sgRNA vector template and the corresponding sgRNA assembly method. Toolbox includes binary vector pHSE401 for dicotyledonous plants, pBSE401, PKSE401 and for monocotyledon binary vector pHUE411, pBUE411. through the functional verification of the CRISPR/Cas9 toolbox in maize and Arabidopsis, prove that the CRISPR/Cas9 toolbox can effectively induce genome editing in dicotyledonous and monocotyledonous plants, so it can be for the relevant researchers to provide more simple, convenient tool platform, implementation efficient creation. Mutants in the application of CRISPR/Cas9 tools, we found that the embryogenic callus as transformation receptor monocots maize can be efficiently generated homozygous or double mutant allele in T0 generation, but the transformation of the most simple and efficient method of Arabidopsis T1 mutant generation has seriously affected the chimeric, mutation the efficiency of transfer to the next generation. We hypothesized that the transgenic T1 generation of chimera formation may be associated with the use of the CaMV 35S constitutive promoter in oocytes or Single cell embryos in lower activity. Therefore, this study uses the egg cell specific promoter Cas9, the creation of the egg cell specific expression of CRISPR/Cas9 system, namely EPC (egg cell-specific promoter-controlled) CRISPR/Cas9 system, and then tested the EPC CRISPR/Cas9, the result shows that the system of T1 in transgenic Arabidopsis thaliana the generation successfully realized two gene (CHLI1 CHLI2) and three genes (TRY CPC ETC2) the homozygous mutant, T1 mutant chimeric generation solves the application of CRISPR/Cas9 technology in Arabidopsis have the serious problems. The three genes and two genes were homozygous or biallelic Mutation Strains accounted for all T1 corresponding transgenic plants at the same time the proportion can reach 8.3% and 13.1%. respectively through three kinds of promoter (EC1.2p/35Sp/EC1.1p) and two (E9t/Nost) of the terminator group, compared with corresponding Find the vector targeting efficiency, Cas9 terminator selection play a crucial role for the EPC CRISPR/Cas9. In addition, in order to further improve the gene targeting efficiency of EPCCRISPR/Cas9 system, using the promoter fusion strategy was optimized to try EPC CRISPR/Cas9 system. Through fusion to try different EC1.2 or Ec1.1 enhancer and promoter, enhancer and discovery the EC1.1 EC1.2 promoter fused significantly improved EPC CRISPR/Cas9 system on target gene homozygous mutation efficiency (17%), to achieve the purpose of.CRISPR/Cas9 and the optimization of cytosine deamination enzyme mediated DNA Editing System, the first is successfully applied in the animal. In order to test the application effect of base editing system in plants, this study created a suitable for dicotyledonous plants (pHSE901, pBSE901, pKSE901, pHEE901) and Monocotyledoneae (pHUE911, pBUE911) of the base Edit the toolbox and tested in Arabidopsis. Based on T1 and T2 generation AtALS target gene editing effects analysis showed that the obtained target gene editing strains accounted for 1.7% lines in T1 generation (4/240); get the target of editing can be efficiently transferred to T2 generation in T1 generation, the three strains of T1 plants in T2 generation, the average proportion of herbicide resistant plants obtained base edit up to 84.1%. in addition, the results of the study suggest that in plants, PAM may be a new type of "ADA window." in short, application prospect in this paper plant genome editing and editing system is both the base in the theoretical research and crop improvement are.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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相關(guān)期刊論文 前5條

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本文編號(hào)：1670450