【作者】 李波；

【导师】 金双侠；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2021， 博士

【摘要】 棉花(Gossypium hirsutum)是我国最主要的农业经济作物之一,是主要的工业纺织原料和农业榨油原料的来源。在棉花中建立有效的研究基因组学的技术方法,对深入研究棉花功能基因的分子机制具有重大的意义。本研究旨在建立多种适用于棉花的植物CRISPR技术,为棉花功能基因的深入研究提供更多的工具。首先,利用菜豆矮缩病毒(BeYDV)滚环复制的功能,结合棉花CRISPR-Cas9基因编辑系统,共同构建了高效的基因编辑敲除载体;在此基础上,将棉花内源的供体模板构建到BeYDV的复制环中,建立了在棉花中基于BeYDV双生病毒的基因编辑敲入系统。此外,优化了CRISPR-LbCpf1系统在棉花中的编辑效率,同时利用CRISPR-LbCpf1编辑系统结合PTC(多顺反子t RNA-crRNA串联)和TDT(Target-donor-Target)结构的方法,建立了基于CRISPR-LbCpf1的基因编辑敲入系统。本研究取得的主要结果如下:第一,在棉花中构建并比较了基于Cas9的不同基因敲除载体的编辑效率。为了提高CRISPR-Cas9基因编辑系统在棉花中的编辑效率,本研究在棉花基因编辑系统pRGEB32-Gh U6.7的基础上构建了两种不同的基因敲除载体,一种是将启动子及靶标位点的sgRNA构建在BeYDV双生病毒的复制环(LIR-Donor-SIR-Rep-LIR)中的pBeYDV-Cas9-KO基因编辑系统,第二种是将驱动Cas9蛋白的Ubi启动子替换为组成型35s启动子的pRGEB32-35s基因编辑系统。将叶绿体合成相关基因Gh CLA1作为靶标基因构建到这3个载体后,通过农杆菌遗传转化方法转入棉花后比较它们在棉花基因编辑中的编辑效率。结果表明:pBeYDV-Cas9-KO系统在棉花靶标基因中表现出最好的编辑效率,同时编辑At Dt亚组的效率高达73.3%;编辑类型主要以片段缺失为主,达到了84.0%,碱基缺失长度的范围主要在1 bp-10 bp之间;编辑位点主要位于PAM上游11-17位;对sgRNA对应的脱靶位点检测没有产生脱靶效应。表明pBeYDV-Cas9-KO基因编辑系统在棉花中对靶标基因的编辑特异性更强,编辑范围更大。第二,在棉花中构建了基于BeYDV双生病毒和Cas9蛋白的基因敲入载体。为了开发在棉花中能够进行基因敲入的载体,本研究在BeYDV双生病毒介导的pBeYDV-Cas9-KO基因编辑载体的基础上,将同源臂,Cas9蛋白构建在BeYDV双生病毒的复制元件中,同时选择棉花叶绿体合成相关基因Gh CLA1为靶标基因,共构建了12个BeYDV介导的基因敲入载体。分别在Gh CLA1基因的多个位点进行外源基因RFP敲入的验证。结果表明:筛选到两个能够在棉花中进行基因敲入的载体Be KI-3和Be KI-4,所获得的阳性植株可以检测到外源基因的敲入,两个载体在棉花中的敲入效率分别达到5.6%和6.5%。第三,温度对CRISPR-LbCpf1基因编辑系统在棉花中编辑效率的影响。对CRISPR-LbCpf1系统编辑Gh CLA1基因的T1代材料和编辑Gh PGF基因的愈伤组织分别进行不同温度的处理并检测在不同温度条件LbCpf1蛋白的活性,同时对各个靶标位点进行编辑效率分析。结果表明:温度对LbCpf1核酸酶的活性具有一定的影响,在34℃的条件下LbCpf1蛋白的活性最高,编辑效率最高,达到了91.5%;同时编辑2个crRNA的效率更高,达到65%;温度的提高对第二个crRNA的编辑效率也有所提高;并且温度处理后没发现脱靶效应,此外,创造了敲除Gh PGF基因的无腺体DNA-free棉花材料,并且可以稳定遗传到下一代。第四,开发了基于CRISPR-LbCpf1的棉花基因编辑敲入系统。利用LbCpf1蛋白同时具有DNA和RNA双重切割能力的特性,串联PTC(Polycistronic t RNA-crRNA)和TDT(Target-Donor-Target)结构建立pRGEB3-Cpf1-KI基因敲入系统,在Gh PGF基因上设计不同靶点,分别在同源臂中间插入Sbf I酶切位点和RFP基因,共构建了4个基因敲入载体。结果表明:基于CRISPR-LbCpf1的基因敲入系统可以将外源片段或基因精确的插入到棉花基因组中,筛选到两个能够在棉花中进行基因敲入的载体KI-Sbf I-1CR和KI-RFP-1CR,敲入效率分别达到6.3%和2.7%。综上所述,本研究分别利用CRISPR-Cas9结合BeYDV双生病毒复制功能,CRISPR-Cpf1的双重切割特性结合TDT结构同源模板等方式,优化了在棉花中进行基因敲除的基因编辑载体,同时提供了能够在棉花中实现基因敲入的基因编辑系统。为未来棉花基因敲除提供了新的技术手段,为基因敲入技术的优化提供了实验基础。在未来可以结合多种基因编辑技术对棉花中重要基因的功能进行解析。更多还原

【Abstract】 Cotton(Gossypium hirsutum)is one of the important economic crops and the main source of raw materials for industrial textile and agricultural oil.It is of great significance to study the molecular mechanism of functional genes though establishing effective technical methods in cotton.In this study,the replication initiation protein(Rep)was been used to recognize the long intergenic region(LIR)structure and initiate rolling-circle replication in the bean dwarf virus,then a highly efficient gene knockout vector was construct combining with CRISPR-Cas9 gene editing system.On this basis,the cotton endogenous homologous templates were constructed into the replication loop and a gene editing knock-in system based on BeYDV Geminivirus was been constructed in cotton.In addition,we optimized the editing efficiency of the CRISPR-Cpf1 system and established a CRISPR-Cpf1 gene editing knock-in system based on the method of combining the CRISPR-Cpf1 editing system with PTC(Polycistronic t RNA-crRNA)and TDT(Target-Donor-Target)structures.The main results of this study are as follows.First,we constructed three knockout vectors based on CRISPR-Cas9 and compared their editing efficiency in cotton.In order to improve the editing efficiency of the CRISPR-Cas9 gene editing system in cotton,two different gene knockout vectors were constructed based on the cotton gene editing system pRGEB32-Gh U6.7.One is the pBeYDV-Cas9-KO gene editing system which the promoter and sgRNA were constructed in the replication loop(LIR-Donor-SIR-Rep-LIR)of the BeYDV geminivirus virus.The second is to replace the UBI promoter driving Cas9 protein with the pRGEB32-35 s gene editing system of constitutive 35 S promoter.The three vectors that the chloroplast synthesis related gene Gh CLA1 were as the target gene were transformed into cotton by Agrobacterium tumefaciens genetic transformation.The results showed that the efficiency of editing At and Dt subgroup simultaneously in cotton by BeYDV-Cas9-KO system was as high as 73.3%;the editing type is mainly fragment deletion,and the length of base deletion mainly ranges from 1 bp to 10 bp.The editing sites were mainly located at positions 11-17 upstream of PAM.We did not find the off-target editing of BeYDV-Cas9-KO by detecting the miss site corresponding to sgRNA.It shows that the pBeYDV-Cas9-KO gene editing system has stronger editing specificity for target genes in cotton,and the editing range is more than larger.Second,we constructed three knock-in vectors based on Cas9 and the BeYDV Geminivirus virus in cotton.In order to establish the knock-in system in cotton,we constructed the homology arms and Cas9 protein into the replication elements of the BeYDV geminivirus on the basis of the BeYDV geminivirus-mediated pBeYDV-Cas9-KO gene editing vector.At the same time,the cotton chloroplast synthesis-related gene Gh CLA1 was selected as the target gene,and 12 BeYDV-mediated knock-in vectors were constructed.Foreign gene RFP was knock in multiple loci of Gh CLA1 gene to verify the knock in system.The results showed that two vectors Be KI-3and Be KI-4 that could knock in cotton were screened,and the knock-in efficiency of the two vectors in cotton was 5.6% and 6.5% respectively.Third,temperature had an effect on the editing efficiency of CRISPR-LbCpf1 in cotton.The T1 generation materials of Gh CLA1 gene and the callus of Gh PGF gene was treated with different temperature,and the expression profile of LbCpf1 protein and editing efficiency were tested under different temperature conditions.The results show that the activity of LbCpf1 nuclease is sensitive to temperature.The activity of LbCpf1 protein is the highest under the condition of 34℃,the CRISPR-LbCpf1 system showed better editing efficiency and reached 91.5%;the editing efficiency that At Dt subfamily was edited simultaneously was as high as 65.0%;the editing efficiency of the second crRNA was better;no off-target effect was found after temperature treatment.In addition,we knocked out Gh PGF gene to obtain a glandfree and nontransgenic cotton material,which can be stably inherited to the next generation.At last,we constructed the gene knock-in vectors based on CRISPR-Cpf1 system in cotton.We utilized the characteristic of Cpf1 protein with double cleavage ability of DNA and RNA,and established the pRGEB3-Cpf I-KI gene knock-in system with PTC(Polycistronic t RNA-crRNA)and TDT(Target-Donor-Target)structure.We designed two targets on Gh PGF gene.Sbf I restriction site or RFP gene was inserted in the middle of the homology arms of pRGEB3-Cpf I-KI vector.We have constructed four knock-in vectors successfully.The results showed that the gene knock-in system based on CRISPR-Cpf1 could accurately insert foreign fragments or genes into the cotton genome.Two vectors KI-Sbf I-1CR and KI-RFP-1CR were screened,and the knock-in efficiency was 6.3% and2.7% respectively.In summary,the vector of gene knockout in cotton was optimized by using CRISPR-Cas9 combined with the replication function of BeYDV Geminivirus virus,the double cleavage characteristics of CRISPR-Cpf1 and the homologous template of TDT structure.At the same time,a gene editing system which can realize gene knock-in in cotton is provided.It provides new technical method for cotton genome gene editing,and provides an experimental basis for the optimization of gene knock-in technology.In the future,multiple gene editing technologies will be applied to analyze the gene functions in cotton.更多还原