核桃CRISPR/Cas9-GA20ox基因定点突变信号的砧穗应答机制

Tranagenic fruit crop plants have been developed using Agrobacterium mediated transformation mostly now. However, the application of these transgenic fruit crops has been greatly challenged because traditional genetically modification has potential risk which derive from uncertainty and random of exogenous gene transferring. Grafting can reduce the risk of genetic modification dramatically because even though transferring of genetic materials including RNA improves the traits such as resistance, economic characteristics etc. between rootstocks and scions during grafting, the DNA sequences of rootstocks and scions are not exchanged respectively. Clustered regularly interspaced short palindromic repeats (CRISPRs) system provides an alternative genome editing strategy. Processing by the endogenous DNA repair machinery generates small indels that, when located within a coding sequence, can disrupt the reading frame and render the gene nonfunctional. The CRISPR/Cas9 system has been applied in several crops successfully. The gibberellin 20-oxidase played an important role in the biosynthesis of gibberellic acid as one of the key oxidase enzymes regulating the generation of biological GAs directly. Studies have been showed GA20ox knockout results in semi-dwarf in rice and wheat. . However, the exchange of genetic material which targeted GA20ox gene knockout of walnut somatic embryos by CRISPR/Cas9 between rootstocks and scions is not sure till now. This project will carry out GA20ox gene knockout signal exchange between rootstocks and scions on the basis of gene transformation system of walnut somatic embryos and CRISPR/Cas9-GA20ox vector construction. The aims are as follows: ①To construct CRISPR/Cas9-GA20ox vectors and screen the suitable ones; ②To optimize efficient walnut CRISPR/Cas9 genome editing system; ③To analysis the knockout effect of walnut CRISPR/Cas9-GA20ox transformants; ④To confirm genetic material exchange initiate from GA20ox gene editing by CRISPR/Cas9 system between rootstocks and scions. These results will illustrate the mechanism of genetic material exchange between rootstocks and scions which mediated by CRISPR/Cas9 system together with establish a semi-dwarf rootstocks and scions grafting system. The findings will provide a brand new strategy for using CRISPR/Cas9 on walnut dwarf germplasm enhancement as well as intiate safety application of CRISPR/Cas9 genome editing rootstocks in important fruit crops.

传统基因转化由于外源基因插入的随机性和不确定性可能带来的潜在巨大风险，使果树转基因应用受到了极大挑战。研究表明，嫁接不改变砧穗DNA序列，可在很大程度上降低转基因风险，包含RNA等在内遗传物质的传递是定向改良嫁接砧穗性状的主要机制。然而，CRISPR/Cas9基因定点突变信号是否能在核桃嫁接砧穗间交流与传递？本课题在近年研究基础上，探寻CRISPR/Cas9定点突变核桃GA20ox基因信号在嫁接砧穗间的传递与应答机理，包括①定点突变载体构建；②高效CRISPR/Cas9转化体系的建立与优化；③CRISPR/Cas9再生植株的定点突变效应分析；④明确核桃CRISPR/Cas9基因定点突变信号在嫁接砧穗间交流与应答的时空特征，阐明砧木基因定点突变信号在野生型接穗中的应答机制。为CRISPR/Cas9应用于核桃矮化种质创新提供全新策略，并为推动果树基因定点突变砧木的安全应用提供科学依据。

传统基因转化方法由于外源基因插入的随机性和不确定性可能带来的潜在巨大风险，已使传统转基因果品安全成为当今民众关注的重要课题，同时也使果树转基因应用受到了极大限制。嫁接在提高果树品质的同时，不改变砧穗DNA序列，却可极大降低转基因风险。然而，研究表明，包含RNA等在内的遗传物质传递与交流是定向改良嫁接砧穗性状的主要机制。本课题在近年研究基础上，探寻CRISPR/Cas9定点突变核桃GA20ox基因信号在嫁接砧穗间的传递与应答机理，包括CRISPR/Cas9定点突变载体构建；高效CRISPR/Cas9转化体系的建立与优化；CRISPR/Cas9再生株系的定点突变效应分析；明确核桃CRISPR/Cas9基因定点突变信号在嫁接砧穗间交流与应答的时空特征，阐明砧木基因定点突变信号在野生型接穗中的应答机制，为CRISPR/Cas9应用于核桃种质资源创新提供全新策略。本研究具体研究结果如下：克隆获得了核桃赤霉素氧化酶基因全长（包括KO, GA20ox, GA3ox, GA2ox），构建稳定表达的核桃赤霉素氧化酶基因过量表达载体、RNA干扰载体以及CRISPR/Cas9-JrGA20ox突变载体；开展了农杆菌介导的核桃体细胞胚遗传转化，构建了稳定表达的遗传转化体系， 将结构良好、生长健壮的阳性核桃体胚快速脱水处理后，转入DKW培养基中萌发培养，建立了高效的包括CRISPR/Cas9-JrGA20ox基因定点突变转化株系在内的培养体系；通过CRISPR/Cas9-JrGA20ox定点突变载体、pTCK303-JrGA20ox干扰载体与pCAMBIA1300-JrGA20ox过表达载体的遗传转化，深入验证了核桃JrGA20ox基因功能，发现其表达量与植株株高正相关、与抗旱性呈现负相关；开展了核桃CRISPR/Cas9-JrGA20ox与野生型植株互为砧穗进行嫁接，采用qRT-PCR检测了砧穗mRNA的变化趋势，发现CRISPR/Cas9-JrGA20ox定点突变砧木可远距离调控野生型接穗中JrGA20ox mRNA的表达量，使其显著降低。进一步验证发现，该种传递是双向的，但砧木对接穗的影响较大。本研究结果为核桃等木本果树矮化/高抗新品种的创制提供理论依据和技术支持，极大程度地降低了核桃转基因的风险，并将为CRISPR/Cas9基因编辑技术应用于果树育种提供全新的思路。

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