利用CRISPR/Cas9技术敲除转运蛋白基因降低油菜种子硫代葡萄糖苷含量的研究

Glucosinolates function as main defense chemicals for Brassica crops to defend against herbivores and pathogens. However, the existence of glucosinolates in oilseeds limits the application of rapeseed meal as feedstuff. With the rapid development of China's rapeseed industry, the planting area of double-low (low erucic acid and low glucosinolate content in seed) rapeseed (Brassica napus L.) varieties has expanded rapidly. However, the plants are more sensitive to herbivores and diseases with the lower content of glucosinolates in these cultivars. GTR1 and GTR2 are glucosinolate-specific transporters, determine the accumulation of glucosinolates in Arabidopsis seeds. The gtr1 gtr2 double mutant do not accumulate glucosinolates in seeds. Therefore, using the B. napus cultivars with strong disease resistance and high seed glucosinolate content as transformation receptors, we aim to knock out the glucosinolate transporter genes BnGTR1 and BnGTR2. First, in order to obtain homozygous mutants in the early generation, we aim to improve the mutagenesis efficiency of CRISPR/Cas9 system by using endogenous promoter of B. napus with higher transcriptional activity during genetic transformation to drive the expression of Cas9 gene. Then, knock out multiple copies of BnGTR1 and BnGTR2 genes by updated CRISPR/Cas9 system to verify the effect of GTR1 and GTR2 on plant disease resistance and seed glucosinolates accumulation and creating new B. napus germplasm with low glucosinolates in seed and high glucosinolates in vegetative organ. The study will provide the important theory basis for the creation of elite rapeseed germplasm.

硫代葡萄糖苷（硫苷）是一种对抗病虫害的防御物质，但其在油菜种子中的积累会降低菜籽饼粕的饲用价值。随着我国甘蓝型油菜产业的“双低”（低硫苷、低芥酸）化，油菜营养体中硫苷含量伴随着菜籽硫苷含量的下降也大幅降低，导致油菜的抗病虫性也显著降低。研究表明拟南芥种子中硫苷的积累是由硫苷转运蛋白GTR1和GTR2将其从营养体中运入引起的，敲除GTR1和GTR2基因会阻断这一过程。本项目以抗病性强但种子硫苷含量高的甘蓝型油菜品种为材料，敲除其硫苷转运蛋白，首先通过筛选更高效的Cas9启动子来提高CRISPR/Cas9系统的突变效率，实现在转基因早期世代得到纯合突变体；然后对甘蓝型油菜BnGTR1和BnGTR2基因进行多重敲除，验证GTR1和GTR2功能缺失对植株抗病性和种子硫苷积累的影响。创建营养体高硫苷、种子低硫苷性状的油菜新种质，从而实现兼顾高抗性和品质改良，为甘蓝型油菜优质种质资源创建提供理论依据。

硫代葡萄糖苷（硫苷）是一种对抗病虫害的防御物质，但其在油菜种子中的积累会降低菜籽饼粕的饲用价值。随着我国甘蓝型油菜产业的“双低”（低硫苷、低芥酸）化，油菜营养体中硫苷含量伴随着油菜籽硫苷含量的下降也大幅降低，导致油菜的抗病虫性也显著降低。研究表明拟南芥种子中硫苷的积累是由硫苷转运蛋白GTR1和GTR2将其从营养体中运入引起的，敲除GTR1和GTR2基因会阻断这一过程。本项目首先筛选甘蓝型油菜中转录活性强的启动子用于驱动Cas9基因的表达，提高CRISPR/Cas9系统介导的多重突变效率，然后对高种子硫苷品种或品系进行遗传转化，最后对甘蓝型油菜的硫苷转运蛋白BnGTR1和BnGTR2基因进行多重敲除，验证在GTR1和GTR2蛋白功能缺失后种子营养品质的变化，解析甘蓝型油菜GTR1和GTR2转运蛋白在种子发育时期硫苷积累中的功能。..首先，本项目选择了5种高硫苷甘蓝型油菜品种/品系，最终选择再生苗分化率为49.68%和64.42%的两种材料作为转化的遗传背景。..随后，本项目筛选了11个在遗传转化过程中表达水平较高的候选基因，结合其在甘蓝型油菜下胚轴、叶片、根等部分的表达水平进行分析后选择MT2-22基因的启动子用于CRISPR/Cas9系统改造。表达水平检测发现，新启动子驱动Cas9的表达水平远远高于35S驱动下的表达，说明改造后的CRISPR/Cas9系统显著提高了Cas9的表达水平，可能对CRISPR/Cas9系统介导的基因编辑效率有较大提高。..第三，甘蓝型油菜中GTR1和GTR2基因各有3个在种皮中表达水平相对较高的拷贝被选为候选敲除基因。本项目各设计了4个靶位点用于CRISPR/Cas9系统介导的基因编辑，构建了多重突变载体并进行遗传转化，最后获得了共计47棵转基因阳性苗。对其进行基因编辑检测发现各靶位点的基因编辑效率从3.3%-42.8%不等。. 最后，对BnaGTR1和BnaGTR2基因敲除株系进行种子硫苷含量检测发现，2个基因同时敲除后种子总硫苷含量显著降低，实现了油菜种质资源的改良。

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