绿豆GR基因生物信息学分析及其CRISPR/Cas9重组载体的构建与转化  

Bioinformatics Analysis of GR Gene and Construction and Transformation of CRISPR/Cas9 Recombinant Vectors in Mung Bean

作　　者：李晓驹 方淑梅[2] 王庆燕 梁喜龙[1] 郑殿峰[1,3] Li Xiaoju;Fang Shumei;Wang Qingyan;Liang Xilong;Zheng Dianfeng(College of Agronomy,Heilongjiang Bayi Agricultural University,Daqing163319;College of Life Science and Technology;College of Coastal Agricultural Sciences,Guangdong Ocean University)

机构地区：[1]黑龙江八一农垦大学农学院,大庆163319 [2]黑龙江八一农垦大学生命科学技术学院 [3]广东海洋大学

出　　处：《黑龙江八一农垦大学学报》2023年第4期1-10,25,共11页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省农垦总局科技攻关项目(HNK135-02-10);黑龙江省杂粮现代农业产业技术协同创新体系项目;黑龙江省杂粮生产与加工特色学科建设项目;黑龙江省研究生创新科研项目(YJSCX2021-Y52)。

摘　　要：绿豆(Vigna radiata L.)是一种严格闭花授粉作物,其人工授粉效率低、成本高,通过杂交获取新品种较为困难。目前,国内外绿豆遗传转化体系鲜见报道,这极大地限制了绿豆基因功能的研究和新品种的开发,因此尝试建立绿豆遗传转化途径具有重要意义。首先对绿豆谷胱甘肽还原酶基因(GR)进行生物信息学分析,并以此为基础,构建基因编辑重组体,同时进行发根农杆菌的遗传转化。结果表明,GR蛋白相对分子质量为58.38 kDa,稳定性较好,为亲水蛋白,有多个磷酸化位点,不含跨膜结构,定位于细胞质中,二级结构主要包括ɑ-螺旋和延伸链,且含有Pyr_redox_2和Pyr_redox_dim两个结构域;系统进化分析发现该蛋白与赤豆(Vigna angularis)亲缘关系最近。在此基础上构建四靶点CRISPR/Cas9-GR-gRNA基因编辑载体,通过发根农杆菌遗传转化绿豆子叶节获取毛状根20株,后经PCR检测获取阳性克隆毛状根12株,其毛状根转化率约为60%,其研究结果可为绿豆后续基因编辑研究奠定重要基础。Mung bean(Vigna radiata L.)is a strictly closed-flowered pollination crop,which has low artificial pollination efficiency and high cost,and it is difficult to obtain new varieties through hybridization.There were few reports on the genetic transformation system of mung bean at home and abroad,which greatly limited the research of mung bean gene function and development of the new varieties.Therefore,it was of great significance to try to establish the genetic transformation method of mung bean.Bioinformatics analysis of the mung bean glutathione reductase gene(GR)was carried out first,a gene editing recombinant was constructed,and the genetic transformation of Agrobacterium rhizogenes was carried out at the same time.The results showed that the relative molecular mass of GR protein was 58.38 kDa with good stability.It was a hydrophilic protein with multiple phosphorylation sites and no transmembrane structure.It was located in the cytoplasm.The secondary structure mainly includedɑ-helix and extended chain.It contained two domains,Pyr_redox_2 and Pyr_redox_dim.Phylogenetic analysis found that this protein was most closely related to Vigna angularis.On this basis,a four-target CRISPR/Cas9-GR-gRNA gene editing vector was constructed.Twenty hairy roots were obtained by genetically transforming mung bean cotyledon nodes by Agrobacterium rhizogenes,and then twelve positive clones were obtained by PCR detection.The hairy root transformation rate was about 60%,and the results could lay an important foundation for subsequent gene editing research of the mung bean.

关 键 词：GR基因 绿豆 生物信息学分析 CRISPR/Cas9重组体构建 毛状根遗传转化 

分 类 号：Q78[生物学—分子生物学]