大豆NIN和NLP基因生物信息学分析及敲除载体构建  

Bioinformatics Analysis of Soybean NIN and NLP Genes and Knockout Vector Construction

作　　者：廖春梅 陈丽玉 杨涔 刘宝辉 孔凡江 LIAO Chun-mei;CHEN Li-yu;YANG Cen;LIU Bao-hui;KONG Fan-jiang(School of Life Sciences/Innovative Center of Molecular Genetics and Evolution,Guangzhou University,Guangzhou 510030,China)

机构地区：[1]广州大学生命科学学院/分子遗传与进化创新研究中心,广东广州510030

出　　处：《大豆科学》2023年第1期1-11,共11页Soybean Science

基　　金：广东省基础与应用基础研究重大项目(2019B030302006)。

摘　　要：为促进大豆NLP家族基因突变大豆材料的获得及大豆NIN和NLP基因功能的研究,本研究对大豆NIN和NLP基因进行生物信息学分析,通过CRISPR/Cas 9基因编辑技术构建基因敲除载体,并且通过大豆毛根转化实验验证靶点的有效性。结果表明:大豆基因组中一共存在4个NIN基因和10个NLP基因家族成员,这些基因都具有RWP-RK和PB1两个保守结构域。亚细胞定位预测表明所有成员都定位在细胞核中,此外GmNIN1b和GmNIN2a还定位于叶绿体。GmNIN1a/b、GmNIN2a/b、GmNLP2a/b及GmNLP3b在根瘤中的表达量相对较高,推测这些基因可能对结瘤过程具有重要的调控功能。成功构建了NLP4和NLP5两个敲除载体,得到可敲除GmNLP4a/b的3个有效靶点和敲除GmNLP5a/b的2个有效靶点。本研究获得了大豆NIN和NLP基因家族的生物信息学依据和创制GmNLP4a/b和GmNLP5a/b基因突变体的技术依据。For further promoting the obtaining of NLP gene mutant soybean materials and studying the functions of GmNINs and GmNLPs, this study used bioinformatics methods to analyze GmNINs and GmNLPs, and constructed the knockout mutation vectors by CRISPR/Cas 9 technology, and then verified the effectiveness of the targets by soybean hair root transformation experiments. A total of 4 GmNINs and 10 GmNLPs were obtained by bioinformatics analysis, and each had two conserved domains, RWP-RK and PB1, and conserved gene structure. Subcellular localization prediction indicated all these NIN and NLP members were located in the nucleus, and GmNIN1 b and GmNIN2 a are also located in chloroplasts. In nodules, the expression of GmNIN1 a/b, GmNIN2 a/b, GmNLP2 a/b and GmNLP3 b was relatively high, predicting that these genes may have important functions in nodules. Two knockout vectors NLP4 and NLP5 were successfully constructed, and three effective targets for knocking out GmNLP4 a/b and two effective targets for knocking out GmNLP5 a/b were obtained. This study provides a biological information basis for the research of soybean NIN and NLP family gene, and also provides technical support for the mutants of soybean NLP4 a/b and NLP5 a/b genes.

关 键 词：大豆 根瘤 NLP基因 生物信息学分析 CRISPR/Cas 9基因编辑技术 基因敲除载体 GmNLP4a/b GmNLP5a/b 

分 类 号：S565.1[农业科学—作物学]