珍稀植物华木莲SRK基因的克隆与亚细胞定位  

Molecular Cloning and Subcellular Localization of SRK Gene in Sinomanglietia glauca

作　　者：邱珊姗 邓凌帆 刘苗苗 张建强[1] 王颜波 刘齐元[3] 王建革[1] QIU Shanshan;DENG Lingfan;LIU Miaomiao;ZHANG Jianqiang;WANG Yanbo;LIU Qiyuan;WANG Jiange(College of Forestry,Jiangxi Agricultural University,Nanchang 330045,China;College of Water Conservancy and Ecological Engineering,Nanchang Institute of Technology,Nanchang 330029,China;College of Agronomy,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学林学院,江西南昌330045 [2]南昌工程学院水利与生态工程学院,江西南昌330029 [3]江西农业大学农学院,江西南昌330045

出　　处：《江西农业大学学报》2021年第6期1339-1347,共9页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31960418)。

摘　　要：【目的】利用杂种优势是植物育种的重要目的,但对于自交不亲和物种来说,因为亲本杂合,很难充分利用最大杂种优势。自交不亲和是开花植物避免近交的重要繁育机制,自交不亲和与自交亲和相互转换在自交不亲和物种中非常普遍,这种转换可为自交不亲和物种育种开辟新的途径。华木莲(Sinomanglietia glauca)为木兰科珍稀物种,有重要科学价值和良好应用前景,但适应能力差,自我更新能力弱。华木莲呈现迟发性自交不亲和特征,自交不亲和机理尚不清楚。从华木莲转录组中鉴定出了与孢子体自交不亲和反应关键基因S位点受体激酶(S-locus receptor kinase,SRK)基因高度同源基因SgSRK,这是唯一一类与已知自交不亲和基因高度同源的迟发性自交不亲和基因,因此了解该基因的作用成为研究华木莲自交不亲和机理的出发点。克隆基因、分析其编码蛋白理化性质及亚细胞定位是进行基因功能分析的前提,因此了解SgSRK基因克隆与亚细胞定位可为揭示该基因在华木莲自交不亲和中的作用提供参考。【方法】以自花授粉后3 h华木莲心皮为材料,根据转录组扩增出了SgSRK基因,对其编码蛋白分析了理化性质,鉴定了结构域,推断了疏水性、信号肽、跨膜结构,并对其亚细胞定位进行了预测。为探讨SgSRK基因与已知的不同物种SRK基因演化关系,基于蛋白序列利用最大似然法构建了系统发育树。将目的基因和GFP基因构建了重组表达载体并转化农杆菌,然后将含有目的基因重组表达载体的农杆菌直接注射烟草叶片,3 d后利用荧光显微镜观察叶片注射部位基因瞬时表达情况,确定其编码蛋白的亚细胞定位。【结果】SgSRK基因共计2463 bp(GenBank登录号:MW139903),编码蛋白含有820个氨基酸,编码蛋白具有4个结构域:B;ectin、S;ocus;lycop、PAN-2、Pkinase Tyr结构域,有跨膜结构,存在信号肽,因此编码蛋白质可�[Objective]Utility of heterosis is a very important aim for plant breeding,but its maximum utility is difficult to achieve for self-incompatible species because their parents are heterozygous.Self-incompatibility(SI)is a major genetic mechanism that prevents self-fertilization in flowering plants.The transition between SI and self-compatibility(SC)exists widely in SI species,and can become a new breeding method for SI species.Sinomanglietia glauca is a rare Magnoliaceae plant with important scientific value and good application prospects,but its adaptability is poor and its self-renewal is weak.The SI in S. glauca is late-acting SI,and its mechanism was unclear.SgSRK,a highly homologous gene of plant S-locus receptor kinase(SRK)gene,the key gene in sporophytic SI(SSI),was identified from S. glauca transcriptome.This is the only kind of late-acting SI(LSI)gene highly homologous to the known SI genes.Therefore,studies of the function of SgSRK will help deep understanding the SI mechanism in S. glauca.Cloning of the gene,physical and chemical properties analysis and subcellular localization of its coding protein are the premise of gene function analysis.Therefore,the cloning and subcellular localization of SgSRK gene can provide a reference for revealing its role in SI in S. glauca.[Methods]SgSRKgene was cloned from the S. glauca carpel 3 h after self-pollination,based on the transcriptomic results,the physical and chemical properties of its coding protein were analyzed,and the motifs were identified.The hydrophobicity,signal peptide and transmembrane structure of this protein were inferred,and its subcellular localization was predicted.In order to explore the evolutionary relationship between SgSRK and SRKs of different known plant species,a phylogenetic tree was constructed by maximum likelihood method based on protein sequences.The recombinant expression vector was constructed with the target gene and GFP gene and then transformed into Agrobacterium tumefaciens.The Agrobacterium tumefaciens containing recombinant

关 键 词：华木莲 自交不亲和 S位点受体激酶 分子克隆 亚细胞定位 生物信息学 

分 类 号：Q945.5[生物学—植物学]