甘蓝型油菜GPDH基因克隆、表达分析及植物过表达载体构建  

Cloningandexpressionanalysisof GPDHgenein Brassicanapus L. and construction of the plant over-expression vectors

作　　者：张超 付三雄[3] 周小婴[3] 唐容[1] 黄莎 杨克相 戚存扣 ZHANG Chao;FU San-xiong;ZHOU Xiao-ying;TANG Rong;HUANG Sha;YANG Ke-xiang;QI Cun-kou(Institute of Oil Crops,Guizhou Academy of Agricultural Sciences,Guiyang 550006,China;College of Agriculture,Nanjing Agricultural University,Nanjing 210015,China;Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]贵州省农业科学院油料研究所,贵阳550006 [2]南京农业大学农学院,南京210015 [3]江苏省农业科学院经济作物研究所,南京210014

出　　处：《南方农业学报》2019年第7期1399-1407,共9页Journal of Southern Agriculture

基　　金：贵州省科学技术基金项目（黔科合LH字〔2014〕7685号）;贵州省科技计划项目（黔科合NY〔2015〕3019-1号）;贵州省油菜现代农业产业技术体系建设项目（GZCYTX2018-08)

摘　　要：【目的】克隆甘蓝型油菜的甘油-3-磷酸脱氢酶(GPDH)基因(BnGPDH),分析其表达特性并构建植物过表达载体,为研究该基因在油菜种子三酰甘油(TAG)合成和积累中的作用机制提供理论参考。【方法】以甘蓝型油菜H105为材料,采用同源克隆技术克隆与At3G07690同源的BnGPDH基因,对其进行生物信息学分析,利用实时荧光定量PCR(qRT-PCR)检测BnGPDH基因在甘蓝型油菜不同组织[根、茎、叶、蕾、花及花后(DAF)7、14、21、30和40 d角果皮和种子]及高、低油含量自交系角果皮和种子中的表达情况,并将克隆获得的BnGPDH基因连接至携带napin启动子的pCAMBIA1390-NAPIN载体上构建植物过表达载体。【结果】克隆获得的BnGPDHc2-1和BnGPDHc2-2基因编码区(CDS)长度均为1338 bp,编码445个氨基酸,其编码蛋白仅有5个氨基酸差异,均具有NAD_Gly3P_dh_N和NAD_Gly3P_dh_C结构域,定位于细胞质,分别与白菜细胞质型GPDH蛋白(XP_009147040.1)和甘蓝细胞质型GPDH蛋白(XP_013585317.1)的氨基酸序列具有较高相似度,对应的相似度为100.00%和99.78%,且二者与双子叶植物GPDH蛋白亲缘关系较近,与单子叶植物GPDH蛋白亲缘关系较远。BnGPDHc2-1和BnGPDHc2-2基因在甘蓝型油菜不同组织中均有表达,且表达模式总体上相似,在21DAF和30DAF种子中的表达量显著高于其他组织部位(P<0.05,下同),表明21DAF和30DAF是油菜种子TAG合成和积累的高峰期,但在角果生长发育不同时期这2个基因的表达略有不同,且在茎、蕾、种子(7DAF、21DAF、30DAF和40DAF)和角果皮(7DAF和40DAF)中,BnGPDHc2-1基因的表达量均显著高于BnGPDHc2-2基因,而在14DAF角果皮中BnGPDHc2-2基因的表达量显著高于BnGPDHc2-1基因。21DAF种子中BnGPDHc2-1基因在3个高油含量自交系中的表达量显著高于3个低油含量自交系,BnGPDHc2-2基因在高油含量自交系IL130和IL594中的表达量显著高于3个低油含量自交系;在30DAF种子中,BnGPDHc2-1和BnGPDHc2-【Objective】Glycerin-3-phosphate dehydrogenase(GPDH)gene(BnGPDH)were cloned from Brassica napus L.,and their expression were analyzed,in addition,the plant over-expression vectors were constructed,in order to lay a foundation for studying the role of BnGPDHs in triacylglycerol(TAG)synthesis and accumulation of rape seeds.【Method 】BnGPDH genes homologous to At3G07690 were cloned from B. napus cultivar H105 by homology cloning,and their basic biological information were analyzed by bioinformatics. Expression patterns of BnGPDH gene in different tissues [roots,stems,leaves,buds,flowers,silique pericarps and seeds at 7,14,21,30,40 d after flower(DAF)]and different oil content inbred lines(silique pericarps and seeds)were detected by real-time fluorescence quantitative PCR(qRTPCR). BnGPDH gene was linked to pCAMBIA1390-NAPIN vector carrying napin promoter to construct plant over-expression vector.【Result】BnGPDHc2-1 and BnGPDHc2-2 have been cloned and their coding sequence(CDS)both were 1338bp encoding 445 amino acids. The encoded proteins of BnGPDHc2-1 and BnGPDHc2-2 had slightly different with five amino acids,but both of them located in the cytoplasm and included NAD_Gly3P_dh_N and NAD_Gly3P_dh_C domains. BnGPDHc2-1 and BnGPDHc2-2 encoded proteins shared 100.00% and 99.78% homology with B. rapa cytoplasmic GPDH protein(XP_009147040.1)and B. oleracea cytoplasmic GPDH protein(XP_013585317.1)respectively,which was high. In addition,both of the cloned BnGPDH had higher homology with dicotyledons GPDH proteins than monocotyledons GPDH proteins. BnGPDHc2-1 and BnGPDHc2-2 were expressed in all tested tissues with similar expression patterns in B. napus and were significantly higher(P<0.05,the same below)expressed in 21 DAF and 30DAF seeds than that of other tissues,which suggested that 21DAF and 30DAF were the peak of TAG accumulation in seeds. However,the expression of BnGPDHc2-1 and BnGPDHc2-2 genes were slightly different at different stages in siliques,BnGPDHc2-1 were higher expressed in stems,buds,seeds[7DA

关 键 词：甘蓝型油菜 甘油-3-磷酸脱氢酶(GPDH) 表达特性 生物信息学 植物过表达载体 

分 类 号：S565.403.53[农业科学—作物学]