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

【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BnGPDHc2-2在3个高油含量自交系中的表达量均显著高于3个低油含量自交系。将BnGPDHc2-1和BnGPDHc2-2基因分别连接至携带napin启动子的pCAMBIA1390-NAPIN载体上可成功构建获得植物过表达载体。【结论】BnGPDHc2-1和BnGPDHc2-2基因在甘蓝型油菜不同发育期角果中行使的功能存在一定分化,在进化过程中可能分别来源于白菜基因组和甘蓝基因组,但均在甘蓝型油菜种子TAG合成和积累发挥重要调控作用。因此,构建的植物过表达载体可用于BnGPDH基因功能分析及油菜种子油含量基因工程改良研究。

【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[7DAF,21DAF,30DAF and 40DAF]and silique pericarps[7DAF and 40DAF] than BnGPDHc2-2,but the expression pattern exhibited the opposite in silique pericarps of 14DAF. In 21 DAF seeds, BnGPDHc2-1 was significantly higher expressed in three high seed oil content(H-SOC)inbred lines than three low seed oil content(L-SOC)inbred lines,but BnGPDHc2-2 was significantly higher expressed only in two H-SOC inbred lines than three L-SOC inbred lines in the seeds of 21 DAF. Additionally,BnGPDHc2-1 and BnGPDHc2-2 were significantly higher expressed in three H-SOC inbred lines than three L-SOC inbred lines in the seeds of 30DAF. Therefore,BnGPDHc2- 1 and BnGPDHc2-2 being linked to pCAMBIA1390-NAPIN vector carrying napin promoter could construct the plant over-expression vector.【Conclusion】BnGPDHc2-1 and BnGPDHc2-2 are cloned and they may play different roles in the development of siliques at various growth stages of B. napus. Although BnGPDHc2-1 and BnGPDHc2-2 originate from B. rapa and B. oleracea genomes,they play an important role in TAG synthesis of rapeseed seeds. The constructed plant overexpression vectors can be used for the functional analysis of BnGPDH gene and the genetic engineering research to improve seed oil content of rape seed.