西瓜谷氨酸脱羧酶GADs的克隆与表达分析

Cloning and Expression Analysis of Glutamate Decarboxylase GADs from Watermelon

谷氨酸脱羧酶(GAD)是催化谷氨酸脱羧的裂解酶,对GABA合成通路起关键作用,并对植物抵御非生物胁迫有重要调控作用。前期通过代谢组和转录组关联分析筛选得到3个西瓜GAD基因家族成员(Cla97C01G007270、Cla97C01G007290和Cla97C04G079700)。在此基础上克隆了3个西瓜GAD家族基因,分别命名为ClGAD1、ClGAD2和ClGAD3,并对其进行生物信息学及表达模式分析。结果显示,ClGAD1、ClGAD2和ClGAD3的CDS分别为1524,1497,1497 bp,其编码的氨基酸数量分别为507,498,498。3个蛋白均为亲水性蛋白;亚细胞定位于线粒体中;具有高度的保守性,保守结构域为谷氨酸脱羧酶;其启动子区域含有大量CAAT-box和TATA-box,以及与逆境胁迫相关的MYB、MYC等相应元件。3个GAD蛋白的二级结构均以α-螺旋、无规卷曲为主;三级结构均为同源六聚体结构。系统进化树显示,GAD基因家族成员与苦瓜、拟南芥的GAD亲缘关系最近。qRT-PCR分析显示,ClGAD1和ClGAD2在茎中表达量最高,而ClGAD3在花中表达量最高。ClGAD1、ClGAD2、ClGAD3分别在盐胁迫12,24,48 h表达量最高;冷和干旱胁迫下3个GAD基因的表达模式较为相似,均在胁迫早期发生大量表达,且ClGAD3在非生物胁迫下的表达量均高于其他2个家族成员。获得了西瓜3个GAD基因的cDNA全长,并对其进行生物信息学和表达模式分析,为丰富西瓜抗非生物胁迫基因资源提供了试验依据。

Glutamate decarboxylase(GAD)is a cleavage enzyme that catalyzes the decarboxylation of glutamic acid,plays a key role in GABA synthesis pathway and has an important influence on plants′resistance to abiotic stress.Three watermelon GAD gene family members(Cla97C01G007270,Cla97C01G007290 and Cla97C04G079700)were screened by the association analysis of metabolomics and transcriptomes.On this basis,three watermelon GAD family genes were cloned,named ClGAD1,ClGAD2 and ClGAD3 respectively,and their bioinformatics and expression patterns were analyzed.The results showed that the CDS of ClGAD1,ClGAD2 and ClGAD3 were 1524,1497 and 1497 bp,respectively,and the number of amino acids encoded by them was 507,498 and 498,respectively.All three proteins were hydrophilic proteins and were located in mitochondria.It was highly conserved,and the conserved domain was glutamic acid decarboxylase.Its promoter region contained a large number of CAAT-box and TATA-box,as well as corresponding elements related to adversity stress such as MYB and MYC.The secondary structures of the three GAD proteins were mainlyα-helix and random coil.The tertiary structures were all homohexamer structures.Phylogenetic tree showed that members of GAD gene family were closest to GAD of Momordica charantia and Arabidopsis thaliana.qRT-PCR analysis indicated that the highest expression levels of ClGAD1 and ClGAD2 were showed in stems,while that of ClGAD3 was showed in flowers.The expression levels of ClGAD1,ClGAD2 and ClGAD3 were the highest under salt stress for 12,24 and 48 h,respectively.Under cold and drought stress,the expression patterns of the three GAD genes were similar,and all of them were expressed in large quantities in the early stage of stress,and the expression of ClGAD3 under abiotic stress was higher than that of the other two family members.The full-length cDNA of three GAD genes in watermelon was obtained,and their bioinformatics and expression patterns were analyzed,which provided an experimental basis for enriching the abiotic stress resistance gene resources of watermelon.