杧果MGAPDH同源基因的克隆及其表达分析  

Molecular cloning and expression analysis of a MGAPDH homologous gene from mango

作　　者：罗聪[1] 何新华[1,2] 胡颖[1] 谭超[1] 欧世金[1] 

机构地区：[1]广西大学农学院,南宁530004 [2]广西作物遗传改良与生物技术重点实验室,南宁530007

出　　处：《果树学报》2011年第6期1019-1024,F0003,共7页Journal of Fruit Science

基　　金：广西高等学校优秀人才资助计划项目(桂教人201065);广西自然科学基金(2011GXNSFA018115);广西教育厅研究生创新计划项目(105931001011)

摘　　要：甘油醛-3-磷酸脱氢酶(GAPDH)是糖代谢和能量代谢过程中的关键酶。根据GAPDH基因保守序列设计PCR引物,利用RT-PCR和改良RACE技术,首次从杧果中分离克隆出MGAPDH同源基因的cDNA全长序列。该序列全长为1 356 bp,开放阅读框为1 203 bp,编码401个氨基酸。利用分子生物学软件对MGAPDH蛋白进行生物信息学分析,结果显示:MGAPDH蛋白分子量为42.8 ku,等电点为8.9;该蛋白包含2个功能域,即NADB_Rossmannsuperfamily和Gp_dh_C superfamily;MGAPDH蛋白不含信号肽序列和跨膜结构,是非分泌蛋白,位于叶绿体中。氨基酸序列进化分析结果显示,杧果MGAPDH蛋白可能与光合作用有关。半定量分析显示,杧果MGAPDH同源基因在杧果不同组织中均表达,并且表达水平差异不明显,说明杧果MGAPDH同源基因可作为杧果基因差异表达的内参基因。Glyceral-dehyde-3-phosphate dehyrogenase （GAPDH） is a key enzyme in sugar metabolisn and energy metabolism. PCR primers were designed by the conserved sequenees of GAPDH gene,and full-length cDNA sequence of MGA PDH homologous gene was firstly cloned from Mango by employin RT-PCR and modified RACE technique. The full-length cDNA is 1 356 bp and contains an ORF witl 1 203 bp, corresponding to a deduced protein of 401 amino acids. The results of bioinformatics analysis showed that the estimated molecular weight and isoelectric point of the putative protein were 42.8 ku am 8.9. The protein had two conserved domains, named as NADB_ Rossmann superfamily and Gp dh C superfamily. Bioinformatics analysis also indicated that the protein of mango MGAPDH is a non-secretin protein without any signal peptide and transmembrane domain,MGAPDH gene was located in the chloro plast. The phylogenetic analysis based on the sequence of amino acids showed that MGAPDH protein ma, relative to photosynthesis. Semiquantitative analysis showed that the MGA PDH homologous gene expressec in all tissues of mango, no significant differences in expression levels were founded among the tested tissues; the result indicted that MGAPDH homologous gene may be used as an internal standard for differential gene expression analysis in mango.

关 键 词：杧果 甘油醛-3-磷酸脱氢酶(GAPDH) 表达分析 序列分析 

分 类 号：S667.7[农业科学—果树学]