玉米ZmGAPDH2基因的克隆及表达特性分析

Cloning and Analysis of Expression Characteristics of Glyceraldehyde-3-Phosphate Dehydrogenase 2(ZmGAPDH2)Gene in Maize

研究通过同源比对查找GAPDH基因的保守区,运用逆转录PCR和同源克隆技术从玉米叶片中克隆出GAPDH2基因的全长cDNA序列。采用qRT-PCR分析两个玉米品种登海605和蠡玉35幼苗叶片中GAPDH2基因在非生物胁迫和ABA处理下的表达特性,利用二维凝胶电泳(2-DE)和基质辅助激光解吸电离/飞行时间质谱法(MAL⁃DI-TOF),对登海605和蠡玉35幼苗在干旱胁迫下叶片中的GAPDH蛋白表达水平进行分析。结果表明,ZmGAP⁃DH2基因开放阅读框(ORF)全长1014 bp,编码337个氨基酸。ZmGAPDH2蛋白分子量为36.53 kDa,属于亲水性蛋白。生物信息分析发现,玉米ZmGAPDH2蛋白和单子叶植物小米GAPDH2蛋白的亲缘关系最近,同源性高达94.66%。qRT-PCR分析表明,ZmGAPDH2在登海605和蠡玉35叶片中均能被ABA和PEG处理诱导表达,登海605在NaCl和蠡玉35幼苗在低温处理下其表达量均有所下降。干旱对登海605和蠡玉35幼苗叶片中ZmGAPDH2蛋白表达量也有影响。

In this study,a GAPDH gene,ZmGAPDH2,was isolated from maize cultivar Liyu 35 using reverse transcription polymerase chain reaction method.Moreover,the expression patterns of ZmGAPDH2 were examined using RT-qPCR method under abiotic stress and ABA treatments,and the ZmGAPDH2 protein abundance were studied under control and drought stress conditions using two-dimensional gel electrophoresis(2-DE)and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry(MALDI-TOF)method in leaves of the two maize cultivars LY35 and Denghai605.The results showed that the full-length cDNA of ZmGAPDH2 was 1014 bp in length and it encoded 337 amino acid residues.ZmGAPDH2,with protein molecular weight of 36.53 kDa,was predicted to be a hydrophilic protein.Bioinformatic analyses revealed that ZmGAPDH2 was highly homologous to other plant GAPDHs,especially to FmGAPDH2(94.66%).The ZmGAPDH2 transcription could be in⁃duced by PEG and ABA treatments both in LY35 and DH605 seedling leaves.However,ZmGAPDH2 expression lev⁃el was reduced during NaCl treatment in DH605 and cold treatment in LY.Furthermore,drought stress also affected the protein level of ZmGAPDH2 in seedling leaves of DH605 and LY35.