葡萄肌醇半乳糖苷合成酶基因VvGolS2-4的克隆及表达特性分析

Gene Cloning and Expression Analysis of Galactinol Synthase Gene VvGolS2-4 in Grape

为了研究葡萄肌醇半乳糖苷合成酶基因家族的特性,以抗低温品种左优红组培苗为材料,通过同源克隆技术,获得VvGolS2、VvGolS3和VvGolS4全长CDS,对其序列进行了生物信息学分析,同时利用实时定量PCR技术探究了3个基因的组织表达特性和响应逆境因子及相关信号分子的特性。结果显示:3个基因c DNA大小分别为954,978,1 011 bp,分别编码317,325,336个氨基酸序列;3个蛋白分子量分别为36.65,36.97,38.12 k Da;等电点分别为5.12,5.33和5.16,且均在C端含有一个疏水的五肽APXAA;实时荧光定量PCR证明,3个GolSs在葡萄不同组织中表达情况不同,VvGolS2和VvGolS4在叶片中表达量最高,而VvGolS3则在花和卷须中表达量最高;不同逆境因子及逆境信号分子均会影响VvGolS2、VvGolS3和VvGolS4基因的表达量,其中VvGolS2和VvGolS4基因的表达受盐诱导最明显,VvGolS3基因的表达受低温诱导最明显;另外,胁迫信号分子乙烯、过氧化氢和脱落酸均可诱导VvGolS2基因表达量上升,而VvGolS3基因的表达受脱落酸和乙烯的诱导,乙烯、过氧化氢和硫化氢则均可诱导VvGolS4基因表达量上升。综上推测,3个Vv GolSs均与葡萄抵御逆境胁迫相关,但各自功能可能有所不同。

Using homology cloning method,the full-length cDNA of galactinol synthase gene VvGolS2,VvGolS3 and VvGolS4 were cloned from Vitis vinifera cultivar Zuoyouhong tissue culture seedling.The analysis of VvGolS2,VvGolS3 and VvGolS4 sequencing indicated that three genes were 954,978 and 1 011 bp ranging in size,coded as 317,325,336 amino acids respectively with molecular weight were 36.65,36.97,38.12 kDa and the isoelectric point were 5.12,5.33,5.16 respectively.Three VvGolSs all possessed a conserved hydrophobic APXAA pentapeptide domain in the C-terminal region.Real-time PCR analysis showed that three VvGolSs were expressed different in all tested tissues,with the highest expression of VvGolS2 and VvGolS4 in leaves,and VvGolS3 was highly expressed in flowers and tendrils.Using tissue culture seedling,the relative expression levels of VvGolS2,VvGolS3 and VvGolS4 after different stresses or different stress singling factors treatment were checked.The VvGolS2 and VvGolS4 were highly induced by salt stress,while the VvGolS3 was only induced by low temperature.Moreover,VvGolS2 was significantly induced by stress-related signal molecules,such as abscisic acid（ABA）,ethylene（ACC）,and hydrogen peroxide（H2O2）.The VvGolS4 was induced by ACC,H2O2 and H2S,and the VvGolS3 was induced by ABA and ACC.Together,these results suggested that these three VvGolSs were involved in abiotic stresses resistance in grape with different functions.