中国野生葡萄病原菌诱导响应VqSAP基因的克隆及表达分析

【目的】为了验证SAP基因在葡萄抗病防御机制中的作用,【方法】在前期通过抑制消减杂交技术(SSH)获得中国野生葡萄毛葡萄株系‘商-24’衰老相关蛋白(SAP)基因EST序列的基础上,采用RT-PCR从毛葡萄‘商-24’中分离到SAP基因cDNA序列,并通过实时定量PCR和半定量PCR分析了SAP基因的表达特异性。【结果】序列分析表明该基因具有完整的的开放阅读框,序列长度为819 bp,编码272个氨基酸残基,相对分子质量为30.56 ku,等电点为8.78,将其命名为VqSAP。多重比较结果显示VqSAP基因与油棕、玉米、萱草、拟南芥、水稻等植物SAP基因的同源性为65%~74%。实时定量结果表明,在白粉菌诱导初期,抗病的毛葡萄株系‘商-24’和感病的华东葡萄株系‘湖南-1’VqSAP基因表达量均升高,这可能是因为它参与了病原菌诱导的细胞程序性死亡过程。水杨酸(SA)、茉莉酸甲酯(MeJA)、乙烯(Eth)处理表明衰老相关蛋白基因表达受激素分子调节,但在抗病和感病株系中基因的表达量有所差异。在嫩叶、嫩茎、花、果皮、卷须等不同葡萄组织中的表达表明VqSAP基因的表达具有空间差异性。【结论】这些结果暗示着VqSAP的表达响应病原菌侵染并受生物胁迫相关激素的调控,很有可能在葡萄防御病原菌侵染的机制中发挥重要作用。

Cloning and expression analysis of a pathogen-responsive VqSAP gene in Chinese wild Vitis quinquangularis

【Objective】 The objective of the study is to verify the role of SAP gene in grape disease resistance.【Method】 Based on a EST sequence of SAP gene in the subtractive suppression hybridization(SSH) cDNA library from young leaves of Chinese wild Vitis quinquangularis clone 'Shang-24' inoculated with Uncinula necator(Schw.) Burr,we isolated the cDNA sequence of that SAP gene using the RT-PCR technology,which was designated as VqSAP.Then we studied its expression patterns by Semi-Quantitative RT-PCR and Quantitative RT-PCR.【Result】 The sequence analysis indicated that its complete ORF was 819 bp,which encoded 272 amino acids,with a predicted molecular weight of 30.56 ku and the isoelectric point of 8.78.The qRT-PCR showed that the expression of VqSAP was induced by U.nector in both the resistant and the susceptible materials,it may be involved in the programmed cell death.VqSAP was also regulated by ethylene(Eth),methyl jasmonate(MeJA) and salicylic acid(SA),but its expression was different between the resistant and susceptible lines.VqSAP was also expressed differentially in leaves,stems,flowers,pericarps and tendrils.【Conclusion】 These results revealed that VqSAP was a pathogen-responsive gene and it was regulated by certain biotic stress-related hormones,indicating it may play an important role in grape pathogen resistance.