中国野生毛葡萄白粉菌诱导响应VqAP基因的克隆及表达分析

Cloning and expression analysis of a powdery mildew-responsive VqAP gene in Chinese wild Vitis quinquangularis

【目的】克隆中国野生毛葡萄"商-24"天冬氨酸蛋白酶(AP)基因的cDNA序列,明确其在葡萄抗病防御机制中的作用。【方法】在前期获得的中国野生毛葡萄株系"商-24"AP基因(VqAP)EST序列的基础上,采用RT-PCR克隆AP基因的cDNA序列,对其序列及编码产物进行生物信息学分析,并通过实时定量PCR和半定量PCR,分析VqAP基因在白粉菌诱导不同时间(0,6,12,24,48,72,96和120h),不同激素(100μmol/L水杨酸,50μmol/L乙烯和0.5g/L茉莉酸甲酯)刺激及不同组织(嫩叶、嫩茎、花、果皮、卷须)中的表达情况。【结果】序列分析表明,VqAP基因序列长度为1 377bp,具有开放阅读框,编码458个氨基酸残基,相对分子质量为50.48ku,等电点为8.73。保守结构域分析表明,VqAP编码产物含有2个保守的具有催化活性的天冬氨酸残基,位于Asp-Thr/Ser-Gly(DT/SG)结构域,该基因编码的氨基酸序列属于植物天冬氨酸蛋白酶A1家族,为非典型的天冬氨酸蛋白酶。荧光定量PCR结果表明,接种白粉菌后6h,VqAP基因表达量为0h的6倍多,之后迅速下调;不同激素处理后,该基因也均诱导表达,这可能是由于VqAP基因参与了植物早期的抗病反应,以及由不同激素诱导的发病相关基因的调节作用。半定量PCR结果表明,在葡萄嫩叶、嫩茎、花、果皮、卷须等不同组织中VqAP基因的表达量不同,这可能与它参与了植物不同组织中功能蛋白的合成与降解有关。【结论】VqAP的表达响应病原菌侵染并受生物胁迫相关激素的调控,可能在葡萄防御病原菌侵染的机制中发挥重要作用。

[Objective] This study aimed to clone a cDNA sequence of aspartic protease （AP） in Vitis quinquangularis ＇ Shang-24＇ to verify the role of AP gene in grape disease resistance mechanism. [Method] Based on a EST sequence of AP gene in the subtractive suppression hybridization （SSH） cDNA library of young leaves of Chinese wild Vitis quinquangularis clone ＇Shang-24 ＇ inoculated with Uncinula necator （Schw.） Burr,we isolated the cDNA sequence of AP gene using the RT-PCR technology,which was designated as VqAP, and analyzed it with bioinformatics method. Real-time quantitative PCR and semi-quantitative PCR were used to detect the expression trends after inoculation with pathogen at different times （0,6, 12,24,48,72,96 and 120 h）, treated with different hormones（100 μmol/L Salicylic acid, 50 μmol/L Ethylene and 0.5 g/L Methyl jasmonate） in different tissues （leaves, stems, flowers, pericarps and tendrils）. [Result] The sequence analysis indicated that the complete ORF was 1 377 bp,encoding 458 amino acids. The predicted molecular weight was 50.48 ku and the isoelectric point was 8.73. Conserved domains analysis of VqAP deduced amino acid showed that the amino acid encoded by VqAP had two conserved aspartic acid residues located in Asp-Thr/Ser-Gly （DT/SG） of these two domains and it was atypical aspartic proteases belonging to elan A1 of plant aspartic proteases gene family. QRT-PCR analysis showed that the expression of VqAP was induced six hours later after infected with U. necator and treatmented with different hormones. The gene may be involved in defense response at the early period of pathogen infection and it may be regulated some pathogen-related genes. Different expression levels of VqAP were observed in leav- es,stems,flowers, pericarps and tendrils which indicated that it involved in synthesis and degradation of different proteins in plants tissues. [Conclusion] VqAP was a pathogen-responsive gene and it was regulated by certain biotic stress-related hormones, indicating that it may play an important role in grape pathogen resistance.