刺葡萄类钙调蛋白基因VdCML8克隆与表达分析及其启动子转录活性测定

Cloning and expression analysis of calmodulin-like protein gene VdCML8 in Vitis davidii and determination of its promoter transcriptional activity

  • 摘要: 【目的】克隆刺葡萄类钙调蛋白基因VdCML8基因及其启动子序列,并对VdCML8基因进行表达分析,对其启动子进行转录活性测定,为深入探究该基因在葡萄抗炭疽病中的生物学功能提供理论参考。【方法】以刺葡萄紫秋为材料,采用RT-PCR技术克隆VdCML8基因及其启动子序列,对VdCML8蛋白的理化性质和二级结构进行生物信息学分析,并采用实时荧光定量PCR检测刺葡萄紫秋和欧洲葡萄红地球CML8基因在接种胶孢炭疽菌及外施水杨酸(SA)和茉莉酸(JA)处理后的表达特征,通过构建β-葡萄糖苷酶(GUS)融合载体转化烟草进行转录活性检测。【结果】VdCML8基因的开放阅读框(ORF)长度为450 bp,编码149个氨基酸残基,具有EF-hand结构域,其二级结构中α-螺旋占65.10%,延伸链占4.70%,无规则卷曲占20.13%,β-转角占10.07%,该蛋白定位于细胞膜中。由系统发育进化树可知,刺葡萄VdCML8蛋白与欧洲葡萄VvCML8和河岸葡萄VrCML8的亲缘关系较近。VdCML8基因的启动子序列(pVdCML8)长度为1050 bp,除含有大量的CAAT-box和TATA-box外,还含有一些光响应元件(L-box、chs-CMALa和TCT-motif)、脱落酸(ABA)响应元件(ABRE)、厌氧诱导响应元件(ARE)、防御和应激元件(TC-rich repeats)、伤害响应元件(WUN-motif)等。构建pVdCML8的瞬时表达载体pVdCML8::GUS,瞬时转化烟草后发现pVdCML8具有转录活性,且能驱动VdCML8基因表达。在接种胶孢炭疽菌后,刺葡萄紫秋VdCML8基因和欧洲葡萄红地球VdCML8基因表达均上调,均在接种后12 h达峰值,二者的相对表达量是对照组(清水处理)的22.08和9.30倍。SA处理3 h时,VdCML8基因的相对表达量是对照组的7.68倍,是VdCML8基因的2.76倍。JA处理6 h时,VdCML8基因达峰值,是对照组的22.25倍,是VdCML8基因的9.04倍。【结论】VdCML8基因是SA和JA信号途径的下游调控基因,SA和JA可诱导其高效表达,参与葡萄炭疽病响应过程,对提高植株抗病性具有一定作用。

     

    Abstract: 【Objective】Calmodulin-like protein gene(VdCML8)in Vitis davidii and its promoter sequence were cloned,and the expression of VdCML8 gene was analyzed,and the transcriptional activity of its promoter was determined to provide theoretical reference for further exploring the anti-anthracnose biological function of this gene in grapes. 【Method】V. davidii Ziqiu was as mate-rials. The VdCML8 gene and its promoter sequence were cloned by RT-PCR,and the physicochemical properties and se-condary structure of VdCML8 protein were analyzed by bioinformatics. Real-time fluorescence quantitative PCR was used to detect the expression characteristics of CML8 gene in V. davidii Ziqiu and V. vinifera cv. Red Globe after inoculation with Colletotrichum gloeosporioides and external salicylic acid(SA)and jasmonic acid(JA)treatments. β-glucosidase(GUS)fusion vector was constructed and transformed tobacco for transcriptional activity detection.【Result】The open reading frame(ORF)of VdCML8 gene was 450 bp,encoded 149 amino acids residues, and had an EF-hand domain. In the secondary structure,the proportion of α-helix was 65.10%,that of extension chain was 4.70%,that of random coil was 20.13%,and that of β-turn was 10.07%. The protein was localized in the cell membrane. According to phylogenetic tree,VdCML8 protein in V. davidii was closely related to V. vinifera VvCML8 and V. vinifera VrCML8. The VdCML8 gene promoter sequence(pVdCML8)was 1050 bp. It contained a large number of CAATbox and TATA-box,and also contained some light response elements(L-box,chs-CMALa and CTT-motif),episisic acid (ABA)response element ABRE,anaerobic induction response element(ARE),defense and stress element(TC-rich repeats),wounding response element(WUN-motif). pVdCML8::GUS,a transient expression vector of pVdCML8,was constructed. It was found that pVdCML8 had transcriptional activity and could drive the expression of VdCML8 gene after transient transformation of tobacco. The expression of V. davidii Ziqiu VdCML8 gene and V. vinifera VdCML8 gene were up-regulated and reached the peak at 12 h after inoculation. The relative expression levels of both were as 22.08 times and 9.30 times as that of control group(water treatment). After SA treatment for 3 h,the relative expression of VdCML8 gene was as 7.68 times as that of control and as 2.76 times as that of VdCML8 gene. After JA treatment for 6 h,VdCML8 gene reached its peak and was as 22.25 times as that of control group and as 9.04 times as that of VdCML8 gene.【Conclusion】 VdCML8 gene is the downstream regulatory gene of SA and JA signaling pathways. SA and JA can induce its efficient expression,participate in the response process of grape anthracnose,and have certain effects in improving plant disease resistance.

     

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