普通菜豆镰孢菌枯萎病抗病相关基因PvCaM1的克隆及表达分析

doi:10.3724/SP.J.1006.2012.00606

摘要/Abstract

摘要： 钙调素蛋白(calmodulin，CaM)作为植物细胞内介导多种功能的Ca²⁺结合蛋白，在调节植物的生长发育和抗病性方面具有重要作用。利用普通菜豆(Phaseolus vulgaris L.)表达序列标签(EST)克隆了含有编码普通菜豆CaM基因的cDNA序列。序列分析表明，cDNA片段长713 bp，命名为PvCaM1，具有一个453 bp的开放阅读框(ORF)，GenBank登录号为JN418801，该基因编码150个氨基酸，预测蛋白质分子质量为17.16 kD。蛋白质结构分析表明，PvCaM1蛋白含有4个Ca²⁺结合结构域(EF-hand)。同源分析结果显示，PvCaM1基因与百脉根、西瓜的CaM基因亲缘关系最近，分别达到77%和76%。荧光定量PCR分析表明，PvCaM1基因受尖孢镰孢菌菜豆专化型FOP-DM01菌株诱导表达，接种病原菌96 h，抗病品种260205根中PvCaM1基因的表达量达到最高，而感病品种BRB-130达到最低，260205叶中PvCaM1基因的表达量均高于BRB-130，而且叶中的表达量高于根和茎中的表达量。PvCaM1基因表达量也受外源植物激素脱落酸、茉莉酸甲酯和乙烯利诱导上调，在根、茎、叶中均有不同程度的表达。本研究表明PvCaM1基因可能通过脱落酸、茉莉酸和乙烯等信号途径参与菜豆对FOP-DM01菌株的防御反应，推测菜豆PvCaM1基因与镰孢菌枯萎病的抗病性有一定关联。

关键词: 普通菜豆, 镰孢菌枯萎病, PvCaM1基因, 基因表达分析

Abstract: Calmodulin (CaM) is a multifunctional Ca²⁺-binding protein in plant cells. It plays important roles in regulating the growth, development and disease resistance in plants. A full-length cDNA sequence coding for CaM in common bean was cloned based on expressed sequence tags from common bean. Sequence analysis showed that the isolated fragment was 713 bp, it contained an open reading frame (ORF) of 453 bp encoding 150 amino acids with a theoretical molecular weight of 17.16 kD, designated PvCaM1 (GenBank accession number JN418801),. Online ScanProsite tool analysis showed that PvCaM1 had four Ca²⁺-binding domains with the function of combining with free Ca²⁺. Homology analysis indicated that PvCaM1 gene was similar to CaM genes in other plant species including Lotus japonicus (CAB63264.3), watermelon (BAI52955.1), Populus (ADC80735.1) and castor bean (XP_002533357.1)．Phylogenetic analysis based on the amino acids sequence of PvCaM1 with other nine species showed that the protein encoded by this gene had the closest relationship with the CaM in Lotus japonicus and watermelon, the homology were 77% and 76%, respectively. Real time-PCR analysis indicated that the expression level of PvCaM1 in the interactions between the resistant cultivars and Fusarium wilt pathogen FOP-DM01 (Fusarium oxysporum f. sp. phaseoli) increased significantly and reached the peak at 96 h, however, the susceptible one touched the bottom under the same conditions．The expression level of PvCaM1 in 260205 leaves was higher than that in BRB-130 at all different time points. PvCaM1 expressed differently in the leaves, stems and roots, the expression level in leaves was higher than that in roots and stems. Transcriptional level of PvCaM1 was up-regulated by exogenous abscisic acid, methyl jasmonate and ethephon, but not increased significantly by salicylic acid and 3-indoleacetic acid. These results suggested that PvCaM1 is probably involved in the abscisic acid, jasmonic acid and ethylene-regulated resistant response pathways, but not closely related to the salicylic acid and 3-indoleacetic acid pathway. The study indicated that the function of PvCaM1 should be closely related to the resistant response pathways against Fusarium wilt pathogen FOP-DM01 with involvement of abscisic acid, jasmonic acid and ethylene in common bean.

Key words: Common bean, Fusarium wilt, PvCaM1 gene, Expression analysis

薛仁风，朱振东，王晓鸣，王兰芬，武小菲，王述民. 普通菜豆镰孢菌枯萎病抗病相关基因PvCaM1的克隆及表达分析[J]. 作物学报, 2012, 38(04): 606-613.

XUE Ren-Feng,ZHU Zhen-Dong,WANG Xiao-Ming,WANG Lan-Fen,WU Xiao-Fei,WANG Shu-Min. Cloning and Expression Analysis of Fusarium Wilt Resistance-related Gene PvCaM1 in Common Bean (Phaseolus vulgaris L.)[J]. Acta Agron Sin, 2012, 38(04): 606-613.

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