普通菜豆镰孢菌枯萎病抗病相关基因PvCaM1的克隆及表达

Cloning and Expression Analysis of Fusarium Wilt Resistance-Related Gene PvCaM1 in Common Bean (Phaseolus vulgaris L.)

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 vulgarisL.)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713bp,命名为PvCaM1,具有一个453bp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17.16kD。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基因受尖镰孢菌菜豆专化型FOP-DM01菌株诱导表达,接种病原菌96h,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。

Calmodulin （CAM） is a multifunctional Ca^2＋-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 con- tained an open reading frame （ORF） of 453 bp encoding 150 amino acids with a theoretical molecular weight of 17.16 kD, desig- nated PvCaM1 （GenBank accession number JN418801）. Online ScanProsite tool analysis showed that PvCaM1 had four Ca^2＋-binding domains with the function of combining with free Ca^2＋. Homology analysis indicated that PvCaM1 gene was similar to CaM genes in other plant species including Lotusjaponicus （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 interac- tions 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 ex- pression level of PvCaM1 in 260205 leaves was higher than that in BRB-130 at all different time points. PvCaM1 expressed dif- ferently 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 path- way. The study indicated that the function of PvCaM1 should be closely related to the resistant response pathways against Fusa- rium wilt pathogen FOP-DM01 with involvement of abscisic acid, jasmonic acid and el：hylene in common bean.