棉花四个C2H2锌指蛋白基因的克隆与表达分析

Cloning and Expression Analysis of Four C2H2 Zinc Protein Gene in Cotton(Gossypium hirsutum L.)

水稻广谱抗病基因Bsr-d1编码C2H2型锌指蛋白,本研究基于此通过RT-PCR技术从棉花中克隆4个与水稻Bsr-d1同源的C2H2型锌指蛋白基因并对其进行了生物信息学分析和表达分析,结果表明这些基因氨基酸序列与Bsr-d1的一致性为25%-35.7%,故命名为GhBsr-d1L1-GhBsr-d1L4;GhBsr-d1Ls基因的开放阅读框大小为663-813 bp,编码的氨基酸数为220-270,蛋白质分子量为23.82-28.64 kD,理论等电点为7.22-8.56,不稳系数为47.7-67.17,亲水平均数为-0.755--0.544,无信号肽和跨膜结构域;GhBsr-d1Ls均定位于细胞核,编码N端的保守L-box结构域,C2H2型锌指结构域以及C端的转录抑制结构域EAR/DLN-box,属于陆地棉中第Ⅱ类C2H2型锌指蛋白亚家族。系统发育树分析表明,GhBsr-d1L1与GhBsr-d3L3的遗传距离最近,GhBsr-d1L2与大豆SCOF-1遗传距离最近,GhBsr-d1L4与长果种黄麻的同源基因遗传距离最近。荧光定量PCR分析表明,GhBsr-d1Ls基因在根、下胚轴及真叶中均表达,但表达量各有差异,同时在不同程度上GhBsr-d1Ls均受到大丽轮枝菌的诱导。推测GhBsr-d1Ls基因可能在棉花响应大丽轮枝菌时起重要作用。

Rice broad spectrum resistance gene Bsr-d1 encodes C2 H2 zinc finger protein.In this study four homologous genes of rice Bsr-d1,was cloned from cotton by RT-PCR,and bioinformatics analysis and expression analysis were performed.The results showed that four homologous genes have 25%~35.7%amino acid sequence identity with Bsr-d1.So they were named GhBsr-d1 L1~GhBsr-d1 L4.GhBsr-d1 Ls encode protein of 220~270 amino acid residues with a predicted molecular mass of 23.82~28.64 kD,the instability index of 47.7~67.17 and no signal peptides and transmembrane domains.Subcellular localization predicted that GhBsr-d1 Ls were all located in the nucleus.They all have a L-box domain at the N-terminal,a C2 H2-type zinc finger domain and a putative transcription repression domain(EAR/DLN-box)at the C-terminal,belong to the classⅡC2 H2-type zinc protein subfamily in upland cotton.Phylogenetic analysis revealed that GhBsr-d1 L1 shares high identity with GhBsr-d1 L3,GhBsr-d1 L2 shares high identity with C2 H2-type zinc finger proteins from Glycine max,and GhBsr-d1 L4 shares high identity with C2 H2-type zinc finger protein from Corchorus olitorius.Fluorescence quantitative PCR analysis showed that,GHBSR-D1 LS gene was expressed in roots,hypocotyls and true leaves,but the expression levels were different.At the same time,GHBSR-D1 LS gene was induced by Verticillium dahliae.to different degrees.These results suggest that GhBsr-d1 Ls may play an important role in the response of cotton against Verticillium dahliae.