海岛棉GbPR10基因的克隆及其抗枯萎病表达分析

Cloning and Expression Analyses of Furasium Wilt Resistance Gene GbPR10 in Gossypium barbadense

【目的】为棉花抗枯萎病分子育种的基因来源提供依据。【方法】根据前期转录组测序和抗病表达数据,从棉花EST数据库中筛选出抗枯萎病有关的基因(登录号为CD486053)序列,在NCBI搜索与该基因同源性为94%的海岛棉抗病相关PR10基因(登录号为AY588276)并设计引物,从枯萎病接菌的抗病海岛棉材料"06-146"克隆一个海岛棉同源基因,命名为GbPR10基因。进行生物信息学和在枯萎病菌、乙烯、水杨酸处理下基因表达量分析。【结果】GbPR10基因有480 bp的ORF序列,编码159个氨基酸。该蛋白序列中具有PR10蛋白特有的Bet-v1结构域和改变的甘氨酸环P-Loop(GXGGXG)。蛋白质序列同源比对表明该蛋白与其他生物PR10蛋白有较高的一致性。亚细胞定位预测表明GbPR10分布于细胞质。qRT-PCR表达分析表明,GbPR10基因在不同抗病海岛棉品种的不同组织上表达量不均匀而较高于感病海岛棉品种;在乙烯和水杨酸处理的1对抗/感海岛棉根系中,抗病品种出现先上调后下调趋势,感病材料后期诱导表达,抗病品种的表达量几乎高于感病品种。【结论】GbPR10基因在海岛棉抗枯萎病信号途径中起重要作用。

【Objective】To provide strong support for the gene sources of cotton wilt resistance molecular breeding.【Method】Based on the previous transcriptome sequencing and disease resistance expression data,the Fusarium wilt related genes(accession number CD486053)were screened from cotton EST database.A homologous gene named GbPR10 gene was cloned from Fusarium wilt resistant in Gossypium barbadense"06-146"by NCBI search for PR10 gene(accession number AY588276)and primer design.Bioinformatics gene expression analysis was carried out under the treatment of Fusarium oxysporum f.sp,ethylene,and salicylic acid.【Result】GbPR10 gene had the ORF sequence of 480 bp and encoded 159 amino acids and the Bet-v1 domain of PR10 protein and the modified glycine ring P-Loopus(GXGGXG)were found in the protein sequence.The homologous alignment of protein sequence indicated that the protein was highly consistent with other biological PR10 protein plants.Subcellular localization analyses pinpointed that GbPR10 was distributed in cytoplasm.QRT-PCR analysis showed that the expression of GbPR10 gene in different tissues of different resistant island cotton varieties was uneven and higher than that of susceptible island cotton varieties.In the roots of one pair of resistance/susceptible island cotton treated with ethylene and salicylic acid,the resistant varieties showed a tendency of up-regulation first and then down-regulation.The expression of resistant varieties was almost higher than that of susceptible varieties at later stage.【Conclusion】It can be concluded in this study that GbPR10 gene plays a significant role in the signal pathway of resistance to Furasium oxysporum f.sp in Gossypium barbadense.