结球甘蓝花粉类钙调素蛋白基因BoCML49的克隆及表达分析  

Molecular Cloning and Expression Analysis of CaM-Like Protein Genes (BoCML49) from Cabbage (Brassica oleracea L. var. capitata)

作　　者：宋明[1] 许俊强[1] 孙梓健[1] 汤青林[1] 王志敏[1] 王小佳[1] 

机构地区：[1]西南大学园艺园林学院/南方山地园艺学教育部重点实验室,重庆市蔬菜学重点实验室,重庆400715

出　　处：《作物学报》2012年第12期2162-2169,共8页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31071802,31000908);重庆市自然基金重点项目(2011BA1002);中央高校基本科研业务费专项(XDJK2012B020,XDJK2009C126);教育部高等学校博士点基金(20090182120003);国家重点基础研究发展计划(973计划)项目(2012CB113900)资助

摘　　要：以结球甘蓝E1为材料,提取花粉萌发前和萌发后的混合花粉总蛋白,总蛋白双向电泳后通过MALDI-TOF-MS鉴定分析差异点,根据差异点分析结果,利用同源克隆得到甘蓝BoCML49基因707bp的cDNA片段。通过对BoCML49基因的qPCR分析表明其表达量在花粉萌发前约为萌发后的2.73倍,表明BoCML49基因在花粉萌发后表达下调。通过5-Race和3-Race分别得到550bp和721bp大小cDNA片段,最终得到结球甘蓝BoCML49全长cDNA序列,开放阅读框位于125~1078bp处,加尾信号（AATAAA）位于第1222bp处。通过cDNA推导得到的氨基酸序列分析表明,BoCML49编码317个氨基酸残基,预测分子量为33.51kD,pI为6.93,经Smart-embl预测其含2个重要的EF-hand结构,分别位于序列第150~178和第216~244位氨基酸残基处,预测结果显示其含有7个α-螺旋和10个β-折叠结构。系统发育树表明结球甘蓝BoCML49与拟南芥AtCML49和AtCML50的亲缘关系较近。BoCML49基因的原核表达得到37.55kD的融合蛋白。Calcium sensor proteins in plants play important roles in pollen germination and pollen tube growth processes. Calmodnlin-like （CML） protein of cabbage （Brassica oleracea L. var. capitata） was identified in the process of pollen germination in cabbage line E1 by two-dimensional electrophosis of the pollen total protein. The sequence of BoCML49 cDNA fragment was 707 bp. The expression analysis by qPCR showed that the BoCML49 gene was down-regulated when pollen germinated, 550 bp and 721 bp DNA fragments were obtained by 5＇-Race and 3＇-Race, respectively. We obtained BoCML49 gene with a total length of 1 343 bp by splicing, its open reading frame was located in the region from 125 to 1 078 bp, and contained a 124 bp 5＇ untranslated region （5＇UTR） and a 265 bp 3＇UTR, the polyadenylation signal （AATAAA） was located at 1 222 bp. The deduced BoCML49 protein contained 317 amino acids, with a MW of 33.51 kD and pI of 6.93. The structural analysis of BoCML49 though Smart-embl showed that it contained two critical functional domain EF-hand modifs, which located at the position of 150–178 and 216–244 amino acid residues, at the same time the ORF might contain seven α-helixes and ten β-sheets. The phylogenetic tree indicated that the BoCML49 had close genetic relationship with AtCML49 and AtCML50. Prokaryotic expression showed that the molecular mass of BoCML49 protein was about 37.55 kD.

关 键 词：结球甘蓝 花粉 类钙调素蛋白49 表达分析 

分 类 号：S635.1[农业科学—蔬菜学]