| 结球甘蓝花粉类钙调素蛋白基因BoCML49的克隆及表达分析 |
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| 引用本文： | 宋明,许俊强,孙梓健,汤青林,王志敏,王小佳.结球甘蓝花粉类钙调素蛋白基因BoCML49的克隆及表达分析[J].作物学报,2012,38(12):2162-2169. |
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| 作者姓名： | 宋明 许俊强 孙梓健 汤青林 王志敏 王小佳 |
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| 作者单位： | 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 |
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| 基金项目： | 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 |
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| 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707 bp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550 bp和721 bp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~
1 078 bp处，加尾信号(AATAAA)位于第1 222 bp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33.51 kD，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37.55 kD的融合蛋白。
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| 关 键 词： | 结球甘蓝 花粉 类钙调素蛋白49 表达分析 |
| 收稿时间： | 2012-04-18 |
Molecular Cloning and Expression Analysis of CaM-Like Protein Genes (BoCML49) from Cabbage (Brassica oleracea L. var. capitata) |
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| SONG Ming
,
XU Jun-Qiang
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SUN Zi-Jian
,
TANG Qing-Lin
,
WANG Zhi-Min
,
WANG Xiao-Jia.Molecular Cloning and Expression Analysis of CaM-Like Protein Genes (BoCML49) from Cabbage (Brassica oleracea L. var. capitata)[J].Acta Agronomica Sinica,2012,38(12):2162-2169. |
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| Authors: | SONG Ming XU Jun-Qiang SUN Zi-Jian TANG Qing-Lin WANG Zhi-Min WANG Xiao-Jia |
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| Institution: | Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education / Chongqing Key Laboratory of Olericulture / College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China |
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| Abstract: | 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 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 obtain 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. cDNA |
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| Keywords: | Cabbage Pollen BoCML49 Expression analysis |
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