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作物学报 ›› 2012, Vol. 38 ›› Issue (12): 2162-2169.doi: 10.3724/SP.J.1006.2012.02162

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

结球甘蓝花粉类钙调素蛋白基因BoCML49的克隆及表达分析

宋明**,许俊强**,孙梓健,汤青林,王志敏,王小佳*   

  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
  • 收稿日期:2012-04-18 修回日期:2012-08-15 出版日期:2012-12-12 网络出版日期:2012-10-08
  • 通讯作者: 王小佳, E-mail: wxj@swu.edu.cn, Tel: 023-68251093
  • 基金资助:

    本研究由国家自然科学基金(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)和教育部高等学校博士点基金(20090182120003)和国家重点基础研究发展计划(973计划)项目(2012CB113900)资助。

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

SONG Ming**,XU Jun-Qiang**,SUN Zi-Jian,TANG Qing-Lin,WANG Zhi-Min,WANG Xiao-Jia*   

  1. 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
  • Received:2012-04-18 Revised:2012-08-15 Published:2012-12-12 Published online:2012-10-08
  • Contact: 王小佳, E-mail: wxj@swu.edu.cn, Tel: 023-68251093

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 bpcDNA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¢-Race3¢-Race分别得到550 bp721 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 kDpI6.93,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7α-螺旋和10β-折叠结构。系统发育树表明结球甘蓝BoCML49与拟南芥AtCML49AtCML50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。

关键词: 结球甘蓝, 花粉, 类钙调素蛋白49, 表达分析

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 150178 and 216244 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

Key words: Cabbage, Pollen, BoCML49, Expression analysis

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