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农业生物技术学报  2019, Vol. 27 Issue (3): 402-410    DOI: 10.3969/j.issn.1674-7968.2019.03.003
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
不结球白菜抗病相关基因BcSGT1的克隆及表达分析
刘东让, 侯喜林, 肖栋*
南京农业大学 作物遗传与种质创新国家重点实验室/江苏现代园艺工程技术中心/农业部华东地区园艺作物生物学与种质创新重点实验室,南京 210095
Cloning and Expression Analysis of Disease-related Gene BcSGT1 in Non-heading Chinese Cabbage (Brassica rapa ssp. chinensis)
LIU Dong-Rang, HOU Xi-Lin, XIAO Dong*
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Jiangsu Engineering and Technology Center for Modern Horticulture/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in Eastern China, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
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摘要 SGT1(suppressor of the G2 allele of skp1)基因作为植物抗病信号途径中的重要元件,广泛参与植物细胞周期、逆境反应和信号转导等过程。为研究不结球白菜(Brassica rapa ssp. chinensis)抗病相关基因BcSGT1的结构和表达特征,本研究通过RACE (rapid-amplification of cDNA ends)技术,以抗病品种‘苏州青’叶片为材料克隆到BcSGT1基因的全长cDNA序列;采用qRT-PCR分析该基因在霜霉病菌(Peronospora parasitica)及黑斑病菌(Alternaria brassicicola)诱导处理条件下的表达模式;用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)技术分析该基因的原核表达特征。序列分析结果表明,BcSGT1基因的cDNA全长为1 418 bp,其中开放阅读框长度为1 074 bp,共编码358个氨基酸(GenBank No. AB495003)。氨基酸同源性的系统进化分析表明,不结球白菜BcSGT1基因与同科植物的进化关系相近,其中与大白菜(Brassica rapa ssp. pekinensis)第3号染色体的基因(Bra000741)同源性最高(97%)。qRT-PCR分析表明,霜霉病菌侵染处理下,BcSGT1基因在抗病品种‘苏州青’和感病品种‘矮脚黄’中的表达量分别于24、48 h达到峰值,且在‘苏州青’中的表达量峰值约为‘矮脚黄’中的2.1倍(P<0.01);黑斑病菌侵染处理下,BcSGT1基因在抗病品种‘苏州青’和感病品种‘矮脚黄’中的表达量分别于12、24 h达到峰值,且在‘苏州青’中的表达量峰值约为‘矮脚黄’中的2.0倍(P<0.01);霜霉病菌侵染处理24和48 h后,黑斑病菌侵染处理12、24和48 h后,BcSGT1基因在抗病品种‘苏州青’中的表达量均显著高于感病品种‘矮脚黄’中的表达量(P<0.01)。原核表达载体经异丙基-β-D-硫代半乳糖苷(isopropyl β-D-thiogalactoside, IPTG)诱导表达出相对分子质量约为39 kD的融合蛋白。BcSGT1基因是不结球白菜霜霉病菌及黑斑病菌抗病反应的重要组成部分,且本研究成功将BcSGT1融合表达进大肠杆菌(Escherichia coli)中。本研究为高产、优质及抗病不结球白菜新品种的选育提供重要的理论依据。
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刘东让
侯喜林
肖栋
关键词 不结球白菜BcSGT1霜霉病菌黑斑病菌qRT-PCR原核表达    
AbstractSGT1 (suppressor of the G2 allele of skp1) as an important element for plant disease resistance, is involved widely in plant cell cycling, stress response, protein ubiquitination and signal transduction processes, and it plays an important role in plant disease resistance. In order to study the structure and expression characteristics of the disease-related gene BcSGT1 in non-heading Chinese cabbage (Brassica rapa ssp. chinensis), the full-length cDNA sequence of BcSGT1 gene was cloned from the resistant variety 'Suzhouqing' by RACE technique. The expression analysis of gene was used by qRT-PCR. The expression pattern of Peronospora parasitica and Alternaria brassicicola induced treatment conditions, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique was used to analyze the prokaryotic expression characteristics for the gene. The analysis results of sequence indicated that the full-length cDNA of BcSGT1 gene was 1 418 bp, and the open reading frame was 1 074 bp in length, encoding a total of 358 amino acids. The relative molecular weight was 39.77 kD, and the protein theoretical isoelectric point was 5.05 (GenBank No. AB495003). The evolutionary analysis of amino acid homologous system showed that the BcSGT1 gene of the non-heading Chinese cabbage had similar evolutionary relationship with the same family plant, and the highest homology (97%) was found with the chromosome 3 gene of Brassica rapa (Bra000741). The qRT-PCR analysis showed that under the infection of P. parasitica, the expression level of BcSGT1 gene in the resistant variety 'Suzhouqing' peaked at the 24 h, while the expression level in the susceptible variety 'Aijiaohuang' reached the peak at the 48 h. The peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.1 times higher than that in the susceptible variety 'Aijiaohuang' (P<0.01). With the infection of A. brassicicola, the expression level of BcSGT1 in 'Suzhouqing' reached its peak at 12 h, while the expression level in the susceptible variety 'Aijiaohuang' peaked at 24 h, and the peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.0 times higher than the peak expression in the susceptible variety 'Aijiaohuang' (P<0.01). The expression level of BcSGT1 in 'Suzhouqing' is significantly higher than the expression level in 'Aijiaohuang' after 24 and 48 hours of infection by P. parasitica, and 12 h, 24 h, 48 h infection by A. brassicicola (P<0.01). The prokaryotic expression vector was induced by isopropyl β-D-thiogalactoside (IPTG) to express a fusion protein with a relative molecular mass of about 39 kD. The BcSGT1 gene of non-heading Chinese cabbage played an important role in the infection of P. parasitica and A. brassicicola, and BcSGT1 successfully achieved fusion expression in Escherichia coli, which provided the conditions for further protein level research and transgenic function research. It will also provide an important theoretical value for the selection of high-yield, high-quality and disease-related non-heading Chinese cabbage varieties.
Key wordsNon-heading Chinese cabbage    BcSGT1    Peronospora parasitica    Alternaria brassicicola    qRT-PCR    Prokaryotic expression
收稿日期: 2018-07-31     
ZTFLH:  S634.3  
基金资助:国家重点研发计划(No. 2016YFD0101701)
通讯作者: *通信作者,dong.xiao@njau.edu.cn   
引用本文:   
刘东让, 侯喜林, 肖栋. 不结球白菜抗病相关基因BcSGT1的克隆及表达分析[J]. 农业生物技术学报, 2019, 27(3): 402-410.
LIU Dong-Rang, HOU Xi-Lin, XIAO Dong. Cloning and Expression Analysis of Disease-related Gene BcSGT1 in Non-heading Chinese Cabbage (Brassica rapa ssp. chinensis). 农业生物技术学报, 2019, 27(3): 402-410.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.03.003     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I3/402
 
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