白菜型冬油菜β-1,3-葡聚糖酶基因在低温胁迫下的表达  

Expression Analysis of β-1,3-Glucanase Gene from Winter Brassica rapa Under Low Temperature Stress

作　　者：马骊 孙万仓 袁金海 刘自刚 武军艳 方彦 许耀照[2] 蒲媛媛 白静 董小云 何辉立 Li Ma Wancang Sun Jinhai Yuan Zigang Liu Junyan Wu Yan Fang Yaozhao Xu Yuanyuan Pu Jing Bai Xiaoyun Dong Huili He(Gansu Key Laboratory of Crop improvement and Germp/asm Enhancement, Rapeseed Engineering Research Center of Gansu Province, College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, China)

机构地区：[1]甘肃农业大学农学院,甘肃省油菜工程技术研究中心,甘肃省作物遗传改良与种质创新重点实验室,兰州730070 [2]河西学院农业与生物技术学院,张掖734000

出　　处：《植物学报》2017年第5期568-578,共11页Chinese Bulletin of Botany

基　　金：国家现代农业产业技术体系(No.CARS-13);国家自然科学基金(No.31460356,No.31560397,No.3166040,No.31660404);国家重点基础研究发展计划(No.2015CB150206);科技部油菜杂种优势利用技术与强优势杂交种创制(No.2016YFD0101300);甘肃省教育厅科研专项(No.2015A-077)

摘　　要：为探明β-1,3-葡聚糖酶基因(β-1,3-glucanase)对油菜(Brassica campestris)抵御低温胁迫能力的作用,通过蛋白质谱分析得到β-1,3-葡聚糖酶蛋白,采用RT-PCR技术克隆白菜型冬油菜(B.rapa)陇油6号和天油4号β-1,3-葡聚糖酶的c DNA序列;并对该序列进行生物信息学分析;进而采用实时荧光定量PCR及半定量PCR检测β-1,3-葡聚糖酶基因在低温胁迫下的表达模式。结果获得长度为1 032 bp的陇油6号β-1,3-葡聚糖酶基因开放阅读框,编码343个氨基酸,相对分子量为38.102k Da,理论等电点为6.63,其与菜心(B.rapa subsp.chinensis)和甘蓝型油菜(B.napus)的蛋白质氨基酸序列同源性高达93.94%。该基因编码的酶是一个主要由α-螺旋组成的亲水性稳定蛋白,含有1个信号肽,存在2个跨膜结构域。该基因在进化上高度保守,其保守序列属于植物的糖基水解酶家族17特有的保守结构域。β-1,3-葡聚糖酶基因表达模式分析显示,4°C时该基因上调表达,继续低温(–4°C)胁迫处理,该基因上调表达至峰值,至–8°C时其表达下调。研究表明从白菜型冬油菜中克隆的β-1,3-glucanase在冬油菜品种陇油6号抗寒过程中发挥作用。This study examined the function of β-1,3-glucanase in Brassica rapa with low temperature stress, whose encoding protein was identified by protein mass spectrometry analysis. The cDNA sequence of β-1,3-glucanase was cloned by RT-PCR of winter rapeseed from Longyou6 and Tianyou4 cultivars. Semi-quantitative PCR and real-time fluorescence quantitative PCR were used to study the expression pattern of β-1,3-glucanase in response to low temperature stress. The open reading frame of the β-1,3-glucanase gene was obtained at a length of 1 032 bp, and encoded 343 amino acid. The molecular weight was 38.102 kDa and the isoelectric point 6.63, with 93.94% amino sequence similarity to B. rapa subsp, chinensis and B. napus. The protein encoded by this gene is a hydrophilic protein with one signal peptide and two transmembrane domains. The prediction of the secondary structures indicated that β-1,3-glucanase is a protein with more α-helices. The predicted β-1,3-glucanase contains a conserved amino acid sequence corresponding to the plant Glycol-Hydro 17 superfamily. RT-PCR and semi-quantitative RT-PCR results showed that β-1,3-glucanase was upregulated in response to 4℃, and the gene was upregulated to peak in expression at -4℃; However, the expression was inhibited at lower temperature （-8℃）. The β-1,3-glucanase gene cloned from the winter B. rapa cv. ＇Longyou6＇ might play a role in cold tolerance.

关 键 词：Β-1 3-葡聚糖酶基因 低温 分子克隆 表达分析 

分 类 号：S565.4[农业科学—作物学]