白菜型冬油菜β-1,3-葡聚糖酶基因在低温胁迫下的表达
收稿日期: 2016-11-23
录用日期: 2017-03-24
网络出版日期: 2017-07-10
基金资助
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)和甘肃省教育厅科研专项(No.2015A-077)
Expression Analysis of β-1,3-Glucanase Gene from Winter Brassica rapa Under Low Temperature Stress
Received date: 2016-11-23
Accepted date: 2017-03-24
Online published: 2017-07-10
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cDNA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38.102 kDa, 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。
马骊, 孙万仓, 袁金海, 刘自刚, 武军艳, 方彦, 许耀照, 蒲媛媛, 白静, 董小云, 何辉立 . 白菜型冬油菜β-1,3-葡聚糖酶基因在低温胁迫下的表达[J]. 植物学报, 2017 , 52(5) : 568 -578 . DOI: 10.11983/CBB16225
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°C, and the gene was upregulated to peak in expression at -4°C; However, the expression was inhibited at lower temperature (-8°C). The β-1,3-glucanase gene cloned from the winter B. rapa cv. ‘Longyou6’ might play a role in cold tolerance.
Key words: β-1; 3-glucanase gene; low temperature; molecular cloning; expression analysis
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