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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。
To explore the effect of β-1,3-glucanase on the ability of Brassica campestris to resist cold stress, β-1,3-glucan The c DNA sequence of β-1, 3-glucanase from B. rapa Longyou 6 and Tianyou 4 was cloned by RT-PCR. The sequence of the cDNA was analyzed by bioinformatics Then the expression patterns of β-1,3-glucanase gene under low temperature stress were detected by real-time fluorescence quantitative PCR and semi-quantitative PCR. Results The open reading frame of β-1,3-glucanase gene from Longyou No.6 with the length of 1 032 bp was obtained, encoding 343 amino acids. The relative molecular weight was 38.102 kDa and the theoretical isoelectric point was 6.63. (B.rapa subsp. Chinensis) and B. napus (B.napus) shared 93.94% amino acid sequence homology. The gene encodes a hydrophilic stable protein consisting mainly of α-helix, containing 1 signal peptide and 2 transmembrane domains. This gene is highly conserved in evolution and its conserved sequence belongs to a conserved domain unique to the glycosyl hydrolase family 17 of plants. β-1,3-glucanase gene expression pattern analysis showed that the gene up-regulated expression at 4 ° C, continue low temperature (-4 ° C) stress treatment, the gene up-regulated to its peak, to -8 ° C Down regulation. Studies have shown that β-1,3-glucanase cloned from Brassica campestris var. Chinensis plays a role in the cold tolerance of Longyou 6, a winter rape cultivar.