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作 者:李雪丹[1] 魏昌赫 邵欢欢[1] 吴燕[1] 张义正[1] 王海燕[1]
机构地区:[1]四川大学生命科学学院,四川省分子生物学及生物技术重点实验室,成都610064
出 处:《植物科学学报》2016年第4期583-592,共10页Plant Science Journal
基 金:supported by grants from the National Science and Technology Pillar Program of China(2007BAD78B03);the “Eleventh-Five” Key Project of Sichuan Province;China(07SG111-003-1)
摘 要:非特异脂质转移蛋白(ns LTP)是植物界普遍存在的一类涉及多种胁迫反应的可溶性蛋白。为了阐明甘薯中非特异性脂质转移蛋白基因IbLTP1和IbLTP2在盐胁迫反应中的功能,本研究运用PCR技术,对IbLTP1和IbLTP2基因进行了克隆,并通过生物信息学方法分析了序列结构、蛋白质保守结构域和系统进化关系;利用q RT-PCR方法检测了这两个基因在不同组织中的表达模式以及盐胁迫条件下的表达差异;将IbLTP1和IbLTP2基因克隆到大肠杆菌的原核表达载体p ET32a中,对重组菌BL21(p ET32a-LTP)的耐盐性进行分析。序列分析表明:IbLTP1和IbLTP2编码区均不含内含子并都具有等位基因。IbLTP1和IbLTP2基因的蛋白质序列分别包括114和94个氨基酸残基并且不含色氨酸残基,蛋白序列N端含有信号肽序列。保守结构域和系统进化分析结果表明:IbLTP1和IbLTP2均含有ns LTP蛋白的保守结构域,IbLTP1属于TypeⅠ而IbLTP2属于TypeⅡ。实时荧光定量PCR分析表明:IbLTP1在幼叶中表达量最高,根中表达量最低;而IbLTP2在茎中表达量最高,成熟叶中表达量最低。在Na Cl胁迫条件下,IbLTP1和IbLTP2表达量在根中基本无变化而在茎和叶中上调。大肠杆菌BL21(DE3)中异源表达IbLTP1和IbLTP2基因能够提高转基因菌株对Na Cl的耐受性。因此,本研究推测IbLTP1和IbLTP2基因可能在甘薯盐胁迫反应中发挥了作用。Nonspecific lipid transfer proteins( ns LTPs) are widely distributed in the plant kingdom and are involved in various stress responses. To clarify the function of ns LTP genes,IbLTP1 and IbLTP2 were cloned by PCR technology,and the sequence structures,conserved domains,and evolutionary relationships were analyzed. Sequences of c DNAs and genomic genes showed that neither gene had introns,but both had several homologous isoforms.IbLTP1 and IbLTP2 encode proteins of 114 and 94 amino acid residues respectively,without any Trp. These proteins contain a signal peptide at the N- terminal and have conserved domains of ns LTP1 and ns LTP2, respectively. The expression patterns and expression differences of IbLTP1 and IbLTP2 in different tissues and under stress were determined byreal-time RT-PCR. Results showed that IbLTP1 and IbLTP2 had higher relative expression levels in young leaves and stems,respectively,and were highly induced under sodium chloride( Na Cl) stress. The coding sequences of IbLTP1 and IbLTP2 were cloned into expression vector p ET32 a and expressed in Escherichia coli BL21( DE3),respectively. The maximal OD600 values of strains harboring p ET32a-IbLTP1 and p ET32a-IbLTP2 were higher than those of the p ET32 a transformed strain under Na Cl stress.
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