烟草NtHPL1基因克隆、表达及功能分析

Cloning,expression and functional analysis of NtHPL1 gene in tobacco

为探究脂氢过氧化物裂解酶(hydroperoxide lyase,HPL)在烟草抗逆境胁迫中的生物学功能,从普通烟草K326中克隆1个脂氢过氧化物裂解酶基因并命名为NtHPL1,对NtHPL1蛋白进行生物信息学分析,并采用qPCR方法分析NtHPL1基因的相对表达量,通过创制NtHPL1过表达转基因材料预测NtHPL1基因的抗逆境胁迫功能。结果表明:(1)NtHPL1基因编码区全长1365 bp,编码454个氨基酸;蛋白质结构和系统发育分析结果显示,NtHPL1蛋白属于13-HPL亚家族。(2)NtHPL1基因在烟草根、茎、叶、花和腋芽中均有表达,且花中相对表达量最高;NtHPL1基因在烟草各发育时期烟叶中的相对表达量不同,盛花期最低,成熟期最高;盐和干旱胁迫均能诱导NtHPL1基因表达。(3)盐和干旱胁迫处理后,野生型烟草叶片发黄,但NtHPL1过表达烟草正常生长,且NtHPL1过表达烟草叶片的叶绿素和脯氨酸含量(质量分数)高于野生型烟草,丙二醛含量比野生型烟草低,表明过表达NtHPL1基因可提高烟草对盐和干旱胁迫的抗性。

To investigate the biological function of hydroperoxide lyase(HPL)in tobacco resistance to adversity stress,a hydroperoxide lyase gene was cloned from Nicotiana tabacum K326 and designated NtHPL1.Bioinformatic analysis was conducted on the NtHPL1 protein,and the relative expression level of the NtHPL1 gene was analyzed by qPCR method.The function of NtHPL1 gene in stress resistance was predicted by creating transgenic materials with NtHPL1 overexpression.The results showed that:1)The total length of the coding region of NtHPL1 gene encoding 454 amino acids was 1365 bp.The results of protein structure and phylogenetic analysis showed that NtHPL1 belongs to 13-HPL subfamily.2)The NtHPL1 gene was expressed in tobacco roots,stalks,leaves,flowers and axillary buds,with the relative expression level in flowers being the highest.The relative expression level of NtHPL1 gene in tobacco lcaves varied at different developmental stages,with the lowest expression level at full bloom stage and the highest expression level at maturity stage.Both salt and drought stress could induce the expression of NtHPL1 gene.3)After salt and drought stress treatments,the leaves of wild-type tobacco turned yellow,while the NtHPL1-overexpressed tobacco grew normally.The chlorophyll and proline contents(mass fractions)in leaves of NtHPL1-overexpressed tobacco were higher than those of wild-type tobacco,and the malondialdehyde content was lower than that of wild-type tobacco,indicating that overexpression of the NtHPL1 gene could improve the resistance of tobacco to salt and drought stress.