转麻疯树甜菜碱醛脱氢酶基因提高烟草耐盐性

Enhancing Salt Tolerances of Tobacco by Transformation of Jatropha curcas Betaine Aldehyde Dehydrogenase Gene

【目的】研究麻疯树甜菜碱醛脱氢酶(JcBD1)的功能。【方法】通过RT-PCR和RACE的方法从麻疯树中克隆甜菜碱醛脱氢酶基因(JcBD1)的全长cDNA序列。将从麻疯树中克隆的JcBD1 cDNA与CaMV 35S组成型启动子融合,构建植物表达载体pBI-JB,利用根癌农杆菌介导转入烟草。对获得抗性的植株用PCR、RT-PCR及Western杂交检测分析,同时测定转基因烟草植株的电导率、JcBD1酶活性及抗盐性等特性。【结果】由JcBD1 cDNA序列推测的JcBD1氨基酸序列与已知的BADH具有很高的同源性,包括BADH家族绝对保守区域十肽"VSMELGGKSP"和半胱氨酸残基。这两部分区域在甜菜碱醛脱氢酶底物特异性结合过程中起着重要作用,与酶的催化活性有关。PCR及RT-PCR检测证明外源JcBD1已整合到烟草基因组中并成功表达,转化率为56.7%。转基因植株的电导率明显低于未转基因植株。盐胁迫处理后,在转基因植株的蛋白提取样中能检测到Western杂交信号,而在未转基因植株中没有检测到杂交信号;转基因植株的蛋白提取样中能检测到甜菜碱醛脱氢酶活性,而对照没有活性,表明JcBD1在转基因植株中得到了表达。转JcBD1烟草在盐胁迫下,生长势明显强于未转基因植株。【结论】JcBD1能在异源植物中正常翻译、表达;JcBD1是盐害胁迫相关的重要基因。

【Objective】JcBD1 gene was cloned from Jatropha curcas L.to research its function.【Method】The full-length cDNA of JcBD1 was isolated from Jatropha curcas L.by RT-PCR and RACE techniques.The expression plasmid pBI-JB was constructed by fusing the cDNA of JcBD1 with the constitutive promoter CaMV 35S and introduced into tobacco plants by Agrobacterium tumefaciens mediated transformation.Transgenic assays were performed using PCR,RT-PCR and Western blot analysis.At the same time,JcBD1 enzyme activity,relative conductivity and salt resistance of transgenic tobacco were measured.【Result】The amino acid sequence deduced from JcBD1 cDNA sequence showed substantialy high similarities to BADH enzyme presented in other plants,including the absolute conservative decapeptide ＂VSMELGGKSP＂ and the cysteine residue in the possible active site.Both the decapeptide motif and the cysteine residue are proposed to play a role in substrate specificity and implicated in catalysis of BADH.The JcBD1 gene was confirmed to be integrated into the genome of tobacco by PCR and RT-PCR analysis with an average frequency of 56.7%.The electrical conductivity of the transgenic lines was obviously lower than wild type both in natural condition and after salt stress.JcBD1 specific activity and hybridization signal were detectable in transgenic plant leaves,while it was not in the control plants.Transgenic tobacco plants grew better than the untransformed plants under salt stresses.【Conclusion】The JcBD1 can be expressed accurately in the exogenous transgenic plants and that transformation of JcBD1 into tobacco plants can confer them more tolerance to salinity.JcBD1 is likely related to salt resistances.