金发草LEA3基因2个剪接体转化烟草的抗干旱胁迫能力分析

摘要

[Objective] The study aimed to identity the functional differences of the two alternatively spliced isoforms of Pogonatherum paniceum group 3 LEA gene with different hydrophilicity and structure under drought stress.[Method] The function of the two isoforms was analyzed by using a plant expression system in tobacco.Specifically,the two isoforms of PpLEA3 gene were PCR amplified and cloned into eukaryotic expression plasmid to generate pBI121-PpLEA3.a and pBI121-PpLEA3.b for tobacco transformation.The establishment of transgenic plant was confirmed with genomic DNA analysis and RT-PCR.Their response to drought stress were studied by using a medium containing 150 mM mannitol.[Result] PpLEA3 transformed tobacco grew better comparing to the wild type tabacco.The non-transformed tabacco plants were relatively shorter,showed leaf wilting and growth deformities,comparing with the transgenic ones.The physiological indexes of stress resistance in the transgenic tobaccos were determined.[Conclusion] The current study showed that tobacco transformed with PpLEA3 gene had better drought stress tolerance than the wild type,which is even stronger for the PpLEA3.a transformed plants.Difference in the nucleic acid sequences of the two PpLEA3 gene isoforms led to altered physiological function.%[目的]为进一步鉴定金发草LEA3基因2个剪接体蛋白亲水能力和结构上的改变导致在抗逆能力方面的差异.[方法]本研究利用植物表达系统分析了2个剪接体在烟草体内的功能以及功能之间的差异.以PpLEA3基因2个剪接体(PpLEA3.a和PpLEA3.b)的重组载体pMD19-T-PpLEA3.a和pMD19-T-PpLEA3.b为模板,PCR法构建植物表达载体pBI121-PpLEA3.a和pBI121-PpLEA3.b,并分别转化烟草得到转基因烟草Nt-PpLEA3.a和Nt-PpLEA3.b.[结果]首先对转基因烟草进行分子检测,发现其基因组DNA和RT-PCR检测均表现为阳性,说明外源基因已整合到烟草基因组并可以正常转录表达.随后筛出转基因烟草并收获T2代种子后,对其进行抗旱生理特性分析,发现在遭受甘露醇150 mM的模拟干旱胁迫后,转基因植株均比野生植株生长状况好,野生植株相对矮小,叶片萎蔫,生长畸形,而转基因植株整体受损情况要好很多.同时分析了此模拟干旱条件下转基因植株与野生型植株的丙二醛含量、叶绿素含量和相对电导率的变化.[结论]转基因烟草在干旱胁迫下表现出良好的耐受性,耐受能力PpLEA3.a＞PpLEA3.b.进一步说明序列上的差异对基因的抗干旱能力功能起着重要作用.