逆境胁迫下柽柳脂质转运蛋白基因(ThLTP)的克隆与功能初步分析  

Cloning and Function Analysis of Tamarix hispida Lipid Transfer Protein under Stress

作　　者：林琳[1] 李健[1] 李慧玉[1] 穆怀志[1] 姜静[1] 

机构地区：[1]东北林业大学,林木遗传育种国家重点实验室,黑龙江哈尔滨150040

出　　处：《林业科学研究》2012年第4期492-499,共8页Forest Research

基　　金：转基因生物新品种培育科技重大专项(2009ZX08009-098B)

摘　　要：从以刚毛柽柳为材料构建的NaCl胁迫的根cDNA文库中测序得到1条LTP基因序列,其全长为635 bp、编码116个氨基酸,命名为ThLTP基因。采用实时荧光定量RT-PCR的方法,分别对0.2 mol.L-1NaCl、150μmol.L-1CdCl2、20%(W/V)PEG6000、100μmol.L-1ABA及4℃下ThLTP基因在柽柳中的表达模式进行了分析,结果表明:ThLTP基因除了在4℃条件下根组织中为下调表达外,在其它处理中均表现为上调表达。将ThLTP基因克隆到大肠杆菌(Escherichia coli)的原核表达载体pET32a中,对重组菌Escherichia coli BL21(pET32a-LTP)的抗旱耐盐性进行分析。结果表明:在0.8%(W/V)NaCl和20%(W/V)PEG6000条件下,对照菌E.coli BL21(pET32a)无对数生长期,而重组菌E.colii BL21(pET32a-LTP)经过3 h的延迟生长后,进入到对数增长期,表明ThLTP可受盐、碱、低温诱导并能提高重组菌的抗旱耐盐能力。In this study, a novel LTP gene which was named ThLTP was cloned from the cDNA library of Tamarix hispida root treated by 0.4 mol·L^-1 NaCl. The sequence of ThLTP was 635 bp in full length, a protein with 116 amino acids residues. The authors examined the expression pattern of the ThLTP gcne in leaf and root of T. hispida treated with 0.2 mol·L^-1 NaCl, 150 μmol·L^-1CdCl2, 20% （W/V） PEG6000, 100 μmol·L^-1 ABA and 4 ℃ stresses for different time using real time RT-PCR. The results showed that the ThLTP gene was induced by all the treatments in leaf and root except the treatment of 4℃ treated in root. ThLTP was inserted into a prokaryotic expression vector （pET32a） to produce the recombinant expression vector pET32a-LTP. The Escherichia coli BL21 （pET32a） could not live but the E. coli BI21 （pET32a-LTP） came to the maximum after 3 h delayed growth under the stress of 0.8% （W/V） NaCl and 20% （W/V） PEG6000. The results showed that ThLTP may be a saltand drought-tolerant gene and enhance the stress tolerance of the recombinant.

关 键 词：刚毛柽柳 脂质转移蛋白 抗旱耐盐性 表达分析 

分 类 号：S793.5[农业科学—林木遗传育种]