柠条锦鸡儿CkLEA4-2基因的克隆与功能分析

Cloning and functional characterization of CkLEA4-2 from Caragana korshinskii

胚胎发育晚期丰富(LEA)蛋白在植物抵抗非生物胁迫过程中起着重要作用。利用RACE技术从柠条锦鸡儿(Caragana korshinskii)中克隆得到一个LEA_4家族基因Ck LEA4-2,其cDNA全长963 bp,编码240个氨基酸。实时荧光定量PCR检测发现CkLEA4-2的表达受干旱、脱水、NaCl、冷、高pH、脱落酸(ABA)处理的诱导。通过构建35S启动子驱动的pCanG-CkLEA4-2植物过表达载体,研究转基因拟南芥并发现,过表达Ck LEA4-2提高了拟南芥在ABA处理及NaCl和甘露醇胁迫下的萌发率。在幼苗生长阶段,转基因拟南芥的抗旱性增强,失水率低于野生型,并且积累了较少的丙二醛,脯氨酸和谷胱甘肽含量高于野生型。同时,CkLEA4-2过表达后使ABA2、AAO3等ABA信号途径基因以及胁迫响应基因RD22、RD29A和MYB2的表达量升高。结果表明,Ck LEA4-2基因参与了植物逆境应答过程,并提高了转基因拟南芥的抗旱能力。

Previous studies have shown that late embryogenesis abundant（LEA） proteins play important roles in plant adaptation to abiotic stresses.However,reports that demonstrate their biological functions in Caragana korshinskii are still very limited.In this study,a LEA_4 encoding gene CkLEA4-2 was isolated from C.korshinskii by rapid amplification of cDNA ends（RACE）.The full-length cDNA of CkLEA4-2 is 963 bp and the putative open reading frame（ORF） encodes a polypeptide of 240 amino acid residues.Quantitative real-time PCR analysis indicated that the transcription of CkLEA4-2 was induced by drought,dehydration,NaCl,cold,high p H and abscisic acid（ABA） treatments.Transgenic Arabidopsis plants overexpressing CkLEA4-2 under the control of the CaMV35S promoter were generated for further analysis.Overexpression of CkLEA4-2 enhanced Arabidopsis tolerance to ABA treatment,osmotic and salt stresses during seed germination,and exhibited resistance to drought stress during seedling development,with lower water loss rates,less accumulation of malondialdehyde（MDA）,increased proline as well as glutathione（GSH） contents.In addition,the expression level of some ABA signaling genes including ABA2 and AAO3 was elevated,and stress-responsive genes such as RD22,RD29A and MYB2,was also up-regulated in CkLEA4-2 overexpression lines.Taken together,these results indicate that CkLEA4-2 is involved in abiotic stress responses and improves drought tolerance of Arabidopsis.