甘薯LEA2基因的克隆与表达分析

Cloning and expression analyses of LEA2 gene from Ipomoea batatas

胚胎发育晚期丰富蛋白(Late embryogenesis abundant,LEA)是植物在逆境胁迫下产生的一种应激蛋白质,具有较高的亲水性和热稳定性,与植物抗逆功能密切相关,研究LEA家族基因对于甘薯抗逆性分子育种具有重要意义.通过对甘薯(徐薯18)转录组数据库搜寻时发现编码LEA2的转录本,随后对该转录本进行了克隆和测序分析,利用数字表达谱(Digital Gene Expression Profi ling,DGE)和半定量RT-PCR分析了Ib-LEA2在不同组织和不同发育阶段的表达图谱.为进一步了解Ib-LEA2在生物体响应盐胁迫中的作用,将Ib-LEA2连接原核表达载体pET-32a,用大肠杆菌BL21(DE3)菌株进行盐胁迫的耐受分析.结果显示:Ib-LEA2完整的开放阅读框(ORF)长度为942 bp,编码313个氨基酸残基.进一步研究发现,在甘薯组织内同时存在3个高度同源的LEA 2基因型(核苷酸序列>99%),分别命名为Ib-LEA2a、Ib-LEA2b和Ib-LEA2c.数字表达谱和半定量RT-PCR的结果显示Ib-LEA2在不同组织和发育阶段具有不同的表达图谱,在茎中的表达量远远高于叶和根.大肠杆菌BL21(DE3)菌株盐胁迫的结果显示,较之对照菌株,表达Ib-LEA2的菌株能够更好地耐受高浓度的NaCl.研究表明,Ib-LEA2对盐胁迫有一定的响应能力,能够保护细胞免受高盐的毒害.

Late embryogenesis abundant （LEA） proteins, which are produced in plant under stress, have high hydrophilic activity and thermal stability. They are closely related to plant defense reactions. Researches on LEA proteins are important for molecular breeding of sweet potato under stress. This research analyzed the expression pattern of Ib-LEA2 in vivo, and investigated the function of LEA2 in defense of salt stress. Transcripts encoding LEA2 proteins were found when searching the transcriptome database of sweet potato. Subsequently, Ib-LEA2 was cloned and sequenced. Digital gene expression profiling （DGE） and semi-quantitative RT-PCR were chosen for study expression oflb-LEA2 in different tissues and under different developmental stages. In order to further study the function in response to salt stress, Ib-LEA2 coding sequence was cloned into pET-32a expression vector and then expressed in E. coli BL21（DE3）. Result shows that Ib-LEA2 had an open reading frame （ORF） of 942 bp in length, which encoded 313 amino acids. Further analysis revealed three highly homologous genotypes for Ib-LEA2 （nucleotide sequences similarity 〉 99%）, named Ib-LEA2a, Ib-LEA2b and Ib-LEA2c. Digital gene expression profiling （DGE） and semi-quantitative assay showed that Ib-LEA2 proteins were differentially expressed in different tissues and under different developmental stages, and that the expression in stems was much higher than in leaves and roots. The growth curves of E. coli BL21（DE3） showed that the strain expressing lb-LEA2 can better tolerate high concentration of salt than the control. Our work revealed that Ib-LEA2 could response to stress, protecting cells from high concentration of salt. The results laid a theoretical foundation to the molecular breeding of sweet potato, promoting the researches in salt-tolerance in plants.