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水稻OsLEA5c基因的克隆、表达和抗逆性分析

         

摘要

To study the resistance of the late embryogenesis abundant protein gene OsLEA5c,the cDNA sequence of OsLEA5c from rice was cloned by RT-PCR.Sequence analysis showed that OsLEA5c consisted of 456 bp open reading frame(ORF)that encoded 151 amino acids with a calculated molecular mass of 16.55 kD and a theoretical pI of 5.07.The deduced OsLEA5c protein was a strongly hydrophobic protein with the grand average of hydropathy(GRAVY)value of 0.020 and had a Pfam:LEA_2 domain architecture at position 44-140.The OsLEA5c protein shared 45.14%-61.59% identity with other members of subgroup 5C LEA proteins from plants.The phylogenetic relationship between related group 5 LEA proteins from different plants was analyzed,indicating that OsLEA5c belonged to subgroup 5C.Recombinant expression plasmid pET32a-OsLEA5c was constructed,and then was transformed into E.coli BL21(DE3)pLysS to obtain the recombinant strains.A fusion protein with a molecular weight of about 34 kD was expressed in the recombinant strains.The results of stress tolerance assay demonstrated that the recombinant strains producing OsLEA5 c fusion protein improved the resistance against diverse abiotic stresses,such as heat,high salinity,hyperosmosis and freeze-thaw.The findings illustrated that OsLEA5c plays an important role in resistance against diverse abiotic stresses during the maturation and desiccation phases of rice seed development.%为了研究水稻胚胎发育后期丰富蛋白基因OsLEA5c 的抗逆性,利用 RT PCR技术从水稻中克隆到OsLEA5c的 cDNA。序列分析表明,OsLEA5c基因的读码框为456 bp,编码1个由151个氨基酸残基组成的蛋白。蛋白分子量为16.55 kD,等电点为5.07。OsLEA5c蛋白的平均亲水系数(GRAVY)为0.020,为疏水蛋白。OsLEA5c蛋白的44-140位氨基酸残基形成 LEA_2结构域。进化树分析表明,OsLEA5 c属于第5组LEA蛋白的C亚组。OsLEA5 c与5 C亚组LEA蛋白的氨基酸一致性为45.14%~61.59%。利用基因重组技术构建的原核表达载体 pET32a Os-LEA5c,转化到E.coli BL21 pLysS菌株中,诱导得到34 kD的融合蛋白。OsLEA5c在大肠杆菌中的表达增加了大肠杆菌对高温、高盐、高渗透压和反复冻融的抗性。OsLEA5c 基因在水稻抗逆和种子成熟脱水过程中发挥重要作用。

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