燕麦AsKUP1的克隆与功能分析

植物钾离子转运蛋白中的Na~+不敏感性KUP/HAK/KT转运蛋白对植物耐盐胁迫起着重要作用。为了阐明钾离子转运蛋白基因AsKUP1在燕麦中响应盐胁迫的表达模式和鉴定其生物学功能,从燕麦中克隆了AsKUP1,利用RACE方法获得AsKUP1全长序列,并对其进行生物信息学分析;构建了pCAMBIA1301-AsKUP1植物过表达载体,转化拟南芥,并运用实时荧光定量PCR方法分析AsKUP1在拟南芥中的表达情况。结果表明,AsKUP1全长2 951bp,包含2 334bp的ORF序列,预测编码777个氨基酸,等电点为8.55,分子量为87.0k D。序列分析表明,AsKUP1与山羊草和小麦KUP/HAK/KT家族亲缘关系较近,预测该蛋白有14个疏水跨膜结构域,位于细胞质膜的概率较大。此外,盐胁迫下,T2转基因种子萌发率为57%,野生型为43%;经潮霉素筛选分离比为3∶1的2个转基因株系后代T3转基因株系根长分别是野生型的1.46倍和1.34倍,T3转基因植株鲜重、干重分别是野生型的1.56倍和1.44倍,Na~+含量无显著差异,而K~+含量为野生型的1.25倍,说明AsKUP1的表达提高了拟南芥植株的耐盐性。本研究结果为揭示钾离子转运蛋白家族基因AsKUP1在植物中的耐盐机制和通过转基因技术提高植物耐盐能力奠定了基础。

Cloning and Functional Analysis of AsKUP1 in Oat

The insensitive Na+ KUP/HAK/KT transport protein played an important role in K + transport protein of plant.In order to elucidate the expression pattern of AsKUP1 in Oat in response to salt stress and its biological function,the K + transport protein in Oats was cloned with RACE method to obtain the full-length sequence of AsKUP1,and its bioinformatics were analyzed.The plant overexpression vector of pCAMBIA1301-AsKUP1 was successfully constructed,and was transformed into Arabidopsis thaliana,then the expression of AsKUP1 in Arabidopsis thaliana was analyzed with real-time fluorescence quantitative PCR method.The results showed,AsKUP1 full-length was 2 951 bp including an ORF of 2 334 bp and predicted to encode 777 amino acid,its isoelectric point and molecular weight was 8.55 and 87.0kD,respectively.The sequence analysis showed AsKUP1 was closer related to the KUP/HAK/KT family of the Aegilops tauschii and the triticum aestivum,and this protein was predicted to contain 14 transmembrane domains and located on cell membra with greater probability.Under the salt stress conditions,the T2 germination rate of transgenic seeds was 57％,while the wild type was 43％.The two transgenic lines with the ratio of 3:1 were screened by hygromycin,and the root length of T3 transgenic plant of their progeny was 1.46 and 1.34 times of the wild type plant.The fresh and dry weight of T3 transgenic plant was 1.56 and 1.44 times of the wild type plant respectively,the difference of Na + content was not significant,and K + content of transgenic plant was 1.25 times of the wild type plant,which indicated that the expression of AsKUP1 improved the salt tolerance of Arabidopsis thaliana.This study laid the foundation for revealing the salt tolerance mechanism of family gene for the family gene of K + transport protein in plant,and for improving the salt tolerance ability of plant by transgenic technology.