马铃薯硝态氮转运蛋白基因的克隆及表达分析
    点此下载全文
引用本文:南运有,吕婷婷,曹敏轩,刘恒志,陈 越,陈 勤.马铃薯硝态氮转运蛋白基因的克隆及表达分析[J].西北植物学报,2019,39(4):588~594
摘要点击次数: 0
全文下载次数: 0
作者单位
南运有1,吕婷婷1,曹敏轩1,刘恒志1,陈 越1,陈 勤2* (1 西北农林科技大学 农学院/旱区作物逆境生物学国家重点实验室 陕西杨陵 7121002 西北农林科技大学 食品科学与工程学院/旱区作物逆境生物学国家重点实验室陕西杨陵 712100) 
基金项目:国家重点研发项目(2018YFD0200805)
中文摘要:该研究以马铃薯双单倍体‘DM’为材料,克隆到高亲和性硝态氮转运蛋白基因 StNRT2.1的全长cDNA(JGI登录号PGSC0003DMT400002924),并对其进行表达模式和生物信息学分析,为深入探索StNRT2.1基因的生物学功能以及提高马铃薯对氮素的利用效率奠定理论基础。结果表明:(1)通过同源克隆与PCR扩增获得StNRT2.1基因cDNA全长片段,并构建pCEGFP StNRT2.1表达载体;测序结果显示其实际所编码的蛋白质序列与数据库中目的基因蛋白质序列完全一致,表明成功克隆到StNRT2.1基因且未出现错义突变。(2)StNRT2.1基因位于马铃薯第11号染色体,cDNA序列全长1 593 bp,编码530个氨基酸,预测蛋白相对分子质量约为57.60 kD, 理论等电点为9.36。(3)生物信息学分析显示,StNRT2.1由20种氨基酸组成,其中甘氨酸(Gly)所占比例最多,达到10.8%,并且主要由228个α 螺旋、27个β 折叠、87个延伸链和188个无规则卷曲构成;StNRT2.1存在功能保守结构MFS_1(PF07690)和12个跨膜螺旋结构域,且N端和C端均位于细胞膜内; StNRT2.1位于质膜上且不具有信号肽,可能为非分泌型膜蛋白。(4)以氮充足(7.5 mmol/L)水平作为对照,马铃薯幼苗经无氮(0 mmol/L)和低氮(0.75 mmol/L)处理3周后呈现出叶片发黄及植株矮化等明显表型差异。(5)qRT PCR结果显示,在无氮条件下,马铃薯根组织中StNRT2.1基因表达量升高3.98倍,说明StNRT2.1可能为诱导型高亲和转运蛋白。
中文关键词:马铃薯  硝态氮  硝态氮转运蛋白2(NRT2)  生物信息学分析  qRT PCR
 
Cloning and Expression Analysis of Potato Nitrate Transporter Gene
Abstract:In this study, we used potato double haploid ‘DM’ as material to clone the complete cDNA of high affinity nitrate transporter gene StNRT2.1(JGI accession number PGSC0003DMT400002924). Expression pattern and bioinformatics analysis of the StNRT2.1 gene were constructed in this study, which provided a theoretical foundation for further exploration of the biological functions of the StNRT2.1 gene and improving the efficiency of nitrogen utilization in potato. The results showed that: (1) complete cDNA fragment of StNRT2.1 gene was obtained by homologous cloning and PCR amplification, and the expression vector pCEGFP StNRT2.1 was constructed. From the sequencing results, it was found that the actual encoded protein sequence was identical with the target protein sequence in the database, indicating that StNRT2.1 gene was cloned successfully without missense mutation. (2) StNRT2.1 gene is located on the chromosome 11 of potato and cDNA sequence was 1 593 bp in length encoding 530 amino acids. The relative molecular mass of the protein is about 57.60 kD, and the theoretical isoelectric point is 9.36. (3) Bioinformatics analysis showed that StNRT2.1 consisted of 20 different amino acids, of which glycine (Gly) accounted for the most, reaching 10.8%, and mainly consisted of 228 α helix, 27 β sheet, 87 extended chain and 188 irregular coils. Moreover, the functionally conserved structure MFS_1 (PF07690) and 12 transmembrane helix domains are present, and the N terminus and C terminus are located in the cell membrane. StNRT2.1 is predicted to be located in the cytoplasm without signal peptide. Therefore, it may be a non secret membrane protein. (4) Compared with sufficient nitrogen level (7.5 mmol/L), potato seedlings showed obvious phenotypic changes including yellowed leaves and shorter plant with the three week treatments of nitrogen free (0 mmol/L) and low nitrogen (0.75 mmol/L) level. (5) The results of real time PCR showed the expression of StNRT2.1 gene in root tissue increased 3.98 times with the nitrogen free treatment, which suggesting that StNRT2.1 may be an inducible high affinity transporter.
keywords:potato  nitrate  nitrate transporter 2 (NRT2)  bioinformatics analysis  qRT PCR
查看全文  查看/发表评论  下载PDF阅读器
   今日访问:902    总访问量:8998885

版权所有:《西北植物学报》编辑部

技术支持:北京勤云科技有限公司