葡萄NIP基因家族的鉴定与表达分析

Genome-Wide Identification and Expression Analysis of the NIP Gene Family in Grape

类根瘤菌26膜内在蛋白(nodulin 26-like intrinsic proteins,NIPs)是水通道蛋白的亚类,在植物营养获取和胁迫应答过程中发挥着重要作用。该研究利用多种生物信息学软件,对葡萄NIP家族基因进行分析,并采用RT-PCR方法克隆得到4个NIP家族基因,利用qRT-PCR方法分析非生物胁迫下NIP基因的表达特征。结果显示:(1)在葡萄基因组中,共鉴定到8个NIP基因,分布于葡萄4条染色体上,主要定位在质膜中;结构上含有6个跨膜结构域和两个典型的保守结构域NPA;氨基酸序列中存在很多个可能的磷酸化位点。(2)进化分析表明葡萄和拟南芥NIP基因具有较高的同源性,基因结构包含外显子数4~6个,保守基序种类和数量相似;基因启动子上游2 kb包含多种应答逆境和激素的顺式调控元件,其数量差异可能与基因本身功能相关。(3)NIP家族基因在不同组织中表达水平差异较大,多数成员在叶中表达水平较高,在茎中较低;成功克隆得到4个葡萄VvNIP基因,其长度分别为789 bp、606 bp、897 bp、789 bp,分别编码262、201、298、293个氨基酸。(4)qRT-PCR结果显示,不同胁迫处理下NIP基因在葡萄叶片中的表达水平不同:低温处理下葡萄NIP基因大多呈显著下调表达;盐胁迫下,除VvNIP2-1、VvNIP4-2外其余家族基因均呈下调表达;干旱胁迫下VvNIP4-2显著上调。研究表明,VvNIP基因对多种胁迫均有响应,为葡萄逆境胁迫机制研究提供了参考。

The nodulin 26-like intrinsic proteins(NIPs)are a subclass of aquaporin,which plays an important role in the process of plant nutrient acquisition and stress response.In this study,we used a variety of bioinformatics software to conduct bioinformatics analysis of grape NIP family genes,and cloned four VvNIP genes by reverse transcription PCR.We used qRT-PCR to analyze the expression characteristics of NIP genes under abiotic stress.The results showed:(1)a total of 8 NIP genes were identified in the grape genome and they were distributed on the 4 chromosomes of grapes,mainly located in the plasma membrane.The structure contains 6 transmembrane domains and two typical conserved NPA domains.Besides,there were many possible phosphorylation sites in the amino acid sequences.(2)Evolutionary analysis showed that the NIP genes of grape and Arabidopsis thaliana had high homology,the gene structure contained 4-6 gene structures with exons and the type and quantity of gene conserved sequences were similar.The 2 kb upstream of the gene promoter contains a variety of cis-regulatory elements that responded to stress and hormones,the quantity difference may be related to the function of gene itself.(3)The expression levels of NIP family genes in different tissues were quite different.Most members had higher expression levels in leaves,and lower expression levels in stems.In addition,four VvNIP genes were cloned and their lengths were 789 bp,606 bp,897 bp and 789 bp,encoding 262,201,298 and 293 amino acids.(4)qRT-PCR results showed that the expression levels of NIP genes in grape leaves were different under different stress treatments:most of the grape NIP genes were significantly down-regulated under low temperature treatment;under salt stress,the other family genes except VvNIP2-1 and VvNIP4-2 were down-regulated;VvNIP4-2 was significantly up-regulated under drought stress.The results showed that VvNIP genes were responsive to multiple stresses,it provides a reference for the study of grape stress mechanism.