楸子MpRZ 1基因的克隆及响应干旱胁迫表达分析  

Cloning of MpRZ1 gene in Malus prunifolia and expression analysis of its response to drought stress

作　　者：王顺才 杨镇菱 檀可馨 王红明 WANG Shuncai;YANG Zhenling;TAN Kexin;WANG Hongming(College of Bioengineering and Technology,Tianshui Normal University,Tianshui,Gansu 741000,China;College of Horticulture,Northwest A&F University,Yangling,Shaanxi 712100,China;Gansu Key Laboratory for Utilization of Agricultural Solid Waste Resources,Tianshui,Gansu 741000,China)

机构地区：[1]天水师范学院生物工程与技术学院,甘肃天水741000 [2]西北农林科技大学园艺学院,陕西杨凌712100 [3]甘肃省农业固体废弃物资源化利用重点实验室,甘肃天水741000

出　　处：《干旱地区农业研究》2024年第4期52-61,88,共11页Agricultural Research in the Arid Areas

基　　金：国家自然科学基金(31660565);甘肃省自然科学基金(20JR10RA795,21JR7RE179);天水师院伏羲科研创新团队项目(FXD2020-11);天水师院产业支撑引导项目(CYZ2019-03);天水师院教育教学研究项目(TYXM2112)。

摘　　要：基于干旱胁迫下楸子(Malus prunifolia)转录组得到的4条RZs转录本,通过电子延伸和RT-PCR方法克隆得到1个新基因MpRZ 1,利用生物信息学方法对该基因编码蛋白的结构及功能特性进行预测,并与苹果(Malus domestica)RZ家族成员进行同源性比较,利用qRT-PCR技术验证干旱胁迫下MpRZ 1基因的表达模式。结果表明,MpRZ 1基因cDNA全长930 bp,开放读码框为843 bp,编码280个氨基酸残基,分子量为30.97 kDa,等电点为9.37,无N端信号肽,无跨膜结构阈,推测该蛋白可能定位于细胞核中,是一种不稳定的碱性亲水蛋白,含有38个潜在的磷酸化修饰位点,13个O-GlcNAc和5个N-Glyc潜在的糖基化修饰位点。该蛋白二级元件主要以无规卷曲和α-螺旋组成,两者占比达87.1%。MpRZ1蛋白N-端含有1个保守的RNA识别基序,由5个反向平行的β-折叠与2个α-螺旋排成β_(1)α_(1)β_(2)β_(3)α_(2)β_(4)β_(5)拓扑结构,C-端的甘氨酸富含区含有1个CCHC型锌指和一系列(GGX)n和(DRX)n重复序列。序列比对和聚类分析显示,MpRZ 1编码蛋白与拟南芥、水稻RZs蛋白有较高的序列相似性和亲缘关系,属于RZ基因家族成员。通过苹果基因组搜索得到9个推测的MdRZs基因,其编码蛋白均为碱性亲水蛋白,其中2个MdRZs编码蛋白(MdP0000088428、MdP0000272138)与MpRZ 1编码蛋白的序列相似性超过96%。楸子和平邑甜茶叶片中RZ基因的表达水平均在干旱胁迫9 d时达到峰值且与对照差异显著(P<0.05),表明MpRZ 1基因参与干旱胁迫的应答过程。Based on four RZs transcripts from the transcriptome library of Malus prunifolia under drought stress,a new gene MpRZ 1 was cloned by electren extension and RT-PCR methods.The structural and functional characteristics of the deduced amino acid sequence of MpRZ 1 gene was analyzed using bioinformatics methods,and a comparative analysis of MpRZ1 protein with predicted apple(Malus domestica)RZ homologous members was conducted.The expression pattern of MpRZ 1 gene under drought stress was conducted by qRT-PCR technology.The results indicated that,cDNA sequence of MpRZ 1 gene was 930 bp in length contained an intact open reading frame of 843 bp,encoding a polypeptide of 280 amino acid residues with a predicted molecular mass of 30.97 kDa and pI of 9.37.N-terminal signal peptide and transmembrane topology were not found in MpRZ1 protein,which was probably located in the nucleus and an unstable alkaline hydrophilic protein containing 38 potential phosphorylation sites,13 O-GlcNAc and 5 N-Glyc potential glycosylation sites.The secondary element of MpRZ1 protein was mainly characterized by random curling andα-helice,accounting for up to 87.1%.The predicted MpRZ1 protein contained one conserved RNA-recognition motif(RRM)with five antiparallelβ-strands and 2α-helices arranged inβ_(1)α_(1)β_(2)β_(3)α_(2)β_(4)β_(5)topology at the N-terminus,and a CCHC type zinc finger domain as well as a series of(GGX)n and(DRY/F)n repeated at the C-terminal glycine-rich region.Sequence alignment and phylogenetic analysis indicated that the predicted MpRZ1 protein shared higher sequence similarity and homology with Arabidopsis and rice RZ proteins,suggesting this MpRZ 1 gene belonged to a homologous member of RZ genes family.Nine different MdRZs transcripts found in the apple genome uniformly encoded alkaline hydrophilic proteins,and this MpRZ1 protein had more than 96%homology with two MdRZs proteins encoded by MdP0000088428 and MdP0000272138 transcripts.The expression level of RZ genes in leaves of M.prunifolia and M.hupehensis at

关 键 词：楸子 RZ基因 克隆 干旱胁迫 平邑甜茶 基因表达 

分 类 号：S661.1[农业科学—果树学] Q786[农业科学—园艺学]