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MpRZ1,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MpRZ1基因的表达模式.结果表明,MpRZ1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β1α1β2β3α2β4β5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,MpRZ1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MpRZ1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MpRZ1基因参与干旱胁迫的应答过程.
Abstract
Based on four RZs transcripts from the transcriptome library of Malus prunifolia under drought stress,a new gene MpRZ1 was cloned by electren extension and RT-PCR methods.The structural and functional characteristics of the deduced amino acid sequence of MpRZ1 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 MpRZ1 gene under drought stress was conducted by qRT-PCR technology.The results indicated that,cDNA sequence of MpRZ1 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 phosphoryl-ation sites,13 O-GlcNAc and 5 N-Glyc potential glycosylation sites.The secondary element of MpRZ1 protein was mainly characterized by random curling and α-helice,accounting for up to 87.1%.The predicted MpRZ1 protein contained one conserved RNA-recognition motif ( RRM) with five antiparallel β-strands and 2 α-helices arranged in β1α1β2β3α2β4β5 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 a-nalysis indicated that the predicted MpRZ1 protein shared higher sequence similarity and homology with Arabidopsis and rice RZ proteins,suggesting this MpRZ1 gene belonged to a homologous member of RZ genes family.Nine dif-ferent 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 9 days of drought stress and showed significant differences compared to the control (P<0.05),indicating that the MpRZ1 gene was involved in the drought stress response process.
基金项目
国家自然科学基金(31660565)
甘肃省自然科学基金(20JR10RA795)
甘肃省自然科学基金(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)
万方数据