| 王顺才,杨镇菱,檀可馨,王红明.楸子MpRZ1基因的克隆及响应干旱胁迫表达分析[J].干旱地区农业研究,2024,(4):52~61 |
| 楸子MpRZ1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 |
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| DOI：10.7606/j.issn.1000-7601.2024.04.06 |
| 中文关键词: 楸子 RZ基因 克隆 干旱胁迫 平邑甜茶 基因表达 |
| 英文关键词:M. prunifolia RZ gene gene cloning drought stress M. hupehensis gene expression |
| 基金项目: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） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 phosphorylation 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 analysis 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 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 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. |
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