甜菜M14品系BvM14-RIN4基因的克隆及应答盐胁迫分析

doi:10.11924/j.issn.1000-6850.casb2020-0848

中国农学通报 ›› 2021, Vol. 37 ›› Issue (14): 27-33.doi: 10.11924/j.issn.1000-6850.casb2020-0848

所属专题：生物技术

甜菜M14品系BvM14-RIN4基因的克隆及应答盐胁迫分析

贾惠旭¹^,²(), 李海英¹^,²(), 端木慧子¹^,²()

¹黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨 1500500
²黑龙江大学生命科学学院/黑龙江省普通高校分子生物学重点实验室,哈尔滨 150080

收稿日期:2020-12-29 修回日期:2021-02-25 出版日期:2021-05-15 发布日期:2021-05-19
通讯作者: 李海英,端木慧子
作者简介:贾惠旭,女,1996年出生,黑龙江牡丹江人,硕士研究生,研究方向:植物分子生物学。通信地址:150080 黑龙江省哈尔滨市学府路74号黑龙江大学生命科学学院320室,Tel:0451-86608586,E-mail: jiahuixu0453@163.com。
基金资助:
国家自然科学基金“转录因子BvGT-1调控乙二醛酶I基因参与甜菜单体附加系耐盐的分子机制”(32072122);国家自然基金青年项目“甜菜M14品系BvM14-RIN4蛋白盐胁迫响应蛋白互作网络的研究”(31801426);黑龙江省省属高等学校基本科研业务费基础研究项目(科技创新类专项重点项目)“甜菜M14品系与二倍体栽培甜菜的比较转录组学研究”(KJCXZD201717);中国博士后科学基金面上项目“甜菜M14品系BvM14-RIN4基因响应盐胁迫的分子机理研究”(2019M651314);黑龙江省普通高等学校青年创新人才培养计划“甜菜M14品系BvM14-RIN4基因耐盐功能及其蛋白调控机理研究”(UNPYSCT-2018008)

BvM14-RIN4 Gene of Sugar Beet M14 Line: Cloning and Response to Salt Stress

Jia Huixu¹^,²(), Li Haiying¹^,²(), Duanmu Huizi¹^,²()

¹Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500
²Key Laboratory of Microbiology, College of Heilongjiang Province/School of Life Science, Heilongjiang University, Harbin 150080

Received:2020-12-29 Revised:2021-02-25 Online:2021-05-15 Published:2021-05-19
Contact: Li Haiying,Duanmu Huizi

摘要/Abstract

摘要：

为了研究甜菜单体附加系M14品系RIN4基因耐盐功能,本研究以甜菜M14品系为实验材料,通过PCR技术获得BvM14-RIN4基因cDNA全长序列,对BvM14-RIN4基因进行了生物信息学分析、组织特异性和应答盐胁迫分析。BvM14-RIN4基因ORF长度为264 bp,编码87个氨基酸,蛋白分子量为 9.67 kDa,理论等电点为9.64,初步鉴定该蛋白为亲水性蛋白;BvM14-RIN4蛋白质二级结构主要由延伸链和无规卷曲组成;BvM14-RIN4蛋白质三级结构预测出该蛋白的保守结构域和疏水区;系统进化树分析结果表明BvM14-RIN4蛋白与菠菜SoRIN4和藜麦CqRIN4蛋白亲缘关系最近;荧光定量PCR结果显示,BvM14 -RIN4基因在根中表达量是叶的2.26倍,说明其具有组织特异性;该基因在200 mmol/L NaCl处理后的根中上调表达,说明它参与甜菜M14品系应答盐胁迫过程。本研究在挖掘甜菜M14品系优质基因和开展甜菜遗传改良工作等方面具有重要意义,为后续该基因的功能研究提供了重要参考。

关键词: 甜菜M14品系, BvM14-RIN4, 基因克隆, 生物信息学分析, 荧光定量PCR

Abstract:

To study the salt-resistant function of RIN4 gene in sugar beet monosomic additional line M14 line, sugar beet M14 line was used as experimental material, the full-length cDNA sequence of the BvM14-RIN4 gene was obtained by PCR, and the BvM14-RIN4 gene was analyzed by bioinformatics and its tissue specificity and response to salt stress were explored. The results showed that the ORF length of BvM14-RIN4 gene was 264 bp which encoded 87 amino acids, the molecular weight of the protein was 9.67 kDa, the theoretical pI was 9.64, and the protein was preliminarily identified as a hydrophilic protein. The secondary structure of BvM14-RIN4 protein was mainly composed of extension chain and random curl; the tertiary structure of BvM14-RIN4 protein predicted the conserved domain and hydrophobic region of the protein; the phylogenetic tree showed that BvM14-RIN4 protein was closely related to RIN4 protein of Spinacia oleracea L. and Chenopodium quinoa L.. The results of quantitative RT-PCR showed that the expression level of BvM14-RIN4 gene in roots leaves was 2.26 times that of leaves, which indicated that it had tissue specificity; the expression of BvM14-RIN4 gene was up-regulated in roots under 200 mmol/L NaCl, which indicated that it was involved the response of sugar beet M14 line to salt stress. This study is of certain significance in mining high quality genes of sugar beet M14 line and carrying out genetic improvement of sugar beet, the results could provide important references for studying the function of BvM14-RIN4 gene.

Key words: sugar beet M14 line, BvM14-RIN4, gene clone, bioinformatics analysis, quantitative RT-PCR

中图分类号:

S566.3

贾惠旭, 李海英, 端木慧子. 甜菜M14品系BvM14-RIN4基因的克隆及应答盐胁迫分析[J]. 中国农学通报, 2021, 37(14): 27-33.

Jia Huixu, Li Haiying, Duanmu Huizi. BvM14-RIN4 Gene of Sugar Beet M14 Line: Cloning and Response to Salt Stress[J]. Chinese Agricultural Science Bulletin, 2021, 37(14): 27-33.

图/表 10

引物名称	序列
BvM14-RIN4-S	5'-TGTTTCTTGTATTACTTTTGAGAGC-3'
BvM14-RIN4-AS	5'-TCAAAGCCACCTTCTACTACAAAT-3'
qRT-PCR-RIN4-S	5'-GCAGGTAACAGATCCTTGCCA-3'
qRT-PCR-RIN4-AS	5'-TTAGGGCTCTGGTTAACGCC-3'

表1. PCR扩增用到的所有引物

图1. 甜菜M14品系根部总RNA提取1%琼脂糖凝胶电泳结果

图2. BvM14-RIN4基因cDNA全长产物的1%琼脂糖凝胶电泳图 M:DL 2000 DNA Marker;1-4:BvM14-RIN4基因的cDNA全长

图3. BvM14-RIN4基因cDNA全长的开放阅读框

图4. BvM14-RIN4蛋白序列的亲/疏水性预测结果

图5. BvM14-RIN4蛋白的二级结构红色e代表延伸链,黄色c代表无规卷曲,绿色t代表β-转角

图6. BvM14-RIN4蛋白的三级结构预测结果

图7. BvM14-RIN4与其他植物RIN4蛋白多重序列比对

图8. BvM14-RIN4蛋白的系统发育树

图9. 甜菜M14品系叶和根BvM14-RIN4基因的应答盐胁迫的表达情况

参考文献 23

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甜菜M14品系BvM14-RIN4基因的克隆及应答盐胁迫分析

BvM14-RIN4 Gene of Sugar Beet M14 Line: Cloning and Response to Salt Stress

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