甜菜M14品系BvM14-Dof3.4基因的克隆及响应盐胁迫表达分析

doi:10.11924/j.issn.1000-6850.casb20191200897

中国农学通报 ›› 2020, Vol. 36 ›› Issue (13): 36-41.doi: 10.11924/j.issn.1000-6850.casb20191200897

所属专题：生物技术；园艺

甜菜M14品系BvM14-Dof3.4基因的克隆及响应盐胁迫表达分析

马春泉¹^,², 黄园园¹^,², 李海英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 150500
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:2019-12-02 修回日期:2020-03-09 出版日期:2020-05-05 发布日期:2020-04-16
通讯作者: 李海英
作者简介:马春泉,男,1980年出生,黑龙江呼兰人,博士,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,Tel：0451-80064372,E-mail：chqm@hlju.edu.cn。
基金资助:
国家自然科学基金资助项目“甜菜M14品系抗氧化酶系统响应盐胁迫应答过程的研究”(31471552);国家自然科学基金资助项目“甜菜M14品系乙二醛酶I基因的转录因子参与响应盐胁迫的调控机制研究”(3167100696)

BvM14-Dof3.4 Gene in Sugar Beet M14 Line: Cloning and Expression in Response to Salt Stress

Ma Chunquan¹^,², Huang Yuanyuan¹^,², Li Haiying¹^,²()

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

Received:2019-12-02 Revised:2020-03-09 Online:2020-05-05 Published:2020-04-16
Contact: Haiying Li

摘要/Abstract

摘要：

为进一步研究BvM14-Dof3.4基因响应盐胁迫的功能,以甜菜M14品系根为材料,通过PCR技术获得BvM14-Dof3.4基因cDNA全长序列并进行生物信息学以及盐胁迫应答分析。结果表明：该基因最大ORF为408 bp,编码135个氨基酸,蛋白分子量为12646.63 Da,理论等电点为4.68,为亲水性蛋白;BvM14-Dof3.4蛋白质二级结构和三级结构主要由延伸链和无规卷曲组成;BvM14-Dof3.4蛋白氨基酸序列与NCBI数据库中甜菜(Beta vulgaris)的氨基酸序列相似度为99.26%;系统进化分析表明BvM14-Dof3.4蛋白与菠菜(Spinacia oleracea)Dof3.4蛋白亲缘性较高。结合实验室前期盐胁迫下甜菜M14品系根的转录组数据分析,BvM14-Dof3.4基因参与甜菜M14品系应答盐胁迫过程,在200 mmol/L NaCl处理下上调2.05倍,在400 mmol/L NaCl处理下上调1.41倍。实验成功获得BvM14-Dof3.4基因cDNA全长,并确定该基因响应盐胁迫,该结果对挖掘甜菜M14品系优质基因和提高栽培甜菜抗逆性等方面具有重要意义,为后续进一步开展BvM14-Dof3.4基因响应盐胁迫功能研究提供参考。

关键词: 甜菜M14品系, BvM14-Dof3.4基因, 克隆, 盐胁迫, 响应

Abstract:

The objective is to study the function of BvM14-Dof3.4 gene in response to salt stress. The full-length sequence of BvM14-Dof3.4 gene from the root of sugar beet M14 line was obtained by PCR and analyzed by bioinformatics. The response mode of BvM14-Dof3.4 gene under salt stress was also verified. The results showed that the largest ORF of the gene was 408 bp which encoded 135 amino acids, the molecular weight of the protein was 12646.63 Da, the theoretical pI was 4.68, the protein was hydrophilic protein; the secondary and tertiary structure of the protein were mainly composed of extension chain and random curl; the amino acid sequence similarity between BvM14-Dof3.4 and Dof3.4 from Beta vulgaris database was up to 99.26%. The phylogenetic tree showed that BvM14-Dof3.4 protein was highly homologous with Dof3.4 protein from Spinacia oleracea. According to the transcriptome database from the root of sugar beet M14 line under salt stress, BvM14-Dof3.4 gene was involved in the response to salt stress, which was up-regulated by 2.05 times under 200 mmol/L NaCl and 1.41 times under 400 mmol/L NaCl. The full cDNA length of BvM14-Dof3.4 gene was successfully obtained in this experiment and its response to salt stress was determined. The results have certain significance for the exploration of high quality genes in sugar beet M14 line and the improvement of stress resistance of cultivated sugar beet, and lay a foundation for further research on the function of BvM14-Dof 3.4 gene in response to salt stress.

Key words: sugar beet M14 line, BvM14-Dof3.4 gene, clone, salt stress, response

中图分类号:

S566.3

马春泉, 黄园园, 李海英. 甜菜M14品系BvM14-Dof3.4基因的克隆及响应盐胁迫表达分析[J]. 中国农学通报, 2020, 36(13): 36-41.

Ma Chunquan, Huang Yuanyuan, Li Haiying. BvM14-Dof3.4 Gene in Sugar Beet M14 Line: Cloning and Expression in Response to Salt Stress[J]. Chinese Agricultural Science Bulletin, 2020, 36(13): 36-41.

图/表 9

引物名称	序列
BvM14-Dof3.4-S	5'-ATGGGTGGAGGCGGAGGC-3'
BvM14-Dof3.4-AS	5'-TCACTTGAGACCATTGGCTGATG-3'

表1. 扩增BvM14-Dof3.4基因的引物

图1. 盐胁迫下甜菜M14品系根总RNA的1%琼脂糖凝胶电泳图

图2. BvM14-Dof3.4基因cDNA全长产物的1%琼脂糖凝胶电泳图 M：DL 2000 DNA Marker;1,2：BvM14-Dof3.4基因的PCR扩增产物

图3. BvM14-Dof3.4基因cDNA全长的开放阅读框

图4. BvM14-Dof3.4蛋白序列的亲/疏水性预测结果

图5. 预测的BvM14-Dof3.4蛋白的二级结构蓝色h代表α螺旋,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. 预测的BvM14-Dof3.4蛋白的三级结构

图7. BvM14-Dof3.4蛋白的系统发育树

图8. 甜菜M14品系根中BvM14-Dof3.4基因响应盐胁迫的表达分析

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甜菜M14品系BvM14-Dof3.4基因的克隆及响应盐胁迫表达分析

BvM14-Dof3.4 Gene in Sugar Beet M14 Line: Cloning and Expression in Response to Salt Stress

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