银杏GbASR基因的克隆、生物信息学及表达分析

doi:10.11924/j.issn.1000-6850.casb2021-0162

中国农学通报 ›› 2021, Vol. 37 ›› Issue (32): 34-41.doi: 10.11924/j.issn.1000-6850.casb2021-0162

所属专题：生物技术

银杏GbASR基因的克隆、生物信息学及表达分析

谢道龙¹^,²(), 谭智¹, 李虹烨¹, 付小侠¹, 王帆¹, 刘晓霞¹^,³, 覃佐东¹^,³, 何福林¹^,³, 骆鹰¹^,³()

¹湖南科技学院化学与生物工程学院,湖南永州 425199
²湖南大学生物学院,长沙 410082
³湖南省银杏工程技术研究中心,湖南永州 425199

收稿日期:2021-02-20 修回日期:2021-06-13 出版日期:2021-11-15 发布日期:2022-01-07
通讯作者: 骆鹰
作者简介:谢道龙,男,1998年出生,湖南娄底人,硕士研究生,主要从事植物分子生物学方面的研究。通信地址：425199 湖南省永州市零陵区湖南科技学院化学与生物工程学院,Tel：0746-6381164,E-mail： 1912359023@qq.com。
基金资助:
湖南省自然科学基金面上项目“CRISPR/Cas9基因编辑技术在银杏MYB基因功能研究中的应用”(2018JJ2144);湖南省自然科学基金青年项目“银杏内酯生物合成关键miRNAs及其调控网络的研究”(2018JJ3199);湖南科技学院重点项目“银杏MYB家族相关基因的克隆及功能分析”(17XKY005);湖南科技学院大学生研究性学习和创新性实验计划项目“银杏ASR基因的克隆、生物信息学及表达分析”(湘科院教发[2018]32号)

GbASR gene from Ginkgo biloba: Cloning, Bioinformatics and Expression Analysis

Xie Daolong¹^,²(), Tang Zhi¹, Li Hongye¹, Fu Xiaoxia¹, Wang Fan¹, Liu Xiaoxia¹^,³, Qin Zuodong¹^,³, He Fulin¹^,³, Luo Ying¹^,³()

¹College of Chemistry and Bioengineering, Hunan University of Science and Engineering, Yongzhou Hunan 425199
²College of Biology, Hunan University, Changsha 410082
³Hunan Provincial Engineering Research Center for Ginkgo biloba, Yongzhou Hunan 425199

Received:2021-02-20 Revised:2021-06-13 Online:2021-11-15 Published:2022-01-07
Contact: Luo Ying

摘要/Abstract

摘要：

为了明确银杏ASR(ABA-stress-ripening)在应答非生物逆境胁迫中的作用,以便为ASR基因功能、环境胁迫研究提供材料。本研究以银杏(Ginkgo biloba)为材料,利用分子生物学技术对ASR基因进行了克隆,构建pCAMBIA1300-ASR-GFP融合表达载体,进行亚细胞定位分析。采用序列比对方法分析GbASR蛋白结构域及系统进化;利用qRT-PCR方法进行组织特异性表达及非生物逆境胁迫下的基因表达分析。结果表明,银杏ASR基因编码区全长546 bp,共编码182个氨基酸;蛋白质等电点为5.33,分子量大小为20111.24 Da,为亲水性稳定蛋白;GbASR蛋白含有1个高度保守的ABA/WDS结构域,且与火炬松PtASR蛋白同源性较高;GbASR蛋白定位于细胞核。此外,GbASR基因在银杏幼苗根、茎、叶各组织中均有表达,且在叶部有较高的表达;在高盐、干旱和高温胁迫下,GbASR基因均受诱导,且在不同胁迫处理时间0 h、6 h、24 h和48 h的表达存在显著的差异。以上分析说明GbASR基因参与响应高盐、干旱和高温等非生物胁迫,这为植物的育种及解释ASR基因功能提供依据。

关键词: 银杏, GbASR基因, 生物信息学分析, 亚细胞定位, 非生物胁迫

Abstract:

The aims are to clarify the role of ASR (ABA-stress-ripening) gene from Ginkgo biloba in response to abiotic stress, and provide materials for studying ASR gene function and environment stress. In this study, Ginkgo biloba was used as material, GbASR gene was cloned and the fusion expression vector pCAMBIA1300-ASR-GFP was constructed by molecular biology techniques, then the subcellular localization of GbASR protein was analyzed. Protein domains and phyletic evolution were analyzed by sequence alignment method. Tissue-specific expression and expression level of GbASR gene under abiotic stress were analyzed by qRT-PCR method. The results showed that the total length of GbASR coding region was 546 bp, encoding a 182 amino acid protein. The theoretical isoelectric point was 5.33 and the molecular weight of GbASR protein was 20111.24 Da, which was a stable hydrophilic protein. Sequence alignment and phylogenetic analysis showed that GbASR protein contained a highly conserved ABA/WDS domain and had high homology with PtASR protein. The subcellular localization analysis showed that GbASR protein was a nucleoprotein. In addition, GbASR gene was widely expressed in different tissues such as root, steam and leaf from Ginkgo biloba, and the GbASR gene was induced under salt, drought and heat stresses, and there were significant different expressions at 0 h, 6 h, 24 h and 48 h under salt, drought, and heat stresses. These results suggest that GbASR might participate in response to abiotic stresses such as salt, drought and heat stresses, and this study could provide a basis for plant breeding and explaining ASR gene function.

Key words: Ginkgo biloba, GbASR gene, bioinformatics analysis, subcellular localization, abiotic stress

中图分类号:

S718

谢道龙, 谭智, 李虹烨, 付小侠, 王帆, 刘晓霞, 覃佐东, 何福林, 骆鹰. 银杏GbASR基因的克隆、生物信息学及表达分析[J]. 中国农学通报, 2021, 37(32): 34-41.

Xie Daolong, Tang Zhi, Li Hongye, Fu Xiaoxia, Wang Fan, Liu Xiaoxia, Qin Zuodong, He Fulin, Luo Ying. GbASR gene from Ginkgo biloba: Cloning, Bioinformatics and Expression Analysis[J]. Chinese Agricultural Science Bulletin, 2021, 37(32): 34-41.

图/表 6

引物名称	引物序列 (5’-3’)	用途
GbASR-F	GCGTTATCCATTGCTTCTGC	ASR PCR扩增引物
GbASR-R	ACCAACACATGCTGTCAACCT	ASR PCR扩增引物
GbASR-GFP-F	AGAACACGGGGGACGAGCTCGGTACCATGTCTCAGGAGCATCATCA	亚细胞定位引物
GbASR-GFP-R	CCCTTGCTCACCATGTCGACTCTAGAGAACAGATGGTGATGATGCTT	亚细胞定位引物
Gb18S-F	CGAAGACGATCAGATACCG	qPCR内参基因
Gb18S-R	TCAGCCTTGCGACCATAC	qPCR内参基因
q-ASR-F	CCGGCTCTGGCTATGGTTATG	qPCR检测引物
q-ASR-R	ATGGTTCCAAGCTCCCCAAC	qPCR检测引物

表1. 本研究PCR扩增所用引物信息

图1. 银杏叶片组织总RNA提取及GbASR基因PCR扩增电泳图 A表示总RNA提取,B、C分别目的基因PCR扩增、重组载体菌落PCR扩增。M分别表示DL2000 DNA Marker(A)和DL2000 Plus DNA Marker(B、C)

图2. GbASR蛋白与其他植物ASR蛋白序列比对

图3. 不同植物ASR蛋白氨基酸序列构建的系统进化树

图4. GbASR蛋白亚细胞定位分析

图5. GbASR基因组织特异表达及响应非生物胁迫的表达分析

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银杏GbASR基因的克隆、生物信息学及表达分析

GbASR gene from Ginkgo biloba: Cloning, Bioinformatics and Expression Analysis

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