大蒜几丁质酶基因AsCHI1的鉴定及其对盐胁迫的响应

doi:10.11924/j.issn.1000-6850.casb2021-0275

中国农学通报 ›› 2022, Vol. 38 ›› Issue (5): 23-29.doi: 10.11924/j.issn.1000-6850.casb2021-0275

所属专题：生物技术；园艺

大蒜几丁质酶基因AsCHI1的鉴定及其对盐胁迫的响应

张宇阳¹(), 周雪¹, 刘灵艺¹, 许吴俊², 任旭琴¹, 王广龙¹(), 熊爱生³

¹淮阴工学院生命科学与食品工程学院,江苏淮安 223003
²淮阴工学院应用技术学院,江苏淮安 223003
³南京农业大学园艺学院/作物遗传与种质创新国家重点实验室/农业农村部华东地区园艺作物生物学与种质创制重点实验室,南京 210095

收稿日期:2021-03-18 修回日期:2021-06-13 出版日期:2022-02-15 发布日期:2022-03-17
通讯作者: 王广龙
作者简介:张宇阳,女,2001年出生,江苏无锡人,本科,主要从事蔬菜生理与分子生物学研究。通信地址：223003 江苏省淮安市枚乘东路1号淮阴工学院,Tel：0517-83559044,E-mail： zhangyuyang0415@163.com。
基金资助:
淮阴工学院博士科研启动基金“主要蔬菜作物重要性状形成的基因调控机制”(Z301B16531);青海省科技厅重点实验室项目“青海省蔬菜遗传与生理重点实验室”(2020-ZJ-Y02);青海省蔬菜遗传与生理重点实验室开放课题“脱落酸和钙信号参与调控大蒜抗旱的分子机制研究”(2020-SCSYS-01)

Garlic Chitinase Gene AsCHI1: Identification and Its Response to Salt Stress

ZHANG Yuyang¹(), ZHOU Xue¹, LIU Lingyi¹, XU Wujun², REN Xuqin¹, WANG Guanglong¹(), XIONG Aisheng³

¹School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, Jiangsu 223003
²Faculty of Applied Technology, Huaiyin Institute of Technology, Huaian, Jiangsu 223003
³State Key Laboratory of Crop Genetics and Germplasm Enhancement/ Ministry of Agriculture Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China/ College of Horticulture, Nanjing Agricultural University, Nanjing 210095

Received:2021-03-18 Revised:2021-06-13 Online:2022-02-15 Published:2022-03-17
Contact: WANG Guanglong

摘要/Abstract

摘要：

本研究旨在研究大蒜几丁质酶基因的序列特征及其对盐胁迫的响应,鉴定其抗逆功能。以大蒜品种‘苍山四六瓣’为研究材料,通过RT-PCR技术分离得到AsCHI1基因。采用BioXM 2.6、ProtParam、DNAMAN、SignalP 5.0、SOPMA、SWISS-MODEL、NCBI和MEGA5等软件分析核苷酸及其编码的氨基酸序列特征,利用荧光定量PCR技术检测其在不同组织以及盐胁迫下的表达特性。该基因开放阅读框全长933 bp,编码310个氨基酸,其编码的蛋白属于几丁质酶Class I类,同时隶属于GH19家族成员。氨基酸序列分析显示,AsCHI1在N端含有大多数几丁质酶具备的信号肽区域,在C端与其他物种CHI氨基酸序列一致性较高。在亲缘关系上与同属百合科的麝香百合LlCHI (QBZ68892.1)以及山茶科的茶CsCHI(XP_028075045.1)较为接近。实时荧光定量PCR技术分析表明,AsCHI1基因在大蒜根、蒜瓣和叶片中均有表达,但在根中表达最高。另外,盐胁迫能显著诱导AsCHI1基因在各组织内的表达。说明该基因可能在大蒜植株抵御盐胁迫的过程中发挥重要作用。

关键词: 大蒜, 盐胁迫, AsCHI1基因, 基因克隆, 响应

Abstract:

The study aims to investigate the sequence characteristics of garlic chitinase gene and its response to salt stress, and to identify its function in resistance to stresses. The AsCHI1 gene was isolated by RT-PCR technology using garlic cultivar ‘Cangshan siliuban’ as the research material. BioXM 2.6, ProtParam, DNAMAN, SignalP 5.0, SOPMA, SWISS-MODEL, NCBI and MEGA5 were adopted to analyze the sequence characteristics of nucleotides and its encoded amino acids. Fluorescent quantitative PCR technology was introduced to detect its expression in different tissues and under salt stress. The open reading frame of the gene was 933 bp in length encoding 310 amino acids. The protein encoded by AsCHI1 gene belonged to chitinase Class I and GH19 family. Amino acid sequence analysis showed that AsCHI1 contained a signal peptide region at the N-terminus possessed by most chitinases, whereas the C-terminus was highly consistent with the amino acid sequences of CHI from other species. In terms of genetic relationship, it was relatively close to Lilium longiflorum LlCHI (QBZ68892.1) in the Liliaceae family and tea CsCHI (XP_028075045.1) in the Theaceae family. Real-time fluorescent quantitative PCR analysis showed that the AsCHI1 gene was expressed in garlic roots, garlic cloves and leaves, but the highest expression was observed in the roots. In addition, salt stress could significantly induce the expression of AsCHI1 gene in various tissues. This gene may play an important role in the resistance to salt stress in garlic plants.

Key words: Allium sativum, salt stress, AsCHI1 gene, gene cloning, response

中图分类号:

S633.4

张宇阳, 周雪, 刘灵艺, 许吴俊, 任旭琴, 王广龙, 熊爱生. 大蒜几丁质酶基因AsCHI1的鉴定及其对盐胁迫的响应[J]. 中国农学通报, 2022, 38(5): 23-29.

ZHANG Yuyang, ZHOU Xue, LIU Lingyi, XU Wujun, REN Xuqin, WANG Guanglong, XIONG Aisheng. Garlic Chitinase Gene AsCHI1: Identification and Its Response to Salt Stress[J]. Chinese Agricultural Science Bulletin, 2022, 38(5): 23-29.

图/表 8

图1. 大蒜AsCHI1基因的RT-PCR扩增图谱

图2. AsCHI1的核苷酸序列及其预测的氨基酸序列

图3. 大蒜AsCHI1氨基酸序列保守域分析

图4. AsCHI1基因推导的氨基酸序列与其他物种CHI序列比对

图5. AsCHI1基因编码的蛋白二级结构预测蓝色：α螺旋;红色：延伸链;绿色：β转角;紫色：无规则卷曲

图6. AsCHI1基因编码的蛋白三级结构预测

图7. 大蒜与其他植物CHI氨基酸序列的系统进化树

图8. 盐胁迫下AsCHI1基因的表达分析不同字母表示在0.05水平上差异显著

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大蒜几丁质酶基因AsCHI1的鉴定及其对盐胁迫的响应

Garlic Chitinase Gene AsCHI1: Identification and Its Response to Salt Stress

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