大蒜己糖激酶基因AsHXK2的克隆及其参与根际促生菌缓解干旱胁迫的表达分析

doi:10.3969/j.issn.1004-1524.2022.09.11

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (9): 1925-1934.DOI: 10.3969/j.issn.1004-1524.2022.09.11

大蒜己糖激酶基因AsHXK2的克隆及其参与根际促生菌缓解干旱胁迫的表达分析

郭春倩¹(), 田洁¹^,²^,^*()

1.青海大学农林科学院, 青海省蔬菜遗传与生理重点实验室, 青海西宁 810016
2.青海大学省部共建三江源生态与高原农牧业国家重点实验室, 青海西宁 810016

收稿日期:2021-08-24 出版日期:2022-09-25 发布日期:2022-09-30
通讯作者: 田洁
作者简介:*田洁,E-mail: tiantian8092001@163.com
郭春倩(1997—),女,青海海东人,硕士研究生,主要从事蔬菜分子生物学研究。E-mail: 1984563279@qq.com
基金资助:
2019年度中国科学院“西部之光”人才培养计划(1-2);国家自然科学基金(31960590);青海省科技厅重点实验室项目(2020-ZJ-Y01)

Cloning of garlic hexokinase gene AsHXK2 and expression analysis of its involvement in rhizosphere growth-promoting bacteria alleviating drought stress

GUO Chunqian¹(), TIAN Jie¹^,²^,^*()

1. Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
2. State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China

Received:2021-08-24 Online:2022-09-25 Published:2022-09-30
Contact: TIAN Jie

摘要/Abstract

摘要：

为明确AsHXK2基因在根际促生菌缓解干旱胁迫下的作用,采用TA克隆方法克隆得到乐都紫皮大蒜AsHXK2基因序列,并对其基本特性进行生物信息学分析。通过实时荧光定量PCR(qRT-PCR)分析了AsHXK2基因在大蒜不同组织和干旱胁迫、根际促生菌等条件下的表达情况。结果表明,AsHXK2开放阅读框全长1 500 bp,可编码499个氨基酸,分子量约为57.77 ku,理论等电点为6.37;亚细胞定位预测结果显示,AsHXK2蛋白主要定位于叶绿体,含有一个特异位点,属于己糖激酶2家族。在进化关系上,大蒜AsHXK2与天门冬科的芦笋HXK2亲缘关系最为接近。qRT-PCR分析表明,AsHXK2基因表达具有明显的组织特异性,其中鳞芽中的表达量最高。此外,不同组织中AsHXK2对根际促生菌作用下的干旱胁迫表现出不同的响应模式。干旱胁迫能够显著上调大蒜根、叶和鳞芽的AsHXK2表达水平;而在干旱胁迫施加根际促生菌的处理下,仅在叶中AsHXK2表达量显著高于干旱胁迫,表明叶片中AsHXK2对根际促生菌缓解干旱胁迫的响应较为明显。本研究为进一步探究根际促生菌缓解大蒜干旱胁迫的作用机制奠定了基础。

关键词: 大蒜, 己糖激酶, 基因克隆, 表达分析

Abstract:

In order to study the alleviating role of AsHXK2 gene by plant growth promoting rhizobacteria under drought stress, TA cloning method was used to clone the sequence of AsHXK2 gene in Ledu purple garlic, and the basic characteristics were analyzed by bioinformatics. qRT-PCR was used to analyze the expression of AsHXK2 gene in different tissues of garlic, drought stress and rhizosphere growth promoting bacteria. The results showed that the open reading frame of AsHXK2 was 1 500 bp in length, encoding 499 amino acids. The molecular weight was about 57.77 ku, and the theoretical isoelectric point was 6.37. The subcellular localization prediction results showed that AsHXK2 protein was mainly located in chloroplast, containing a specific site, belonging to the hexose kinase 2 family. In terms of evolutionary relationship, garlic AsHXK2 was close to asparagus HXK2 in Asparagacea, qRT-PCR analysis showed that the expression of AsHXK2 gene had obvious tissue specificity, and the expression level in scale buds was the highest. In addition, AsHXK2 in different tissues showed different response patterns to drought stress induced by plant growth promoting rhizobacteria. Drought stress could significantly up-regulate AsHXK2 expression in roots, leaves and scale buds of garlic. However, in the treatment of rhizosphere growth-promoting bacteria and drought stress, the expression of AsHXK2 in leaves was significantly higher than that of drought stress, indicating that AsHXK2 in leaves was more obviously responsive to rhizosphere growth-promoting bacteria and drought stress. The study would provide the foundation for further exploring the mechanism of alleviating role by plant growth promoting rhizobacteria under drought stress.

Key words: garlic, hexokinase, gene cloning, expression analysis

中图分类号:

S633.4

郭春倩, 田洁. 大蒜己糖激酶基因AsHXK2的克隆及其参与根际促生菌缓解干旱胁迫的表达分析[J]. 浙江农业学报, 2022, 34(9): 1925-1934.

GUO Chunqian, TIAN Jie. Cloning of garlic hexokinase gene AsHXK2 and expression analysis of its involvement in rhizosphere growth-promoting bacteria alleviating drought stress[J]. Acta Agriculturae Zhejiangensis, 2022, 34(9): 1925-1934.

图/表 9

图1 大蒜AsHXK2基因的RT-PCR扩增 M,DNA分子量标准(2 000 bp)。

Fig.1 AsHXK2 gene amplified by RT-PCR from garlic M, DNA marker (2 000 bp).

图2 大蒜AsHXK2基因的核苷酸序列及其编码的氨基酸序列

Fig.2 Nucleotide sequences and predicted amino acid sequences of AsHXK2 gene from garlic

图3 大蒜AsHXK2蛋白跨膜结构域的预测

Fig.3 Prediction of the transmembrane domain of AsHXK2 protein

图4 大蒜AsHXK2氨基酸序列的疏水性和亲水性分析

Fig.4 Analysis of hydrophilicity and hydrophobicity of amino acid sequences of AsHXK2 from garlic

图5 AsHXK2蛋白二级、三级结构预测

Fig.5 Secondary and tertiary structure prediction of AsHXK2 protein

图6 大蒜AsHXK2氨基酸序列保守域预测

Fig.6 Prediction of conserved domain of amino acid sequences of AsHXK2 from garlic

图7 AsHXK2氨基酸序列的系统进化树

Fig.7 Phylogenetic tree of AsHXK2 amino acid sequence

图8 不同大蒜组织AsHXK2基因表达量不同处理间没有相同字母表示差异显著(P<0.05)。下同。

Fig.8 AsHXK2 gene expression in different tissues of garlic The bars with different letters showed the significant difference (P<0.05). The same as below.

图9 不同处理下各大蒜组织AsHXK2基因表达量 A,叶;B,根;C,假茎;D,鳞芽。

Fig.9 AsHXK2 gene expression in various tissues of garlic under different treatments A, Leaf; B, Root; C, Pseudostem; D, Scale bud.

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大蒜己糖激酶基因AsHXK2的克隆及其参与根际促生菌缓解干旱胁迫的表达分析

Cloning of garlic hexokinase gene AsHXK2 and expression analysis of its involvement in rhizosphere growth-promoting bacteria alleviating drought stress

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