大蒜类钙调蛋白基因<i>AsCML15</i>和<i>AsCML42</i>的分离及其在渗透胁迫中的功能分析

doi:10.7668/hbnxb.20194748

摘要/Abstract

摘要：

类钙调素蛋白(CMLs)是植物中的Ca²⁺感受器之一,参与植物的生长发育和适应环境变化的过程。为了解大蒜CML的序列特征及其对渗透胁迫的响应,从大蒜品种苍山四六瓣中克隆得到AsCML15和AsCML42基因,并对其在干旱以及盐胁迫条件下的表达模式进行了分析。结果表明,AsCML15和AsCML42基因开放阅读框长度分别为498,543 bp,编码165,180个氨基酸残基。AsCML15和AsCML42分别含有4,3个EF-hand结构域。AsCML42与拟南芥AtCML42和AtCML43在进化关系上更为接近,而AsCML15与拟南芥AtCML15、AtCML16亲缘关系较近。荧光定量PCR结果表明,AsCML15和AsCML42在鳞茎、叶片、根都有表达,且均能被200 mmol/L NaCl和15% PEG6000所诱导。AsCML15和AsCML42基因可能参与了大蒜抵御盐胁迫和干旱胁迫的过程,可进一步鉴定其生物学功能。

关键词: 大蒜, 类钙调蛋白, 基因克隆, 表达分析, 渗透胁迫

Abstract:

Calmodulin-like proteins(CMLs),one of the Ca²⁺ receptors in plants,are involved in the process of plant growth and development,as well as adaptation to environmental changes.To understand the sequence characteristics of garlic CMLs and their responses to osmotic stress,AsCML15 and AsCML42 genes from garlic variety Cangshan siliuban were cloned,and their expression patterns under drought and salt stress conditions were determined.The results showed that the open reading frame of AsCML15 and AsCML42 genes were 498 and 543 bp in length,respectively,encoding 165 and 180 amino acid residues.AsCML15 and AsCML42 harbored four and three EF-hand domains,respectively.AsCML42 was closer to Arabidopsis AtCML42 and AtCML43 in evolutionary relationship,whereas AsCML15 was more closely related to Arabidopsis AtCML15 and AtCML16.Real-time Quantitative PCR technology showed that AsCML15 and AsCML42 were expressed in bulbs,leaves,and roots,and these two genes can be induced by 200 mmol/L NaCl and 15% PEG6000.The AsCML15 and AsCML42 genes may be involved in the process of garlic resisting salt and drought stress,and their biological functions can be further identified.

Key words: Garlic, Calmodulin-like protein, Gene cloning, Expression analysis, Osmotic stress

王广龙, 许吴俊, 陈洋洋, 胡珍珠, 孙敏, 熊爱生. 大蒜类钙调蛋白基因AsCML15和AsCML42的分离及其在渗透胁迫中的功能分析[J]. 华北农学报, 2024, 39(4): 80-86. doi: 10.7668/hbnxb.20194748.

WANG Guanglong, XU Wujun, CHEN Yangyang, HU Zhenzhu, SUN Min, XIONG Aisheng. Isolation and Functional Analysis of Garlic Calmodulin Genes AsCML15 and AsCML42 under Osmotic Stress[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 80-86. doi: 10.7668/hbnxb.20194748.

http://www.hbnxb.net/CN/Y2024/V39/I4/80

图/表 8

表1 AsCML15和AsCML42克隆和荧光定量PCR引物

Tab.1 Primer sequences applied for AsCML15 and AsCML42 cloning and Quantitative Real-time PCR

用途 Function	基因名称 Gene name	上游引物(5'—3') Forward primer(5'—3')	下游引物(5'—3') Reverse primer(5'—3')
克隆	AsCML42	ATGTCTCCAGCTCCTCCATCAAAGA	CTAAGATTGAGCAACAACACTCCTC
Gene cloning	AsCML15	ATGGTTGGAAAATGCAAGCTACGGA	TCAATTAGCGACGGCATGAACGCCC
荧光定量PCR	AsCML42	GAGCACATCCTCCACCTCATTCC	CATCCACACTTCCGATCATCACCAT
Quantitative	AsCML15	GATCACCTGAACCAACTCCGAGAG	AGCTCCGCCGCCGATATGTA
Real-time PCR	AsSAND	GCGTCAACGAATGTTCCAATTACCA	TCTCTTCAGTCTCAACTTCATCAGCAT

图1 大蒜AsCML15和AsCML42基因的RT-PCR扩增图谱 M.DNA Marker;1.AsCML42基因;2.AsCML15基因。

Fig.1 RT-PCR amplification pattern of AsCML15 and AsCML42 genes from garlic M.DNA Marker;1.AsCML42 gene;2.AsCML15 gene.

图2 大蒜AsCML15和AsCML42基因的核苷酸序列及其推测的氨基酸序列

Fig.2 Nucleotide acid and predicted amino acid sequences of AsCML15 and AsCML42 genes from garlic

图3 大蒜AsCML15和AsCML42氨基酸的亲水性预测

Fig.3 Prediction of hydrophilicity of AsCML15 and AsCML42 from garlic

图4 不同物种的CML15和CML42的氨基酸序列比对 ^*.Ca²⁺结合位点。

Fig.4 Alignment of amino acids of CML15 and CML42 from different species ^*.Ca²⁺ binding sites.

图5 AsCML15和AsCML42蛋白与拟南芥CML的系统进化树分析

Fig.5 Phylogenetic tree of AsCML15 and AsCML42 from garlic and CML from Arabidopsis

图6 大蒜AsCML15和AsCML42基因在模拟干旱下的表达水平相同组织内不同小写字母表示在P<0.05水平差异显著。图7同。

Fig.6 Expression patterns of AsCML15 and AsCML42 genes in garlic under simulated drought stress Different lowercase letters within the same tissues indicate significant differences at the 0.05 level.The same as Fig.7.

图7 大蒜AsCML15和AsCML42基因在盐胁迫下的表达水平

Fig.7 Expression patterns of AsCML15 and AsCML42 genes in garlic under salt stress

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大蒜类钙调蛋白基因AsCML15和AsCML42的分离及其在渗透胁迫中的功能分析

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大蒜类钙调蛋白基因AsCML15和AsCML42的分离及其在渗透胁迫中的功能分析

Isolation and Functional Analysis of Garlic Calmodulin Genes AsCML15 and AsCML42 under Osmotic Stress

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