西瓜响应非生物胁迫关键基因<i>CDPK</i>克隆、表达分析及表达载体构建

doi:10.7668/hbnxb.20195255

摘要/Abstract

摘要：

为探讨西瓜钙依赖蛋白激酶(CDPK)在嫁接以及非生物胁迫环境下的功能,利用RT-PCR技术从西瓜嫁接苗中克隆了ClCDPK(Cla97C01G019720)基因,对其进行了生物信息学分析。进一步根据ClCDPK序列设计带有Kpn Ⅰ和Sal Ⅰ酶切位点的特异引物,进行扩增,双酶切后与pCAMBIA1300连接,成功构建目标基因的表达载体pCAMBIA1300-35S-ClCDPK。利用 RT-qPCR 技术,测定ClCDPK在自根苗(ZG)与嫁接苗(JJ)分别遭受盐、干旱胁迫后的基因表达量。结果表明,ClCDPK基因ORF为1 647 bp,编码548个氨基酸。其蛋白存在STKc_CAMK和FRQ1功能结构域,为亲水性蛋白,亚细胞定位预测该蛋白位于细胞核,对ClCDPK与其他6种植物的CDPK进行进化树分析发现,其与葫芦科甜瓜、南瓜的CDPK亲缘关系较近,蛋白序列同源比对超过92.64%,具有较高的同源性。RT-qPCR表达结果显示,ClCDPK在嫁接苗表达量显著高于自根苗,随着胁迫时间的持续,嫁接苗和自根苗的ClCDPK表达量先升后降,并且同一胁迫处理下,嫁接苗的ClCDPK表达量高于自根苗。试验表明,ClCDPK正响应盐和干旱胁迫,并且嫁接苗抵御胁迫的能力高于自根苗。推测ClCDPK是西瓜响应嫁接的关键因子之一,从而提高了西瓜嫁接苗的抗盐抗旱能力。

关键词: 西瓜, ClCDPK, 非生物胁迫, 载体构建, 表达分析

Abstract:

In order to investigate the function of watermelon calcium-dependent protein kinase (CDPK) in grafted seedlings and abiotic stress environments, this study used RT-PCR technology to clone the ClCDPK(Cla97C01G019720) gene from watermelon grafted seedlings and performed bioinformatics analysis on it. Further designed specific primers with Kpn Ⅰ and Sal Ⅰ enzyme cleavage sites based on the ClCDPK sequence,conducted amplification and double enzyme cleavage, and connected with pCAMBIA1300 to successfully construct the expression vector pCAMBIA1300-35S-ClCDPK for the target gene.Using RT-qPCR technology, the gene expression levels of ClCDPK were measured in self rooted seedlings (ZG) and grafted seedlings (JJ) after being subjected to salt and drought stress, respectively.The results showed that the ORF of ClCDPK gene was 1 647 bp, encoding 548 amino acids. Its protein contained STKc_CAMK and FRQ1 functional domains, and was a hydrophilic protein. Subcellular localization prediction showed that the protein was located in the nucleus. Evolutionary tree analysis of ClCDPK with CDPK from six other plants revealed that it was closely related to CDPK from Cucurbitaceae melons and pumpkins, with protein sequence homology alignment exceeding 92.64%, indicating high homology.The RT-qPCR expression results showed that the expression level of ClCDPK in grafted seedlings was significantly higher than that in self rooted seedlings. With the duration of stress, the expression levels of ClCDPK in grafted and self rooted seedlings first increased and then decreased, and under the same stress treatment, the expression level of ClCDPK in grafted seedlings was higher than that in self rooted seedlings.This study indicated that ClCDPK responded positively to salt and drought stress, and the ability of grafted seedlings to resist stress was higher than that of self rooted seedlings. It is speculated that ClCDPK is one of the key factors in watermelon's response to grafting, thereby improving the salt and drought resistance of watermelon grafted seedlings.

Key words: Watermelon, ClCDPK, Abiotic stress, Vector construction, Expression analysis

宋嘉欣, 李明轩, 李爱, 粟柴静, 张卫华, 蔡泽宇, 吴颖. 西瓜响应非生物胁迫关键基因CDPK克隆、表达分析及表达载体构建[J]. 华北农学报, 2024, 39(6): 40-46. doi: 10.7668/hbnxb.20195255.

SONG Jiaxin, LI Mingxuan, LI Ai, SU Chaijing, ZHANG Weihua, CAI Zeyu, WU Ying. Cloning,Expression Analysis and Expression Vector Construction of CDPK,a Key Gene in Response to Abiotic Stress in Watermelon[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 40-46. doi: 10.7668/hbnxb.20195255.

http://www.hbnxb.net/CN/Y2024/V39/I6/40

图/表 10

表1 RT-qPCR 内参与特异性基因引物

Tab.1 Reference and gene-specific primers for RT-qPCR

基因名称 Gene name	引物序列(5'—3') Primer sequence (5'—3')
ClCDPK	F: TTCAAGTCAGCCAAAGAG
	R: GACATAGTATGCACTACCG
ACTIN	F: CCATGTATGTTGCCATCCAG
	R: GGATAGCATGGGGTAGAGCA

图1 ClCDPK基因的ORF扩增 M.DL2000 DNA Marker;1.ClCDPK的片段;2.阴性对照(H₂O)。

Fig.1 ORF amplification of ClCDPK M.DL2000 DNA Marker;1.Fragment of ClCDPK;2.Negative control(H₂O).

图2 ClCDPK 蛋白的保守结构域分析

Fig.2 Conserved domain analysis of ClCDPK protein

图3 ClCDPK蛋白二级(A)、三级(B)结构模型

Fig.3 The secondary(A)and tertiary(B)structure model of ClCDPK protein

图4 ClCDPK蛋白的互作网络预测

Fig.4 The sinteraction network prediction of ClCDPK protein

图5 西瓜ClCDPK与其他植物CDPK同源蛋白的多重序列比对

Fig.5 Comparison of multiple sequences between ClCDPK and other plant CDPK homologous proteins

图6 ClCDPK 与其他植物的CDPK进化树分析

Fig.6 The phylogenetic tree analysis between other plants CDPK and ClCDPK

图7 ClCDPK基因的双酶切验证 M.DL10000 plus DNA Marker;1—2.使用Kpn Ⅰ和Sal Ⅰ对重组质粒进行双酶切验证。

Fig.7 Recombinant plasmid double digestion verification of ClCDPK M.DL10000 plus DNA Marker;1—2.Double digestion validation of recombinant plasmids using Kpn Ⅰ and Sal Ⅰ.

图8 ClCDPK在盐(A)和干旱(B)胁迫不同时间的表达分析不同小写字母表示同一时间不同处理下差异显著(P<0.05)。

Fig.8 Expression analysis of ClCDPK gene under salt(A)and drought(B)stress at different times Different lowercase letters indicate significant differences under different treatments at the same time (P<0.05).

图9 ClCDPK基因在盐(A)和干旱(B)胁迫不同处理中的表达分析不同的小写字母表示同一处理不同时间下的差异显著(P<0.05)。

Fig.9 Expression analysis of ClCDPK gene in different treatments of salt(A)and drought(B)stress Different lowercase letters indicate significant differences in the same treatment at different times(P<0.05).

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西瓜响应非生物胁迫关键基因CDPK克隆、表达分析及表达载体构建

Cloning,Expression Analysis and Expression Vector Construction of CDPK,a Key Gene in Response to Abiotic Stress in Watermelon

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西瓜响应非生物胁迫关键基因CDPK克隆、表达分析及表达载体构建

Cloning,Expression Analysis and Expression Vector Construction of CDPK,a Key Gene in Response to Abiotic Stress in Watermelon

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