蓖麻类钙调蛋白基因<i>RcCML42</i>克隆与表达载体构建

doi:10.7668/hbnxb.20192783

摘要/Abstract

摘要：

为了探究类钙调蛋白基因家族成员在蓖麻体内的作用及表达模式,在蓖麻体内挖掘家族基因CML42,并对其进行克隆与表达分析。以哲蓖三号植株为试验材料,克隆获得RcCML42完整编码区序列(CDS),利用生物信息学方法对RcCML42基因序列进行分析,包括编码氨基酸、蛋白分子量及等电点、跨膜结构域、氨基酸序列一致性等内容。通过荧光定量PCR(qRT-PCR)技术,检测RcCML42基因在蓖麻植株根、茎、叶位置不同时间点的特异性表达情况。结果显示,RcCML42 CDS长度为597 bp,编码198个氨基酸,含有3个EF-hand钙结合结构域。蛋白分子量为22.27 ku,等电点为4.55,不存在跨膜结构域。氨基酸序列一致性分析表明,RcCML42与巴西橡胶树一致性最高,达86.84%。qRT-PCR结果显示,RcCML42基因在蓖麻根、茎、叶组织中均有表达,在经NaCl处理后,RcCML42在根中的表达量呈现先下降后上升的趋势。RcCML42在茎中12 h高度表达,在叶中为8 h高度表达。且在茎中的表达量显著高于其在根、叶中的表达。CML是植物体内最重要的一类钙感受器,通过与钙调素结合蛋白结合进而对植物细胞进行调控。当植物受到环境中NaCl侵害时,CML可以参加体内钙离子的调节,进而减轻盐胁迫对植物体的损害。综上,推测RcCML42在盐胁迫条件下的蓖麻信号转导中起重要作用。

关键词: 蓖麻, 类钙调蛋白, 耐盐, 钙离子, 生物信息学

Abstract:

In order to explore the role and expression patterns of members of the Calmodulin-like proteins gene family in castor, the family gene CML42 was extracted from castor, and its cloning and expression were analyzed. Zhebi No.3 plant was used as the material to clone and obtain the complete coding region sequence (CDS) of RcCML42, bioinformatics method was used to analyze RcCML42 gene sequence, included amino acid coding, protein molecular weight and isoelectric, transmembrane domain, amino acid sequence consistency and so on. Using qRT-PCR to analyze the changes of expression of castor RcCML42 gene in root, stem and leaf tissues at different time points.The results showed that RcCML42 CDS was 597 bp in length, encoded 198 amino acids, contained three EF-hand calcium-binding domains, the protein molecular weight was 22.27 ku, the isoelectric point was 4.55, and there was no transmembrane domain. Analysis of amino acid sequence identity showed that RcCML42 had the highest identity with Hevea brasiliensis, reaching 86.84%.The results of qRT-PCR analysis showed that the expression of RcCML42 in the roots after NaCl treatment showed a trend of first decreasing and then rising. The expression level of RcCML42 was highly expressed at 12 h in the stem and at 8 h in the leaf. And expression in stems was significantly higher than its expression in roots and leaves CML is one of the most important calcium receptors in plants. It regulates plant cells by binding with calmodulin binding protein. When plants are affected by NaCl in the environment, CML can participate in the regulation of calcium ions in the body, thus reducing the damage to plants caused by salt stress.In summary, RcCML42 plays an important role in the signal transduction of castor under salt stress.

Key words: Castor, Calmodulin-like protein, Salt tolerance, Calcium ion, Bioinformatics

孙德慧, 王晓宇, 王莹, 霍红雁, 刘海臣, 徐惠, 张继星. 蓖麻类钙调蛋白基因RcCML42克隆与表达载体构建[J]. 华北农学报, 2023, 38(1): 94-101. doi: 10.7668/hbnxb.20192783.

SUN Dehui, WANG Xiaoyu, WANG Ying, HUO Hongyan, LIU Haichen, XU Hui, ZHANG Jixing. Cloning and Expression Vector Construction of Castor Calmodulin-like Proteins Gene RcCML42[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 94-101. doi: 10.7668/hbnxb.20192783.

http://www.hbnxb.net/CN/Y2023/V38/I1/94

图/表 16

表1 引物序列

Tab.1 Sequences of primers

引物名称 Primer	上游引物(5'—3') Forward primer(5'—3')	下游引物(5'—3') Reverse primer(5'—3')
RcCML42	TCCCCCGGGGGAATGGAAGTAGAACCAGCAGCA	GCTCTAGAGCTCAAGAGCTGCGAACAAGAAC
qRcCML42	GTTCAAGACCTCAGCCAAGC	AGCCTCCATCTCCTCATCG
qRcSKIP	CATTGCTGAGGTCAGTGCAT	GACGGCCATGCTCTTCTAAC
qRcADP	GAGAGATGCTGTGCTGCTTG	CTCCTGCTCCATTTCCAT

表2 PCR反应体系及程序

Tab.2 PCR reaction system and procedure

PCR反应体系 PCR reaction system		PCR反应程序 PCR reaction procedure
试剂 Reagent	用量/μL Consumption	温度/℃ Temperature	时间 Time
cDNA 2×Es Taq Master Mix ddH₂O RcCML42-F RcCML42-R	2.0 12.5 8.5 1.0 1.0	94 94 55 72 72	10 min $30 s 30 s 30 s$ 35循环 5 min

表2 PCR反应体系及程序

Tab.2 PCR reaction system and procedure

PCR反应体系 PCR reaction system		PCR反应程序 PCR reaction procedure
试剂 Reagent	用量/μL Consumption	温度/℃ Temperature	时间 Time
cDNA 2×Es Taq Master Mix ddH₂O RcCML42-F RcCML42-R	2.0 12.5 8.5 1.0 1.0	94 94 55 72 72	10 min $30 s 30 s 30 s$ 35循环 5 min

表3 酶切体系及连接体系

Tab.3 Enzyme digestion system and linking system

试剂 Reagent	用量/μL Consumption	试剂 Reagent	用量/μL Consumption
重组质粒	10	质粒酶切条带	7.5
Recombinant		Plasmid enzyme
plasmid		cut bands
SmaⅠ	1	PCHF-3300酶切条带	7.5
XbaⅠ	1	Enzyme cut band
10×T Buffer	2	of PCHF-3300
0.1% BSA	2	10×T₄ Ligase Buffer	1.0
		T₄ Ligase	1.0

表4 qRT-PCR反应体系及程序

Tab.4 qRT-PCR reaction system and procedure

qRT-PCR反应体系 qRT-PCR reaction system		qRT-PCR反应程序 qRT-PCR reaction procedure
试剂 Reagent	用量/μL Consumption	温度/℃ Temperature		时间 Time
cDNA TB Green Ⅱ ddH₂O qRcCML42-F qRcCML42-R qRcSKIP-F qRcSKIP-R qRcADP-F qRcADP-R	1.00 6.25 4.25 0.50 0.50 0.50 0.50 0.50 0.50	95 95 55 72	30 s $5 s 30 s 30 s$ 40循环

表4 qRT-PCR反应体系及程序

Tab.4 qRT-PCR reaction system and procedure

qRT-PCR反应体系 qRT-PCR reaction system		qRT-PCR反应程序 qRT-PCR reaction procedure
试剂 Reagent	用量/μL Consumption	温度/℃ Temperature		时间 Time
cDNA TB Green Ⅱ ddH₂O qRcCML42-F qRcCML42-R qRcSKIP-F qRcSKIP-R qRcADP-F qRcADP-R	1.00 6.25 4.25 0.50 0.50 0.50 0.50 0.50 0.50	95 95 55 72	30 s $5 s 30 s 30 s$ 40循环

图1 PCR扩增产物 M.DL2000 DNA Marker;1—3.PCR结果。

Fig.1 PCR products M.DL2000 DNA Marker;1—3.PCR results.

图2 氨基酸序列

Fig.2 Amino acid sequence

图3 蛋白保守区预测

Fig.3 Prediction of conserved domains

图4 序列一致性对比

Fig.4 Sequence consistency comparison

图5 二级结构预测 h.α-螺旋;e.延伸链;t.β-转角;c.无规则卷曲。

Fig.5 Secondary structure prediction h.Alpha helix;e.Extended strand;t.Beta turn;c.Random coil.

图6 三级结构预测

Fig.6 Tertiary structure prediction

图7 跨膜结构预测

Fig.7 Prediction of transmembrane

图8 蛋白的疏水性分析

Fig.8 Hydrophobicity analysis of protein

图9 测序结果比对

Fig.9 The sequencing results compared

图10 质粒电泳检测

Fig.10 Plasmid electrophoresis detection

图11 质粒酶切检测

Fig.11 Plasmid digestion detection

图12 RcCML42在蓖麻根(A)、茎(B)、叶(C)中的特异性表达不同小写字母表示差异显著(P<0.05)。

Fig.12 Specific expression of RcCML42 in castor root(A),stem(B)and leaf(C) Different small letters indicate significant differences(P<0.05).

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蓖麻类钙调蛋白基因RcCML42克隆与表达载体构建

Cloning and Expression Vector Construction of Castor Calmodulin-like Proteins Gene RcCML42

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蓖麻类钙调蛋白基因RcCML42克隆与表达载体构建

Cloning and Expression Vector Construction of Castor Calmodulin-like Proteins Gene RcCML42

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