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

doi:10.7668/hbnxb.20192783

Abstract

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

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.

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Figures/Tables 16

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

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

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

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

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

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

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

Fig.2 Amino acid sequence

Fig.3 Prediction of conserved domains

Fig.4 Sequence consistency comparison

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

Fig.6 Tertiary structure prediction

Fig.7 Prediction of transmembrane

Fig.8 Hydrophobicity analysis of protein

Fig.9 The sequencing results compared

Fig.10 Plasmid electrophoresis detection

Fig.11 Plasmid digestion detection

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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