Cloning and Functional Analysis of CsWAK8 Gene from Camellia sinensis during Cold Stress

doi:10.13560/j.cnki.biotech.bull.1985.2024-0542

Abstract

Abstract:

Objective The wall-associated kinase (WAK) is a unique class of receptor-like kinase (RLK) that plays an important role in regulating plant growth and responding to both biotic and abiotic stresses. Exploring the function of CsWAK8 in responding to cold stress may provide a theoretical basis for further analysis of the cold resistance mechanisms in Camellia sinensis. Method The CsWAK8 gene was cloned from the leaves of C. sinensis. The quantitative real-time PCR method was used to analyze the expression pattern of the CsWAK8 gene in different tissues and different cold-resistant tea tree varieties during the wintering period. An agrobacterium-mediated method was used to heterologously express the CsWAK8 gene in Arabidopsis thaliana. The cold-treated phenotypic observation, enzyme activity determination, and cold-response-related gene expression detection of transgenic plants were also carried out. Result The CDS of CsWAK8 gene is 2 307 bp, and encodes a protein of 768 amino acids, which contained a conserved domain unique to the WAK family. CsWAK8 was highly expressed in the mature leaves of C. sinensis, and its expression in the leaves and roots of cold-sensitive tea tree varieties was significantly higher than that in cold-tolerant ones during the wintering period. Nine transgenic lines of A. thaliana were acquired through the heterologous over-expressions of the CsWAK8 gene, and three of these lines were analyzed for their cold resistances. Under cold stress, the root lengths and survival rates of the transgenic lines were significantly lower than those of the wild type. Additionally, the degree of wilting in the rosette leaves of potted seedlings was higher in the transgenic lines compared to the wild type, and the MDA content in the transgenic variety L48 was significantly higher than that of the wild type after 6 h of freezing treatment. Furthermore, qPCR analysis revealed that the relative expressions of AtCBFs in the CsWAK8 gene transgenic A. thaliana under cold treatment were significantly lower than those in the wild type. Conclusion The transgenic A. thaliana is more sensitive to cold treatment than wild-type A. thaliana. CsWAK8 may play a negative regulatory role in response and tolerance to cold stress through the CBF-mediated cold-signaling pathway.

Key words: Camellia sinensis, wall associated kinase (WAK), gene cloning, functional verification, cold stress

JIAO Xiao-yu, WU Qiong, LIU Dan-dan, SUN Ming-hui, RUAN Xu, WANG Lei-gang, WANG Wen-jie. Cloning and Functional Analysis of CsWAK8 Gene from Camellia sinensis during Cold Stress[J]. Biotechnology Bulletin, 2025, 41(2): 210-220.

Figures/Tables 7

Table 1 The primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
CsWAK8-F	ATGGCTTTCTCGCATGGAATGCAAT	CsWAK8 CDS 扩增 CsWAK8 CDS amplification
CsWAK8-R	TCACCTCCCACCATCCATTGGCAAA	CsWAK8 CDS 扩增 CsWAK8 CDS amplification
CsWAK8-qF	AATGGAATTGGAGGGGTTGAT	CsWAK8 qPCR分析 CsWAK8 qPCR analysis
CsWAK8-qR	ACATGTTCCCTCACACTATCATATC	CsWAK8 qPCR分析 CsWAK8 qPCR analysis
β-actin-qF	GCCATCTTTGATTGGAATGG	茶树内参基因qPCR分析 qPCR analysis of reference genes in C. sinensis
β-actin-qR	GGTGCCACAACCTTGATCTT
CsWAK8-3301-F	TATGACCATGATTACGAATTCATGGCTTTCTCGCATGGAATGCAAT	pCAMBIA3301-CsWAK8载体构建 pCAMBIA3301-CsWAK8 vector construction
CsWAK8-3301-R	CAGGTCGACTCTAGAGGATCCTCACCTCCCACCATCCATTGGCAAA
3301-35S pro-R	TGTTCTCTCCAAATG AAATGAACTTCCTT	转基因拟南芥分子鉴定 Molecular identification of transgenic A. thaliana
AtCBF1-qF	GCCACGAGTTGTCCGAAGAA	AtCBF1 qPCR分析 AtCBF1 qPCR analysis
AtCBF1-qR	AAGCCGAGTCAGCGAAGTTG	AtCBF1 qPCR分析 AtCBF1 qPCR analysis
AtCBF2-qF	ATTTCGCTGACTCGGCTTGG	AtCBF2 qPCR分析 AtCBF2 qPCR analysis
AtCBF2-qR	ACGCATCTTGGCTCTGTTCC	AtCBF2 qPCR分析 AtCBF2 qPCR analysis
AtCBF3-qF	GCCGATCAGCCTGTCTCAAT	AtCBF3 qPCR分析 AtCBF3 qPCR analysis
AtCBF3-qR	GCTCTGTTCCGCCGTGTAA	AtCBF3 qPCR分析 AtCBF3 qPCR analysis
AtUBQ10-qF	AGTCCACCCTTCATCTTGTTCTC	拟南芥内参基因qPCR分析 qPCR analysis of reference genes in A. thaliana
AtUBQ10-qR	GTCAGCCAAAGTTCTTCCATCT

Fig. 1 PCR amplification product of CsWAK8 gene in C. sinensisM: DL 5 000 DNA marker; 1-2: PCR product

Fig. 2 Protein structure analysis of CsWAK8 in C. sinensisA: Domain architecture analysis of CsWAK8 proteins. B: Prediction of transmembrane structure in CsWAK8 proteins. C: Prediction of secondary structures in CsWAK8 proteins

Fig. 3 Expressions of CsWAK8 in different tissues of C. sinensis and different cold-resistant varieties （lines） of C. sinensisA: Phenotypes of different tea tree varieties under cold damage, SXPX1, SXPX2, SXPX4, SXPX5, SXPX10 and SXPX11 are different C. sinensis varieties in the Shexian experimental zone. B: Expressions of CsWAK8 in different tissues of C. sinensis. C: Expressions of CsWAK8 in the leaves of tea tree varieties （lines） with varied cold damage symptoms. D: Expressions of CsWAK8 in the roots of tea tree varieties with varied cold damage symptoms. Different lowercase letters indicate significant difference （P<0.05）. The same below

Fig. 4 GUS staining and molecular identification of wild-type and transgenic A. thalianaA: PCR detection of transgenic A. thaliana; M: DL 5000 DNA marker; +: pCAMBIA3301-CsWAK8 plasmid; WT: wild-type A. thaliana. L0-L48: pCAMBIA3301-CsWAK8 transgenic A.thaliana. B: The GUS staining of transgenic A.thaliana seedling; C: qPCR detection of transgenic A.thaliana. N.D.: Not detected

Fig. 5 Phenotypic analysis of wild-type and transgenic A. thaliana under low-temperature stressA, B: Phenotypes and root lengths of wild-type and transgenic A. thaliana seedlings under （4±1）℃ treatment. C, D: Phenotypes and survival rate of wild-type and transgenic A. thaliana seedlings after recovery from （-8±1）℃ treatment. E: Phenotypes of 30-day-old pot-grown wild-type and transgenic A. thaliana after recovery from low-temperature treatment

Fig. 6 Analysis of physiological indicators and AtCBFs gene expression of wild-type and transgenic A. thaliana under freezing stress

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