茶树CsWAK8克隆及其在响应冷胁迫过程中的功能分析

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

摘要/Abstract

摘要：

目的细胞壁关联蛋白激酶（wall associated kinase，WAK）是一类特殊的类受体激酶（receptor like kinase， RLK），其在调节植物生长和应对生物或非生物胁迫等方面发挥着重要作用。探究CsWAK8在响应冷胁迫过程中的功能，为今后解析茶树抗寒机理提供理论依据。方法从茶树叶片中克隆了CsWAK8。采用实时荧光定量PCR（qPCR）分析CsWAK8在不同组织以及越冬期不同抗寒性茶树品系中的表达模式。通过农杆菌介导法在拟南芥中异源表达CsWAK8，并对转基因植株进行冷处理表型观察、酶活测定和冷响应相关基因表达检测。结果 CsWAK8的CDS全长2 307 bp，编码768个氨基酸，具有WAK家族特征保守结构域。CsWAK8在茶树成熟叶片中高表达，在越冬期，冷敏感型茶树品系叶片和根中CsWAK8表达量多显著高于冷耐受型茶树品系。通过异源过表达获得了9株转CsWAK8拟南芥纯合株系，对其中3个株系进行了耐冷性分析，发现在冷胁迫下，转基因株系的根长和存活率显著低于野生型，盆栽苗莲座叶的枯死程度较野生型更高，且转基因株系L48在冷冻处理6 h时的MDA含量显著高于野生型。此外，qPCR分析结果显示，在冷胁迫下，转CsWAK8拟南芥中AtCBFs的相对表达量大多显著低于野生型。结论转CsWAK8拟南芥相较于野生型拟南芥对冷处理更敏感。CsWAK8可能通过CBF介导的冷信号途径在响应和耐受冷胁迫过程中起负调节作用。

关键词: 茶树, 细胞壁关联蛋白激酶（WAK）, 基因克隆, 功能验证, 冷胁迫

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

焦小雨, 吴琼, 刘丹丹, 孙明慧, 阮旭, 王雷刚, 王文杰. 茶树CsWAK8克隆及其在响应冷胁迫过程中的功能分析[J]. 生物技术通报, 2025, 41(2): 210-220.

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.

图/表 7

表1 本实验用到的引物

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

图1 茶树CsWAK8的PCR扩增产物M: DL 5 000 DNA marker; 1-2: PCR产物

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

图2 茶树CsWAK8的蛋白结构分析A：CsWAK8蛋白结构域架构分析；B：CsWAK8蛋白的跨膜结构预测；C：CsWAK8蛋白的二级结构预测

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

图3 茶树不同组织及不同抗寒性茶树品种（系）中CsWAK8的表达分析A：不同茶树品系受冷害表型，SXPX1、SXPX2、SXPX4、SXPX5、SXPX10和SXPX11分别为歙县试验园区中不同茶树品系；B：CsWAK8在茶树不同组织中的表达；C：CsWAK8在受冷害症状轻重不同的茶树品种（系）叶中的表达情况；D：CsWAK8在受冷害症状轻重不同的茶树品种（系）根中的表达情况。不同小写字母表示差异显著（P<0.05）；下同

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

图4 野生型和转基因拟南芥GUS染色和分子鉴定A：转基因拟南芥PCR检测电泳图，M：DL 5000 DNA marker，+：pCAMBIA3301-CsWAK8质粒，WT：野生型拟南芥，L0-L48：转pCAMBIA3301-CsWAK8拟南芥；B：转基因拟南芥幼苗GUS染色；C：转基因拟南芥qPCR检测，N.D.：未检出

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

图5 低温胁迫下野生型和转基因拟南芥表型分析A、B：野生型和转基因拟南芥幼苗在（4±1）℃处理下的表型和根长；C、D：野生型和转基因拟南芥幼苗（-8±1）℃处理恢复后的表型和存活率；E：盆栽30 d的野生型和转基因拟南芥在低温处理恢复后的表型

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

图6 冷冻胁迫下野生型和转基因拟南芥生理指标和AtCBFs基因表达分析

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

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