茶树CsCML24的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0797

摘要/Abstract

摘要：

类钙调素（calmodulin-like protein,CML）是植物体内一类钙受体蛋白,介导Ca²⁺与下游靶蛋白的相互作用,在植物抗逆反应中发挥重要作用。探究茶树中CML蛋白在逆境胁迫中的功能,为进一步研究茶树CsCML24对逆境胁迫的响应机理提供理论依据。以龙井43一年生茶树扦插苗为材料,克隆得到类钙调蛋白基因CsCML24（GenBank登录号为MZ325391）,并进行了生物信息学分析。同时,利用实时荧光定量PCR技术分析茶树CsCML24组织表达特异性以及不同非生物胁迫处理下的表达模式。结果显示,CsCML24的CDS长为480 bp,编码159个氨基酸。CsCML24为无信号肽及跨膜结构的稳定性亲水蛋白,含有EF-hand保守结构域,能与Ca²⁺结合。qRT-PCR表明,CsCML24在茶树各组织中均表达,在成熟叶的表达量显著高于其他组织,茎中表达量较低;该基因在低温（10℃）、干旱（20% PEG 6000）、高盐胁迫（200 mmol/L NaCl）和ABA（100 μmol/L）处理均能诱导表达,且在不同胁迫下表达差异显著,推测该基因可能通过ABA信号途径调节对低温和干旱的耐受性。

关键词: 茶树, 类钙调蛋白, 非生物胁迫, 表达分析

Abstract:

Calmodulin-like protein（CML）is a type of calcium receptor protein,which mediates the interaction of Ca²⁺ with downstream target proteins,and plays an important role in plant abiotic stress response. Exploring the function of CML in tea plant(Camellia sinensis) under adversity stress could provide theoretical references for further studying the molecular regulation mechanism of CsCML24 in stress response of tea plants. The CsCML24 gene（GenBank accession number：MZ325391）of CML was cloned from the leaves of tea cultivar ‘Longjing 43’ and analyzed by bioinformatics. Meanwhile,the relative tissue-specific expressions and expression patterns under different abiotic stresses were analyzed by real-time quantitative PCR. Results of sequence analysis indicated that the full-length CDS sequence of the CsCML24 gene was 480 bp,encoding 159 amino acids. The CsCML24 had no signal peptide and transmembrane region,and belonged to hydrophilic protein,contained EF-hands,which can be combined with Ca²⁺. Tissue-specific expression results from qRT-PCR showed that the CsCML24 gene expressed in various tissues,and highly expressed in the mature leaves,and the least in the stem. The expression of CsCML24 was significantly induced under low temperature（10℃）,drought stress（20% PEG 6000）,salt stress（200 mmol/L NaCl）and ABA,and the expression varied in different stress. It is speculated that this gene regulates the low temperature and drought tolerance of plants via ABA signaling pathway.

Key words: Camellia sinensis, calmodulin-like protein, abiotic stress, expression analysis

康芮, 刘春晖, 陈思文, 赵仁亮, 周琼琼. 茶树CsCML24的克隆及表达分析[J]. 生物技术通报, 2022, 38(3): 22-30.

KANG Rui, LIU Chun-hui, CHEN Si-wen, ZHAO Ren-liang, ZHOU Qiong-qiong. Cloning and Expression Analysis of CsCML24 Gene in Camellia sinensis[J]. Biotechnology Bulletin, 2022, 38(3): 22-30.

图/表 12

表1 引物序列

Table 1 Primer sequences

引物名称 Primer name	序列 Sequence（5'-3'）	用途 Function
CsCML24-F	CATACAACAACAATGTGTCCTACC	基因克隆 Gene cloning
CsCML24-R	AAAGGCACCGAACAATACTA	基因克隆 Gene cloning
CsCML24qRT-F	AGGAGGCGTTTCGGTTTATG	实时荧光定量 Quantitative real-time PCR
CsCML24qRT-R	CCTCATCAGTAGCATCAAAACCAG	实时荧光定量 Quantitative real-time PCR
CsPTBqRT-F	ACCAAGCACACTCCACACTATCG	茶树内参基因 Reference gene
CsPTBqRT-R	TGCCCCCTTATCATCATCCACAA	茶树内参基因 Reference gene

图1 CsCML24的克隆 A：CsCML24扩增产物电泳图;B：CsCML24核苷酸序列与推测的氨基酸序列

Fig. 1 Cloning of CsCML24 gene A：Electrophoresis of PCR product for CsCML24. B：Nucleotide sequence and deduced amino acid sequence of the CsCML24

表2 CsCML24蛋白的基本理化性质

Table 2 Basic physicochemical properties of CsCML24 protein

蛋白 Protein	分子量 Molecular weight/Da	分子式 Formula	原子总数 No. of atoms	等电点 pI	脂肪系数 Aliphatic index	不稳定系数 Instability index	总亲水性 Total hydrophilicity
CsCML24	38 248.08	C₁₃₈₆H₂₂₉₄N₄₈₀O₅₉₃S₉₆	4 849	5.23	22.08	38.05	-0.596

图2 茶树CsCML24氨基酸序列亲疏水性分析

Fig. 2 Hydrophilicity and hydrophobicity analysis of amino acids in CsCML24 from C. sinensis

图3 茶树CsCML24二级结构预测蓝色：α-螺旋;红色：β-折叠;绿色：β-转角;粉色：不规则卷曲

Fig. 3 Secondary structure prediction of CsCML24 in C. sinensis Blue：Alpha helix. Red：Extended strand. Green：β-turn. Pink：Random coil

图4 茶树CsCML24三维结构预测

Fig. 4 3-dimension structure prediction of CsCML24 in C. sinensis

图5 茶树CsCML24磷酸化位点预测

Fig. 5 Phosphorylation site prediction of CsCML24 in C. sinensis

图6 茶树CsCML24保守结构域预测

Fig. 6 Conserved domain prediction of CsCML24 in C. sinensis

图7 CsCML24氨基酸序列与其他植物的多重序列比对

Fig. 7 Multiple sequence alignment of amino acid sequences among CsCML24 and other plants

图8 CsCML24与其他物种氨基酸序列的系统进化树

Fig. 8 Phylogenetic tree analysis of deduced amino acid sequences of CML24 from C. sinensis and other plant species

图9 CsCML24在茶树不同组织部位中的表达模式不同字母表示在0.01水平上有显著差异。下同

Fig. 9 Expression patterns of CsCML24 in various tissues of C. sinensis plant Different letters indicate significant differences at the 0.01 level. The same below

图10 CsCML24在不同胁迫条件下的表达情况

Fig. 10 Expression analysis of CsCML24 gene under different stress treatments

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