Cloning and Expression Analysis of CsCML24 Gene in Camellia sinensis

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

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

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

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

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

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

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

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

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