西瓜ClPP2C3克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0709

摘要/Abstract

摘要：

【目的】蛋白磷酸酶2C（protein phosphatase 2C, PP2C）是动植物中均存在的一类蛋白磷酸酶，拟探究其在西瓜果实成熟过程中发挥的重要作用。【方法】通过逆转录PCR（reverse transcription-polymerase chain reaction, RT-PCR）从栽培类型西瓜‘97103’中克隆ClPP2C3，并对其进行生物信息学、表达模式和亚细胞定位分析。【结果】西瓜ClPP2C3的cDNA序列长度为1 317 bp，编码438个氨基酸，其分子量大小为47.81 kD，蛋白质等电点为5.12。ClPP2C3包含1个PP2C保守结构域，与负调控果实成熟的番茄SlPP2C3、草莓FaABI1具有较高的同源性。西瓜ClPP2C3在细胞核表达。ClPP2C3在含糖量高的栽培品种中表达量显著高于含糖量低的野生品种。栽培品种ClPP2C3的2 kb片段长度启动子活性显著高于野生品种，而1 kb片段长度启动子活性之间无显著差异。【结论】由1-2 kb区间SNP导致的启动子活性差异对不同含糖量品种ClPP2C3表达量存在影响。

关键词: 西瓜, ClPP2C3, 基因克隆, 亚细胞定位, 启动子活性差异分析

Abstract:

【Objective】Protein phosphatase (PP2C) is a type of protein phosphatase that exists in both animals and plants, In order to explore its important role in the ripening process of watermelon fruits.【Method】In this study, a PP2C gene ClPP2C3 was cloned in watermelon (Citrullus lanatus) by reverse transcription- polymerase chain reaction (RT-PCR), and its bioinformatics, expression pattern and subcellular localization analysis were conducted. 【Result】The cDNA sequence length of ClPP2C3 in watermelon was 1 317 bp and it encoded 438 amino acids. The molecular weight of ClPP2C3 protein was 47.81 kD and its isoelectric point was 5.12. The ClPP2C3 protein contained one PP2C conserved domain and it had high homology with tomato SlPP2C3 in tomato and FaABI1 in strawberry that negatively regulated fruit ripening. ClPP2C3 in the watermelon was located in the nucleus. The gene quantitative expression analysis showed that the expression of ClPP2C3 in high-sugar-containing watermelons was significantly higher than that in low-sugar-containing watermelons. The 2 kb promoter (up 2 kb to ATG) activity of cultivated watermelon was significantly higher than that in ancestral watermelons, while there was no significant difference in the 1 kb promoter (up 1 kb to ATG) activity detected between these two species. 【Conclusion】The SNPs in 1-2 kb promoter region might result in the difference of promoter activity in different varieties.

Key words: Citrullus lanatus, ClPP2C3, gene cloning, subcellular localization, analysis of promoter activity differences

朱毅, 柳唐镜, 宫国义, 张洁, 王晋芳, 张海英. 西瓜ClPP2C3克隆及表达分析[J]. 生物技术通报, 2024, 40(1): 243-249.

ZHU Yi, LIU Tang-jing, GONG Guo-yi, ZHANG Jie, WANG Jin-fang, ZHANG Hai-ying. Cloning and Expression Analysis of ClPP2C3 in Citrullus lanatus[J]. Biotechnology Bulletin, 2024, 40(1): 243-249.

图/表 9

表1 引物序列信息

Table 1 Primer sequence information

功能Function	正向引物序列 Forward primer sequences（5'-3'）	反向引物序列Reverse primer sequences（5'-3'）
克隆基因 Cloning of genes	ATGGCGGAGATTTGCTGTA	TCAAATTAAATTCTTTTGTACTAAATT
荧光定量PCR Quantitative real-time PCR	AGTGGCTAGACGTCGGAGAA	AAATCTCCCGACCGTTTCTT
亚细胞定位 Subcellular localization	TGTGCGATGGATATTTCGCGAATGGCGGAGATTTGCTGTAAA	GATTTCTGGATCCGCAGGCCTAATTAAATTCTTTTGTACTAAATTAAAAA
启动子活性分析 Activity analysis of promotor	0.5k-GTCGACGGTATCGATAAGCTTTAGAAAAAAAACAATTATTTAAATGGGG	TTGTCTAGAACTAGTGGATCCTACTAAGTTCTTTAATCTCCCCTCCC
	1.0k-GTCGACGGTATCGATAAGCTTAGGTTTCATAGAACATTACACATTCAAA
	2.0k-GTCGACGGTATCGATAAGCTTTTTTAATTTTTATTAGCAATTAGCAATTAGA

图1 西瓜ClPP2C3的CDS序列

Fig. 1 CDS sequence of ClPP2C3 in watermelon

图2 西瓜ClPP2C3与其他植物PP2C蛋白氨基酸序列比对分析 AtAHG3：拟南芥（At3G11410）；AtHAI2：拟南芥（At1G07430）；AtHAI3：拟南芥（At2G29380）；SlPP2C3：番茄（Solyc06g076400.2.1）；方块区域代表PP2C保守结构域

Fig. 2 Alignment analysis of amino acid sequences between ClPP2C3 in watermelon and the PP2C proteins in other plants AtAHG: Arabidopsis（At3G11410）; AtHAI2: Arabidopsis（At1G07430）; AtHAI3: Arabidopsis（At2G29380）; SlPP2C3: tomato（Solyc06g076400.2.1）. The block area indicates the conserved domain of PP2C

图3 ClPP2C3蛋白与其他植物PP2C蛋白的进化树分析 SlPP2C3（Solyc06g076400.2.1）、SlPP2C1（Solyc03g096670）：番茄；AtAHG3（At3G11410）、AtHAI2（At1G07430）、AtHAI3（AtG29380）：拟南芥；FaABI1（AEP67941）：草莓

Fig. 3 Phylogenetic analysis of ClPP2C3 protein and PP2C protein in other plants SlPP2C3（Solyc06g076400.2.1）, SlPP2C1（Solyc03g096670）: tomato; AtAHG3（At3G11410）, AtHAI2（At1G07430）, AtHAI3（AtG29380）: Arabidopsis; FaABI1（AEP67941）: strawberry

图4 ClPP2C3亚细胞定位分析

Fig. 4 Subcellular localization analysis of ClPP2C3

表2 西瓜ClPP2C3在不同含糖量品种中的表达量分析

Table 2 Expressions of ClPP2C3 in watermelon varieties with different sugar contents

分类 Classification	品种名称 Variety name	含糖量（Brix） Sugar content	基因相对表达量 Relative gene expression
C.lanatus.cultivar	97103	9.4±0.648	1.19±0.098ns
C.lanatus.cultivar	WDM	11.6±1.068	1.28±0.124ns
C.lanatus.cultivar	ZZJM	10.6±0.294	1.24±0.102ns
C.lanatus.cultivar	JMT	11.0±0.355	1.39±0.149*
C.lanatus.cultivar	MG728	11.4±1.471	1.25±0.036ns
C.lanatus.cultivar	JX1M	10.9±0.455	1.36±0.136*
C.mucosospermus	PI595203	2.5±0.432	0.36±0.045****
C.mucosospermus	PI248178	2.8±0.216	0.42±0.029****
C.mucosospermus	PI254740	2.1±0.294	0.32±0.054****
C.colocvynthis	PI220778	1.1±0.216	0.12±0.036****
C.colocvynthis	PI632755	1.7±0.141	0.15±0.022****
C.colocvynthis	PI549161	1.3±0.374	0.13±0.022****
C.colocvynthis	PI296337	1.8±0.432	0.19±0.037****
C.colocvynthis	PI296341-FR	0.7±0.245	0.10±0.029****

图5 西瓜ClPP2C3在不同含糖量品种中的表达量分析 CL：栽培类型；CM：半野生类型；CC：野生类型；**P<0.01；***P<0.001；****P<0.000 1

Fig. 5 Expressions of ClPP2C3 in watermelon varieties with different sugar contents CL: Cultivar. CM: Semi-wild type. CC: Wild type. **P< 0.01; ***P< 0.001; ****P< 0.000 1

图6 栽培和野生品种中西瓜ClPP2C3的3个片段长度启动子活性差异分析 ns：无显著差异，P>0.05；**P<0.01

Fig. 6 Difference analysis of promoter activities at three fragment lengths of ClPP2C3 between cultivated and wild watermelon ns: No significant difference, P>0.05; **P<0.01

图7 不同品种西瓜ClPP2C3的转录调控模型 A：栽培品种启动子序列分析；B：祖先品种启动子序列分析。W-box、MYC、ABRE、TATA：启动子上的结合元件；WRKY：转录因子；ABA：脱落酸

Fig. 7 Transcription regulation models of ClPP2C3 in different watermelon varieties A: Promoter subsequence analysis of cultivated varieties. B: Analysis of promoter subsequence of ancestral varieties. W-box, MYC, ABRE, TATA: Binding element on promoter. WRKY: Transcription factor. ABA: Abscisic acid

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