浙江红花油茶CchABCG5基因的功能

doi:10.3969/j.issn.1004-1524.20240746

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (7): 1407-1416.DOI: 10.3969/j.issn.1004-1524.20240746

浙江红花油茶CchABCG5基因的功能

缪百灵^a(), 陈娟娟^a, 李亮杰^a, 楚宗丽^b, 董向向^a^,^*()

信阳农林学院 a. 园艺学院; b. 农学院,河南信阳 464000

收稿日期:2024-08-19 出版日期:2025-07-25 发布日期:2025-08-20
作者简介:缪百灵(1994—),女,河南信阳人,硕士研究生,研究方向为植物遗传育种。E-mail:bailingm@163.com
通讯作者: ^*董向向,E-mail:dong_xiangx@163.com
基金资助:
河南省科技攻关项目(24210211165);河南省高等学校重点科研计划项目(24B210011);信阳农林学院青年教师科研基金项目(QN2021048);信阳农林学院青年教师科研基金项目(QN2021045);信阳农林学院园艺植物资源与利用科技创新团队项目(XNKJTD-012)

The function of CchABCG5 gene in Camellia chekiangoleosa Hu

MIAO Bailing^a(), CHEN Juanjuan^a, LI Liangjie^a, CHU Zongli^b, DONG Xiangxiang^a^,^*()

College of Horticulture; b. College of Agriculture, Xinyang Agriculture and Forestry University, Xinyang 464000, Henan, China

Received:2024-08-19 Online:2025-07-25 Published:2025-08-20

摘要/Abstract

摘要： ABCG5是参与植物生长发育的转运蛋白。为研究ABCG5基因的功能,以浙江红花油茶为材料,利用RT-PCR技术克隆浙江红花油茶CchABCG5基因的编码序列(CDS),通过生物信息学分析、亚细胞定位和拟南芥遗传转化,对该基因功能进行初步分析。结果表明,CchABCG5基因CDS长度为1 935 bp,编码644个氨基酸,有1个核苷酸结合结构域和1个跨膜结构域,是WBC型半分子转运蛋白。亚细胞定位分析显示,CchABCG5蛋白定位在细胞膜上。系统发育分析表明,CchABCG5蛋白与茶树的亲缘关系最近。CchABCG5过表达拟南芥表现出莲座叶减少和早花表型,相关开花基因表达量显著升高,种子和叶片的脂质含量升高。因此,推测CchABCG5基因在浙江红花油茶开花调控和脂质转运中发挥作用。

关键词: 浙江红花油茶, ABCG5, 转运蛋白, 基因克隆, 亚细胞定位, 遗传转化

Abstract:

ABCG5 is a transporter involved in plant growth and development. To investigate the function of the ABCG5 gene, Camellia chekiangoleosa Hu was used as experimental material. The coding sequence(CDS) of the CchABCG5 gene was obtained by RT-PCR amplification, and the gene function was preliminarily analyzed by bioinformatics analysis, subcellular localization and genetic transformation of Arabidopsis thaliana. The results showed that the CDS length of CchABCG5 gene was 1 935 bp, encoding 644 amino acids, with one nucleotide binding domain and one transmembrane domain, and it was a WBC-type half-sized transporter. Subcellular localization analysis showed that CchABCG5 protein was localized in the cell membrane, and phylogenetic analysis indicated that the CchABCG5 protein shares the closest evolutionary relationship with that of tea plant (Camellia sinensis). CchABCG5-overexpression Arabidopsis thaliana exhibited a reduced number of rosette leaves, an early flowering phenotype, and significantly elevated expression levels of flowering-related genes, as well as increased lipid content in seeds and leaves. Therefore, it was speculated that CchABCG5 gene played a role in flowering regulation and lipid transport in Camellia chekiangoleosa Hu.

Key words: Camellia chekiangoleosa Hu, ABCG5, transporter, gene cloning, subcellular localization, genetic transformation

中图分类号:

S794.4

缪百灵, 陈娟娟, 李亮杰, 楚宗丽, 董向向. 浙江红花油茶CchABCG5基因的功能[J]. 浙江农业学报, 2025, 37(7): 1407-1416.

MIAO Bailing, CHEN Juanjuan, LI Liangjie, CHU Zongli, DONG Xiangxiang. The function of CchABCG5 gene in Camellia chekiangoleosa Hu[J]. Acta Agriculturae Zhejiangensis, 2025, 37(7): 1407-1416.

图/表 10

表1 引物信息

Table 1 Primer information

基因Gene	正向引物Forward primer(5’-3’)	反向引物Reverse primer(5’-3’)	用途Usage
CchABCG5	GGTACCATGGAGACACAAGGTTGTGAGATTG	GTCGACAAGAAAAGCACCCTTAAGGCCC	基因克隆Gene cloning
CchABCG5	ACAGCAATCACTGCACCAAG	TGCTTCTACACTCACCTTCATCA	相对表达量分析
			Relative expression analysis
AtACT2	AAGCTGGGGTTTTATGAATGG	TTGTCACACACAAGTGCATCAT	相对表达量分析
			Relative expression analysis
AtCO	ATTCTGCAAACCCACTTGCT	CCTCCTTGGCATCCTTATCA	相对表达量分析
			Relative expression analysis
AtFT	CTGGAACAACCTTTGGCAAT	AGCCACTCTCCCTCTGACAA	相对表达量分析
			Relative expression analysis
AtFUL	GGAGAAGAAAACGGGTCAGC	ACTCGTTCGTAGTGGTAGGAC	相对表达量分析
			Relative expression analysis

图1 CchABCG5基因的克隆与过表达载体酶切结果 M, DL 5 000 marker;1,目的基因片段;2,pRI 101-AN-CchABCG5质粒的酶切条带。

Fig.1 Cloning of the CchABCG5 gene and restriction enzyme digestion of its overexpression vector M, DL 5 000 marker; 1, Target gene fragment; 2, Restriction enzyme digestion bands of pRI 101-AN-CchABCG5 plasmid.

图2 CchABCG5蛋白的编码序列和氨基酸序列 *,终止子。

Fig.2 Coding sequence and amino acid sequence of CchABCG5 protein *, Terminator.

图3 不同植物ABCG5蛋白的进化树

Fig.3 Phylogenetic tree of ABCG5 proteins from different plant species

图4 Cch5ABCG5蛋白的结构域与多序列比对

Fig.4 Structural domain and multiple sequence alignment of CchABCG5 protein

图5 CchABCG5基因过表达拟南芥的卡那霉素筛选(A)与PCR鉴定(B) M, DL 5 000 marker;1~3,过表达株系;-,阴性对照;+,阳性对照。

Fig.5 Kanamycin screening (A) and PCR verification (B) of CchABCG5-overexpression Arabidopsis thaliana M, DL 5 000 marker; 1-3, Overexpression lines; -, Negative control, +, Positive control.

图6 CchABCG5蛋白在拟南芥原生质体中的亚细胞定位 GFP,绿色荧光;mCherry,细胞膜marker;Bright,明场;Merged,叠加场。

Fig.6 Subcellular localization of CchABCG5 protein in Arabidopsis thaliana protoplasts GFP, Green fluorescence; mCherry, Cell membrane marker; Bright, Bright field; Merged, Superposition field.

图7 拟南芥的表型 A,拟南芥的开花表型;B,拟南芥的抽薹时间;C,拟南芥的莲座叶数量。Col-0,野生型拟南芥;0E1~OE3,3个CchABCG5基因过表达拟南芥株系。采用Duncan新复极差法比较差异显著性,无相同小写字母表示不同株系间差异显著(P<0.05)。下同。

Fig.7 Phenotype of Arabidopsis thaliana A, The flowering phenotype of Arabidopsis thaliana; B, Bolting time of Arabidopsis thaliana; C, The number of lotus leaves of Arabidopsis thaliana. Col-0, Wild-type Arabidopsis thaliana; OE1-OE3, three CchABCG5-overexpression Arabidopsis thaliana lines. Duncan’s multiple range test was used to compare the significance of differences, the bars marked without the same lowercase letters indicated significant difference between different lines (P<0.05). The same as below.

图8 拟南芥中开花相关基因的相对表达量采用t检验进行差异显著性分析,**表示P<0.01。

Fig.8 Relative expression levels of flowering-related genes in Arabidopsis thaliana t-test was used to analyze the statistical significance of differences, ** indicated P<0.01.

图9 拟南芥种子(A)和叶片(B)的脂质含量

Fig.9 The lipid content in seeds (A) and leaves (B) of Arabidopsis thaliana

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浙江红花油茶CchABCG5基因的功能

The function of CchABCG5 gene in Camellia chekiangoleosa Hu

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