苦蘵P450家族基因鉴定与表达分析

doi:10.3969/j.issn.1004-1524.2021.11.02

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (11): 2009-2016.DOI: 10.3969/j.issn.1004-1524.2021.11.02

苦蘵P450家族基因鉴定与表达分析

蒋智芳¹^,²(), 韩怡蝶¹, 楼盼盼¹, 郭宏¹, 冯尚国¹^,², 沈晨佳¹^,², 王慧中¹^,²^,^*()

1.杭州师范大学生命与环境科学学院,浙江杭州 311121
2.浙江省药用植物种质改良与质量控制技术重点实验室,浙江杭州 311121

收稿日期:2020-12-22 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 王慧中
作者简介:*王慧中,E-mail: whz62@163.com
蒋智芳(1991—),女,江西上饶人,硕士,研究实习员,主要从事药用植物次生代谢方面的研究。E-mail: jiangzf1991@163.com
基金资助:
国家自然科学基金(31970346);浙江省自然科学基金(LY20H280012);浙江省自然科学基金(LY19C160001)

Identification and expression analysis of cytochrome P450 family genes from Physalis angulata L.

JIANG Zhifang¹^,²(), HAN Yidie¹, LOU Panpan¹, GUO Hong¹, FENG Shangguo¹^,², SHEN Chenjia¹^,², WANG Huizhong1¹^,²^,^*()

1. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
2. Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou 311121, China

Received:2020-12-22 Online:2021-11-25 Published:2021-11-26
Contact: WANG Huizhong1

摘要/Abstract

摘要：

细胞色素P450酶(cytochrome P450)广泛参与植物次生代谢,是当前的研究热点。基于苦蘵转录组数据,分析并鉴定了部分苦蘵P450家族基因。共鉴定得到795个可能为P450家族基因的序列片段,并将它们聚类到25个不同的聚类(cluster)中,其中,有12个聚类具有组织特异性表达。最终拼接得到30个苦蘵P450家族基因的全长序列,它们与其他物种P450基因高度同源。进化分析表明,这30个苦蘵P450基因可归为9个P450亚家族。通过定量PCR技术,分析了多个P450基因在果实不同成熟阶段的表达变化。结果表明,9个P450基因随着果实成熟表达量上升,另外9个P450基因表达量则逐渐降低。以果实为材料,分析了P450家族基因在乙烯(ACC)与乙烯抑制剂(1-甲基环丙烯,1-MCP)处理下的表达变化。研究表明,部分P450家族基因在ACC和1-MCP处理下表现出相反的表达模式,这说明P450可能参与乙烯介导的果实成熟过程。克隆了3个果实特异表达的P450家族基因(comp164210、comp131532、comp152181),它们主要定位于细胞质和细胞膜中。本研究为苦蘵药用性状遗传改良提供了有价值的遗传信息。

关键词: 细胞色素P450酶, 苦蘵, 转录组, 乙烯

Abstract:

Cytochrome P450 enzymes are widely involved in plant secondary metabolism. Based on the transcriptome data of Physalis angulata, several P450 family members had been identified. 795 possible sequence fragments of P450 gene had been identified, and they were clustered into 25 different clusters, including 12 clustering with tissue-specific expression. By bioinformatics splicing, full-length sequence of 30 Physalis angulata P450 gene family, which was highly homologous with other P450 species genes, were obtained. Phylogenetic analysis showed that 30 P450 genes could be classified into nine P450 subfamilies. By qRT-PCR method, expression of 30 P450 genes under different developmental stages of fruits was analyzed. The expression of nine P450 genes increased with fruit maturation, while expression of other nine P450 genes decreased gradually. The expression of P450 family genes under ethylene (ACC) and ethylene inhibitor (1-methylcyclopropene, 1-MCP) treatments were also analyzed. Part of the P450 family genes showed the opposite expression pattern under ACC and 1-MCP treatments, suggesting P450 family genes might be involved in ethylene-mediated fruit ripening process. Further, three fruit-specific expressed P450 family genes (comp164210, comp131532, comp152181) had been cloned, and they were mainly located in the cytoplasm and cell membrane. This study provided valuable genetic information for the genetic improvement of medicinal characters of P. angulata.

Key words: cytochrome P450 enzyme, Physalis angulate, transcriptome, ethylene

中图分类号:

S567.21

蒋智芳, 韩怡蝶, 楼盼盼, 郭宏, 冯尚国, 沈晨佳, 王慧中. 苦蘵P450家族基因鉴定与表达分析[J]. 浙江农业学报, 2021, 33(11): 2009-2016.

JIANG Zhifang, HAN Yidie, LOU Panpan, GUO Hong, FENG Shangguo, SHEN Chenjia, WANG Huizhong1. Identification and expression analysis of cytochrome P450 family genes from Physalis angulata L.[J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2009-2016.

图/表 7

表1 苦蘵P450基因扩增引物

Table 1 Primer sequence for P450 gene of Physalis angulata L.

P450基因 P450 gene	上游引物(5'→3') Forward primer sequence(5'→3')	下游引物(5'→3') Reverse primer sequence(5'→3')
comp164210	GGTACCATGGTAGATATTCATGGAT	GTCGACAGTGGATGATATTGTTGAT
comp131532	GGTACCATGGATTTCTCTCTTGCC	GTCGACTATATCGTTGTACAAATCTG
comp152181	GGTACCATGGTGCTTTCTCTTTTTTC	GTCGACACGGTGGCTTATTGGTTTC

图1 苦蘵P450家族基因表达谱分析绿色表示低表达,红色代表高表达。

Fig.1 Expression spectrum analysis of P450 genes in Physalis angulate Green meant low expression, red meant high expression.

图2 P450家族基因同源进化分析

Fig.2 Homologous and phylogenetic analysis of P450 genes

图3 P450家族基因在苦蘵果实不同成熟阶段的表达水平

Fig.3 Expression level of P450 genes at different ripe stages of fruit

图4 P450家族基因在ACC和1-MCP处理下的表达差异分析

Fig.4 Expression analysis of the P450 family genes under ACC and 1-MCP treatments

图5 三个果实特异表达P450基因的克隆

Fig.5 Clone of three fruit-specific expression P450 genes

图6 苦蘵3个P450蛋白的亚细胞定位情况

Fig.6 Subcellular localization of three P450 protein

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苦蘵P450家族基因鉴定与表达分析

Identification and expression analysis of cytochrome P450 family genes from Physalis angulata L.

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