甜瓜细胞色素P450家族基因的鉴定及其响应光辐射强度的表达分析

doi:10.16420/j.issn.0513-353x.2024-0990

摘要/Abstract

摘要：

为探究甜瓜细胞色素P450单加氧酶（cytochrome P450 monooxygenase，CYP450）基因家族的功能，利用生物信息方法分析甜瓜基因组数据，鉴定出195个全长CYP450基因，归属于10个家族簇（clan），不均匀分布于甜瓜的12条染色体上。MEME分析鉴定甜瓜CYP450家族包含15个保守基序。理化性质分析表明绝大多数甜瓜CYP450蛋白为疏水性蛋白，主要定位于叶绿体/质体膜系统。该家族基因启动子区存在多种顺式作用元件，包括核心启动子元件（如TATA-box、CAAT-box）、转录因子结合位点（如MYB、MYC），光响应元件（如GT1-motif、Box 4）和激素响应元件（如ABRE、CGTCA-motif）。通过对不同强度光辐射处理下甜瓜黄绿叶突变体Cmygl-1（MT）和野生型（WT）植株叶片的RNA-seq分析，共鉴定出69个差异表达的CmCYP450基因；其中有16个在叶片中表达水平较高，且其表达受不同强度光辐射差异调控，提示这些基因可能与光辐射诱导的突变体叶色表型相关。

关键词: 甜瓜, P450基因家族, 黄绿叶, 突变体, 光辐射强度, 差异表达分析

Abstract:

To investigate the functions of cytochrome P450 monooxygenase（CYP450）gene family in Cucumis melo L.，the genomic data was analyzed using bioinformatics methods to identify the CYP450 family members in C. melo. The results revealed that a total of 195 full-length CYP450 genes were identified，belonging to 10 clan family clusters，and they were unevenly distributed on 12 chromosomes，contained 15 conserved motifs：The results of physical and chemical property analysis showed that the vast majority of CmCYP450 proteins were hydrophobic proteins，mainly localized in chloroplasts and plastid membranes：A large number of eukaryotic RNA polymerase Ⅱ binding sites，transcription factor binding sites，as well as photoresponsive and hormone responsive elements are present in the promoter regions of family genes. There were 69 CmCYP450 genes differentially expressed between yellow green leaf mutant Cmygl-1 and wild-type plants in response to different light intensities by RNA-seq. Among them，16 CmCYP450 genes are relatively highly expressed in melon leaves and show differential expression trends induced by different light radiation intensities，which may be involved in the regulation of leaf color variation of mutants.

Key words: Cucunis melo, CYP450 gene family, yellow-green leaf, mutant, light intensity, expression analysis

韩宏伟, 常亚南, 刘会芳, 庄红梅, 邢嘉怡, 张晓达, 祖丽皮耶·阿卜杜米力科, 王浩, 王强. 甜瓜细胞色素P450家族基因的鉴定及其响应光辐射强度的表达分析[J]. 园艺学报, 2025, 52(12): 3271-3287.

HAN Hongwei, CHANG Yanan, LIU Huifang, ZHUANG Hongmei, XING Jiayi, ZHANG Xiaoda, ZULIPIYE · Abudumilike, WANG Hao, WANG Qiang. Identification of Cytochrome P450 Family Genes in Melon and Expression Analysis in Response to Light Intensity[J]. Acta Horticulturae Sinica, 2025, 52(12): 3271-3287.

图/表 13

表1 不同处理的透光率及光辐射强度

Table 1 Transmittance and light radiation intensity of different treatments

处理 Treatment	方法 Method	平均透光率/% Average transmittance	光辐射强度（× 10⁴lx） Light radiation intensity
强光（SW） Strong light	自然光（无遮光） Natural light（without shading）	100.0	6.08
亚强光（SN） Sub-strong light	距离植株生长点上方0.30 m覆盖一层棚膜 A layer of greenhouse film was covered 0.30 m above the plant growth point	78.2	4.75
遮荫（ZY） Shading	在SN处理的基础上再覆盖一层遮阳网 Based on the SN treatment，an additional layer of shading net was covered	28.7	1.74

表2 qRT-PCR引物序列

Table 2 Pirmer sequences used for qRT-PCR analysis

引物名称Primer name	上游引物（5′-3′）Forward primer	下游引物（5′-3′）Reverse primer
MELO3C021434.2	GGTTCAGGCTTACAAGGCTAAT	CCACTCTCAAGGCATTCTACG
MELO3C009884.2	GGTGCCACTATTGTGTCTCTTC	GCCTATTCGGTTAGCAACTCTT
MELO3C002338.2	AAGCTCTTCCAATGCACCAAG	TGATGCCACAACACCAACAC
MELO3C018724.2	AGCATGGCAGGATTAGGAATTG	TTGTGGCGTTGGAGGAGTT
MELO3C011682.2	CATCTCGGCGTGGAAGGTAT	CGTGGCTGTCGTTCAAGTG
MELO3C011976.2	CGGCGGAGTATTGTCAGATG	CAACCAATCCAAGAACAACACA
MELO3C007154.2	TGAGCCACCATCCTACCTAAC	CACTTCCAACTCACGGTTCTT
MELO3C004214.2	GGAGAAGTGCCAGAGAACCT	TTTCACCCAGTTGCCCAATC
MELO3C022113.2	CGAGGCTGTCTGGTTCAAGA	CAATGACGGCGACGATGAG
MELO3C027057.2	GCAGACGCCAACGCCATTA	GCACGATCCAATTACGACGATT
MELO3C004135.2	CGGCAGCAACATTGAAACAA	TCTCACAAGCAGCAACAACA
MELO3C004201.2	ACACGACTTGGACTTGGAGAG	CCACAATGCTCAACAGAGACAA
MELO3C002187.2	CAATAGTCAGTGGCAACAACCT	GCGTAGCAGTTTCCGAATCAT
MELO3C006499.2	GGACAACGACTAATCGCCTAC	ATGATGGAATCGCTTGCTCTC
MELO3C026749.2	GGATGTGGTAGACTCTGTAGCA	CTTGACTGTCCTCCTGAGATTC
MELO3C026019.2	TTCCATTAACCAGTGCTGCTC	GCTCTTGACGAAGTGCTTGTA
MELO3C016167.2	TGTTGCCGTCCGATCCTTAT	CTTCCTCTTGCTCCTCTCCTT
MELO3C035538.2（CmCYP195）	TTGTCCAACTTGTTAGCGATGT	CTTGAATCCCGTGAAGATGGTA
MELO3C004801.2（CmCYP24）	GTCCAATCCTTCGCCTTCATT	GCCATTCGACAGATCACATCAT
MELO3C026260.2（CmCYP174）	TGCTTCCTCTTCTTCTTCTTCC	TTGGTGGAGATGGAGGTGAA
MELO3C011869.2（CmCYP79）	CACCGCTTCTCTGTCAACCT	TGAGCAATGGCAGCAAGATTC
MELO3C021842.2（CmCYP144）	ATCACTTCCAACCAACACAACA	CTGTAGGACCTGACACGAGAA
MELO3C015058.2（CmCYP101）	CCAGCACCACTGAGATATTGAG	TGAGAACATGAGAAGCAAGGAT
MELO3C013868.2（CmCYP95）	GCTTCGACGGCTGATAACAAT	GCAACTCCAATGGCTTCTCC

图1 甜瓜（Cm）、拟南芥（At）与水稻（Os）CYP450的系统发育树红色：CmCYP450；蓝色：AtCYP450；粉色：OsCYP450

Fig. 1 Phylogenetic tree of CmCYP450s，AtCYP450s and OsCYP450s Red：CmCYP450s；Blue：AtCYP450s；Pink：OsCYP450s

图2 甜瓜CmCYP450家族成员的基因结构

Fig. 2 The Gene structure of CmCYP450 gene family members in Cucumis melo

图3 甜瓜CmCYP450家族成员的保守基序示意图

Fig. 3 The conserved motifs of CmCYP450 gene family members in Cucumis melo

图4 甜瓜CmCYP450家族启动子的顺式作用元件

Fig. 4 Analysis of cis-acting elements of the CmCYP450 family promoter in Cucumis melo

图5 甜瓜CmCYP450基因的染色体定位

Fig. 5 Chromosomal localization of CmCYP450 in Cucumis melo

图6 CmCYP450家族基因的复制与共线性分析基因组中P450基因对的共线性关系用彩色线表示。不同的颜色代表不同的染色体

Fig. 6 Gene replication and collinearity analysis of CmCYP450 The collinearity relationships of gene pairs are represented by colorful lines. Different chromosomes are represented by different colors

图7 甜瓜（Cm）、拟南芥（At）与水稻（Os）CYP450的共线性分析灰色线为不同基因组之间的共线关系，彩色线为CYP450家族基因之间的共线关系

Fig. 7 Synteny analysis of P450 genes between the genomes of Cucumis melo，Arabidopsis thaliana and Oryza sativa The gray lines are all collinear relationships among different genomes，and the colored lines are collinear relationships among P450 gene family genes

图8 甜瓜P450蛋白互作网络示意图

Fig. 8 Protein interactions network diagram of P450 genes in Cucumis melo

图9 不同光辐射强度处理差异表达CmCYP450热图（A）、突变体与野生型植株的表型（B）、RNA-seq和qRT-PCR相对表达量的相关性分析（C） B图左侧植株为突变体Cmygl-1，右侧植株为野生型植株

Fig. 9 The heat map of differential expression of CmCYP450 under different light radiation intensities（A），Phenotypes of mutants and wild-type plants（B），Correlation analysis of relative expression levels between RNA seq and qRT PCR（C） The plant on the left in Figure B is the mutant Cmygl-1，and the plant on the right is the wild type

图10 部分CmCYP450基因的qRT-PCR相对表达量（fold change）与转录组测序结果（FPKM）的表达趋势分析 SWWT：强光处理下野生型植株；SNWT：亚强光处理下野生型植株；ZYWT：遮阴处理下野生型植株；SWMT：强光处理下突变体植株；SNMT：亚强光处理下突变体植株；ZYMT：遮阴处理下突变体植株

Fig. 10 Analysis of expression trends of some CmCYP450 genes using qRT-PCR（fold change）and RNA-seq（FPKM） SWWT：The wild type plants with strong light treatment；SNWT：The wild type plants with sub intense light treatment；ZYWT：The wild type plants with shade treatment；SNMT：The mutants with strong light treatment；SNMT：The mutants with sub intense light treatment；ZYMT：The mutants with shade treatment

图11 黄绿叶突变体中16个叶片高表达CmCYP450基因响应不同光辐射强度的表达趋势 FPKM：基因表达量。不同小写字母表示0.05水平差异显著

Fig. 11 The expression trends of 16 CmCYP450 genes highly expressed in mutant leaves in response to different light radiation intensities FPKM：Fragments Per Kilobase of transcript per Million mapped reads. Different lowercase letter indicate significant differences at 0.05 level

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