基于加权关联网络分析的辣椒(Capsicum annuum L.)果实颜色转变相关基因的鉴定

Weighted Correlation Network Analysis Reveals Candidate Genes Related to Fruit Color Transition in Pepper(Capsicum annuum L.)

辣椒属于茄科(Solanaceae)辣椒属(Capsicum L.)一年生或多年生植物,其播种面积和产值在中国均居蔬菜首位。果色是辣椒众多经济性状中最直观的性状之一,直接影响人们的购买选择。辣椒果色受多种因素调控,相关色素生物合成的分子调控还需进一步解析。本研究以果实发育过程中呈现5种颜色的辣椒种质HNCA0076为实验材料,进行了生理检测、转录组测序以及WGCNA分析。在果实5个不同颜色时期,叶绿素含量变化较大,类胡萝卜素含量整体呈现递增趋势,类黄酮含量无明显变化,花色苷含量在紫色果期后逐渐降低,橙色果期后又逐渐升高。RNA测序数据质量满足进一步分析要求。4个转色期的上调(下调)表达基因数分别为826(276)、390(588)、1248(1800)和1528(2485),随着果实的逐步发育,相邻比较组合中的差异基因的数量逐渐增加。差异表达基因经GO富集分析,主要集中在分子功能中的四吡咯结合、血红素结合、转移酶活性、氧化还原酶活性、铁离子结合和水解酶活性等过程;KEGG富集分析主要富集在类胡萝卜素生物合成、类黄酮生物合成和淀粉、蔗糖代谢等信号通路,其中类胡萝卜素代谢途径的3个基因在紫色时期向黄色时期的转变过程中表达上调,类黄酮代谢途径的11个基因在黄色时期向红色时期的转变过程中表达下调。4个对比组合差异基因分别注释到8877、8736、8689、8573个转录因子,共同注释到8049个转录因子,包括AP2、F-box、MYB和UDPGT等类型。利用非冗余的差异表达基因进行了加权关联网络分析,关联到4个与叶绿素、类胡萝卜素、花色苷、类黄酮含量相关的模块,进一步分析表明p450(LOC107859314)、Pkinase(LOC107839192、LOC107875415)、F-box(LOC107863894)和zf-MYND(LOC107852593)等29个转录因子可能参与辣椒果实转色的调控。本研究为进一步探索辣椒果实色素形成过程中的关键调控因子以及分析相关色素生物合成的调控提供了重要数据。

Pepper belongs to the annual or perennial pepper genus(Capsicum L.) in the Solanaceae. Amid all vegetables grown in China, pepper is ranked first as per its sowing area and output value. Fruit color is one of the most intuitive economic traits of pepper, which directly affects people’s purchase choice. Nevertheless, the regulation of pepper fruit color is affected by many factors, especially the key regulatory factors in the process of fruit pigment formation and the regulation of related pigment biosynthesis are not clear. To understand the mechanism of pepper fruit color formation, physiological testing, transcriptome sequencing and weighted gene co-expression network analysis(WGCNA) was performed using Capsicum germplasm HNCA0076 as the experimental material, presenting five colors during fruit development. During the five different color periods of the fruit, the chlorophyll content was significantly different, the carotenoids content showed an increasing trend, the flavonoid content was relatively stable, and the anthocyanin content decreased gradually after purple fruit stage, and increased after orange fruit stage. The quality of the RNA sequencing data met the requirements for further analysis. The number of differentially expressed genes(DEGs) up-regulated and down-regulated in each period was 826(276), 390(588), 1248(1800) and 1528(2485). As the fruit matures, the number of differential genes in adjacent comparison combinations gradually increased. DEGs were analyzed by gene ontology(GO) enrichment with a main focus on molecular functional such as tetrapyrrole binding, heme binding, transferase activity, oxidoreductase activity, iron ion binding and hydrolase activity. After KEGG enrichment analysis, DEGs were found mainly enriched in carotenoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism. Among these, the expression of the three genes from the carotenoid metabolic pathway were up-regulated during the transition from purple to yellow, and the expression of 11 genes from the flavonoid metabolic pathway were down-regulated during the transition from yellow to red. The differentially expressed genes of four comparative combinations were annotated to 8877, 8736, 8689, and 8573 transcription factors(TFs), and together to 8049 TFs, including AP2, F-box, MYB, and UDPGT. We performed a WGCNA using non-redundant DEGs associated with four modules including chlorophyll, carotenoid, coloring, and flavonoid content. Further analysis indicated that 29 TFs such as p450(LOC107859314), Pkinase(LOC107839192, LOC107875415), F-box(LOC107863894), and zf-MYND(LOC107852593) may be involved in the regulation of pepper fruit color conversion. This study would provide an important data for further exploring the key regulatory factors in the process of pepper fruit coloring and analyzing the regulation of related pigment biosynthesis.