Gene identification of a CYP450 super-gene family in Prunus persica and functional analysis of a key gene, Prupe.6G046800

Abstract:【Objective】Cytochrome P450 (CYP450), a heme-binding protein, plays an important role in the activities of plants. It is mainly involved in the detoxification pathway and biosynthesis, as well as the synthesis and metabolic reactions of alkaloids, sterols, fatty acids, hormones, pigments, among oth- ers. Recently, an increasing number of genes of the CYP450 superfamily have been discovered in crops. However, their functions in peach (Prunus persica) have rarely been reported.【Methods】In this study, the members of the CYP450 superfamily in peaches were systematically analyzed, including their cate- gories, gene structure, expression pattern, and functional analysis of Prupe.6G046800, an important member of CYP450 genes. Firstly, two methods were used to screen the protein sequences of the peach CYP450 gene superfamily, and the Arabidopsis CYP450 genes were used as exogenous genes to con-struct a phylogenetic tree. Then, the existing data were used to analyze the expression of the CYP450 genes in different tissues, gene structure and its specific expression in peach fruit development. Finally,Prupe.6G046800, one of the genes highly expressed in the early stages of fruit development, was select-ed for the analysis of heterogeneously stable transformation of the tomato and the specific expression of target gene tissue. The transient transformation test of Prupe.6G046800 in peach fruit was conducted to study the promoter activity.【Results】A total of 295 members of the CYP450 superfamily were identi-fied from the peach genome. According to the classification principles of clade and Arabidopsis, its clas-sification results were similar to that of Arabidopsis. The members of the CYP450 superfamily were di-vided into nine families, including four polygenic families and five single-gene families. No CYP727 family members were found, of which CYP71 had the highest number of members. Expression analysis of the 295 genes in different tissues of peach showed that 145 genes were expressed in at least one tis-sue. Among these, there were 50, 25, 25, 18, 16, and 11 genes specifically expressed in the root, phlo-em, leaf, flower, seed, and fruit, respectively, of which the number of genes specifically expressed in the root was the highest. The results showed that CYP450 genes were involved in the entire growth and de-velopment of peach. The results of gene structure analysis showed that the number of introns of the CYP450 gene ranged from 0 to 16, 83.4% of the CYP450 genes contained 1-4 introns, and the number of genes without introns reached 10. Among these, Prupe.6G358000 had the highest number of introns,reaching 16, followed by Prupe.3G185900 with 13 introns. In general, the length of peach genes is sub-stantial, and the number of exons is significantly different, which may be related to the functional rich-ness of the CYP450 superfamily. Analysis of CYP450 gene expression during peach fruit development showed that out of the 295 genes, 110 CYP450 genes were expressed in the fruit. Among them, the ex-pression levels of 45 members showed a continuous decline with fruit development. The expression of 36 genes increased with fruit development and reached its peak 105 d after flowering. The remaining genes of other patterns were less; for example, only three genes had lower expression in the middle stage of fruit development and higher expression in the early and mature stages. Analysis of the specific expression of Prupe.6G046800 in tomato tissue showed that the expression of this gene differed signifi-cantly in different tissues of overexpressed tomato plants. Besides, its expression in different tissues was significantly higher than that of wild-type tomato plants. The highest expression was found in the young leaves and fruits, which was lower in the mature leaves and flowers, and almost absent in the roots and stems. The Prupe.6G046800 promoter element and activity analysis showed that the cis-act-ing element contained 51 TATA-box, 34 CAAT-box, and other basic transcriptional elements, as well as elements related to the optical signal, abscisic acid response, drought induction, defense and stress, and meristem expression. This implies that Prupe.6G046800 may not only be affected by light, drought, and other environmental conditions but may be regulated by hormones, jointly determining growth and de-velopment of peach fruit as well. It was found that there were four single nucleotide polymorphism (SNPs) sites in the two promoters by cloning the promoter sequence of Prupe.6G046800 and compari-son with the reference genome‘Lovell’. Construction of the GUS reporter drive overexpression vector associated with the two promoter genotypes of Prupe.6G046800, and utilization of the transient trans-formation of peach flesh were mediated by the agrobacterium. GUS chemical staining results showed that GUS genes driven by the two promoter genotypes had expression activity, but the difference was not significant.【Conclusion】A total of 295 members of the CYP450 superfamily was identified from the peach genome, which could be divided into nine families, namely CYP71, CYP97, CYP74,CYP711, CYP51, CYP85, CYP710, CYP86, and CYP72. Transcriptome data showed that the CYP450  gene was expressed in the different tissue parts of the peach, but the most specific genes were expressed in the root, indicating that CYP450 gene was involved in the entire growth and development of peach. Overexpression of Prupe.6G046800 resulted in typical auxin accumulation symptoms such as plant dwarfing, decreased single fruit weight, and seed sterility. Promoter cis-acting element analysis showed that the gene might be regulated by light, drought, and hormone signals. The results of this study would provide experimental support for analyzing the functions of CYP450 family genes in fruit crops.