Molecular Mechanism Of Juvenile Red Phenomena In Sweet Potato Leaves Based On Transcriptome

In order to resist all kinds of biological and abiotic stress,the young leaves of plants are often accompanied by color changes from red to green during the development process.This phenomenon is of great significance to the adaptive evolution of plants.The juvenile red phenomena in leaves is mainly related to the accumulation pattern of anthocyanins,but the molecular regulation mechanism is not clear.In this study,sweet potato[Ipomoea batatas(L.)Lam.]was used as a material to reveal the transcription factor regulatory network of juvenile red phenomenon based on SSR molecular markers,transcriptome,bioinformatics analysis and qPCR,and provide a preliminarily molecular basis for further analysis of plant juvenile red phenomenon.In order to select suitable sweet potato varieties as test materials,on the basis of phenotype,sweet potato germplasms were collected from Hainan and the surrounding areas,and 64 sweet potato varieties with fine growth situation were selected.Thirty pairs of SSR primers were used to conduct PCR amplification on DNA of the 64 sweet potato materials.UPGMA clustering and genetic distance analysis were carried out to elucidate their phylogenetic relationship.Using 30 pairs of SSR primers,a total of 161 clear bands were amplified,including 135polymorphic bands.The percentage of polymorphic bands reached 83.9%.Each pair of primers amplified 5.37 alleles on average.The polymorphic information content(PIC)of the primers ranged from 0 to 0.75,with an average PIC of 0.44.UPGMA clustering showed that the genetic similarity coefficient of these sweet potato germplasms was 0.74-0.98,with an average of 0.78.At the coefficient of 0.75,these materials could be divided into three categories:Ⅰ,Ⅱ and Ⅲ.Among them,95.31%materials belonged to classⅠ,and the overall genetic similarity coefficient was high.Most of the unknown germplasms collected from Hainan were far from those collected from Jiangsu.The materials from the same area scattered in different branches,displayed weak regionalism.The genetic distance among the tested 64 materials was 0.02-0.37,with the average value as 0.22,showing close genetic distance.The sweet potato germplasms in Hainan have close phylogenetic relationships and narrow genetic background.This phenomenon might be the results of natural selection of the local climate,such as the high temperature,high humidity and high light conditions in Hainan.High-quality and distantly related germplasms of sweet potato may be introduced to promote germplasm innovation,and enrich the genetic resources in Hainan.Chuanshanzi has a typical juvenile red phenomenon,which was selected as the representative variety in this study,and Xuzicai 8 and Ningzishu 1 were selected as the reference varieties for transcriptome sequencing.In this study,50554 Unigenes were obtained by sequencing and assembly,with an average length of 93 lnt.The assembly quality is good and can be used for subsequent analysis.Based on the annotation analysis of Nr,Swiss-Prot,KOG and KEGG databases,a number of candidate structural genes related to flavonoid metabolism were extracted.Protein sequences annotated by four databases were compared with TF database,and a total of 1,446 transcription factors were obtained,distributed in 56 families.The analysis of gene differential expression between the adjacent leaves of Chuanshanzi showed that the number of down-regulated genes was more than up-regulated genes.Differential expression gene enrichment analysis showed that most of the differential genes were involved in various biological processes such as metabolism,ribosome,splicing,hormone signal transduction and plant-pathogen interaction.Based on the transcription factors obtained above,phylogenetic trees of 62 R2R3-MYBs,112 bHLHs and 2 WDRs were constructed and their functions were preliminarily analyzed.The results showed that some MYBs and bHLHs were involved in metabolic regulation,development and stress tolerance.Among them,Unigene0023598(MYB-A598),Unigene0031237(MYB-A237)and Unigene0031238(MYB-A238)may be involved in transcriptional activation regulation of anthocyanin structural genes.Unigene0001224(MYB-R224),Unigene0021216(MYB-R216),Unigene0003763(MYB-R763),Unigene0009722(MYB-R722),Unigene0001816(R3 MYB-816)and Unigene0018082(R3 MYB-082)are candidate genes of the inhibitory MYB transcription factor.Unigene0041364(bHLH2-364)and Unigene0025722(WDR1-722)may form MBW complex with MYBs to regulate anthocyanin metabolism.The juvenile red phenomenon of Chuanshanzi is caused by the alternate accumulation of anthocyanins and chlorophyll.The expression pattern and correlation analysis indicated that the molecular mechanism behind juvenile red phenomenon may be the feedback regulatory network,which formed by MYB,bHLH and WDR transcription factors.The expression patterns of anthocyanin-related structural genes,bHLH2-364,activated MYB and inhibited MYB except MYB-R763 were similar,mainly expressed in green leaves.This suggested that MYB activators may have a positive regulatory effect on structural genes,bHLH2-364 and partially MYB repressors.The expression pattern of MYB-R763 was unique,mainly expressed in green leaves,which was negatively correlated with the expression pattern of active MYB and structural genes.From the above results,it can be inferred that there were two kinds of feedback regulation modes in Chuanshanzi:First,the MBW complex composed of activated MYBs,bHLH and WDR activated the expression of inhibitory MYBs,thus feedback interfered with the activation regulation and prevented the excessive accumulation of anthocyanins.Second,under the guidance of development signals,the expression patterns of activated MYBs and inhibited MYBs in the leaf development process were in contrast to each other,and the expression of anthocyanin structural genes was actively inhibited in the later stage of leaf development.Based on the above two regulatory models,the molecular mechanism behind the juvenile red phenomenon in the plant is explained to some extent.