Studies On The Molecular Mechanism Of Peel Color Difference Formation Based On RNA-Seq In The Wild Banana(Musa Itinerans)

Musa plant is an important type of ornamental plants in tropical and south subtropical regions.Wild banana Musa itinerans,widely distributed in the southern China,is a kind of flower or fruit ornamentals in Musa genus and widely used in landscaping.Wild banana resources are very rich in Fujian province,some of which have the specific ornamental traits of purple-red fruit,provide a good gene resource for the ornamental traits improvement of Musa plants.However,the mechanism of the color change in the wild banana peel remains unclear.The research on the molecular mechanism of the color change formation in the wild banana peel will lay a foundation for the color improvement of Musa plants.In this study,comparative analysis of the pigment content in different color peels were conducted,which revealed that the color differences of wild banana peel were caused by anthocyanins.The anthocyandin species in the wild banana peels were identified by HPLC.Then,RNA-Seq was performed on the green part?GP?and purple part?PP?in the wild banana peel using the high-throughput sequencing technology.Differentially expressed genes related to anthocyanin biosynthesis were screened by the comprehensive analysis of expression differences in the transcriptome level and miRNA regulation level.Some structural genes and MYB transcription factor regulation genes involved in anthocyanin biosynthesis were isolated by TA cloning,and the expression patterns of differentially expressed genes were verified by qPCR.This study is expected to explain the mechanism of color difference formation in the wild banana peel from the molecular level and to provide a theoretical basis for the regulation of anthocyanin biosynthesis in the wild banana peel and color improvement in Musa plants,etc.The main results are as follows:1.Analysis and identification of pigment in the wild banana peel The content of pigment metabolites in different color parts of wild banana peel were determined using a variety of colorimetric method such as pH differential method.The results showed that the content of anthocyanin,proanthocyanidin and flavonoid in the peel was significantly higher than the content of chlorophyll and carotenoid.The content of anthocyanin,proanthocyanidin and flavonoid in purple peel was significantly higher than those in green peel,and the difference of anthocyanin and proanthocyanidins between the two color peel was very significant.In contrast,the starch and sucrose content in the purple peel was significantly lower than those in the green peel,and the difference of starch was very significant.Pigment determination results confirm that the color difference of wild banana peel is caused by the different content of flavonoid such as anthocyanins in different color parts.The anthocyanins of delphinidin,cyanidin,malvidin,petunidin and peonidin were identified from purple peel of wild banana by HPLC.And the content of delphinidin was the highest and up to 85.057 mg/100g,which was the main reason for the accumulation of purple pigments.2.Analysis on the changes of pigment and related enzyme activities in the wild banana peel under different developmental stagesThe content of anthocyanins,proanthocyanidins,flavonoids,chlorophyll,carotenoids,and the enzyme activity changes of CHS,CHI,F3H,DFR,ANS,UFGT,and GTase in different development stages of banana peel were analyzed by enzyme-linked immunosorbent assay?ELISA?.The results showed that the content of anthocyanin,proanthocyanidin,and flavonoid were highest in the peel of low development degree fruit,followed by the mature fruit peel,and the content was lowest in the peel of moderately developed fruit.The content of chlorophyll and carotenoid gradually decreased with development.The content of anthocyanin in the wild banana peel had a significant positive correlation with the content of proanthocyanidin in different developmental stages.The anthocyanin content did not show significant correlation with the activities of CHS,CHI,F3H,DFR,UFGT and GSTs.But,there was a significant positive correlation between flavonoid content and CHS activity,proanthocyanidin content and CHI,ANS activity.The above results showed that the phenylpropane metabolic pathway in the tested wild banana peel of different developmental levels was mainly shunted toward the proanthocyanidin and flavonoid biosynthesis branch.3.Transcriptomics analysis of different color peel in the wild bananaThe lncRNA libraries were structured from green part and purple part of wild banana peel in the same fruit comb,and deep sequencing of which were performed using the Illumina HiSeqTM2500 platform with a output of 53,982,293 and 58,948,129 clean reads respectively.Using HISAT2 software,sequence comparison between clean reads and reference genome of Musa acuminata and Musa itinerans were carried out.The result showed that the alignment ratios of clean reads with the reference genome of M.itinerans were over 79.45%.According to the M.itinerans genome,a total of 110,348 transcripts with an average length of 3382bp were obtained by using the assembly software StringTie,which construct a more comprehensive and high quality wild banana peel transcriptome database.Compared with the genome annotation information of M.itinerans,a total of 6,732 new genes were excavated from the assembled transcripts.5,101 new genes,accounting for 75.77%of the total new genes,were annotated by blasting with COG,GO,KEGG,KOG,Pfam,Swiss-Prot,eggNOG,and NCBI NR databases.98.61%of the annotated genes were annotated by blasting with NR database,and 89.13%of which were homologous with the genes of M.acuminata,suggesting that wild banana had a more close genetic relationship with A genotype banana.KEGG pathway analysis suggested that the new genes involved in secondary metabolism in the wild banana peel transcriptome were enriched in the phenylpropanoid biosynthesis pathway.4,134 differentially expressed genes were screened out by transcriptome analysis of different color peel on the same fruit comb.Among them,1,558 genes were up-regulated in purple peel,2,576 genes were up-regulated in green peel.And 3,908 differentially expressed genes,accounting for 94.53%,were annotated by public database.1,780 and 1,320 differentially expressed genes were annotated by GO and KEGG databases respectively.The GO function annotation of these differentially expressed genes are mainly enriched in the function categories related to anthocyanin biosynthesis and fruit development,including naringenin-chalcone synthase activity,positive regulation of flavonoid biosynthesis and fruit ripening,etc.The KEGG pathway is mainly enriched in metabolic pathways such as flavonoid biosynthesis,phenylpropanoid biosynthesis,and phenylalanine metabolism.Both of these GO categories and KEGG pathways were closely related to the biosynthesis of plant anthocyanins.Using the annotation information of transcriptomic database,75 structural genes,112 anthocyanin modified genes and translocated genes involved in the anthocyanin synthesis of wild banana peel were screened and identified.All of these genes involved all of the major genes in anthocyanin biosynthesis as well as modifying genes of glycosylation,acylation,and methylation,and three kinds of pigment transport-mediated protein including glutathione transferases?GSTs?,multidrug resistance-associated protein?MRP?and multidrug and toxic compound extrusion protein?MATE?.Among them,69 genes were differentially expressed in different color peel of wild banana.At the same time,1,546 genes encoding for transcription factors were searched from the transcriptome database of wild banana peel.In total,286,179 and 14 genes were annotated as MYB,bHLH and WD40,respectively.Among the differentially expressed transcription factor genes,the bHLH was the most significantly up-regulated transcription factor family,while the most significantly down-regulated transcription factor family was MYB.By homologous aligning with Arabidopsis thaliana database,twenty transcription factor genes,including twelve MYBs,five bHLHs,two WD40s and one WRKY genes,were filtrated because of their potential function in anthocyanin biosynthesis of wild banana peel.The protein interaction network prediction of differentially expressed genes related to anthocyanin biosynthesis indicated that the transcription factor MYB?Mi_g026681?was responsible for regulating the upstream structural gene C4H of the anthocyanin biosynthesis pathway,while another MYB?Mi_g021391?interacted with bHLH?Mi_g014100?to regulate transcription of the downstream structural genes like F3H,DFR,LDOX,and ANR.On this basis,the possible transcription regulatory network of anthocyanin biosynthesis pathway in the wild banana peel had been constructed.Through transcriptomic annotation and domain analysis,29 R2R3-MYBs of differentially expressed genes in the wild banana peel were screened,and the conserved sequences of these genes were analyzed.The domain motif of them was identified as[W]-x?19?-[W]-x?19?-[W]-x?12?-[E]-x?15?-[W]-x?18?-[W]-x?3?-[L].Eight MYB genes,Mi_g026681,Mi_g030747,NewGene₂1582,Mi_g021391,NewGene₁0237,Mi_g011516,Mi_g028070,and Mi_g019957,may participate in phenylpropanoid metabolic pathway such as anthocyanin biosynthesis,were screened by cluster analysis with known function R2R3-MYB gene of Arabidopsis thaliana.Among them,two MYB genes,Mi_g026681 and Mi_g030747,may play an inhibitory role in the gene expression regulation.Mi_g011516,Mi_g028070,Mi_g019957 and NewGene₁0237 may be involved in the flavonoids biosynthesis of wild banana peel.Mi_g021391 may be related to anthocyanin biosynthesis in the wild banana peel.This result was consistent with the prediction result of mRNA protein interaction.4.lncRNA analysis of different color peel in the wild banana3,938 lncRNAs expressed in the wild banana peel were screened and identified,including intergenic lncRNAs?65.9%?,antisense lncRNAs?14.5%?,sense lncRNAs?10.4%?,and the intronic lncRNAs?9.1%?,which provided data resources for the screening of functional lncRNAs in future.Among them,there are 2 lncRNAs aligned to known lncRNAs.The comparative analysis of lncRNA and mRNA showed that the sequence length,the ORF length,the expression level and the exon number of lncRNA were lower than that of mRNA.409 differentially expressed lncRNAs were screened by using green peel library of wild banana as a control,and the expression level of lncRNAs in purple peel was significantly higher than that of green peel.Through the functional enrichment analysis of their target genes,we found that differentially expressed lncRNAs may be involved in biological processes such as protein synthesis,flavonoid biosynthesis and DNA-binding transcription factor activity.Protein interaction network analysis results of differentially expressed IncRNA target genes in the wild banana peel showed that the MYB?Mi_g026681?protein had interactions with the proteins encoded by C4H?Mi_g014079?and 4CL?Mi_g008868?,and another MYB gene?Mi_g003034?interacted with WD40?Mi_g029091?together to regulate the transcription of ANR?Mi_g016749?.All of these indicated that the differential expression of lncRNAs corresponding to these target genes may indirectly regulate wild banana peel anthocyanin biosynthesis by regulating these target genes.5.miRNA analysis of different color peel in the wild banana254 miRNAs,including 132 known miRNAs and 122 new miRNAs were identified to express in the wild banana peels from two small RNA libraries of purple peel?PP?and green peel?GP?by high-throughput sequencing.They were classified into 58 known miRNA families.Of these,244 miRNAs with 3,975 target genes has been predicted and annotated into 40 GO categories and 101 KEGG metabolic pathways.36 miRNAs,differentially expressed in the different color peel,were obtained by differential expression levels analysis of miRNA.Among them,ten miRNAs were up-regulated in purple peel and twenty-six were up-regulated in green peel.GO function and KEGG pathway enrichment analysis results of differentially expressed miRNA target genes showed that the differentially expressed miRNA target genes were enriched in DNA binding,anion transmembrane transporter activity,hyperosmotic response,DNA metabolic process,protein kinase activity,organic anion transport,cell differentiation,and protein phosphorylation categories,and plant-pathogen interactions pathway.Through the correlation analysis between differentially expressed miRNAs and their target genes,the miRNA regulated the MYB transcription factor genes of anthocyanin synthesis are identified mainly from the family of miR159,miR535,miR858 and miR482.Interaction networks prediction of miRNA-mRNA-lncRNA associated with anthocyanins in the wild banana peel further confirmed the interaction role of MYB?Mi_g021391?and bHLH?Mi_g014100,Mi_g009620?in the transcription regulation of anthocyanin biosynthesis downstream genes.And two miRNAs targeted MYB?Mi_g021391?and three lncRNAs targeted the two miRNAs were screened out.6.Cloning and expression analysis of genes related to anthocyanidin biosynthesis in the wild banana peelSpecific primers were designed based on the sequences of differentially expressed genes related to anthocyanin biosynthesis in the wild banana peel transcriptome for TA cloning.A total of 14 cDNA fragments with typical domains and complete ORF sequence were cloned from the total RNA reverse transcription library of different color peel,which including one each of C4H,CHS,CCR,and FLS,two each of CHI and F3H,three each of DFR and MYB.In addition,seven part sequences of CHI,F3H,TT12,and four MYB genes have gained.Sequence analysis showed that as a transporter,TT12 has ten transmembrane structures.Domain analysis showed that two out of seven MYBs belong to R2R3-MYB,one was 3R-MYB and four were 1R-MYB/MYB-like.The clustering results showed that three TT2 homologs?MiTT2-1,MiTT2-2,and MiTT2-3?are clustered in the same branch with AtMYB123/TT2,belong to the 5th subfamily which involved in the regulation of A.thaliana phenylpropanoid metabolic processes,most likely involved in anthocyanin synthesis in the wild banana peel.Relative expression of 9 structural genes and 8 transcription factor genes in anthocyanin biosynthesis pathway of the wild banana peel were analyzed by qPCR.The results showed the same expression trend with the RNA-Seq,which verified the reliability of the sequencing results and expression changes trend of some important genes.In the same way,the expression levels of six miRNAs and their target genes were analyzed,and the expression pattern of 3 couples was also consistent with the result of RNA-Seq.In this paper,through pigment analysis and HPLC identification,it was clarified that the color difference in the wild banana peel was caused by the accumulation of anthocyanins such as delphinidin.On this basis,the anthocyanin biosynthesis structural genes,transcription factors and the lncRNAs and miRNAs that may be targeted to these key genes were screened by the analysis using RNA-Seq?mRNA,lncRNA and miRNA?in different color peel.And the structural genes associated with anthocyanin biosynthesis,the key transcription factor MiTT2?Mi_g021391?were cloned from the wild banana peel.The expression pattern of these genes also been clarified and the possible regulatory network of anthocyanins biosynthesis was constructed in the wild bananas,which provides rich data resources and lays a good foundation for the study of pigment metabolism in the wild bananas and even other Musa plants.