【作者】 邓素芳；

【导师】 赖钟雄； 王天池； 陈敏；

【作者基本信息】 福建农林大学 ， 花卉与景观园艺， 2018， 博士

【摘要】 芭蕉属植物是热带南亚热带重要的观赏植物。华南地区广泛分布的野生蕉——阿宽蕉（Musa itinerans）是一类观叶、观果的芭蕉属植物,在园林绿化上得到广泛应用。福建野生蕉资源非常丰富,部分野生蕉具有紫红色的果实,具有优异的观赏性状,为芭蕉属植物观赏性状改良提供了优异的基因资源。但是,野生蕉果皮颜色变化的机制还不清楚。开展野生蕉果皮颜色变化的分子机制研究,将为芭蕉属植物的颜色改良奠定基础。本研究通过对不同颜色果皮的色素含量比较分析,在明确野生蕉果皮颜色差异是由花青素引起的基础上,利用HPLC法鉴定野生蕉果皮中的花青素种类。然后利用新一代高通量测序技术,对野生蕉果皮色素差异较大的绿色果皮（GP）和紫色果皮（PP）部分进行mRNA、miRNA和1ncRNA的深度测序,通过转录组水平和miRNA调控水平的综合分析,筛选出与花青素合成相关的差异基因,并利用TA克隆分离出花青素合成相关的结构基因和转录调控MYB基因,通过qPCR验证差异基因的表达模式,以期在分子水平上解释野生蕉果皮颜色差异形成的机制,为野生蕉果皮花青素合成调控和芭蕉属植物颜色改良提供理论依据。主要研究结果如下:1.野生蕉果皮色素的分析与鉴定利用pH示差法等多种比色方法测定同一果梳野生蕉不同颜色果皮的色素代谢物含量。结果表明,果皮中的花青素、原花青素和类黄酮含量显著高于叶绿素和类胡萝卜素的含量;紫色果皮中的花青素、原花青素和类黄酮含量显著高于绿色果皮中的花青素、原花青素和类黄酮含量,且花青素和原花青素在两种颜色果皮中的差异达极显著;相反地,紫色果皮中的淀粉和蔗糖含量显著低于绿色果皮中的淀粉和蔗糖含量,且淀粉含量差异达极显著。色素测定结果证实,野生蕉果皮的颜色差异是由果皮的花青素等类黄酮物质含量的显著性差异引起的。试验通过HPLC分析在野生蕉紫色果皮中检出飞燕草色素、矢车菊色素、锦葵色素、矮牵牛素和芍药色素等5种花青素类型,而且含量最高的是飞燕草色素,达85.057 mg/100g,它是紫色色素积累的主要原因。2.不同发育程度野生蕉果皮色素及相关酶活性的变化分析利用酶联免疫法分析了不同发育程度野生蕉果皮花青素、原花青素、类黄酮、叶绿素和类胡萝卜素等色素成分以及花青素合成过程中的 CHS、CHI、F3H、DFR、ANS、UFGT 和 GTase 等酶活性的变化情况,结果表明供试的野生蕉果皮中花青素、原花青素和类黄酮含量在低发育程度的果皮中最高,其次是发育较成熟的果皮,在中度发育的果皮中含量最低;叶绿素和类胡萝卜素的含量随着发育程度的提高,呈逐渐减少的趋势,不同发育程度的野生蕉果皮花青素的含量与原花青素的含量呈显著正相关,叶绿素与花青素、原花青素的含量成负相关。检测的CHS、CHI、F3H、DFR、UFGT和GSTs等酶活性与花青素的含量未表现出显著相关性,但CHS活性却与类黄酮的含量显著正相关,CHI和ANS活性与原花青素的含量显著正相关。说明供试的不同发育程度的野生蕉果皮中苯丙烷代谢主要往原花青素和类黄酮合成分支分流。3.野生蕉果皮不同颜色部位的转录组学分析利用Illumina HiSeqTM2500平台对野生蕉同一果梳上绿色果皮和紫色果皮1ncRNA文库进行转录组深度测序,分别得到53,982,293和58,948,129 条 Clean reads,采用 HISAT2 软件将 Clean reads 与参考基因组Musa acuminata和Musa itinerans分别进行序列比对,与M.itinerans基因组的比对率高达79.45%以上。根据M.itinerans基因组,利用组装软件StringTie对Clean reads进行转录本拼接,共获得110348条平均长度为3382bp的转录本,从而构建了较为全面的、质量较高的野生蕉果皮转录组数据库。将拼接的转录本与原有的基因组注释信息进行比较,发掘出6,732个新基因,共有5,101（75.77%）个新基因注释到COG、GO、KEGG、KOG、Pfam、Swiss-Prot、eggNOG、NCBI NR 等 8 个数据库,有 98.61%的新基因获得NR数据库功能注释,并有89.13%的基因注释到小果野蕉（M.acuminata）的基因组上,提示供试野生蕉与A基因型的小果野蕉具有更近的亲缘关系。KEGG通路分析提示野生蕉果皮转录组中涉及次生代谢的基因在苯丙烷生物合成途径中富集。经过同一果梳上不同颜色果皮转录组基因表达量的比较分析,筛选出4,134个差异表达基因,其中有1,558个基因在紫色果皮中上调表达,2,576个基因在绿色果皮中上调表达,有94.53%的差异表达基因（3,908个）获得公共数据库的注释。分别有1,780个和1,320个差异表达基因注释到GO和KEGG数据库,这些差异表达基因的GO功能主要富集在包括柚苷配基-查尔酮合成酶活性、黄酮类生物合成过程的积极调控和果实成熟等在内的与花青素合成、果实发育相关的功能条目,而KEGG通路则主要富集在类黄酮生物合成、苯丙素生物合成和苯丙氨酸代谢等代谢通路中,这些GO条目和KEGG途径都与植物花青素的合成密切相关。利用转录组数据库的注释信息,筛选和鉴定出野生蕉果皮花青素合成的相关结构基因75个,花青素修饰和转运基因112个,囊括了参与花青素苷合成的所有催化酶以及糖基化、酰基化、甲基化酶基因,和3种色素转运介导蛋白——谷胱甘肽转移酶（GSTs）、多药耐药抗性相关蛋白（MRP）和有毒化合物排出蛋白（MATE）基因,其中69个基因在不同颜色野生蕉果皮中差异表达。同时,从野生蕉果皮转录组中搜索到1546个编码转录因子的基因,其中有286、179和14个基因分别注释为MYB、bHLH和WD40,而差异表达的转录因子基因中bHLH家族上调最显著,下调表达最显著的是MYB。通过与拟南芥数据库同源比对,筛选出可能参与野生蕉果皮花青素合成的转录因子基因20个,包括12个MYB,5个bHLH,2个WD40和1个WRKY基因。花青素合成相关差异表达基因的蛋白互作网络预测表明转录因子MYB（Mi_g026681）负责调控花青素合成途径上游结构基因C4H,而另一个 MYB（Mi_g021391）和 bHLH（Mi_g014100）互作共同调控下游结构基因（F3H、DFR、LDOX和ANR）的转录,并以此构建出野生蕉果皮花青素合成的途径和可能的转录调控网络。利用转录组注释和结构域分析,筛选出29个在野生蕉果皮差异表达的R2R3-MYB基因,并分析了这些基因序列的保守性,确定野生蕉果皮差异表达的R2R3-MYB基因的结构域基序为[W]-x（19）-[W]-x（19）-[W]-x（12）-[E]-x（15）-W]-x（18）-[W]-x（3）-[L]。通过与拟南芥R2R3-MYB基因、已知功能的MYB基因聚类分析,筛选出8个可能参与花青素合成等苯丙烷类代谢途径的MYB基因（Mi_g026681、Mi_g030747、NewGene₂1582、Mi_g021391、NewGene₁0237、Mi_g011516、Mi_g028070 和 Mi_g019957）。其中,Mi_g026681和Mi_g030747两个MYB基因可能在调控基因表达过程中起抑制作用;Mi_g011516、Mi_g028070、Mi_g019957 和NewGene₁0237可能参与野生蕉果皮中黄酮类化合物的合成;Mi_g021391可能与野生蕉果皮中花青素的合成相关。该筛选结果与mRNA蛋白互作预测结果一致。4.野生蕉果皮不同颜色部位的lncRNA分析通过对野生蕉果皮转录组测序数据的进一步挖掘,筛选鉴定出3938条在野生蕉果皮中表达的lncRNA,其中65.9%为基因间lncRNA,还包含了 14.5%的反义、10.4%的正义和9.1%的内含子lncRNA,为后续功能lncRNA的筛选提供数据资源。其中,有2条lncRNA比对到已知lncRNA。lncRNA与mRNA比较分析提示1ncRNA序列长度、序列ORF长度、表达水平、外显子数均低于蛋白质编码基因。以野生蕉绿色果皮为对照,筛选到409条差异表达1ncRNA,其中紫色果皮中lncRNA的表达量显著高于绿色果皮中的1ncRNA表达量,通过对其靶基因的功能富集分析发现差异表达1ncRNA可能参与蛋白质合成、黄酮类生物合成过程以及DNA结合转录因子活性等生物学过程。通过野生蕉果皮差异表达lncRNA靶基因蛋白互作网络分析,明确了差异表达lncRNA靶基因中MYB基因（Mi_g026681）与C4H（Mi_g014079）、4CL（Mi_g008868）的靶向关系,另一个MYB基因（Mi_g003034）与 WD40（Mi_g029091）共同调控ANR基因（Mi_g016749）的转录,说明这些靶基因对应的差异表达1ncRNA可能通过调控这些靶基因,进而间接调控野生蕉果皮花青素苷的生物合成。5.野生蕉果皮不同颜色部位的miRNA分析通过对紫色果皮（PP）和绿色果皮（GP）两个小RNA文库的高通量测序分析,共鉴定出254个在野生蕉果皮中表达的miRNA,分布在58个已知miRNA家族当中,其中包含132个已知的miRNA序列和122个新的miRNA。对254个miRNA进行靶基因预测,共有244个miRNA预测到3,975个靶基因,这些靶基因可注释到40个GO条目和101个KEGG代谢通路中。通过miRNA表达量的差异分析,共得到36个同时在GP和PP中差异表达的miRNA基因。其中10个miRNA在紫色果皮中上调表达,26个在绿色果皮中上调表达。通过对差异表达miRNA靶基因的GO功能和KEGG通路富集分析表明,差异表达miRNA靶基因在DNA结合、离子跨膜运输活动、高渗的响应、DNA代谢过程、蛋白激酶活性、有机离子转运、细胞分化和蛋白磷酸化等8个GO功能条目和植物-病原互作代谢通路富集。通过差异表达miRNA与其靶基因的关联分析,找到调控花青素合成相关MYB转录因子基因的miRNA家族主要是miR159、miR535、miR858和miR482等家族。野生蕉果皮中与花青素相关的miRNA-mRNA-lncRNA互作网络预测进一步证实了 MYB（Mi_g021391）和 bHLH（Mi_g014100、Mi_g009620）互作在调控花青素合成下游基因转录的作用,同时筛选出靶向调控MYB（Mi_g021391）的两个miRNA及其3个1ncRNA。6.野生蕉果皮花青素合成相关基因的克隆及表达分析我们利用野生蕉果皮转录组中与花青素合成相关的差异表达基因序列进行特异引物设计,分别以不同颜色果皮总RNA反转录文库为模板,进行PCR扩增,共获得14条具有典型结构域和完整ORF的基因,包括各1条C4H、CHS、CCR和FLS,2条CHI和F3H,3条DFR和MYB,以及7条基因的部分序列（各1条CHI、F3H和TT12、4条MYB）。序列分析结果表明,TT12作为转运蛋白,具有10处跨膜结构,而获得的7个MYB基因中有2个（MiTT2-2和MiTT2-3）属于 R2R3-MYB,1 个（MiTT2-1）属于 3R-MYB,4 个属于1R-MYB/MYB-like。聚类分析结果表明,有3个TT2同源基因（MiTT2-1、MiTT2-2和MiTT2-3）与参与调控拟南芥苯丙烷代谢过程的第5亚族的AtMYB123/TT2聚在一个分支,可能参与野生蕉果皮花青素的合成。通过qPCR对9个野生蕉果皮花青素合成途径上的结构基因和8个转录因子基因进行相对表达量分析,得到的表达模式与转录组测序结果一致。利用荧光定量的方法对6个miRNA及其6个靶基因的表达水平进行验证分析,有4对得到了与转录组测序一致的结果。本文通过色素分析和HPLC鉴定,明确了野生蕉果皮颜色差异是由于果皮中的飞燕草色素等花青素累积造成的,并在此基础上,利用RNA-Seq（mRNA、1ncRNA和miRNA）分析筛选出果皮颜色差异相关的花青素合成结构基因、转录因子及其可能靶向这些关键基因的1ncRNA和miRNA,克隆出野生蕉果皮花青素合成相关结构基因和关键转录因子MiTT2（Mi_g021391）,明确它们的表达模式,构建出野生蕉果皮花青素合成可能的调控网络,为野生蕉乃至芭蕉属植物的色素代谢研究提供数据资源,奠定良好基础。更多还原

【Abstract】 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.更多还原