Identification And Analysis Of MiRNAs And Target Genes Involved In Blueberry Fruit Ripening

Blueberry has high health care value and economic value.In the process of introduction of blueberry,the flavor of the same variety in different regions becomes weak and the quality declines.Besides,the fruit ripening stage is inconsistent in the process of production.Exploring the molecular mechanism of blueberry fruit ripening can provide a theoretical basis for production.The ripening process of blueberry fruit is very complex,including not only color changes(chlorophyll degradation and anthocyanin synthesis),but also hormone regulation,metabolism of sugars,acids,esters and aromatic substances,and fruit softening.In addition to the well-known transcription factors and so on,micro RNAs are widely participate in the metabolic process of fruit maturation.During this research,transcriptome sequencing,s RNA sequencing and analysis were performed on northern highbush blueberry 'Legacy' by highthroughput sequencing technology,and miRNAs involved in the ripening process of blueberry fruits was identified at the omics level.A series of bioinformatics methods and physiological data were used to analyze and predict the various processes and patterns of fruit ripening that miRNA and its target genes might participate in.The miRNAs and its target genes were mined by combining published degradation data.Among them,this study verified the negative regulatory interaction of Vco-miRNA858 on its target gene Vc TT2-type MYB through the transient expression system of tobacco.Then,the functions of VcomiRNA858 and its target gene Vc TT2-type MYB were studied by apple peel transient expression system and blueberry transgenic system.Specific research results are as follows:(1)The blueberry 'Legacy' has a fruit development cycle of about 77 days.The fruit development curve was "double S shape",showing a trend of "slow-fast-slow-fast".Fruit development cycle has been divided into four stages: slow growth stage,which is 7-28 days after full bloom;Rapid growth stage,namely 29-49 d after full bloom;Slow growth stage,which is 50-70 days after full bloom;The growth stage before maturity is 71-77 days after full bloom.With the maturity of blueberry fruit,the content of chlorophyll decreased gradually,and the content of anthocyanin increased gradually.The contents of total phenols and flavonoids showed a similar trend,both decreasing first and then slightly increasing,but the overall trend was decreasing.The contents of soluble total sugar,glucose and fructose increased continuously,while the content of sucrose was always at a low level with slight fluctuations.(2)Using BGISEQ-500 platform,transcriptome sequencing was performed on three key stages during the development and ripening of blueberry fruits: “UR”(DAF=28),“SR”(DAF=49),and "TR"(DAF=77).Transcriptome analysis of 9 samples was completed,and a total of 186330 transcripts were obtained by assembly.Through screening,functional annotation and enrichment of differential genes,most genes of catalytic activity,metabolic process and cellular process were annotated into GO database.However,Photosynthesis,Flavonoid biosynthesis,Phenylpropanoid biosynthesis and other pathways were enriched in KEGG database.(3)The s RNA sequencing was performed on the key period of blueberry fruit development.9samples were sequenced,and 195.61 M Raw Reads were obtained.A total of 60 known miRNAs and 145 novel miRNAs were identified.Among them,the known miRNAs come from 11 miRNA families,and the miRNA535 family has 13 members,which is the largest number.The expression levels of all the identified miRNAs were analyzed,and 81 differentially expressed miRNAs were screened.Target gene prediction and function annotation was performed on 60 known miRNAs and 145 novel miRNAs.According to the miRNA-target gene pairs predicted by the sequencing results,combined with the published degradome data on of blueberry fruits,miRNA-target gene pairs that may be involved in fruit ripening were screened.(4)Functional analysis of the above Vco-miRNA858 and Vc TT2-type MYB were performed.The subcellular localization experiment of tobacco showed that Vc TT2-type MYB was localized in the nucleus.A negative regulatory interaction between 35S:: Vc MIR858 and 35S:: Vc TT2-type MYB-GUS was verified using the transient expression system of tobacco.In this study,we overexpressed Vc MIR858 and Vc TT2-type MYB by apple pericarp heterologous expression system,respectively.The results showed that Vco-miR858 did not play an obvious role in regulating proanthocyanin biosynthesis and accumulation,while Vc TT2-type MYB played a positive role in proanthocyanin biosynthesis.(5)After infecting blueberry leaves,co-culture and screening culture,they were transformed to the northern high bush 'Sierra' blueberry leaves and regenerated to obtain resistant buds.Through PCR detection,3 buds of Vc MIR858 were obtained with target bands,and 2 buds of its target gene Vc TT2-type MYB were obtained with target bands.Aiming at the rooting of transgenic blueberry stems,an efficient bagged rooting technology was setted up.This technology can greatly improve the rooting effect and shorten the rooting time of blueberry,and the rooting rate can reach more than 84%.It takes 13 days to start rooting,and about 45 days to transplant seedlings,with low time cost.All positive transgenic plants have not produced fruit yet,and the specific fruit phenotype remains to be further observed.