Identification And Functional Study Of Microrna Involved In Regulation Of Fruit Ripening And Climacteric In Melon

Melon(Cucumis melo L.)is a widely cultivated horticultural crop in the world and has great economic value.Melon has a relatively short growth period,and relatively small genome size.Melon fruit has climacteric and non-climacteric types according to different varieties.These characteristics make melons suitable as a model plant for studying fruit development and ripening.miRNA is a kind of noncoding regulatory molecule that widely existing on animal and plant kingdoms.In plants,miRNAs are usually 20-24 nt in length,and its precursor are transcript by RNA polymerase II.miRNA plays an important role in plant growth and development,biotic and abiotic stress,signal transduction and other biological processes.However,the study of miRNA in fruit development and ripening is limited.Therefore,a deeper understanding of the role of miRNA in the fruit ripening will help us to deepen our knowledge of the fruit ripening,and also to improve the quality and economic value of the fruit.Hetao melon was used to the study.Hetao melon was the special product of Bayannur City,Inner Mongolia Autonomous Region,and one of the Agro-product Geographical Indication of China.The seeds of Hetao melon were preserved in our laboratory.After 11 generations of self-pollination breeding,a typical and stable phenotype of Hetao melon inbred line was obtained.In this experiment,the fruit of Hetao melon inbred line was used for the study.The miRNAs were identified using next generation sequencing,functions were studied by transient expression and stable transformation of plants.The main results and conclusions are as follows:(1)The miRNA libraries were constructed from the mesocarp of melon fruit of four developmental stages: growth stage(18 DAP),maturity stage(36 DAP),climacteric stage(~ 40 DAP),and post-climacteric stage(~ 42 DAP).And were sequenced by Illumina Hiseq sequencing platform,61 conserved miRNAs belong to 29 miRNA families were found in four different developmental stages of fruit.Differential expression analysis showed 32 miRNAs was differentially expressed from growth stage to post-climacteric stage.36 novel miRNAs were identified,among which cme-m1429-3p was the highest expressed and a new member of miR399 family.(2)A degradome library was constructed from mixed samples of four the fruits developmental stage samples,and was sequenced by Illumina system.A total of 48 miRNAs corresponding 60 target genes,including 81 transcripts,were identified.Of the 60 target genes,34 belong to transcription factors.Meanwhile,several target genes of miRNA were found to be related to Auxin signaling pathway.The GO enrichment analysis of target genes showed that these target genes were significantly enriched in 13 biological processes,including fruit development,auxin response,cell differentiation and cell proliferation regulation.(3)qRT-PCR was employed to quantify the expression of 23 miRNAs in melon roots,stems,leaves,cotyledons and fruits at 9,18,27 and 36 days after pollination,climacteric stage and postclimacteric stage.six miRNAs were specifically expressed in the fruit,and showed a specific expression pattern in the fruit.The expression of cme-miR393,cme-miR530 and other miRNAs in fruits of climacteric stage and post-climacteric stage was significantly increased.In addition,7 miRNAs were specifically expressed in roots,stems,leaves or cotyledons.(4)The functions of cme-miR393 and cme-m1429-3p in fruit ripening were studied by transient expression.It was found that overexpression of cme-miR393 significantly delayed fruit ripening,while overexpression of cme-m1429-3p did not show significant phenotype.(5)According to the results of transient expression,the stable expression melon line of cmemiR393 was constructed by ovary injection.And phenotypes of the fruits of T3 generation plant lines were observed and analyzed.The results showed that the average fruit ripening days of 67-2,69-1 and 17-2 were 48.5,48.9 and 46.4 days after pollination,respectively.The average fruit ripening time of non-transformed control melon was 42.3 days.The fruit ripening time of over expression lines was 5.6 days later than that of control lines.The expression of cme-miR393 was 2-8 times higher than that of the control,and the expression of target gene CmAFB2 was 0.3-0.7 times lower than that of the control.(6)TIR1/AFB,AUX_IAA,ARF,GH3 and SAUR gene families were identified,and the expression levels of these five families were analyzed using our laboratory data and published data.The results showed that ARF and AUX_IAA family genes played an important role in the growth of melon fruit,among which 7 genes were target genes of miRNA,indicating that miRNA was a potential target for fruit ripening regulation.In conclusion,miRNA plays a regulatory role in melon fruit development and ripening.CmemiR393 regulates fruit ripening by regulating the expression of CmAFB2.