Identification, Expression Characteristic And Functional Analysis Of The Genes Related To Melon Fruit Development

Melon is one of the important economic crops and widely cultivated all over the world. However, it gives rise to huge economic losses because of the over ripeness, softening and rottenness within a short postharvest storage. Melon has become another important model plant to clarify the molecular mechanisms of fleshy fruit development and ripening due to it’s research in-depth and botanical characteristics. Ethylene regulates the ripening process and affects the ripening rate and postharvest storability in climacteric fruit. Therefore, it has become a hot topic to study the molecular mechanism of melon fruit ripening via ethylene.To screen for genes that are differentially expressed at the burst of ethylene climacteric, and help to elucidate the molecular mechanism of ethylene climacteric at the transcriptional level in melon fruit, we performed suppression subtractive hybridization (SSH) to generate forward and reverse libraries, for which2groups of fruit samples, from the typically respiratory climacteric melon fruit of Hetao, at disparate stages were used. Forward subtraction was performed using ethylene climacteric fruit cDNA (poly A+RNA of the fruits—when ethylene production increased, peaked, and decreased respectively—mixed in equal proportions) as the tester and pre-ethylene climacteric (5days before the ethylene climacteric) fruit cDNA as the driver. For the corresponding reverse libraries, the cDNAs were reciprocated. The isolated EST clones were sequenced, and databases were searched for homologous genes. From the forward library, the386EST sequences were assembled into179unigenes, while the417EST sequences were assembled into213unigenes in the reverse library. The identified ESTs encoded proteins that mediate metabolism of energy and substances, transcription, posttranslational modification, protein turnover and chaperones, binding protein, signal transduction, growth and development, transport, body defense and self-rescue, unknown function etc. The expression patterns of the6randomly selected candidate genes in fruit at various stages of development were analyzed by qRT-PCR. The results of showed that the transcription of the Chib1, L-Asp, CSGP and ACO1genes increased dramatically during fruit ripening (40-45DAP) and the peaks of Chibl, L-Asp, CSGP and ACO1mRNAs coincided with the climacteric peak of ethylene production. The XTH2and DAFP mRNAs levels were higher during pre-climacteric stage of ethylene.In this study, the full-length cDNAs of the ethylene signal transduction component genes Cm-CTR1, Cm-EIN2, Cm-EIL1, Cm-EIL2, Cm-ERF1and Cm-ERF2were cloned, and the expression patterns of these genes were analyzed by QRT-PCR. Quantitative RT-PCR analysis indicated as follows:The transcript level of the Cm-CTR1was higher and had few changes from15DAP to45DAP, while slightly reduced at ethylene climacteric stage; Cm-EIN2had similar expression patterns in young leaves and petal tissues—approximately2times higher than in root, the minimum and maximum expression were observed in the stem and ovary, respectively. During fruit development, Cm-EIN2mRNA increased slightly from20DAP, peaked at35DAP, and declined. Leaves that were treated with IAA and ABA had lower levels of Cm-EIN2transcripts. The expression patterns of Cm-EILl, Cm-EIL2, Cm-ERF1and Cm-ERF2genes were highly relative to fruit ripening process and ethylene production. The expression patterns of these genes reveals that Cm-EIL1, Cm-EIL2, Cm-ERF1and Cm-ERF2genes may regulate the climacteric ripening, while Cm-EIN2and Cm-CTR1gene may affect the fruit development.The vector-free and marker-free linear cassettes of a fruit-specific stable expression of ACO1antisense gene was constructed, which was combined with the matrix attachment region sequence of tobacco and fruit-specific expression promoter of Cucumisin gene in melon, for the first time (RB-MAR-CMC-antiACO1-nos-MAR-LB). The melon cultivar Hetao was transformed with these linear gene cassettes via pollen-tube pathway. The PCR data showed that the transgenes were inserted into the recipient’s genome.3strains with improved storage capacity have been selected. The fruits were harvested in normal commercial picking stages. The endogenous ethylene contents of the transgenic T2fruits were significantly lower than non-transgenic ones, and were approximately4%of the control at12days post-harvest. It was not found the peak of endogenous ethylene in transgenic fruits. The fruit firmness between the transgenic T2and non-transgenic control was approximately the same when harvest, however, the transgenic fruit firmness remained basically unchanged, while the control one gradually decreased after storage a number of days. The soluble solids contents between the transgenic T2and non-transgenic control were approximately the same during storage.