Analysis Study Of The Specific Gene CpFAS1 Of Y Chromosome In Papaya(Carica Papaya L.) And The Optimization Of The Papaya Regeneration System

Papaya is a popular and productive fruit in tropical and subtropical regions worldwide,for its outstanding nutritional value.Sex determination in papaya is controlled by an XY sex chromosome system with the genotypes of XX female,XY male and XYh hermaphrodite in the previous study.Sex determination of male and hermaphrodite is controlled by two slightly different Y chromosomes,Y and Yh,respectively.Previous study shows that two male to female sex reversal mutants were generated from gama ray irradiation,and DNA sequence analysis of these two mutant revealed 1 Mb overlapping deletion in the MSY.To obtain male to female sex reversal,both stamen promoting and carpel suppressing sex determination genes have to be knocked out or deleted Annotation of this 1 Mb deleted region,revealed a few Y-specific genes,and CpFAS1 is one of them.CpFAS1 is the only candidate gene that can respond to the environmental changes in the 1 Mb sex determining region in papaya.We analyzed the sequence of CpFAS1 and put forward the hypothesis,having a new sense of pseudogenes.Yeast-Two-Hybrid analysis was conducted to establish protein-protein interaction in order to vertify the previous guess.Moreover,the exploration of the reproduction system of papaya laid the foundation for the papaya callus transformation.Overexpression and gene silencing constructs were transformed into calli of papaya to verify the gene function.Primary results includes:1.The analysis of the sequence of CpFAS1 and the RNA-seq data,detecting that the expression of CpFAS1 in X chromosome.For CpFAS1 is a presudogene in X chromosome,we guess that presudogene may work in some way and work in the process of sex determination.2.The bait vector,pGBKT7-FASl,was hybridized with the cDNA library of papaya,yielding 64 positive clones,and 41 clones have fragment more than 500 bp.They were sent to be sequenced and the sequencing results were blasted in the conserved domain database(CDD)of NCBI,9 positive clones were matched to different conserved domain.3.Analysis of the function of the corresponding protein of different conserved domain,we post two models of the mechanism of action in papaya that may provide certain reference for further research.4.We explored the reproduction system of papaya and found that the best explant for callus induction was the hypocotyl of Kapoho in our test.2,4-D is necessary for the callus inducton and the optimal concentration was 10 mg/L.The optimal concentration of 6-BA and NAA was 0.2 mg/L,respectively.The optimal concentration of IBA was also 0.2 mg/L.The exploration of the reproduction system of papaya will lay the foundation for the papaya callus transformation.5.The overexpression and gene silencing constructs were transformed into the calli of papaya respectively,we got the resistance plants.Because there are a false positive PCR verification,we also need to develop southern blot test for further validation.There was a new phenotype that a small leaf grow on the base of the leaf,which founded in the resistance plants with the overexpression vector.Papaya,as the important material of the research of sex determination,with knowledge of it,if we can find the sex determination gene from the candidate genes and build the network of sex determination,it will bring the research ideas and breakthrough point for the other plants.Moreover,CpFAS1 may regulate the gender of papaya by epigenetic,and presudogene may work in the process.There are a little research results about the epigenetic effects on plant sex so far.Therefore,the research of plant sex determination from the perspective of the epigenetics regulation,will provide the evidence for the role of epigenetic regulation in plant sex determination,and also provides a new research direction.