Functionalanalyses Of CmPI Gene In Melon Flower Development

Melon?Cucumis melo L.?is an annual trailing herb of Cucurbitaceae.It is a nutritious fruit and an important horticultural crop.Melon is an andromonoecious model plant with male and bisexual flowers,and the development of floral organs accords with ABC?D?E development model.PISTILLATA?PI?belongs to MADS-box transcription factor family gene,which plays an important role in the development of Arabidopsis thaliana flower organs.In this study,the CmPI gene was cloned from melon,and the overexpression and CRISPR/Cas9 editing vectors were constructed.The melon was transformed via pollen-tube pathway transformation.The main findings are as follows:?1?The full-length cDNA sequence of the CmPI gene was cloned.The gene has a full length of 1052 bp and ORF was 747 bp,encoding 248 amino acids.The physicochemical properties,hydrophilicity,subcellular localization,signal peptide,promoter and evolutionary relationship of CmPI protein were analyzed.The results showed that CmPI protein was a hydrophilic unstable protein,which was located in the nucleus and contains signal peptide.The promoter region contains ethylene,gibberellin,salicylic acid,anaerobic,light and drought response elements.Phylogenetic analysis showed that they were relatively close to cucumber,zucchini,pumpkin and bitter gourd.?2?The expression characteristics of CmPI gene in different organs and tissues of melon were detected.The expression of CmPI gene was the highest in the stem.The expression was lower in the root and leaf than in the stem,the lowest in the ovary and fruit.Its expression was about 2200 and 4000 times higher in the stamen and petals than in the stigma.?3?The overexpression vector pPZP221-CmPI and CRISPER/Cas9 editing vector pCRI-CmPI were successfully constructed and transformed melon.The obtained T₁ generation plants were detected by PCR,and the positive rates were 40%and 30%respectively.By sequencing analysis,two of the positive plants transformed by editing vector pCRI-CmPI appeared base substitution.?4?The expression level of CmPI gene was significantly higher than that of wild-type lines in the T₃ generation over-expressing transformed plants.?5?The expression levels of CmAP1,CmACS7 and CmACS11 in staminate sepal of T₃ generation transformed plants were detected.It was found that the expression levels of CmAP1,CmACS7 and CmACS11 were significantly lower than those of wild type,suggesting that CmPI gene might have negative regulation to the expression of CmAP1,CmACS7 and CmACS11 genes.?6?The expression levels of CmGNL,CmGNC,CmGATA15,CmSEN1,CmBPE and CmChs genes,which may be regulated by CmPI gene,were detected in T₃ generation transformed plants.The results showed that the expression levels of CmGNL,CmGNC,CmGATA15 and CmSEN1 were significantly lower than those of wild type and the expression of CmBPE and CmChs increased significantly.The expression of CmGNL,CmGNC,CmGATA15 and CmSEN1 might be negatively regulated by CmPI gene,and CmBPE and CmChs might be positively regulated by CmPI gene.?7?The number of male flowers in T₃ generation transgenic plants was significantly more than that in wild type plants,CmPI gene might promote the development of male flowers.