The Role Of CmbHLH93 And CmbHLH130 In Fruit Development Of Melon

Melon,which has been widely cultivated all over the world,is a highly economic horticultural crop.At the same time,it is also one of the model plants of cucurbitaceae to study the process of sex differentiation and fruit ripening.In this study,Hetao melon(Cucumis melo L.cv Hetao melon)was selected as the research material,which was planted in Hetao area of Bayannur City,Inner Mongolia Autonomous region.The CmbHLH family was systematically analyzed.Two highly expressed gene CmbHLH93 and CmbHLH130 were chosen to explore their function accroding to the fruit transcriptome data obtained by previous research.The main results are as follows:(1)A total of 160 bHLH genes were identified in the whole melon genome by bioinformatics methods,which encoding 213 proteins.According to the structure of CmbHLH genes,the intron distribution patterns were divided into 13 types.Chromosome location showed that CmbHLH93 and CmbHLH130 genes were located on chromosome 3 and 1,respectively.Based on the phylogenetic tree constructed byamino acid sequences,the bHLH family was divided into 18 subfamilies,in which CmbHLH93 and CmbHLH130 belonged to the 18 th and 8th subfamilies,respectively.Amino acid sequences analysis revealed that the 7th to 18 th subfamilies were highly conserved.CmbHLH proteins conteined HLH or bHLH-MYC-N domain,and 10 members also had ACT domain.Conserved motif analysis showed that CmbHLH proteins had motif 1 or motif 2.Only 110 bHLH genes were expressed in melon fruit by heat map analysis,which most genes were expressed in the growth of melon fruit.It was predicted that 96% of CmbHLH proteins were located in the nucleus.(2)The cDNA of CmbHLH93 and CmbHLH130 genes were cloned by RT-PCR using total RNA of fruit,and the ORF lengths were 1023 bp and 867 bp,respectively.The overexpression vector and gene editing vector were constructed,named pPZH93 ,pPZH130,pYLH93 and pYLH130,respectively.(3)RT-PCR analysis showed that CmbHLH93 was highly expressed in bisexual flower and the growth stage of fruit,CmbHLH130 was highly expressed in roots and climacteric fruit,and CmBTB/POZ was highly expressed in leaves and he growth stage of fruit.(4)pGBH93Y,pGBH93C,pGBH130Y,pGB130C and pGBBT vectors were constructed and transferred into yeast AH109 to study the phenomenon of self-activation.The results showed that CmbHLH130,CmbHLH93 and CmBTB/POZ proteins had no self-activation phenomenon.(5)The bait and prey vector of CmbHLH130,CmbHLH93 and CmBTB/POZ were constructed to study their interaction.The results showed that there were nohomodimers or heterodimers between CmbHLH130 and CmbHLH93 proteins,but CmbHLH93 and CmBTB/POZ proteins interacted with each other.(6)The fusion expression vector of pCAH93-GFP and pCAH130-GFP were constructed and transformed into Agrobacterium tumefaciens GV3101 for transient expression in tobacco leaves.The results showed that CmbHLH93 and CmbHLH130 proteins were located in the nucleus.(7)The overexpression vectors pPZH93 ,pPZH130 and editing vectors pYLH93 and pYLH130 were transformed into wild-type melon by ovary injection which 1725,1202,1472 and 1184 T1 transformed seeds were obtained and the positive rates of PCR detection were 13.1%?13.3%?12.5% and 14.6%,respectively.T1 transformed seeds were sown in greenhouse,and 6,4,5 and 3 positive fruits were obtained by PCR detection.The ripening day of T1 fruit with overexpression of CmbHLH93 and CmbHLH130 were 39.2 d and 37.8 d,respectively,which were 4.0 d and 5.4 d earlier than that of wild type,which indicated that CmbHLH130 and CmbHLH93 genes could promote fruit ripening.And the fruit firmness of overexpressed CmbHLH93 was lower than that of wild-type in the same developmental stage.