Functional Analysis Of ACC Synthetase Genes CmACS7 And CmACS11 During Fruit Ripening In Melon

Melon?Cucumis melon.?is a diploid annual creeping herb with many species,wide distribution,abundant nutritional value and high economic benefits.The melon which is a typical climateric fruit has become another model plant to study fruit development and ripening.1-aminocyclopropane-1-carboxylic acid synthetase?ACS?is a rate-limiting enzyme in the process of ethylene synthesis,which plays an important role in the regulation of ethylene synthesis and in the whole process of plant growth and development.In this study,we found that CmACS7 and CmACS11could regulate fruit ripening by promoting ethylene synthesis,regulating fruit softening and sugar metabolism.The main findings are as follows:?1?In this study,the T₁ generation transgenic melon of overexpression and CRISPR/cas9 gene editing of CmACS1?CmACS7 and CmACS11 were obtained in the previous work,and they were used as experimental materials for next study.At present,T₄ generation transgenic homozygous lines with overexpression and gene editing of CmACS7 and T₄ generation transgenic homozygous lines with overexpression of CmACS11 have been screened.The expression of target genes in T₄transgenic melon fruits of different lines was analyzed by real-time fluorescence quantitative method.was analyzed by real-time PCR.The results showed that compared with wild-type fruit,CmACS1 expression was up-regulated in 2overexpressed CmACS1 lines,while down-regulated in 3 edited lines.And CmACS7expression was up-regulated in 1 overexpressed CmACS7 line,while down-regulated in 3 edited lines.CmACS11 expression was up-regulated in 2 overexpressed CmACS11 lines,while down-regulated in 3 edited lines.?2?Phenotypic observation showed that there were some phenotypes in the transgenic plants,for example,the T₄ overexpression transgenic plants of CmACS1,CmACS7 and CmACS11 showed that the elongation of root and hypocotyl was inhibited,and the curvature of top hook increased,which indicated that it might promote ethylene synthesis.Phenotypes such as plant dwarfism,reduced leaf area and slow development of floral organs appeared in CmACS7 overexpression transgenic plants.At the same time,T₄ generation overexpressed transgenic fruits of CmACS1,CmACS7 and CmACS11 ripened about 5 days earlier than that of the wild type,while the edited fruit ripened about 11 days later than the wild type.?3?The physiological indexes related to fruit ripening of T₄ generation transgenic melon of CmACS1,CmACS7 and CmACS11 were determined.The results showed that there was no significant difference in fruit size,weight,vertical and horizontal diameter between CmACS1,CmACS7 and CmACS11 overexpression and gene editing fruit,but the firmness of CmACS1,CmACS7 and CmACS11 overexpression fruit was significantly lower than that of wild type,and the firmness of editing fruit was significantly higher than that of wild type.And the sugar content of overexpressed fruit was significantly higher than that of wild type.These results suggested that CmACS7 and CmACS11 can promote melon fruit ripening,and they could regulate fruit ripening by promoting ethylene synthesis,regulating fruit softening and sugar metabolism.?4?In order to study the molecular mechanism of the function of CmACS7 and CmACS11,subcellular localization was performed.The results showed that both CmACS7 and CmACS11 were located in endoplasmic reticulum.?5?According to the analysis of promoter prediction sequence information,CmPI,a transcription factor related to flower development,which interacts with CmACS11gene,was obtained by yeast one hybrid screening,indicating that CmACS11 could be regulated by CmPI transcription factors and thus affect flower development.At the same time,the transcription factors related to fruit ripening which may interact with CmACS11 were selected for yeast one hybrid verification.At present,CmACS11 gene promoters and their mutants with the binding sites of CmMADS1,CmNAC2,CmNAC29,CmRAV2 and these transcription factors have been successfully cloned,and 12 vectors,such as pAbAi-Bait,pGADT7-CmMADS1,pGADT7-CmNAC2,pGADT7-CmNAC29 and pGADT7-CmRAV2,have been successfully constructed and transformed into Y1H Gold.In addition,the lowest AbA concentration to inhibit the expression of AbA^r gene was screened.The purpose of this study is to find the transcription factors that regulate the expression of CmACS11 gene,and then analyze the molecular mechanism of regulating melon fruit ripening through ethylene pathway.