Cloning And Functional Study Of NAC Transc Ription Factors During Papaya (Carica Papaya L.) Fruit Ripening

Papaya(Carica papaya L.)is an economically important fruit in southern China and other tropical and sub-tropical countries.Papaya is a typical climacteric fruit meaning that fruit softening and senescence rapidly occur after harvest,which makes it hard for storage and transportation.These characteristic severely limits the development of papaya industry.So many researchers focused on the study of the key enzyme genes of papaya ripening.Recently,NAC transcription factors,the largest plant-specific transcription factor family have been studied extensively in fruit ripening regulatory role.However,little is known about the function of NAC transcription factors during papaya ripening.Therefore,it is necessary to investigate the papaya NAC transcription factors in fruit regulatory mechanism that mainly contribute to the aging process.In the study,experimental investigation is mainly divided into three parts:the first part is cloning of NAC genes involved in fruit ripening from papaya;the second part is mainly to detected their expression profiles in different organs,including roots,stems,leaves,flowers,and fruits then analyzed their expression profiles under ethylene treatment;the third part,we have investigated the papaya NAC gene transformation of tomato.Thus,this study provided valuable information for further exploration of the functions of NAC genes during papaya development and fruit ripening.During this study,we have achieved the following results:1.A total of eight NAC genes were isolated from ethylene/1-MCP treated papaya through RNA-seq.And the eight genes were cloned using RT-PCR technology successfully,named CpNAC1-CpNAC8 respectively.NAM conservative domain structure were found in these genes,belongs to the NAC transcription factor family.The basic physical and chemical properties,protein structure,gene structure,conserved elements,phylogenetic trees and promoter elements were analyzed systematically.Different species of NAC genes were relatively conservative.CpNAC1-CpNAC5 protein are hydrophilic unstable proteins,CpNAC7 and CpNAC8 belong to the stability of hydrophobic proteins,CpNAC6 belongs to hydrophobic and instable protein.Eight CpNAC proteins do not belong to secretory protein and membrane protein.The CpNAC proteins were predominantly serine-modified.Subcellular localization protein indicated that CpNAC were mostly distributed in the nucleus.Phylogenetic tree analysis showed that CpNAC proteins belonged to different subfamilies.2.We also study the changing of firmness,total soluble solids,and carotenoids in fruit ripening.The firmness of the fruits declined at broken color stage and fruit softening become rapidly.The content of the soluble solids in fruits showed downward trend from half yellow stage to full yellow stage.The level of lycopene and β-carotene showed slightly downward trend after rising first.β-cryptoxanthin indicated the rising trend in the process of fruit ripening and the contents of lutein in papaya pulp are extremely low.The content of lycopene was the highest,the orange red flesh color mainly caused by the accumulation of lycopene and β-carotene.3.Tissue-specific analysis by quantitative real-time PCR(qRT-PCR)revealed that the eight CpNACs exhibited different expression profiles.The level of expression of CpNAC6 and CpNAC7 is low during fruit ripening and there is no noticeable change.Another six genes showed different trend in the process of fruit ripening.Among them,the level of expression of CpNAC1 in the fruit senescence showed the gradual declining trend;CpNAC2 and CpNAC3 expression level is high in fruit senescence;during fruit ripening,CpNAC4,CpNAC5 and CpNAC8 were downward trend after rising first.The same subfamily genes have opposite trends of tissue expression.These findings indicated that NAC transcription factors differentiate into different members during long-term evolution and NAC may play different roles in various organs during papaya growth and development.4.Furthermore,we also investigated the influence of ethylene on the target gene expression.We found that CpNACl,CpNAC5 expression quantity meet minimum during 24 h,significantly lower than control;CpNAC2 showed no significant difference between genes and control;CpNAC3 was up-regulated during short-term treatment with ethylene for 12 h;CpNAC4,CpNAC8 at 6 h were significantly lower than control,stress expression quantity and CpNAC8 expressed in 12 h is significantly higher than control.It’s speculated that CpNAC1,CpNAC5 may through the negative regulation of ethylene biosynthesis and signal transduction involve in fruit mature aging regulation,CpNAC2,CpNAC4 may not be directly through ethylene signal pathways to regulate,during fruit mature CpNAC3,CpNAC8 may be induced by ethylene regulation fruit ripening.5.We constructed overexpression vector pBI121:CpNAC1,pBI121:CpNAC3,and pMDC 140:CpNAC5 by In-Fusion cloning technology,Agrobacterium mediated leaf disc transformation were carried out in tomato.We confirmed that CpNAC genes have been imported into tomato genome,obtained the expression positive CpNAC1 tomato T0 seedling 18 lines,the segregation ratio of T1 generation seedlings was 3:1,and the expression positive CpNAC1 tomato T1 seedling 11 lines.In addition,we obtained expressing positive CpNAC3 tomato T0 seedlings in 4 lines.T0 generation of transgenic tomato plants and phenotypic observation indicated that overexpression of CpNAC1 gene on fruit color phase showed yellow sepals and orange pericarp.The size of fruit and number of seeds of the transgenic plants decreased significantly than that in wild type tomato.It indicated that overexpression of CpNAC1 genes may affect carotene accumulation,seed growth and development.