The CDNA Libraries Construction Of Melon Under Salt Stress And Analysis Function Of NAC Genes

Melon is an important economically crop that is extensively cultivated insemi-arid regions. Cultivation area and yield of melon in China was at the front ranksof the world. Over the past few decades, there has been a dramatic increase in thesalinization of arable land which affects approximately20%of global irrigatedagricultural land. Crops growth and development were lagged even dead and the yield,quality was declined by salinization. Breeding excellent salt-tolerant varieties is agood strategy for the threat of soil salinization to crops, which contributed to plantsresistance to salt stress as a hotspot of research in the world. However, Salt tolerancehas been regarded as a complicated trait with multiple related genes and pathways.Therefore, the pathways and metabolism of plant resistance to salt stress were still notclear so far. Now, Two melon varieties were used to as materials that based on ourprevious research, which included Bingxuecui (salt tolerance material) and Yulu (saltsensitive material). The work of gene identification salt stress, family analysis,functional verification works were performed by the technology of molecular biology,physiological and biochemical. The following were research results.The construction of two cDNA libraries were obtained by SSH using the root ofBingxuecui and Yulu, and604ESTs from Yulu and339ESTs from Bingxuecui weresequencd, which were remove the adaptors and vector sequence by crossmatch.According to function classification in MIPS database, compare with SSH libraryfrom salt-tolerant Bingxuecui cultivar, proportion of transcripts involved inmetabolism, protein fate, cellular communication/signal transduction mechanisms,cell rescue/defense were4%,1.46%,0.94%,0.4%larger in the salt-tolerant cultivarthan the salt-sensitive cultivar, respectively. Quantitative real-time PCR analysis of27transcripts revealed that their expression were induced by salt stress. We selected one transcription factor (NAC) for further research. We carried out ainformatics systematic analysis of the melon NAC TF family base on a draft of melongenomic sequence. Then, we analyzed the phylogeny of all melon NAC genes andchromosomal locations.82NAC genes in the melon genome were identifed duringthis study, most of whic contain highly conserved N-terminal NAC domain, dividedinto five subdomains (A–E) and a highly variable C-terminal transcriptionalregulation domain. However,5CmNACs (CmNAC20, CmNAC22,CmNAC75,CmNAC28and CmNAC62) lack conserve A subdomains,2CmNACs ofwhich do not contain conserve B, C and D subdomains. To further examine thediversity in melon NAC genes, conserved motifs were predicted by using MEMEprogram. In general, NAC proteins clustered in same subgroups, share similar motifcomposition. Interestingly, most of the conserved motifs were found lying within theN-terminal NAC domain. While, none of the conserved motifs were found at thediversified C-terminal ends of the NAC proteins. We identified6(7.3%) CmNACproteins containing transmembrane domain.82CmNACs have the most number ofNAC genes on chromosome11(11), the least number of NAC genes on chromosomethe fifth chromosomes (4NAC genes). There are three NAC genes can’t locate on thespecific chromosomes. Phylogenetic tree made from multiple sequence alignment ofall82CmNAC proteins divided them into seven distinct subgroups. Subgroup I andIV consists of the maximum (19) number of CmNAC proteins, while subgroup VIIcontain minimum five CmNAC proteins. CmNAC genes and NAC genes fromArabidopsis were divided8obvious clades which were named from subgroup1to8.The largest cladewas subgroup6and4, which contained19members, respectively.However, the smallest was subgroup7and it contained3members. By combiningSSH libraries, transcriptome data under salt stress, NAC genes related with salt stressin Arabidopsis, we identified that more CmNACs members related to salt stress in thefirst and fourth group, especially in the second subgroup of the fourth group.Expression analysis showed that eight of nine CmNAC genes from the secondsubgroup of the fourth group were induced, one CmNAC gene was inhibited by saltstress, suggesting that our prediction can be confirmed. CmNAC34and CmNAC14from8CmNAC genes related to salt stress washeterologously expressed in yeast for functional characterization base on its mostexpression under salt stress, which enhanced the sensitivity of the cells tohigh-salinity to salt stress，and CmNAC34inhibited their growth rate. CmNAC14wascloned for its full lengeth because it s expression increased continually under saltstress. Additionally, tissue expression and abiotic stress expression was performed.CmNAC14shows higher expression in flower and is induced by salt, drought, heat,cold, ABA. Then CmNAC14was localized to the nucleus and transformed inArabidopsis for functional verification. The results showed that over expression ofCmNAC14resulted in plants more sensitive to high-salinity and remarkably inhibitedthe growth rate of plants under salt stress. The enhanced hypersensitivity to salt stressof the transgenic plants of CmNAC14could be attributed to lower expression ofADH1and COR47, increased cell membrane integrity unstability, Na+content, lipidperoxidation under salt stress.We identified a large number of genes from melon, which were induced by salt. Itcould be foundation of melon salt-tolerant germplasm innovation in the future. Theanalysis of melon NAC gene family show that the second branch of the IV subgroupparticipate in response to salt. It shown that over expression CmNAC14arabidopsisplant lead to lower stress-related gene expression, more Na+content, damaged cellmembrane and retarded of root development, which enhanced hypersensitivity to saltstress of the transgenic plants.