Cloning And Functional Identification Of Transcription Factor CpNAC8 In Wintersweet (Chimonanthus Praecox (L.) Link)

Wintersweet(Chimonanthus praecox(L.)Link)is a famous traditional species originating in China,and it as an important winter garden flower and cut flower has high economic and ornamental value.To breed abiotic resistance cultivars is a very important task in wintersweet.The NAC gene family which is one of the largest plant-specific transcription factor familie has been reported to be involved in regulation of plant development,biotic and abiotic stress.It can be specifically combined with cis-regulatory elements,so as to activate or inhibit the expression of downstream target genes.Nowdays,the research of NAC genes mainly concentrated on model plant.However,the research in landscape plants is still very limited.This thesis aims to explore the function of transcription factor Cp NAC8 in wintersweet.The main results are as follows:1.Molecular characteristics of CpNAC8 geneBased on part of the NAC transcription factor gene sequence from the transcriptome sequencing database of wintersweet,a pair of specific primers were designed according to the NAC gene sequence.We obtained a new gene,named as Cp NAC8 using RACE cloning method.The c DNA sequence with a full-length of 1583 bp contains an 807 bp open reading frame(ORF)which encodes 134 amino acids.By phylogenetic tree analysis and protein alignment comparison,it indicated that Cp NAC8 gene belongs to II/ONAC4 sub-family and the genetic relationship is close to Solanum tuberosum.2.Expression pattern analysis of Cp NAC8 geneExpression pattern of CpNAC8 gene in different tissues,different flower development stages,and different treatment including drought,salt,high temperature,low temperature,ABA,GA,were analyzed by Quantitative Real-time PCR.The results indicated that Cp NAC8 expressed in all wintersweet tissues,and the expression especially higher in mature leaves and pistil.It speculated that the gene function may be associated with leaf morphogenesis and flower development.During flower development,the Cp NAC8 gene had the highest expression level in sprouted phase,and had no expression in blooming phase.The expression of Cp NAC8 induced under drought,salt,high temperature,ABA,GA treatment.However,the time point of highest expression in different treatment was different.Based on these results,it speculated that this gene may respond to drought,salt,and high temperature stress,and may be involved in signal transduction of ABA and GA.3.Transcriptional activation assay of Cp NAC8We constructed yeast expression vector p GBKT7-Cp NAC8-ORF,p GBKT7-Cp NAC8-VP16,and transferred into the yeast AH109 respectively.The results demonstrated that Cp NAC8 was a transcription activator and had the activity of transcriptional activation.4.Overexpression of CpNAC8 gene in Arabidopsis thalianaWe constructed overexpression vector p CAMBIA1300-Cp NAC8,and then transform it into Arabidopsis thaliana using inflorescence dip method.We obtained homozygous T3 generation of transgenic Arabidopsis thaliana by screening.We chose three transgenic lines,Cp NAC8-7 with the highest expression,Cp NAC8-4 with middle expression and Cp NAC8-3 with lower expression to observe the phenotype and analysis the stress resistance tolerance.The results showed that overexpression of Cp NAC8 gene in Arabidopsis didn’t influenced the growth of transgenic plants.However,the transgenic Arabidopsis seeds were more sensitive to Na Cl than the wild plants.The Cp NAC8 may play a negative role of salt resistance tolerance druing soil salt stress treatment.Moreover,overexpression of Cp NAC8 gene could enhance the drought resistance of transgenic Arabidopsis.The Cp NAC8 gene may be involved in the regulation of salt and drought stress resistance.