Screening And Identification Of NAC Transcription Factors For Seed Development In Tartary Buckwheat(Fagopyrum Tataricum)

Tartary buckwheat is a kind of food and medicine crops,with high nutritional value.Tartary buckwheat has high flavonoid content,which can improve immunity,delay aging,reduce three levels of high blood pressure,and prevent cardiovascular and cerebrovascular diseases.However,the low yield of Tartary buckwheat greatly limits the development of Tartary buckwheat industry.The basic research on Tartary buckwheat is relatively weak.At present,it mainly relies on traditional conventional breeding for variety improvement.In recent years,the completion of genome sequencing of Tartary buckwheat has greatly promoted the development of molecular breeding of Tartary buckwheat.NAC transcription factors are a family of plant specific and most widely studied transcription factors,which are not only involved in the regulation of plant growth and development,such as root development,flower development,seed development and leaf senescence,but also involved in the regulation of biological and abiotic stresses.In our previous study,we conducted transcriptome sequencing on seeds of Tartary buckwheat at different developmental stages,and found that many NAC transcription factors may be involved in the regulation of development of Tartary buckwheat seeds.In this study,based on the genome of Tartary buckwheat,the whole genome identification and analysis of NAC transcription factors in Tartary buckwheat were carried out,and the transcription factors involved in the regulation of Tartary buckwheat seed development were selected for gene cloning and analysis.At the same time,the expression of candidate NAC genes in different tissues,seed development and abiotic stress were analyzed by real-time fluorescent quantitative PCR(q RT-PCR).Subcellular localization,transcriptional activation analysis and transgenic function verification of the two NAC transcription factors were performed.The main conclusions of this study are as follows.(1)Through genome-wide analysis,72 genes of Tartary buckwheat NAC family were identified,distributed on 8 chromosomes of Tartary buckwheat,among which 71 NAC genes contained introns.72 NAC proteins from Tartary buckwheat and 105 NAC proteins from Arabidopsis thaliana were used to construct phylogenetic tree.The results showed that177 NAC proteins could be divided into 9 subfamilies.Through the transcriptome analysis of Tartary buckwheat seed development,33 NAC genes were found to be differentially expressed during seed development,then pick the eight expression and with high abundance of seed development express quantity increased quantity gradually cut of NAC genes and one expression of NAC genes as a candidate gene for subsequent analysis.(2)Nine Tartary buckwheat NAC genes were cloned and named as Ft NAC2,Ft NAC3,Ft NAC4,Ft NAC5,Ft NAC7,Ft NAC8,Ft NAC9,Ft NAC15 and Ft NAC17.The subcellular localization prediction showed that eight NAC genes were localized in the nucleus.The expression of nine Tartary buckwheat NAC genes in roots,stems,leaves,flowers and seeds was detected by q RT-PCR.The results showed that four Tartary buckwheat NAC genes,including Ft NAC2,Ft NAC3,Ft NAC4 and Ft NAC7,were highly expressed in seeds.The expression of these four NAC genes was detected at different stages of seed development(pre filling stage,filling stage and maturity stage),and it was found that the expression levels of Ft NAC2,Ft NAC3 and Ft NAC4 were gradually increased with the development of seed,and the expression level of Ft NAC7 was the highest at the seed development and filling stage.Evolution analysis shows that,with the four Tartary buckwheat NAC proteins closest relative arabidopsis NAC proteins involved in regulating the abiotic stress and active oxygen metabolism,and thus to explore the four Ft NAC genes in response to low temperature,drought and salt three abiotic stress conditions,the results showed that four Ft NAC genes on the trend and degree of three kinds of stress are different response.Ft NAC3 and Ft NAC4 were the most responsive to drought stress,and these two genes were most closely related to the abiotic stress marker gene ATAF1 in Arabidopsis thaliana,so the two genes were screened for functional verification.(3)The pGFP-Ft NAC3 and pGFP-Ft NAC4 vectors were constructed and subcellular localization of Ft NAC3 and Ft NAC4 transcription factors was performed.The results showed that the two transcription factors were localized in the nucleus.The p GBKT7-FTNAC3 and p GBKT7-FTNAC4 vectors were constructed and the transcriptional activation activity was analyzed using yeast system.The results showed that both Ft NAC3 and Ft NAC4 had transcriptional activation activity.Ft NAC3 and Ft NAC4 plant overexpression vectors were constructed and transformed into Arabidopsis thaliana by inflorescence staining method to verify their functions.At present,10 positive strains were screened respectively.