The Clone And Analysis Of Stress Response Related NAC Transcription Factors In Cotton

Adversity stresses as drought, salinity, diseases seriously affect the normal growth and development of plant, and restrict the corp yield. Plant formed many protection machnism in the long evolution process. Through adversity signal perception and regulation of related genes expression, plant could maintain the normal physiological and metabolic level. NAC proteins are plant-sepcific transcription factors which play impotant roles in development, organ formation, plant senescence, hormone siganaling and strss response. However, only little information regarding stress-related NAC genes got further study in modle plant such as Rice and Arabidopisis. NAC genes found in upland cotton is more involved in adversity stress response to drought, high and salt, and part of them are involved in leaf senescence and fiber development. Object: To predict the whole NAC genes in Gossypium arboretum will be contribute to make use of NAC gene resources.This project was focused on function analysis of NAC genes in cotton. We aimed to clone NAC genes in cotton, to research the expression change in the adversity stresses condition and to prove the nature of transcription factors, which is nessesary to reveal the mechanism of NAC TF. Methods: We predicted the whole NAC genes in Gossypium arboretum by bioinformatics methods.Gh SNAC5 and Gh SNAC8 were islated from cotton via RT-PCR; The expression change of Gh SNAC5 and Gh SNAC8 were studied using fluorescence quantitative RT-PCR; We constructed yeast transcriptional activation carrier and subcellular localization carrier to study the nature of transcription factors; Results: We have predicted 138 NAC genes in Gossypium arboretum, and named as Ga NAC001-Ga NAC138. According to the similarity of their protein sequences and the phylogeny analysis, all Ga NAC genes were clustered into 11 distinct subfamilies which have 4-25 Ga NACs, respectively. Gene structure analyses show that 63.7% of Ga NACs gene contains two introns. Sequence analysis shows that the Gh SNAC5 c DNA has a length of 1080 bp, and open reading frame(ORF) encoding a protein of 299 amino acids. The Gh SNAC8 c DNA has a length of 1104 bp, and open reading frame(ORF) encoding a protein of 349 amino acids. Both Gh SNAC5 and Gh SNAC8 contain the NAM domain in the N- end. Gene structure analysis of Gh SNAC5 and Gh SNAC8 show that both them have three exons and two introns which have the characteristics of typical plant introns; According to the q RT-PCR results, the relative expression of Gh SNAC5 and Gh SNAC8 was obviously up regulated in the salinity and drought condition when treated two hours. In the cyanosis VD8 stress, the relative expression of Gh SNAC5 and Gh SNAC8 was obviously up regulated in 6 hours, which show time delay than in salinity or drought stress; According to the growth situation on SD medium and β-gal color test, we determine both Gh SNAC5 and Gh SNAC8 have transcriptional activation. We make Gh SNAC5/ Gh SNAC8 and GFP to fusion proteins which specially localized in nuclear; We useing p CAMBIA3301 and p ROKⅡ constructed a new vector which have a 35 s promotor before MCS and also have a bar gene. Conclusion: We have comparative analysised between the NAC protein of Gossypium raimondii and Gossypium hirsutum with 138 NAC proteins of Gossypium arboretum which is useful to the depth reseach of NAC transcription factors in cotton. We have cloned two NAC transcription factors from upland cottonand named as Gh SNAC5 and Gh SNAC8. And have accomplished the basic research. These results are significant not only to the deepth functional research of Gh SNAC5 and Gh SNAC8, but also to prove NAC gene resources in cotton.