| R2R3-MYB transcription factors in Arabidopsis thaliana contained 2 conserved MYB structural domains, and participated in primary and secondary metabolism, cell morphogenesis and development process, biological and non-biological stress reaction of Arabidopsis thaliana. R2R3-MYB transcription factors were divided into 25 subfamilies based on the conserved amino acid sequences, 4 genes in the 22 nd subfamily were At MYB44, At MYB77, At MYB73 and At MYB70. A large number of previous studies showed that the function of 4 genes in this subfamily were mainly involved in defense biological and abiotic stress responses in Arabidopsis thaliana, and there was an obvious expression phenomenon, so we predicted 22 nd subfamilies in R2R3-MYB family possessed typical redundancy and diversity on structure and function. At MYB44 was researched clearly, previous studies showed that At MYB73 could resist Pseudomonas syringae DC3000 and Sclerotinia sclerotiorum by affecting the salicylic acid and jasmonic acid in our laboratory. In this study, whether At MYB73 playing a role in drought stress was researched from the perspective of drought stress.The research work and the results of this paper were listed as follows:1. Conserved amino acid sequence, secondary structure content, cis-acting element and space-time expression rule in the promoter of 4 genes in 22 nd subfamily of R2R3-MYB family were analyzed and compared, the results showed that genes in 22 nd subfamily possessed redundancy and diversity on structure and function.2. Variation conditions of At MYB73 were detected by using Real-time PCR after treatment at 0 h, 1 h, 3 h, 6 h and 12 h, the results showed that At MYB73 presented a trend of rise first then fall after treatment of high salt, low temperature and mechanical damage, the level of expression of At MYB73 decreased continuously after treatment of drought. The result after treatment of hormone ABA was similar to drought.3. At MYB73 promoter was analyzed to find a series of light response elements, ABA response element ABRE and cis-acting elements of drought and heat stress; At MYB73 promoter was cloned, p At MYB73:GUS was built and transgenic plants were obtained; GUS staining showed that At MYB73 expressed not only in the pod, the top of the pistil, the sepal, the blade edge and around the necrotic disease spot, but also in the seedling leaf stomata and root obviously.4. At MYB73 was cloned and p35S::At MYB73-GFP was constructed, At MYB73 located in the nucleus was proved by using protoplast transformation and transgenic plants respectively.5. The seed germination rate and seedling root growth of knockout mutant were higher than wild type, and the stomatal closure rate of knockout mutant was lower than wild type, when mannitol simulating drought stress. The results showed that knockout mutant was not more sensitive to drought stress than wild type. ABA related genes in plants under drought stress were detected to find that downstream genes of ABA, such as ABI2, and ABI5 in the mutant, were higher than in the wild type.6. Result similar to drought treatment was obtained by treating seeds, seedlings, and leaves of Arabidopsis thaliana with exogenous ABA, showed that mutant was not more sensitive to ABA than the wild type. Arabidopsis thaliana with obvious differences of seed germination after ABA treating were differential gene high-throughput detected to find that 846 genes were up-regulated, 734 genes were down-regulated; 54 genes were associated with hormone, 7 genes in the promoter contained MYB binding sites; 2 up-regulated genes were involved in ABA signaling pathways, and further suggested that At MYB73 negative regulated ABA pathway.In addition, yeast library was screened, the self-activation of bait vector in the yeast two hy brid need to use 140 m M 3-AT to suppress was confirmed; the At MYB73 vector with HA and flag label was constructed, and the transgenic plants were obtained, in order to definit At MYB73 mechanism, which laid a foundation for carrying out CHIP. |
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