Molecular Mechanism Of DBB1α Gene Response To Heat Stress In Arabidopsis And Prokaryotic Expression

Temepreture is one of the most important factor affacting the yield and quality of plant, the research on the physiology and biochemistry basis of plant under heat stress is significative to do some meaures to lighten the damage of plant under heat stress, and to reserch on the basic theory of heat stress.It is not clear about the relationship between DBB1a gene and heat stress signaling. Through analysing the thermortholerance ability of DBB1a over-expression and under-expression mutants, we discovered that the DBB1a under-expression mutunt had the ability of heat-resistant. On that basis, This thesis study the funtion of DBB1a in heat signal pathway detailedly. Besides, we have gained DBB1a using prokaryotae expression. The main results of this thesis are listed as follows:(1) The transcription of DBB1a is heat-induced. We found that DBB1a contains HSE structure needed in heat sress factor; Heat stress can induce the transcriptive expression of DBB1a through q-PCR.(2) The phynotype of DBB1a mutants response to various heat treatments were identified. Through analyzing the difference of gemination, hypocotyl length, basic thermotolerance and acquired thermotolerance in WT,35S:DBB1a, DBB1aRNAi mutants, the results show that 35S:DBB1a is more sensitive to heat stress and DBB1aRNAi is less sensitive. In conclusion, we suspect that DBB1a may be involved in heat signaling and works as a negative regulator.(3) DBB1a regulates the expression of a series of important genes of heat signaling. We detected the expression of key genes in heat signaling of DBB1a over-and under-expression mutants by q-PCR. The result showed that the expression of HsfA2,HsfB1,HsfB2a,HSP70,HSP101,APX2,Zat10 and Zat12 were up-regulated in DBB1a over-expression mutants and the expression of HSP101 and APX2 were down-regulated in DBB1a under-expression mutants.(4) DBB1a takes part in regulating the heat-regulated physiolgic and biochemic character in arabidopsis. Through analysing the heat-regulated physiolgic and biochemic character in DBB1a over- and under-expression mutants, the results showed that over expression of DBB1a could increse the relative conductivity and the concertration of MDA; we further indentified that DBB1a worked as a negative regulator in heat signaling. (5) We constuct pCold-DBB1a fusion vector successfully and purify the fusion protein. The produced fusion protein DBB1a was confirmed by Western blotting, which indicated that the recombinant DBB1a protein had high specificity, which paved a way for further studies on DBB1a.