Functional Analysis Of BHLH Transcription Factor B695 From Maize (Zea Mays L)

A basic helix-loop-helix (bHLH) is a protein structural motif that characterizes a family of transcription factors, plant bHLH transcription factors are often important in development or cell activity. Meanwhile, Manganese (Mn) is an important nutrition material in plant growth; but large accumulation of Mn in plant will cause serious of poison. It is important to understand the mechanism of plant expression of bHLH transcription factors to improving crop resistance.Main research objective of this thesis is based on the B695 whichs belong to bHLH transcription factor family from Maize. The main results were as follows:1. The full length cDNA sequences 749bp of B695 clone from Maize, B695 open reading frame also as the length of 749bp; HLH conserved domain is located at 92-143 amino acids.2. pEGAD-B695 plant expression vector construction for GFP stable expression, fluorescence microscope observated onion epidermal cells. The result showed that B695 had the nuclear localization signal, and its position was sitting in the nucleus.3. pET-30a-B695 vector construction for prokaryotic expression, the vector was constructed successfully and transferred into E.coli to get target protein. The protein had a 6xHis tag on the C-Terminal by IPTG-induced, about 32kDa large of the B695 fusion protein and B695 polyclonal antibody had preparaed, Western Blot result showed that the Polyclonal Antibodies can recognize B695 proteins well.4. Using yeast one-hybrid to validating B695 could activate the expression of report gene in yeast, it had the function of trans-activation in yeast and the functional structure of this domain was at the C-Terminal, its N-terminal did not have the function of trans-activation.5. Real-time PCR results showed the difference of B695 expression in maize under different heavy metal stress. The result was that, B695 was induced by Fe, Mn and Cu, Zn meanwhile, endogenous B695 gene expression could be stimulated by Mn.6. The result of transgenic yeast strains stressed by different impactors showed that, AH109 yeast strains to the adversity stress (high salt, drought, heavy metal) of adaptation ability could significantly improved by B695; while the result of various stress processing of B695 and wild type growed on 1/2 MS plates claimed that, transgenic plants’ tolerance to high Mn2+ could increased by B695.7. According to NBT and DAB staining, the results showed that compared with wild type, B695 over expression transgenic tobacco plants accumulate less ROS; the result of ROS clear related enzyme activities and Real-time PCR displayed B695 had expression of tobacco in Mn stress processing. It was considered that B695 could reduce the accumulation of ROS in plants.8. Real-time PCR results showed that under normal condition and Mn treatment, expression of NtCDPK2, NtAREB, NtERF in B695 transgenic plants were lower than the wild-type ones, meanwhile, expression of NtLEA5 was comparable, Nramp and Irt1 which function related to across membrane transports’expression were less than wild-type’s.9. The result of Fe and Mn content determination in B695 transgenic tobacco and wild type controls by Mn stress processing showed that, the content of Fe in B695 transgenic tobacco was less than wild type controls after Mn stress, while the Mn content was far below wild types, it validated that IRT1 and Nramp could improve plant absorpte Mn in Fe-deficiency environment; and due to the role of B695, it could keep Mn content in a suitable concentration in plant, resulted that it could not disturb plant normal life activities.10. Mn stress processing in short period dealed with wild-type control and B695 transgenic plants, the result of reactive oxygen staining claimed that, B695 was belong to the induced transcription factors, when the plant get signal of Mn, B695 ’turned on’, it would make the accumulation of ROS in plant back to normal levels in a short period.By the results of these experiments, B695 plays as a class of regulatory proteins, takes part in stress signaling pathway involved in Mn, and plays an important role in the regulation of plant ROS levels. This essay tries to figure out the signal transduction and regulation of gene expression in abiotic stress, and it is important for the improvement of plant stress resistance.