The Study On The Functional Of Transcription Factor BHLH30in Glycine Max Tamba

The component of acidic soil is a very complex in which plants are stressed by aluminum, low-phosphorus and acidic damage etc. Aluminum is the major factor of limiting crops'growth in acidic soils, and its form changes with the external environment. Aluminum takes the form of Al3+when pH is lower than5. Al3+can afftect the absorption of water and nutrition of plant, and then cause damages to the roots. There are many studies concerning about aluminum resistance mechanism of plant, however, of which the knowledge is very limited because of its complicated physical and chemical properties and difficulty to be located. So far, the aluminum resistance mechanism of plant is still at the stage of reasoning hypothesis, which needs further validation by experiments.BHLH30gene of Tamba black soybean was upregulated in SSH cDNA library constructed by my research group, which implied that it was closed related to aluminum resistance. The preseent research reported indicate bHLH transcription factors are closely related to plant resistance, secondary metabolism, growth and development, and morphogenesis. However, there are no reports about the functional of transcription factor bHLH in soybean until now. BHLH30gene was isolated from Tamba black soybean, a strong aluminum's resistance cultivar, and its function was verified by overexpressing it in wild type tabacco. These results will lay foundation for the research of plant aluminum resistance mechanism. The main research results are as follows:The expression profilings of bHLH30were analyzed by semi-quantitative RT-PCR. The results showed that the expression of bHLH30increased in both roots with the duration increased after treatment with50μM AlCl3. Generally bHLH30's expression showed firstly upregulated then downregulated trence, which showed that the expressions of bHLH30gene in both black soybeans were induced by aluminium.The bHLH30gene of Tamba black soybean's root treated by50μM AlCl3was obtained by construct the vector pWD18T-GmbHLH30. The phylogenetic analysis was done by the deduced amino acids of bHLH30and other bHLHs in plant. The result showed that bHLH30was closed to bHLH51and bHLH106, and was far from bHLH25. The domains of bHLH30, which contained basic and HLH regions. The result showed that GmbHLH30proteins can be combined DNA sequence.In order to obtain the purified protein GmbHLH30, the prokaryotic expression vector pET28a(+)-GmbHLH30was constructed. And the sequence analysis showed that GmbHLH30gene containd many rare codons, so E.Coli Rosetta(DE3) was used for protein expression. GmbHLH30protein was totally expressed in the form of inclusion, using gel purification method. The pure proteins were used as antigens to produce their antibodys.Transgenic tobacco lines GmbHLH30-10and GmbHLH30-20were obtained by transforming plant expression vector pK2-35S-GmbHLH30into wild type tabacco. These transgenic strains were verified in genomic, transcriptional and protein level, separately. The aluminium resistance of GmbHLH30transgenic tabacco was analyzed. The results showed that the Relative Root Elongation of two lines GmbHLH30-10and GmbHLH30-20were2and1.75times longer than that of the control after treatment with50μM AlCl3; while the content of Pro, MDA, H2O2and PC decreased and the soluble sugar increased in the root in the transgenic strains. These results indicated that GmbHLH30transgenic tabacco improve its aluminium resistance by maintaining osmotic pressure, lower membrane lipid peroxidation.To sum up, GmbHLH30gene of Tamba black soybean may be a candidate gene of Aluminium resistence. As not only there are basic and HLH domain in GmbHLH30gene which implies that it can bind DNA to regulate the downstream genes, but also it was upregulated by AlCl3, and overexpression of it in tabacco confers tabacco's aluminium resistence.