Functional Analysis Of A BHLH Gene From Populus Koreana

Under the natural conditions, plants usually suffer the stresses of drought, salinity or low temperature, which depress the growth rate, even lead to plant death. The destroies caused by different environmental stresses are the main constraints of the agriculture, improvement of ecological environment and sustainable development. Study the function of stress tolerance genes is the base for resistant breeding. The bHLH (basic/helix-loop-helix) transcription factor is a class of important transcription factors whose domain has the ability to bind with DNA and form homologous or heterologous dimers with bHLH protein. Although bHLH family has crucial fuction in both animal and plant, it has been little studied in plant; therefore, their biological function are still unclear. Studies have shown that bHLH paticipated many vital processes of plant cell and related to stress tolerance response of plant. In this study, the expression and fuction of bHLH from Populus koreana were investigatedOne bHLH gene was isolated from Populus koreana by using homology cloning, which was designed as PkbHLH. The length of PkbHLH was 986 bp, with an open reading frame of 894 bp in length, encoding 297 amino acid residues with a molecular weight of 35.46 kDa and isoelectric point of 6.60. The bHLH from Populus koreana shared low similarities with that from other plants.The 1741 bp length promoter of PkbHLH was coloned by homology cloning. Sequence analysis showed that there are seveval cis-acting elements related to stress response, such as ABRE, W-box, MYB, ARR1AT, and GATABOX. The promoter was cloned into pBI121 plasmid to drive the expression of GUS. The recombinant plasmid was transformed into Agrobacterium tumefaciens that was used for tobacco transformation. GUS stained analysis showed that the promoter has the expression avtivities. Both Arabidopsis and tobacco transformants were generated by floral-dip transformation method. The results show that bHLH was mainly expressed in stem of Arabidopsis and little in the leaves. The same result was obtained when tobacco transformants were observed.To investigate the molecular function of the bHLH, cDNA library of Populus koreana was constructed. The promoter of bHLH was cloned into the pHIS2 plasmid and the recombinant plasmid and cDNA library were transformed into yeast by co-transformation. The upstream regulatory gene of bHLH from the cDNA library were isolated by yeast one-hybrid system and 5 candidates (protein phosphatase 2c, cell wall-associated hydrolase, glycolate oxidase, gal 4 activation domain polypeptide, photosystemâ…¡protein D1) were obtained.The cis-acting element ABRELATERD1 that associated with stress response was studied. The sequence was triple in tandem repeat and linked with pHIS2. The interaction of ABRELATERD1 element and ABRE was investigated by yeast one-hybrid system. The result showed that an ABRE can recognize the ABRELATERD1, suggesting that this ABRE may regulate the expression of bHLH.