Cloning And Function Analysis Of BpGT14 Gene Promoter In Betula Platyphylla Suk.

Promoter 2169bp sequence of BpGT14 gene was cloned from birch genomic DNA by SiteFinding-PCR.Then the sequence was analyzed by PLACE.To study the promoter activity,1156bp fragment was chosen to construct plant expression vector.The activity of BpGT14 promoter in tobacco and birch stem suspension cells were analyzed by agrobacterium infection.Promoter 1156bp fragment and a MYBPLANT element were chosen from promoter sequence to study the regulatory protein by Yeast One-Hybrid.Finally,the verification experiment was implemented to determine the binding.The main results are as follows:1.Cloning and Bioinformatics analysis of BpGT14 gene promoter in Betula platyphylla Suk.2169bp sequence was cloned from upstream of BpGT14 gene initiation codon ATG.The analysis of bioinformatics showed that this fragment not only contained transcription core elements TATA box and CAAT box,but also some elements which can respond to abiotic stress and hormones.Abiotic stress responsive elements included 31 dehydration response elements and 23 temperature-responsive elements.Hormones responsive elements included GA,ABA and others.Meanwhile,two important MYB transcription factor binding elements were found which regulate phenylpropanoid and lignin biosynthesis.2.Promoter BpGT14 gene activity analysispBpGT14::GUS plant expression vector was constructed and transformed tobacco.It proved that the promoter had high promoter activity in stem segments.When the tobacco was treated with GA,H202 and 4*C,promoter had no significant response and enzyme activity was downward trend.In contrast,promoter activity was significantly increased by ABA,NaCl and PEG treatment.Further transformation of birch suspension cells indicated that the promoter had a similar response pattern to that of tobacco treated with abiotic stress and hormone.3.Candidate proteins interaction with promoter fragment screened through Y1HPromoter 1156bp element was chosen to analyze its interaction protein through yeast one-hybrid.WRKY1 and Auxin response transcription factor were screened and named BpWRKYl and BpARF2 gene.BpARF2 transcription factor included special domain and its function was related to auxin response.The promoter 1156bp sequence had three TGTCTC motifs which was the binding DNA sequence of BpARF2.The result of RT-PCR indicated that the BpARF2 gene was respond to ABA which can improve the expression of target gene.4.Candidate proteins interaction with promoter MYBPLANT element screened through Y1HIn this study,MYBPLANT element was chosen to analyze its interaction protein through yeast one-hybrid.Multiple interacting candidate proteins were screened including protein glycosylation transcription factor,GPI protein and a couple of unknown function protein.Meanwhile,the study has screened a candidate protein containing PHD and DUF3594 domain belonging to Alfin-like gene family.Its DNA binding site PHD domain and functional site DUF3594 domain may work together to regulate the gene expression.5.The verification of interaction between birch Alfin-like protein and MYBPLANT elementThe study successfully constructed the Alfin-like gene pBI121 plant expression vector and MYBPLANT element reporter vector.The reporter gene expression of co-transformed Alfin-like gene and MYBPLANT element in tobacco indicated that there may be interaction between these two parts.The expression of co-transformed plants was twice reached that of individual transformed MYBPLANT element plants.Stress treatments result determined that birch Alfin-like gene was respond to 4℃ and ABA,which indicated that the gene might be related to adversity response.