Cloning And Gentic Transformation Of Bhlh Transcription Factor Gene In Populus Xiaohei T. S.Hwang Et Liang

In this study Populus xiaohei was used to be materials. We cloned CDS and promoter sequence of the two bHLH （basic helix-loop-helix） transcription factor genes, named PxbHLH01and PxbHLH02. In this paper we analyzed bioinformatics of the CDS sequence, amino acids sequence, protein and promoter of the two genes. The expression vector of plant pBI121-PxbHLH01/PxbHLH02was constructed to transformate explant of plants. Transgenic plants was acquired. Finally, according to the promoter sequences of PxbHLH01and PxbHLH02, we constructed pAbAi-PxbHLH01/PxbHLH02yeast one-hybrid bait vector. The study laid the foundation for the further study of the upstream transcriptional regulation mechanisms of PxbHLH01and PxbHLH02gene. The results were as follows:1Cloning and bioinformatics analyse of the CDS sequence of PxbHLH01and PxbHLH02of P. xiaoheiIn this research, we cloned PxbHLH01and PxbHLH02CDS sequences, which were411bp and417bp.The sequences had been uploaded to the （GenBank database accession number:KJ466370, KJ466371）. NCBI sequence alignment results showed that the consistency with the nucleotide sequence of the bHLH151（UPBEAT1, AT2G47270.1） gene in Arabidopsis were38.85%and40.71%; the consistency with the unknown function gene （XM₀02320932.1） in Populus trichocarpa were97.57%and82.73%; the consistency with the unknown function gene （XM₀02301486.1） in P. trichocarpa were82.38%and98.32%.Biological softwares were used to predict and analysis the function of PxbHLH01and PxbHLH02amino acid sequences. The three-dimensional structure model and phylogenetic trees of PxbHLH01and PxbHLH02were constructed by softwares.Populus Genome Integrative Explorer （PopGenIE）, The Arabidopsis Information Resource （TAIR） and other databases were used to analyse the expression of PxbHLH01and PxbHLH02homologous gene of P. trichocarpa and A. thdiana.2Optimized the tissue culture systems of P. xiaohei and obtained transgenic plantletsIn this study, the culture system of P. xiaohei had been optimized. The optimal condition of P. xiaohei tissue culture was1/2MS+NAA0.5mg/L+IBA0.05mg+L. The best way of regeneration of transgenic P. xiaohei was callus regeneration pathway and successfully obtained PxbHLH01/PxbHLH02overexpressing transgenic plants.PCR analysis showed that PxbHLH01/PxbHLH02gene was integrated in the genome of P. xiaohei. Under normal culture conditions, the non-transgenic plants showed that the leaves were green, full, and the taproots were more slender with more branch. Transgenic P. xiaohei in selected medium showed that the leaves were bright green, full, and the stems were thicker than those of wild-type plants. Compared with non-transgenic P. xiaohei, the transgenic shows that a significantly change of the root:root was very short, enlarged, and with fewer branches and with more root hairs.3Research of the upstream regulatory pathway of PxbHLH01and PxbHLH02geneIn this study, the promoter sequences of PxbHLH01and PxbHLH02gene were obtained, which were1360bp and1913bp, respectively. Sequence alignment showed that the consistency with unknown function genes （XM₀02320932.1） promoter in P. trichocarpa were95.17%and68.91%; Sequence alignment showed that the consistency with unknown function genes （XM₀02301486.1） promoter in P. trichocarpa were69.46%and96.25%.Analysis of cis-acting element showed that the promoter sequence had the essential components to start transcription. Meanwhile, there were a number of cis-acting elements in the promoters, the function of these elements were stress control, hormone regulation, light regulation and metabolism. And there were also a lot of anti-stress and regulatory elements. It is worthwhile to note that the promoter sequences contained a lot of oxygen response element （CURECORECR）.