Cloning And Sequence Analysis Of The PtADC Promoter

At present, the bourgeoning genetic engineering gains popularity in genetic improvement. Promoter plays a crucial role in regulating gene’s temporal and spatial expression. Thus, it’s important to isolate stress-responsive promoters and analyze their functional mechanism, which will pave the way for the identification of stress-responsive transcription factors and set the foundation for stress tolerance improvement of citrus genetically. PtADC which is a key gene in polyamine biosynthesis is responsive to dought and low temperature.The main purpose of this research were the isolation of the promoter of PtADC, and the analysis of the cis-acting elements in it, which will pave the way for identification of transfactor corresponding to the cis-element and better understanding of stress regulation mechaniasm of ADC. The main results are as following:1. Based on the known cDNA sequence, PtADC DNA sequence of coding region was amplified from genomic DNA, which was the same as the cDNA sequence. This indicated that PtADC gene does not contain introns.2. Based on the known nucleotide sequences, primers for specific amplification were designed and 2100 bp 5’ upstream sequence was isolated via four rounds of chromosome walking by Adaptor-PCR and TAIL-PCR. Then PCR and bioinformatics analysis were carried out to verify that the 2100 bp sequence was exactly the 5’ upstream sequence of PtADC.3. Bioinformatics analysis of the upstream region of PtADC via the PLACE and PlantCarire online software predicted several putative stress response cis-elements, namely 2 LTRs,16 MYCs,8 MYBs,1 ABREs,6 G1-Ts,7 W-boxs and so on.4. Four deletions were designed. The full-length promoter and four deletions were inserted into plasmid pBI121 to produce Pg-GUS, Del1-GUS, De12-GUS, De13-GUS, and De14-GUS. Furthermore, the full-length promoter was also fused with GFP. These constructs were transformed into tobacco (Nicotiana nudicaulis) by leaf-discs method. The transgenic tobaccos were identified by PCR. Then T1 plants were obtained.