Analysis Of WND1B Promoter From Populus Trichocarpa

The secondary growth is a significant biological activity for plant development, and the secondary xylem plays an important role for human life. NAC transcription factors are the new transcription regulatory factors discovered in recent years, which are unique to plants. It was reported that NAC transcription factors play important roles in multiple biological functions such as development, responses to stresses and improving the quality of crops. SND1, a NAC domain transcription factor that found in Arabidopsis thaliana, is a key regulator that regulates the secondary wall biosynthesis in fibers. PtrWNDs, NAC transcription factors from Populus trichocarpa may start the entire secondary wall biosynthesis. And PtrWND1B was a homologous to SND1. Here we carried out a number of analysis in the promoter of PtrWND1B and have reached the flowing conclusion:1. PtrWND1B promoter cloning. Based on the sequence of PtrWND1B, The5' regulatory region of WNDIB was clones by PCR. Its full length is2146bp. The sequence was cloned into pMD-19T vector and transformed into E.coli Top10. Result of sequence alignment confirmed the fragment products as the PtrWND1B promoter, which was abbreviated to PtrWND1BP. The core configurations and several sequences similar to eukaryotic cis-regulatory elements were found in this promoter region.2. Truncated sequences of PtrWND1B promoter cloning. Based on the sequence of PtrWNDIBP, four truncated sequences of WNDIB promoter were generated. The sequence was cloned into pMD-19T vector and transformed into E.coli Top10. Result of sequence alignment confirmed the fragment products as the truncated sequences of PtrWND1B promoter, which named as PtrWND1BP-F2, PtrWND1BP-F3, PtrWND1BP-F4and PtrWND1BP-F5.3. Transient Expression of PtrWND1B promoter and truncated sequences. Five pairs of primer were designed and the expression vectors pCAMBIA1301POG-1BP/1BP-F2/1BP-F3/1BP-F4/1BP-F5-GUS were constructed. Transient expression analysis was carried out in leaves of Nicotiana tabacum. GUS activity determination revealed that PtrWNDIBP, PtrWND1BP-F3, PtrWND1BP-F4and PtrWND1BP-F5could drive expression of GUS.4. The expression of PtrWND1B promoter and truncated sequences in Arabidopsis thaliana. The expression vectors pCAMBIA1301POG-1BP/1BP-F2/1BP-F3/1BP-F4/1BP-F5-GUS were expressed in Arabidopsis thaliana. GUS activity determination revealed that the gene activated by PtrWND1B promoter expressed specifically in vascular, and PtrWND IBP and truncated sequences could drive expression of the GUS. And we also find the mini promoter of PtrWND1B.5. Activity comparison of PtrWND1B promoter and truncated sequences. pCAMBIA1301POG-1BP/1BP-F2/1BP-F3/1BP-F4/1BP-F5-GUS were expressed in leaves of Nicoti-ana tabacum. Activity determination of GUS protein was carried out in order to compare the activity of PtrWND1BP, PtrWND1BP-F2and PtrWND1B-F3with the control. Results showed that PtrWND1BP and PtrWND1BP-F3was active, and the fragment deleted from PtrWND1BP-F2had significant impact on promoter activity.