Isolation Of CDNAs Encoding Enzymes Involved In Lignin Biosynthesis And Obtainment Of Transgenic Poplars

Lignin is one of the major secondary products in plants with important biological functions,however which is the source of the pollution of papermaking industry. The purpose of this study is to regulate lignin biosynthesis and reduce the lignin content in the Chinese white poplar (Populus tomentosa) by antisense RNA, which is the important Chinese special local tree species and important papermaking resource tree species. Following is the major related progresses.1 Using RT-PCR technique, two cDNAs encoding caffeic acid O-methyltransferase (COMT) and caffeoyl Co-enzyme A O-methyltransferase (CCoAOMT) had been cloned from Chinese white poplar. Sequence analysis displayed that these two cDNAs shared high homologies with the corresponding cDNAs in the other species listed in the genus of Populus. The results of Northern dot hybridization showed that genes of COMT and CCoAOMT expressed with high level in the developing secondary xylem, and their expressional peak is coincident to the process of lignification. The two genes didn't expressed in the leaf and petiole. COMT and CCoAOMT are involved in the biosynthesis of lignin, the expressional characterization is consistent with their functions. The nucleic sequences of COMT and CCoAOMT gene cloned from P. tomentosa have been deposited in the GenBank with number of AF237777 for COMT and AF240466 for CCoAOMT 2 Through a series of DNA recombination, the antisense expression vectors containing antisense COMT and CCoAOMT cDNAs were constructed, respectively. The antisense expression vector integrating both COMT and CCoAOMT cDNAs was also constructed. They were confirmed by PCR and restriction analysis.3 The cultural system was established with shoot, leaf and young stem as explants from the field-growing triploid Chinese white poplar B19, B331 and B304. To our knowledge, it is the first time that we have obtained the cultural plantlets of triploid Chinese white poplar in vitro. It laid the foundation for the genetic engineering of triploid Chinese white poplar.4 Antisense CCoAOMT cDNA had been transformed into hybrid poplar (P. tremula×P. alba). The transgenic plants were identified by PCR, PCR-Southern and Southern analysis. Probed with Digoxigenin-lablelled single RNA strand corresponding to antisense cDNA strand, Northern dot analysis of total RNAs extracted from transgenic hybrid poplar displayed that the antisense CCoAOMT cDNA integrated in genomic DNA of transgenic plants had expressed.