Cloning And Functional Analysis Of UDPGDH Gene CDNA Of Ramie (Boehmeria Nivea)

Ramie (Boehmeria nivea) is one of the most important fiber crops in China. In textile industry, a pretreatment procedure of degumming is always required to get rid of the non-cellulose materials such as pectin, hemicellulose, lignin, et al. This pretreatment procedure, not only increases the cost, but also polutes the environment. Therefore, it is of great importance to investigate the molecular mechanism(s) of pectin, hemicellulose biosynthesis and improve the molecular composition of the fiber crops.Previous research results suggested that UDP-glucose dehydrogenase (UDPGDH) plays a critical role in the regulation of the biosynthesis of pectin and hemicellulose. In this study, the full length ramie UDPGDH cDNA was cloned. The cloned ramie cDNA was expressed in exogenous plants using transgenic technologies and the functions were analyzed. A in vitro ramie culture system was established whereas a genetic transformation technique was developed. These results will greatly facilitate the further study of the molecular breeding of ramie.The major research results presented in this study are briefly summarized as following:Based on the homology comparision of the known UDPGDH genes, a pair of degenerate PCR primers were designed. Using the cDNA synthesis from the total RNA isolated from ramie strain Xiangzhu No. 3, a fragment of ramie UDPGDH gene was amplified. The RACE procedure was carried out to obtain the full length ramie cDNA, the resulted cDNA contains 1837 bp (Gene Bank No. EF178294), which contains an open reading frame that encodes a protein of 480 amino acid residues. Bioinformatic analysis was performed on the obtained ramie full length UDPGDH cDNA and the deduced protein. The results demonstrated that the gene is highly homologous to the known plant UDPGDH genes.. The deduced protein contains all the functional domains of UDPGDH, the critical amino acid residues of the enzyme activity core and important modification sites are also presented in the deduced protein.The obtained ramie UDPGDH gene was cloned inton vectors pET32 and pMAL and expressed in E. coli. The expressed protein clearly demonstrated the UDPGDH enzyme activity in vitro. In combination with the bioinformatic analysis, it was concluded that the full length ramie UDPGDH cDNA was successfully cloned in this study.To investigate the relationship of the expression level of UDPGDH gene and the formation of fiber, the expression levels of UDPGDH in different ramie tissues were examined using semi-quantitative RT-PCR. The results suggested that in ramie, UDPGDH gene is expressed in stem, phloem, leaf, and root. The expression level of UDPGDH gene in ramie decreases in different tissues in the sequence decribed above.The core region of UDPGDH was cloned into the binary expression vectors (pFGC5941, pWM101) and transformed into tobacco via leaf disc infection with Agrobacterium tumefaciens system. The result demonstrated that UDPGDH gene was successfully integrated into tobacco genome. Tissue analysis indicated that three expression vectors in the tobacco may effect hemicelluloses and pectin synthesis. The cells in cross-section of leaf and stem become swollen and intercellular space of the ground tissue increass when the expression of UDPGDH gene was inhibited in tobacco. When UDPGDH gene was over expresed, opposite physiological changes were observed. These results further suggested that UDPGDH gene may play an important role in the synthesis of pectin and hemicelluloses in ramie.Using ramie leaves as explant, a in vitro culture system of ramie was established. Xiang Zhu No. 3 in MS supplemented with TDZ 0.3 mg/L + 2,4-D 0.01-0.03 mg/L, Cheng Bu Qing Ma in MS with TDZ 0.45 mg/L + 2,4-D 0.03 mg/L, Xiang Zhu No. 2 in MS with TDZ 0.15 mg/L + 2,4-D 0.03 mg/L, they formed the fine callus on the hormone combinations. Induced high frequency shoots of Xiang Zhu No. 3 in MS with TDZ 1 mg/L + 2,4-D 0.1 mg/L, and the Cheng Bu Qing Ma in MS with TDZ 0.5 mg/L + 2,4-D 0.3 mg/L, Zhu Xiang No. 2 in MS with TDZ 0.75 mg/L + 2,4-D0.3 mg/L. PCR positive plants were obtained with leaves disk transformation. The further molecular breeding research of ramie might be carried out based on the results presented in this work.