Cloning Of CDNA Encoding UGPase And 4CL From Amorpha Fruticos And Expression In Transgenic Plants

The two most abundant components of the wood fiber are cellulose and lignin. While the cellulose is the valuable component for the pulp and paper industry, removal of the lignin is the most energy intensive and environmentally damaging step of the pulping process. Ideally we would like to identify and propagate a tree that produces a maximal amount of wood with a reduced lignin content and increased cellulose content by biotechnology.A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGPase), a key enzyme producing UDP-glucose in the synthesis of sucrose and cellulose, was cloned by using this method. Another Amorpha fruticosa cDNA clone encoding 4-coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned ,too.A full-length UGPase cDNA was obtained. The size of the cDNA fragment is 1832 bp, which included an 1416 bp open reading frame and encoded a peptide of 471 amino acids. The deduced amino acid sequence exhibits significant homology with the UGP genes cloned from other plants.A full-length 4CL cDNA was obtained. The size of the cDNA fragment is 1911 bp, which included an 1623 bp open reading frame and encoded a peptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins.The sense expression plasmid of UGPA and the antisense expression plasmid of 4CL gene were constructed with the target gene trans-inserted into plant expression vector pCAMBIA1301 respectively. Transgenic tobaccoand poplar plants were obtained by regeneration of agrobacterium-mediated transformed leaves. PCR and Southern blot analyses confirmed the integration of target gene in transformed tobacco genome. We evaluated lignin content severely altered in the expression of transgenic tobacco with pCA4CL antisense constructs and pCAUGPA constructs. The Klason lignin level of anti-4CL-transformed tobacco was much lower than that of the control. The cellulose content increased in transgenic tobacco with UGPA constructs.We also obtain transgenic tobacco transformed with Acetobacter xylinum UGPase gene. Compared the lignin and cellulose level of these two kinds of transgenic tobacco, we found that the cellulose content increased and the lignin content deduced in transgenic tobacco transformed with Amorpha fruticosa UGPA constructs, both the cellulose content and the lignin content increased in transgenic tobacco transformed with Acetobacter xylinum UGPA constructs.