Cloning And Expression Analysis Of The Genes Involved In The Lignin Synthesization Of Bambusa Multiplex (lour.) Raeuschel

Bambusa multiplex (Lour.)Raeuschel belongs to Bambusoideae subfamily in Gramineae family.China is one of the most abundant bamboo resource countries in the world.and it has the largest cultivation area and highest values of usage in China. Because of its fast-growing and high cellulose content characteristics ,Bambusa multiplex (Lour.)Raeuschel is a good material in paper pulping.However,due to the high level of lignin content in Ph.edulis,a large quantity of chemicals must be used to separate fiber from lignin,.Then,fiber is used in papermaking industry. Moreover,the chemicals for separation have greatly increased the cost of paper making process and have caused serious environmental pollution. Therefore, the most important is how to breed new varieties to meet the making paper.By using of bio-engineering measures, without affecting the growth of bamboo,change the content of lignin or change its pulp performance to cultivat the bamboo suitable for the bamboo board processing have become an important research project in current biotechnological field.4CL is one of the key enzymes in the phenylpropanoid pathway and has significant effects on the lignin content of plant.COMT,CCoAOMT and CAD are another three enzymes in the lignin synthesization pathway. They also plays an important role in lignin synthesis.The cDNA of 4CL,COMT,CCoAOMT,CAD genes were cloned from cDNA prepared from bamboo shoot of Bambusa multiplex (Lour.)Raeuschel using RT-PCR and RACE methods.Its nucleotide sequences and the encoded amino acid sequences were analyzed. Tissue specific expression showed that CAD expressed in leaf,sheath,young stem and bamboo shoots,much higher in bamboo shoots.The results were summarized as follows:(1) The RNA was isolated respectively from Bambusa multiplex (Lour.)Raeuschel shoot by kit and Trizol methods.The result showed that the quality was good enough for further research.(2) A gene of CAD family was cloned by RT-PCR and SMART RACE RT-PCR from the shoots of Bambusa multiplex (Lour.)Raeuschel(GenBank accession number: GU985522). The CAD of it ,with the cDNA length of 1131 bp,contained an openreading frame of 1107 bp which encoded a polypeptide of 368 amino acids with predicted molecular mass of 39 kD.The PI of it is 6.06.(3) The results of sequence analysis showed that the deduced polypeptide contained Alcohol dehydrogenase GroES-like domain which is in 34-149 and Zinc-binding dehydrogenase which is in the 191-315 in the conserved sequences of the CAD family .It was highly homologous to the CAD proteins from O.sativa and Zea mays and its identity to CAD proteins from O.sativa was up to 97.0%.The phylogenetic tree analysis also indiated that CAD was more related to the CAD in O.sativa.(4) Tissue specific expression showed that CAD expressed in leaf,sheath,young stem and bamboo shoots,much higher in bamboo shoots.All the study will provide a theoretical basis for further studying molecular regulation mechanism of the CAD gene and for taking advantage of its function by gene engineering technique.(5) A 278 bp DNA fragment of 4CL,a 429 bp DNA fragment of COMT,and a 257 bp DNA fragment of CAD were cloned from the genomic DNA of Bambusa multiplex (Lour.)Raeuschel which encoded 92 amino acids,282 amino acids and 85 amino acids respectively.The 4CL,CCoAOMTand COMT DNA fragments in Bambusa multiplex (Lour.)Raeuschel shared 90%homology with that of Oryza sativa and Zea mays . GenBank accession numbers were FJ787494,FJ790329 and HM245382 respectively.(6) Utilizing vector pE-SUMO to construct expression vector pE-SUMO-CAD thro- ugh the expression vector induced to express, the result showed that the protein m- olecular of this expressive gene was 39kDa by analysis of SDS-PAGE and its exp- ression products were mostly composed of inclusion body.(7) The pCAMBIA1301-CAD plant expression vector were constructed.It has provided theoretical foundation for the more research of transforming other plants .In sum, the paper had succeeded to clone 4CL,CCoAOMT,COMT and CAD genes in lignin from Bambusa multiplex (Lour.)Raeuschel.The results of the four sequences and amino acids ware analysised by different methods .The paper had succeeded to construct prokaryotic expression vector pE-SUMO-CAD, optimize the induced expressive parameter of project bacterium . The paper had succeeded to construct plant expression vector pCAMBIA1301-CAD.These results had provided theoretical foundation for the more research of lignin in Bambusa multiplex (Lour.)Raeuschel.