Cloning And Expression Analysis Of Cellulose Synthase In Ramie And Transgenic Vectors Construction

Cellulose is the most abundant biological macromolecules in nature. With the development of society, the demand for cellulose has increased phenomenally. However, the mechanism of cellulose biosynthesis has been poorly understood. Ramie [Boehmeria nivea (Linn.) Gaud.] is an important textile-based crop. It encounters the bottleneck states of the study on increase the production and quality of ramie fiber. So it is important to research the molecular mechanism of cellulose biosynthesis, and it is necessary for increase production and quality of ramie fiber based on molecular level.Three ramie cellulose synthase genes were cloned from cDNA of XiangZhu3ramie, and their expression pattern was analysised, and overexpression vector or RNAi expression vector were constructed. The main results of this study are as follows:1.According to the known sequence of BnCesAl(DQ077190),5’RACE PCR groove primers were designed to amplify the5’terminal fragment of BnCesAl using cDNA from ramie selection "Xiangzhu3". Then the gene sequence was assembled. The assembled cDNA of BnCesAl is3891bp in length, including183bp of5’ UTR and459bp of3’ UTR. The coding region is3246bp, encoding a deduced aminoacid sequence of1082amino acid residues. And the complete coding sequence of BnCesAl was cloned from cDNA of "Xiangzhu3".2. The sequence alignment showed that an unigene sequence is highly similar with plant cellulose synthase gene. The unigene expands2438bp, containing incomplete protein coding region. According to the known fragment, the5’and3’RACE PCR groove primers were designed, and a761bp5’ terminal sequence and a740bp3’terminal sequence were amplified separately. The full length cDNA sequence of3849bp was assembled. Then bioinformatics analysis was performed. And the results showed that this gene has an coding region of3237bp, It codes a protein of1079amino acids which weighs120.7KD, and whose isoelectric point is pH6.63. The sequence shared86.5%homology with AtCesA3, but less than69.8%with other AtCesAs, therefore it is identified as BnCesA3gene.3. There was another unigene sequence which is highly similar with plant cellulose synthase gene and expands1711bp with the start codon. The nucleotide sequence and amino acid sequence shared60.7%and75.8%homology respectively with AtCesA6, so it refered as BnCesA6.A1539bp-sequence of it was cloned. 4. Semi-quantitative RT-PCR analysis indicated that the three BnCesAs expressed in bark of four varieties tested ramie. And the transcripts level in the bark is significantly different among varieties of ramie.The expression level of three BnCesAs genes in different tissues from high to low is bark, leaf, top stem and root in Xiangzhu3ramie variation.5. Overexpression vectors of BnCesA1and BnCesA3, RNAi vectors of BnCesAl and BnCesA6were constructed and transferred into Agrobacterium EHA105successfully, which provide a good foundation for gene function study.