CDNA Library Construction And Molecular Cloning Of Cellulose Synthase Genes (PeCesA12 And PeCesA11) From Moso Bamboo

Moso bamboo (Phyllostachys edulis) is one of the most extensive distribution and highest economy value bamboo used for stuff and food. Bamboo grows very fast, and could be the ideal resource of cellulose material for development of biomass energy. The unique structural features of bamboo fiber make them have many superior properties. The exploitation and utilization of bamboo fiber and their excellent genetic resources, not only can solve the problem of quickly aging of post-havrest of bamboo shoot, but also can make great contribution to the development of biomass energy and fine fiber plant species. In order to investigate the mechanisms of rapid aging for post-havrest of bamboo shoot and biosynthsis for bamboo fiber, a cDNA library for the bamboo shoots of Phyllostachys edulis was constructed and two genes involved in cellulose synthesis were isolated by PCR screening method in this study. In subsequent study, the expression pattens of the two genes were carried out by using quantitative Real Time PCR method. Finally, the relationship between expression pattens of the genes and the cellulose contents at different tissues of the bamboo were investigated. The results are as follows:A full-length cDNA library for the shoots of Moso bamboo was constructed by using Gateway technique. The quality detection indicated that the library had a high title of 1.7×10⁶ cfu mL^-1 and the total clones were 8.5×10⁶ cfu. It also had a high ratio of recombination (up to 95%) with the insert at an average size of approximately 1.5kb. These results showed that the library was high quality, which is suitable for the isolation of target genes and provide an important foundation for the studies on the molecular mechanisms involved in cellulose synthesis of plant.Two probable cellulose synthase A catalytic subunit genes with full-length ORF sequence were acquired using rapid and effective PCR screening method. Multiple alignment analyses showed that the two genes of Moso bamboo shared high similarity (over 85%) with the CesA genes from Bambusa oldhamii, Oryza sativa, Triticum aestivum and Hordeum vulgare. Further analyses indicated that they contained highly conserved Zinc finger domains CXXC motif, transmembrance domains and conserved QXXRW domains.The expression profile of PeCesA11 and PeCesA12 was analyzed by using quantitative Real Time PCR analyses. The results showed that the expression of two genes in the upper part of the bamboo shoots was very low, but it was higher in the base part. It was relatively lower in the leaves compared to the roots and stems.The cellulose contents showed that the upper part of bamboo shoots was 2.9%, the base part was 27.89%, root was 48.45%, stem was 46.45%, and leaf was 23.98%, respectively. The relationship between the expression of PeCesA11 and PeCesA12 and the cellulose content at different tissues were observed. The expression of PeCesA11 and PeCesA12 was elevated with the increase of cellulose content, which suggests that the two genes are involved in cellulose synthesis.Two cellulose synthesis genes were cloned in this study, which provides a theoretical foundation for understanding the way of cellulose synthesis as well as exploitation and utilization of bamboo fiber genetic resources, and has important scientific significance in forest cellulose genetic engineering projects and in the development of biomass energy.