The CDNA Cloning And Expression Analysis Of Cellulose Synthase A Gene Family Of Boehmeria Nivea

Ramie is an important stem fiber perennial root crop planted in China. It is also applicable in soil and water conservation. Its industrial use as biofuel material is getting more attractive in recent years. The ramie stem fiber is over27-268mm in length be regarded as the longest fiber with high quality in the plant kingdom. The development process of ramie stem fiber is different from cotton seed coat fiber and the wood fiber in woods. The molecular approach of its development mechanism and composition accumulation or metabolism has profound theoretical significance and practical application value.Based on the ramie transcriptome data previous obtained we run the local BLAST comparison with cellulose synthase gene cDNA of high plants as the probe that are documented in the GenBank. High homology sequences of cellulose synthase gene cDNA of ramie were identified from the transcriptome database. Several pairs of specific primers were designed to clone these fragments by core sequences PCR amplification and then followed with3’RACA and5’RACE. Finally several full-length cDNA sequences were obtained of four ramie cellulose synthases be nominalted as BnCesA2, BnCesA3, BnCesA4, BnCesA5and two partial sequence of BnCesA6, BnCesA7are cloned and sequenced. The all cloned cDNA sequences were confirmed as ramie synthase genes by online GenBank BLAST and analysis of its conserved domains.A phylogenetic tree was constructed with the cloned sequence and most of the CesA sequences of a variety of high plants that were registered in Genbank. It is predicated that the BnCesA2and BnCesA5maybe involved in primary cell wall cellulose biosynthesis, and BnCesA3, BnCesA4maybe involved in secondary cell wall cellulose biosynthesis.The gene expression of the four ramie cellulose synthases BnCesA2, BnCesA3, BnCesA4, BnCesA5were quantitative in phloem and core stick tissues of ramie stems in different stages. The Fluorescence quantitative primers were designed in the hypervariable regions of those genes cDNA respectively and the Real-time quantitative RT-PCR was conducted with plant actin gene Actin1as molecular internal reference. The results showed that the four ramie cellulose synthases gene are expression in phloem and core stick of ramie stems during all the growth stage whereas some differences existed in expression quantity. Thus we conclude that the four cellulose synthase genes are all involved in the synthesis of ramie phloem fiber but there is no evidence that anyone expression is restricted in the phloem.