Construction Of The Expression Interference Vector Of Ramie CCoAOMT Gene And Its Transformation

Caffeoyl-CoA 3-O-methyltransferase(CCoAOMT) plays a key role in lignin biosynthesis in plant.In our previous research,the cDNA sequence of ramie CCoAOMT has been cloned and studied.In this paper,the gene cDNA is cloned into a plant expression interference vector,pFGC5941,as an RNAi recombinant.The recombinant is transformed into Tobacco(Nicotiana tabacurn) "WS38",Flax(Linum usitutissimum L.) and Ramie (Boehmeria nivea) via Agrobacteriurn turnefaciens mediated transformation.It is expected the expression of this interference gene to reduce the activity of CCoAOMT in ramie and flax so as to reduce lignin content in cortex of the crops and improve the fiber quality.The Ramie CCoAOMT cDNA conservative sequence of 624 bp was inserted into pFGC5941 two clone sites border to CHS intron in a reverse direction.Thus the plasmid pFGC5941-BnCCoAOMTi is constructed the gene is controlled by 35S promoter and it can form a 624 bp dsRNA after splicing.It will be the interference molecule to CCoAOMT in the transgenics.For testing the interference fuction of the recombinant the molecule is first transformed into the Tobacco(Nicotiana tabacum) "WS38",a model variety for lignin research,by Agrobacteriurn turnefaciens mediated transformation.Several transgenics obtained and the transformed plantlets show slower growth and one of them shows a willowy stem.In both staining of petiole slice and chemical analysis,an obviously decline of lignin content is detected.Whereas flax shows a higher PPT resistant which is the selection marker(Bar~r) for pFGC5941 transformation.We reconstruct the vector with hygromycin as selection marker (Hyg~r) to replace the PPT resistance(Bar~r).A modified recombinant interference vector (pFH-BnCCoAOMT) was constructed and used to transform flax hypocotyls.The regenerated transgenic flax seedlings were obtained after hygromycin screening.The phenotype of the transgenic are roughly the same as the control on the characters of growing form and configuration while the biopsy of stem straining confirmed that the transgenic flax content less lignin than non-transgenic partner. Based on the study above,we tried the ramie transformation with BnCCoAOMT RNAi.Some PPT tolerance ramie calluses are obtained.But it is difficult to make it differentiation so we did not get transgenic seedling yet.But our researches give an amazing result that the RNAi of BnCCoAOMT can effectively inhibit the CCoAOMT activity in transgenic tobacco and flax and show a reduced lignin contents.Much experience have been extracted from this study to benefit our research on Ramie gene engineering to reduce its lignin contents or improve components of the lignin monopoly for a better fiber quality.