Transformation Of Tobacco And Arabidopsis With AtUGAE4 Antisense Gene And Its Effects On The Intercellular Adhesion

The single cell suspension culture of plant could provide separate cells in the same genetic background and physiological state. Therefore, it has great value in the research of cell biology, mutated cell strain screening, mass cell culturing, functional genes analysising and genetic engineering.However, as the intercellular adhesion is very difficult to eliminate, we can not obtain the single cell of plant at this point. In the growing plant cells, wall is mainly made up of cellulose, hemicellulose, pectin and few structure proteins. UDP-α-D-galacturonic acid is the main precursor for plant and bacteria synthesizing polysaccharide component of cell surface. Thereby, the enzyme which participates in the process of activating GalA may play an important role in regulating the biosynthesis of pectin. UDP-GlcA 4-epimerase, namely UGAE is the key enzyme which catalyzes UDP-α-D-glucuronic acid (UDP-GlcA) transform to UDP-α-D-galacturonic acid (UDP-GalA). So, if we can find a way to control the expression of UDP-GlcA 4-epimerase gene in the plant, it is possible to inhibit the biosynthesis of pectin and eliminate the intercellular adhesion.This study used the conserved sequence of AtUGAE gene family, which was cloned from AtUGAE4 to construct antisense gene expression vector, and transformed it into agrobacterium via freeze-thaw method. After transformed tobacco and arabidopsis media by Agrobacterium, we obtained 4 transgenic tobacco plants and a lot of arabidopsis callus. We hope the antisense expression vector will express in the transformed plants, produce antisense RNA fragments to pair up with mRNA of AtUGAE4, and then inhibit the expression of AtUGAE4 and even the whole gene family. The major research achievement as following: 1. Construction of antisense AtUGAE4 gene expression vectorDigested clone vector pMD-UGAE4 and pV-HSP70bGUS by Xba I and Sac I, then collected AtUGAE4 fragment and big fragment of pV-HSP70bGUS plasmid. Ligased the two fragments, then we got the antisense expression vector pV-70bUGAE4 which contains a HSP70b-reAtUGAE antisense gene expression box. The PCR amplification and enzyme digestion analysis indicated that the expression vector contains the expected fragment. Then we transformed it into Agrobacterium EHA 105 by free-thaw method for the following steps of gene transformation.2. Antisense AtUGAE4 gene transformed tobacco and arabidopsisWe transformed Antisense AtUGAE4 gene into Tobacco and Arabidopsis respectively, and obtained 4 transgenic tobacco plants and a lot of Arabidopsis callus. The target band can be found in the PCR products of transgenic tobacco and arabidopsis through electrophoresis, but can not be found in the non-transgenic plants, this phenomenon indicates that the foreign gene has been integrated into genomes of tobacco and arabidopsis. In the roots of tobacco and callus of arabidopsis which have kanamycin resistance can be found green fluorescence when activated by 395nm light, it proves GFP gene and concatenate HSP70b-reAtUGAE4 antisense gene have integrated into genomes of tobacco and arabidopsis.3.Analysis of the function of antisense AtUGAE4 gene in transgenetic tobaccoHaving been transformed, transgenetic tobacco is hard to regenerate, most of regenerative buds developd as misshapen seedlings, we only obtained 4 transgenic plants. Heat activate in 37℃for 36 hours, no shape change. We hypothesize AtUGAE4 antisense gene inhibited pectin synthesis in tobacco and brought handicap of wall synthesis, then led tobacco dead or misshapen. Multicopy of AtUGAE4 antisense gene might be transformed into the transgenic tobacco plants, co-inhibition effect decreases the interference effect of antisense RNA.4. Analysis of the function of antisense AtUGAE4 gene in callus of transgenetic arabidopsisHeat activate both transformed callus and wild type callus of arabidopsis in 37℃for 36 hours, and then we observed the edge of arabidopsis callus via a 10×40 microscope, it showed that compared to non-transgenic arabidopsis, callus from transformed explants are more incompact,and some single cells and cell clusters appeared. It proves that expression of antisense AtUGAE4 gene can eliminate intercellular adhesion in arabidopsis.