Effects Of AtUGAE4 Antisense Gene On The Intercellular Adhesion In Vitro Of Tomato, Arabidopsis And Tobacco

Plant cell suspension culture, including the best suspension culture systems in tobacco and Arabidopsis,are mainly composed of cell clusters . It is an ideal target to construct single cell suspension culture system. It has great value in areas of cell biology research,mutated cell strain selection,mass cell culture, functional genes analysis,genetic engineering,genetic by usually providing single cells with constant background and physiological state.But single cells are hard to get. Pectin is the main substance that cause cell adhesion under cell suspension culture.It might create a new single cell suspension culture system,providing plant pectin biosynthesis is already functionally inhibited.Pectins are structurally complex plant cell-wall polysaccharides that mainly exist in fruit and plant primary cell walls. They take part in cell adhesion. Pectins mainly exist in intercellular layer,making the cells connected with each other. According to Orellana's research, GalA account for 20%-35% of sugar residue of plant intercellular layer polysaccharides,mainly acting as a major sugar residue of plant pectic polysaccharides. So,the enzymes involed in GalA activation should be important in pectin biosynthesis.Many research results have pointed that UDP-GlcA 4-epimerase (UGlcAE) is the key enzyme which catalyzes the epimerization of UDP-α-D-glucuronic acid (UDP-GlcA) to UDP-α-D-galacturonic acid (UDP-GalA).The amount of UDP-GalA may be reduced by using antisense RNA technology to inhibit expression of UDP-GlcA-4-epimerase gene. Antisense RNA is refers to RNA fragments which are complementary to specific mRNA.It can code or be translated to certain proteins.The sequences of antisense RNA are complementary to certain mRNA,when they connect,they can prevent mRNA being translated to proteins,even the gene is active. Thereby antisense RNA can inhibit gene expression.We cloned the conserved sequence of AtUGAE gene family,and constructed AtUGAE4 gene antisense expression vector,and then transferred it into Agrobactrtium EHA105 by freeze-thaw method.Then used it in genic transformation in tomato, Arabidopsis and tobacco.We got more than 20 transformed tobacco plants,lots of tomato callus and Arabidopsis callus.We hope the antisense expression vector will express in transformed plants,produce mRNA fragments complementary to that of AtUGAE4,and then inhibit the mRNA activity of AtUGAE4 and even the whole gene family. Major research efforts include the following:1. Establishment of suspension culture system of Arabidopsis rootIn this experiment we need lots of Arabidopsis roots. Arabidopsis seeds were germinated in 1/2 MS solid medium.Prepare MS liquid medium supplemented with different concentration of NAA:0 mg/L, 0.05 mg/L,0.1 mg/L,0.2 mg/L,100mL flask with 15mL former MS liquid medium,then sterile them. Chose 20days old seedlings,and cut the over-long roots, leaving 1cm.Put them in flasks with the MS medium above and cultivate.The results showed that 0.05 mg/L NAA was an appropriate selective concentration for Arabidopsis root regeneration. Under this concentration,the amount and length of roots are much higher than that of other concentration.2. Cloning and sequencing analysis of conserved sequence of AtUGAE4 gene family and construction of the antisense expression vectorTotal DNA was isolated from Arabidopsis.The AtUGAE4 gene was amplified by PCR.The PCR products were purified and inserted into pMD18-T vector.The sequencing result showed that one was cloned wrong.The recombinant vector was double cut by Xba I /Sac I ,left AtUGAE4 fragment; pSAU2008 was also double cut by Xba I/Sac I ,left 13481bp fragment. Ligase the two fragments,then we got the antisense expression vector pB+35SrUGAE4 with 35S-reAtUGAE-Tnos antisense gene expression box..The PCR amplification and enzyme digest analysis indicated that the expression vector contained the expected fragment.Then transfer it into Agrobacterium EHA 105 by free-thaw method and used in genic transformation.3. Antisense AtUGAE4 gene transfered into tomato,ArabidopsisAntisense AtUGAE4 gene was transfered into tomato,Arabidopsis.Use a 10×16 microscope to observe the callus orinated from transferred explants. The results showed that callus from transformed explants are tight,and had no apparent differences with that of the untransformed explants .Antisense AtUGAE4 had no apparent effectd in inhibiting pectin biosynthesis in tomato and Arabidopsis.4. Identify the function of antisense AtUGAE4 gene in tobaccoThe tobacco explants were infected with Agrobacterium EHA 105 harboring a plasmid vector pB+35SrUGAE4 with AtUGAE4 gene.The target gene AtUGAE4 was amplified by PCR.The expected amplified product appeared; AtUGAE4 segment aroud 700bp;the non-transgenic plant had no band.The result indicated the gene was transferred into tobacco successfully. We got the regenerated plants,and it indicated that antisense AtUGAE4 had no apparent effects in inhibiting pectin biosynthesis in tobacco.