Transformation And Expression Of A Modified Brazzein Gene In Plant And E.coil

Sweet-tasting proteins, found in the tropical plants, have some merits: intense sweetening profile, low-calorie, not arousing tooth decay and can be used in diabetic foods. Seven proteins have been proved to be sweet-tasting proteins: thaumatin, monellin, mabinlin, brazzein, pentadin, curculin and miraculin. Brazzein was extracted from the fruit of Pentadinplandra brazzein Baillon. Of seven sweet-taste proteins, brazzein has smallest molecular mass, simply molecular structure and is most heat-stable.In order to make use of brazzein, we have studying on the expression of Brazzein gene in E. coli and plants: lettuce and tobacco. The results obtained are summarized below.1. PCR primer including mutative site at 5' terminal of Brazzein gene was designed and synthesized. Modified Brazzein gene was amplified successfully from the wild Brazzein gene by PCR. After digested by Nae I and BanH I , the gene was cloned into Pinpoint Xa vector, which was used for gene expression in E. coli. Recombinant vector was introduced into E. coli strain TG-1. Recombinant protein were induced by IPTG, analysized by SDS-PAGE. Four different concentration of IPTG and five different time course used for gene expression were adopted to produce recombinant protein and best products were obtained when the bacterium culture is induced with lOOmM IPTG for 5 hours. Recombinant protein was detected by Western blot, and identified to include distinctive protein of modified Brazzein gene.2. The modified Brazzein gene was amplified successfully by PCR and cloned into pGEM-T easy vector first, then inserted into pBI221 vector and lead to 35S:mSB:Nos-ter construct. It was inserted into pBI221 vector again and resulted in a fusion of 35S:mSB:Nos-ter and 35S:GUS:Nos-ter. Finally this fusion wasinserted into the binary vector pBinplus. named pBGSB.Leader peptide gene was synthesized and fused with the modified Brazzein gene. The recombinant DNA named LSB was inserted into pBI221 vector and 35S:LmSB:Nos-ter was constructed. The construct was inserted into pBI221 vector again and a fusion of 35S:LmSB::Nos-ter and 35S:GUS:Nos-ter was generated. This fusion was finally inserted into the binary vector pBinplus, named pBGLSB.pBGSB and pBGLSB were introduced into Agrobacterium tumefaciens strain LBA4404 and GV3101 :pMD90 using triparental mating.3. Wounded cotyledons of four-day-old seedlings of lettuce (Lactuca saliva L.). as explants, were infected by Agrobacterium tumefaciens strain LBA4404 and GV3101:pMP90 harboring the expression vectors pBGSB and pBGLSB respectively and co-cultured for 3 days in darkness on the culture medium (MS+0.1 mg/L 6-BA+O.l mg/L NAA+0.7% agor pH 5.7). Explants were then transferred onto the selection medium containing 500mg/L carbenicillin and lOOmg/L Kanamycin and incubated at 25,16/8h light/dark cycle. Small leaves of adventitious shoots differentiated from explants were cut and dipped into GUS staining solution. Positive shoots, GUS tinted, were induced to root. Rooted plants were planted in vermiculite and soil. When plants were grown, DNA were extracted and transgenic lettuce plants were identified by PCR amplifying. Seeds of transgenic plants were obtained.4. Leaves of tobacco (Nicotiana tabacum) were wounded, infected by Agrobacterium tumefaciens strain LBA4404 and GV3101:pMP90 harboring expression vector pBGSB and pBGLSB and co-cultured for 3 days in darkness on the culture medium (MS+0.5mg/L 6-BA+0.7% agor pH 5.7). Explants were then transferred onto the selection medium containing 500mg/L carbenicillin and 150mg/L Kanamycin and incubated at 25, 16/8h light/dark cycle. Leaves of adventitious shoots were detected by GUS staining. Positive shoots were induced to root on medium (l/2MS-f-50mg/L Kan). Rooted plants were planted in vermiculite and soil. When plants were grown, DNA were extracted and transgenic plants were identified by PCR amplifying. Seeds of transgenic tobacco plants were obtained.