| ORF1 and ORF2 cDNA from BYDV-GPV strain were introduced into a plasmid (pwubi.tml) containing the maize ubiquitin promoter sequence which drives high levels of expression in cereal cells successively. Plasmid DNAs were introduced into four Triticum aestivum cultivars Longjian 127, CA8686, Jingdong 8 and Yangmai 158 by pollen tube pathway and microprojectile bombardment. The resistance of some transgenic wheat plants to BYDV-GPV was detected in the fields. It was studied about the effects on the callus induction of mature embryos by three phytohormones. The optimal recipes to Longjian 127 and Shan 160 were established. IPTG-induced expression vector in Escherichia coli (E. coli) was constructed and used to express fusion proteins of ORF1 and ORF2 gene. The results showed that:The plant expression vector pPPI29 was constructed by the following series of steps using standard recombinant DNA techniques. Full-length ORF1 (BamHI and Kpnl sites were added to its upstream and downstream terminal) and ORF2 (KpnI and EcoRI sequence were introduced into its 5' terminal and 3' terminal) cDNA were amplified from RNA extract of BYDV-GPV strain and cloned into pGEM-T-Vector to produce pGEMORFl and pGEMORF2. Several bases of pWubi.tml was replaced by digestion with BamHl and Kpnl and ligation of the same terminal ORF1 cut from recombinant pGEMORFl to yield plasmid pWORFl. Then plasmid pPPI29 was constructed by digestion of pWORFl with Kpnl and EcoRI and ligation of the same terminal ORF2 cut from recombinant pGEMORF2. The results showed that ORF1 and ORF2 were directly inserted into pWubi.tml in series.1652 calluses derived from immature embryos of Yangmai 158 were bombarded with microprojectiles coated with DNA containing pPPI29 and pAHC20 using a PDS 1000/He gun. The bombardment was performed 9 days after embryo induction. Sixty-five resistant wheat plants resist to 1-3 mg bialaphos per litre were obtained and six transgenic lines were confirmed by dot-plot, PCR-Southern hybridization.The transformed frequency was 0.36% by the biolistic method.After excision of the stigmas, a drop of a plasmid DNA solution (containing 1 g pPPI29, 1g p35SNEO and 5g ssDNA in 101 solution) was delivered to the end of the styles of florets from Triticum aestivum cultivar Longjian 127, CA8686 and Jingdong 8. A total 2577 mature wheat seeds were collected from 3551 florets. The seedlings from harvested seeds were screened by dot-blot, PCR-Southernhybridization and resistance assay in fields. The results showed that six CA8686 and one Longjian 127 positive seedlings in TO transgenic wheat were confirmed by molecular analysis. No positive result was determined in the seedlings of transformed Jingdong 8. Resistance assay in the fields showed that some transgenic lines of CA8686 displayed higher resistance to virus infection. When the controls showed serious symptom, the seedlings of transgenic lines have no symptoms or only slightly yellowing on the leaf apex after inoculation for 23 days. A substantial delay in symptom development was shown on the one transgenic line of Longjian 127. When the seedlings of controls all showed yellowing, the seedlings of transgenic wheat displayed milder symptom.The callus induction of Longjian 127 and Shan 160 mature embryos was carried out with quadratic orthogonal rotation combination designs. The results showed that inositol played a leading role on the callus induction than 2, 4-D and Kinetin. The mathematic models were established to fit for each wheat cultivar. The optimal concentrations of the media for Longjian 127 were 2, 4-D 1.57-1.91mg/L, inositol 0.104-0.125g/L and kinetin 0.79-1.02mg/L under the 95% confidence limit, and for Shan 160 it were about 2, 4-D 2.08-2.40mg/L, inositol 0.099-0.112g/L and kinetin 0.933-1.267mg/L.The IPTG-inducible expression vector pETORF1 and pETORF2 were constructed with ORF1 and ORF2 of BYDV-GPV strain, respectively. The two plasmids were transferred into E. coli BL21 (DE3)-plysS and fusion proteins was preliminary expressed. The results showed that the spe...
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