【作者】 刘太国；

【导师】 成卓敏；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2003， 博士

【摘要】 本文将人工合成的BYDV-GPV株系复制酶基因ORF1和ORF2 cDNA片段正向串联到了植物表达载体pWubi.tm1质粒上，成功地构建了植物表达载体pPPI29，利用花粉管通道法和基因枪法分别转化了普通小麦品种陇鉴127、CA8686、京冬8号和扬麦158，并进行了部分转基因小麦的抗病性鉴定；探讨了植物激素对成熟胚愈伤组织诱导的影响，获得了较高成熟胚愈伤组织的优化培养基配方：并对ORF1和ORF2基因进行了原核表达。 研究结果为： 根据已经测定的BYDV-GPV部分序列，设计并合成ORF1和ORF2相应的上、下游引物，在引物中分别引入BamHI、KpnI和EcoRI位点，使得ORF1 5′端BamHI、3′端含有KpnI位点，ORF2基因5′端含有KpnI、3′端含有EcoRI位点，以BYDV-GPV株系病毒RNA为模板，RT-PCR分别扩增了GPV株系的ORF1和ORF2基因片段，并将其分别插入到pGEM-T-vector载体中，获得重组质粒pGEMORF1和pGEMORF2；将pGEMORF1和含有植物特异性表达启动子ubiquitin和终止子tm1的pWubi.tm1用BamHI和KpnI双酶切、回收，使回收的pWubi.tm1和ORF1片段连接成重组质粒pWORF1；然后用KynI和EcoRI双酶切pWORF1和pGEMORF2，并回收pWORF1（7.553kb）和ORF2（1.875kb），同样进行连接、转化和鉴定得到植物表达载体pPPI29（9.428kb），用于小麦的遗传转化。 利用基因枪轰击扬麦158愈伤组织1652个，经过双丙氨膦筛选，获得65株抗性小麦，经过分子检测共获得6株转基因植株，转化率为0.36%。 利用花粉管通道法共转化3个小麦品种（陇鉴127、CA8686和京冬8号），3671朵小花，收获2577粒种子，将收获的种子分别播种于温室（于4℃冰箱中春化21天）和田间，进行检测。分子检测结果表明，检测到6株CA8686阳性材料，其转化效率为0.42%；1株陇鉴127阳性结果，其转化效率为0.1%；京冬8号没有检测到阳性后代。对T₀代部分转基因材料在田间进行抗病性鉴定，CA8686转基因小麦表现出具有对GPV株系的高度抗性，在对照植株严重发病时不表现症状或仅出现轻微的叶尖发黄症状，抗病性鉴定与分子检测结果一致；陇鉴127转基因系可达到减轻症状的效果，在对照下部叶片全部黄化时，下部叶片仍没有表现出严重症状，仅表现轻微症状。 采用二次正交旋转组合试验设计，探讨了2，4-D、肌醇和激动素对陇鉴127和陕160小麦成熟胚的愈伤组织诱导率的影响。结果表明，肌醇浓度对于小麦成熟胚愈伤组织的贡献率最大，其次为2，4-D，再次为激动素，并建立了适合两个小麦品种愈伤组织诱导率的数学模型，筛选出了适合两个小麦品种成熟胚愈伤组刘太国 摘 要2003年6月织诱导的最佳配比培养基配方。适合陇鉴 127小麦成熟胚愈伤组织诱导的培养基配方为：1．57～1．91m旮L的2，4－D、0．79～l＊Zing／L 的激动素和0．104～0．1259／L的肌醇；适合陕 160 ，J’麦成熟胚愈伤组织的诱导的培养基配方为：2刀8～2．40mg／L的二，4D、0．933～1．267mg／L的激动素和 0＊99～0．112g／L的肌醇。 构建了BYDVGPV株系复策酶基因的原核表达载体pETOgyl 和pETOgyZ，并进行了融合蛋白的初步表达，结果 ORF基因有特异性的蛋白产物得到初步表达，而ORFZ基因没有特异性蛋白表达。更多还原

【Abstract】 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更多还原