表达雪花莲外源凝集素基因的油菜转基因植株的获得

利用农杆菌素LBA4404（pCAMBIA3300RG)转化优良甘蓝型油菜恢复系W723的下胚轴节段.pCAM-BIA3300RG含有Rssl启动子引导的雪花莲外源凝集素基因（gna）和CaMV-35S启动子引导的除草剂抗性基因（bar）。经过两轮除草剂（2.5mg/L bialaphos）筛选（两周/轮）,除草剂抗性再生芽被传入生根培养基中生根,对根系旺盛生长的植株中所含gna基因进行PCR分析,PCR分析证实了这些植株确为转基因植株,利用Western印迹法对随机选择的5株含gna基因的转基因植株的分析发现,其中4株表达了gna基因。目前正对这些表达gna基因的转基因植株进行后代遗传分离分析。     

转基因水稻纯系对褐飞虱的抗性研究

利用基因枪法将含潮霉素抗性基因、ＧＵＳ报告基因和雪花莲凝集基因的２个质粒ｐＷＲＧ１５１５和ｐＲＳＳＧＡ１共同转化粳稻品种鄂宜１０５的成熟胚诱导的愈伤组织,从轰击的１５２块愈伤组织中共再生出２６株独立转基因植株,ＰＣＲ／Ｓｏｕｔｈｅｒｎ印迹法分析发现,７３％的转基因植株含有所有３个外源基因,遗传分析证实外泊基因在转基因植株后代中以孟德尔方式遗传,从其Ｒ１代亲本为孟德尔３：１方式遗传的Ｒ２代中,鉴定出     

生物鉴定和喂养试验证明GNA赋予转基因水稻纯系对褐飞虱的抗性

参照Rao等（1998）的褐飞虱生物鉴定和喂养方法,用水稻褐飞虱生物Ⅰ型的－龄若虫食喂,用基因枪法获得2个转基因水稻纯系。这2个纯系均含有并表达潮霉素抗性基因（hpt),gusA报告基因和雪花莲凝集素基因（gna)。褐飞虱生物鉴定和喂养试验表明,水稻纯系对褐飞虱具有显著的抑制作用。具体表现为降低褐飞虱成活率和繁殖力、延缓褐虱发育以及减少褐飞虱进食是。通过褐飞虱生物鉴定和喂养试验证明,表达GNA的转基因水稻纯系对严重危害水稻生产的褐飞虱具有抗性作用。     

Transgenic rice homozygous lines expressing GNA showed enhanced resistance to rice brown planthopper

Mature seed-derived calli from two elite Chinese japonica rice (Oryza sativa L.) cultivars Eyi 105 and Ewan 5 were co-transformed with two plasmids, pWRG1515 and pRSSGNA1, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. 61 independent transgenic rice plants were regenerated from 329 bombarded calli. 79% transgenic plants contained all the three genes, revealed by PCR/Southern blot analysis. Western blot analysis revealed that 36 out of 48 gna-containing transgenic plants expressed GNA (75%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From the R2 generations whose R1 parent plants showing 3:1 Mendelian segregation patterns, we identified five independent homozygous lines containing and expressing all the three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and declining BPH feeding. These BPH-resistant lines have been incorporated into rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.