STUDY ON THE GENOME OF CAULIFLOWER MOSAIC VIRUS (XINJIANG ISOLATE)——RESTRICTION MAPPING OF VIRION AND CLONED VIRAL DNA

The genomie DNA of CaMV (Xinjiang isolate) was mapped on the virion DNA with anumber of restriction endonueleases by double digestions and partial digestions of one-end~(32)P-labelled fragments. It contained the unique sites of BglI, SalI and XhoI. BamHI andHhaI each cut it at two sites, EeoRI at five sites (three of which were located), Hpa Ⅱ atseven sites, BglⅡ at eight sites, and HaeⅢ at ten sites. The sites of all the enzymes usedabove, as well as HindⅢ which cut at ton sites were also mapped on the cloned CaMVDNA. Virion DNA and cloned viral DNA gave the same results in the number and locationof cleavage sites of the restriction enzymes tested on both DNAs. Like most of the otherisolates, CaMV-Xinjiang isolate has three single-stranded discontinuities. From the diges-tion patterns of SalI, XhoI, EcoRI and HhaI, it is concluded that the restriction map ofCaMV-XJ genome is closely related to that of BKMV isolate from East Germany.     

双抗转基因烟草纯合系的选育及田间试验

为了将双抗转基因烟草推向实用,进行了双抗转基因烟草纯合系的选育。在试验田中利用人工攻毒进行了T1代植物的抗病毒特性的分离试验;在抗性良好的T1植株的后代T2代植物上进行了田间的病毒接种试验和转基因表达的分析,从中选育出双抗转基因烟草纯合系。又于试验田中,在这些纯合系后代T3代上验证了抗病毒特性的遗传稳定性。在转基因表达分析中,发现CMV外壳蛋白基因的表达在翻译水平上受到抑制。     

Introducing Bt Gene Into Maize With Ovary Injection

It is reported here that Bt toxin gene has been successfully transferred into maize inbred line by ovary injection for the first time both at home and abroad. One transgenic plant (To) has been confirmed by Southern blotting and PCR test, and 71 progenies (T1) from T0 have been obtained through self-pollination. Of these 71 progenies, seven plants demonstrated positive results in the PCR test; four were used to feed Asian corn borer, and certain effect of insect-resistance was observed. The experiments on the ovary injection in Hainan Province have also been repeated, thus providing new chance to the application of genetic engineering to the maize improvement.     

花椰菜花叶病毒(新疆分离物)基因组的研究——病毒粒体DNA和克隆的病毒DNA的限制性内切酶图的测定

本文用混合酶解和一端以~(32)P标记的DNA的部分酶解测定了花椰菜花叶病毒新疆分离物(CaMV-XJ)基因组DNA的限制性内切酶图.它含有BglⅠ,SalⅠ,XhoⅠ的单一切点.BamHⅠ和HhaⅠ各有2个切点,EcoRⅠ有5个切点,HpaⅡ有7个切点,BglⅡ有8个切点,HaeⅢ有10个切点,HindⅢ有10个切点.在病毒粒体DNA和克隆的病毒DNA上这些酶的切点数目和位置是一样的.同其他大多数CaMV分离物一样,CaMV-XJ DNA上也有3个单链不连续区.从SalⅠ,XhoⅠ,EcoRⅠ,HhaⅠ的酶切情况看,CaMⅤ-XJ基因组的内切酶图谱与东德的CaMⅤ分离物BkMⅤ比较接近。     

VIRUS RESISTANCE OF TRANSGENIC TOBACCO PLANTS EXPRESSING TOBACCO MOSAIC VIRUS COAT PROTEIN GENE

An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco genome via Ti plasmid and a large number of regenerated plants, including both systemic and local lesion hosts for TMV, were obtained. Southern blot analysis revealed that 1-5 copies of the CP gene were integrated into the tobacco genome. RNA and protein analysis demonstrated that the TMV-CP gene was correctly expressed in the transgenic plants. The abundance of TMV-CP mRNA in total leaf RNA accounted for 0.005-0.01%, while the amount of coat proteins reached 0.05-0.2% of the total leaf soluble proteins. Virus challenge experiments showed that the symptom development of virus infection was markedly delayed and the replication as well as the spread of the virus was significantly inhibited in the transgenic plants expressing the TMV-CP gene. Three of these plants were completely protected afte     

表达烟草花叶病毒外壳蛋白的转基因烟草及其对TMV的抗性

利用体外重组DNA技术构建携带烟草花叶病毒(TMV,国内流行的普通株)外壳蛋白基因(CP)的中间表达载体,通过土壤农杆菌(Agrobacterium tumefaciens)Ti质粒,重组TMV-CP基因被转移至烟草细胞,并获得大量再生的转基因烟草。工程烟草的基因组经Southern印迹法分析证明,CP基因在再生工程株染色体中有1—5个拷贝的插入。分子杂交分析确证TMV-CP基因在转基因株中获得正确表达,其mRNA和蛋白产物的丰度分别达0.005—0.01％及0.05—0.2％。攻毒实验表明90％以上的能表达TMV—CP基因的工程烟草能不同程度地抑制病毒的复制、扩散,并显著延缓,减轻系统病状的发生。有3株工程植株直至花期无病状表现,生长正常。这一抗性作用机制在于转化细胞中的外壳蛋白在病毒侵染早期有效地抑制了入侵病毒颗粒的脱壳,从而阻断病毒的复制。     

高效抗虫转基因烟草的研究

苏云金杆菌HD-1的杀虫蛋白基因经5′端改造,3′端进行4种不同长度缺失后插入到含有双增强子的35S启动子,翻译增强子“Ω′”片段的双元载体中,借助土壤农杆菌LBA 4404将在此双元载体上的杀虫蛋白基因及新霉素磷酸转移酶基因(NPTII)转入到生产品种烟草NC89的染色体上,从而获得了抗卡那霉素的转化再生烟草植株。用1—3龄烟青虫对这些转化植株进行大量重复虫试结果表明用4种不同长度B.t.基因转化的再生植株中都有抗虫性高的植株,其中以1.8kb的B.t.Cry IA(c)基因转化的植株杀虫效果最好,这一组转基因植株的平均杀虫率在90—100％的约占该组总虫试植株的50％。对高抗虫性植株的子一代(T1)和子二代(T2)进行遗传分析,分子生物学分析和进一步的抗虫试验表明B.t.基因已遗传到子代并初步选到了高抗虫性的转基因纯合株系D8-14和D19-8等。     

用子房注射法将Bt毒蛋白基因导入玉米的研究

本文在国内外首次报道了用子房注射的方法将Bt毒蛋白基因导入玉米自交系的全过程。获得的一株转基因玉米(T_0)自交留种,得到了71株T_1代的植株。转基因玉米(T_0)经点渍法、Southern吸印/分子杂交以及PCR扩增均获得阳性结果;用T_1代植株叶片DNA进行PCR扩增,在71株中有7株呈阳性反应;对其中4株进行抗玉米螟测试,呈现一定的抗虫效果;在海南岛用农大60玉米再次进行子房注射,也获得了成功,说明这种方法是可重复的,从而为利用基因工程技术改造玉米开拓了新的途径。