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1.
利用农杆菌转化法,将组成型表达β-1,3葡聚糖酶及几丁质酶基因的双价植物表达载体pBLGC转化优质甘蓝型油菜品种H165,并得到了抗卡那霉素(Kanamycin,Kan)的再生植株。我们对所得到的抗Kan的再生苗进行了初步分子生物学检测,结果表明,在K15(Kan15mg/L)培养基上的绿苗中有30%为PCR阳性植株,而在K25(Kan10mg/L)培养基上的绿苗有53%的阳性率。对部分PCR阳性的转基因植株进行了点杂交分析,结果均表现出较强的杂交信号,这初步说明外源基因已整合到油菜基因组中。转基因植株的活体接种油菜菌核病菌(Sclerotiniasclerotiorium)试验表明,部分转基因植株比对照显示较强抗病性。  相似文献   

2.
用冻融法和三亲交配法将具有几丁质酶基因、β—1,3—葡聚糖酶基因和卡那霉素抗性标记基因的质牲pBLGC导入具有Ri质粒的发根农杆菌中。用叶盘法转化烟草的三个品种,经Kan抗性筛选获得126株再生植株,对119株再生植株分别以启动子CaMV35S、几丁质酶基因和β—1,3—葡聚糖酶基因的引物进行PCR检测,其中几丁质酶基因至阳性的植株有72株,β—1,3—葡聚糖酶基因至阳性的有47株,具有几丁质酶基因和β—1,3—葡聚糖酶基因的植株有36株。对转化的24株转基因经PCR-Southern杂交,结果均至阳性。实验结果表明,外源基因的转化频率与植物品种、外源基因片段的大小有关,几丁质酶基因和β—1,3—葡聚糖酶基因分别或共同整合到植物基因组中。  相似文献   

3.
比较了乙酰丁香酮、pH、共培养温度及不同激素配比对根癌农杆菌转化油菜(Brassica napus)的影响,建立了油菜高效转化体系.按该体系,将组成型表达几丁质酶和β-1,3-葡聚糖酶基因转化甘蓝型双低杂交油菜亲本恢复系和保持系,获得了转基因恢复系和保持系植株.对再生植株进行PCR和SouthemBlot检测,结果表明外源基因已整合到油菜基因组中.连续3代的PCR检测及转基因植株抗病性在自交后代中得到保持,证实外源基因已遗传到T3代.对转基因植株T1代离体叶进行菌核病菌丝体接种和T1及T2代大田自然侵染鉴定,结果表明,转基因恢复系棚40-12、棚40-32和保持系96B-2对菌核病的抗性比受体对照大幅度提高,大田鉴定其发病率连续2年均比受体对照减少78%以上,比抗病品种‘中油821'减少75%以上,病情指数比受体对照和‘中油821'减少均达显著水平,其抗病性能在后代稳定遗传,获得了高抗菌核病的转基因育种材料.  相似文献   

4.
将抗真菌病基因导入马铃薯的研究   总被引:2,自引:0,他引:2  
实验采用微束激光穿刺技术和农杆菌介导方法,将携带有Ⅰ型烟草β-1,3-葡聚糖酶基因和Ⅰ型菜豆几丁质酶基因的pBLGC质粒导入马铃薯(Solanum tuberosum L.)品种“津引8号”。微束激光穿刺技术转化中,获得了7株卡那霉素抗性再生苗,分子检测证明,其中有1株为菜豆几丁质酶基因单基因转化植株。在农杆菌介导方法中得到了13株卡那霉素抗性再生苗,其中有3株为烟草β-1,3-葡聚糖酶基因单基因  相似文献   

5.
比较了乙酰丁香酮、pH、共培养温度及不同激素配比对根癌农杆菌转化油菜(Brassica napus)的影响, 建立了油菜高效转化体系。按该体系, 将组成型表达几丁质酶和β-1, 3-葡聚糖酶基因转化甘蓝型双低杂交油菜亲本恢复系和保持系, 获得了转基因恢复系和保持系植株。对再生植株进行PCR和Southern Blot检测, 结果表明外源基因已整合到油菜基因组中。连续3代的PCR检测及转基因植株抗病性在自交后代中得到保持, 证实外源基因已遗传到T3代。对转基因植株T1代离体叶进行菌核病菌丝体接种和T1及T2代大田自然侵染鉴定, 结果表明, 转基因恢复系棚40-12、棚40-32和保持系96B-2对菌核病的抗性比受体对照大幅度提高, 大田鉴定其发病率连续2年均比受体对照减少78%以上, 比抗病品种‘中油821’减少75%以上, 病情指数比受体对照和‘中油821’减少均达显著水平, 其抗病性能在后代稳定遗传, 获得了高抗菌核病的转基因育种材料。  相似文献   

6.
转基因玉米中目的基因的遗传表达及其抗病性研究   总被引:4,自引:0,他引:4  
采用花粉介导方法将几丁质酶基因和潮霉素基因导入玉米(Zea maysL.)自交系海92-1中,以筛选抗玉米丝黑穗病的转基因品种.对转化植株及其后代植株的PCR、Southern blot检测表明,目的基因已导入转化植株并整合到其基因组中,且能够稳定遗传.ELISA分析证明转基因植株中目的基因可高效表达,表达产物量在9.8~16.3 ng?g-1鲜叶左右.统计分析显示,目的基因产物表达水平与转基因植株的抗病性呈极显著正相关(r=0.925,P<0.01).接种病毒鉴定结果揭示转基因株系的抗病性比对照提高3~4级.结合农艺性状筛选,选育到401、403这2个抗丝黑穗病且其它农艺性状优良的转基因纯合株系.  相似文献   

7.
徐香玲  李集临  马兴红 《植物研究》2002,22(3):T002-T003
构建了一个含有多个调控序列的带有几丁质酶基因的植物表达载体,通过发根农杆菌的Ri质粒介导转化烟草的三个品种:K326,RG11,VA116,在卡那霉素抗性培养基上筛选,获得了7株再生植株,以PCR检测、DNA斑点杂交证明表达载体构建成功,几丁质酶基因导入烟草并整合到烟草基因组中。  相似文献   

8.
文摘     
《生物技术通报》2000,(4):58-61
00 0 0 6 5反义PEP基因调控油菜籽粒蛋白质 /油脂含量比率的研究[中 ]/陈锦清… / /农业生物技术学报 .1999,7(4) .- 316~32 0利用根癌农杜菌 (Agrobacteriumtumefaciens)EHA10 1携带反义PEP基因和卡那霉素、潮霉素抗性基因 ,采用共培养法转化甘蓝型油菜 (BrassicanapusL .)浙油 75 8和浙优油1号的下胚轴 ,通过组织培养获得了能在潮霉素培养基上生长的油菜转基因植株。经GUS组织化学染色及PCR扩增检测 ,证明外源基因已插入油菜基因组并得到表达。浙油75 8转基因植株T1代种子平均含…  相似文献   

9.
利用微束激光穿刺法将抗真菌基因导入棉花的研究初报   总被引:4,自引:1,他引:3  
以棉花感受态萌动种胚作为外源基因转化的受体,用激光微束穿刺法将β-1,3-葡聚糖酶及几丁质酶双价基因导入棉花,所构建的植物表达载体pB IBGC携带有筛选标记npt-Ⅱ(新霉素磷酸转移酶)基因。激光转化处理的种胚经卡那霉素筛选,已经获得抗性小植株。研究了微束激光转化法用于棉花感受态萌动胚转化预培养的时间、高渗液对材料的处理等。研究表明:用微束激光转化处理种胚是一种操作简便、重复性好的转化方法,用该法可将外源基因导入植物感受态萌动胚,避开植株离体再生的困难。  相似文献   

10.
为了提高‘云资粳41’和‘云资粳43’的抗稻瘟病能力,利用农杆菌介导法将二价表达载体pCAMBIA1300-Pi-ta+-Bchi转化到水稻愈伤组织中。经组织培养获得再生苗,再通过氯酚红显色法、PCR检测和抗稻瘟病鉴定法获得抗稻瘟病的转基因植株,为进一步创建持久、广谱抗稻瘟病水稻新材料奠定基础。结果显示:(1)抗性愈伤组织经分化和生根培养后,共获得T0代水稻再生苗137株,其中‘云资粳41’14株,‘中花11’82株,‘云资粳43’41株。(2)经氯酚红显色法和PCR对再生苗检测,‘中花11’、‘云资粳41’、‘云资粳43’的转化苗阳性率分别为66%、43%和55%。证明2个外源基因已经整合到水稻基因组中。(3)对转化阳性植株温室接种稻瘟病病菌66b鉴定结果显示,转基因植株较非转基因植株对稻瘟病的抗性明显增强,而且转Pi-ta+基因和几丁质酶基因双价的水稻植株比转单价Pi-ta+基因或几丁质酶基因的水稻植株抗稻瘟病能力强。(4)氯酚红检测结果存在一定的假阳性,PCR检测结果更真实可靠,但氯酚红显色法方便、快速,结果观察直观,可对大量的转基因植株进行初步筛选。研究表明,转Pi-ta+基因和几丁质酶基因双价基因的水稻植株具有更高的抗稻瘟病能力。  相似文献   

11.
Insect resistance of transgenic tobacco expressing an insect chitinase gene   总被引:24,自引:0,他引:24  
Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control  相似文献   

12.
Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.  相似文献   

13.
Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.  相似文献   

14.
昆虫杆状病毒几丁质酶及其应用研究进展   总被引:2,自引:0,他引:2  
杆状病毒几丁质酶基因(chitinase,,ChiA)是晚期表达的非必需基因,高度保守。表达产物几丁质酶分为3个区:N-端信号肽区,中部酶活性区和C-末端酶内质网结合区。该酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的几丁质,促进虫体液化;作为组织蛋白酶原(pro-V-Cath)的分子伴侣,参与其加工和运输过程; 影响多角体的形成,并与细胞的裂解有关;还与病毒侵染机制相关联。杆状病毒ChiA与细菌ChiA源于共同的祖先,而昆虫ChiA则可能直接来自杆状病毒。在害虫生物防治中,杆状病毒ChiA可直接作为杀虫剂,或作为苏云金杆菌和杆状病毒等微生物杀虫剂的增效剂使用;杆状病毒ChiA可转入植物,获得具有持续杀虫及抗病活性的转基因植物;将杆状病毒ChiA的内质网定位序列删除、突变,或在病毒基因组中插入外源ChiA,重组病毒的杀虫活性增强。通过基因工程手段,删除病毒基因组ChiAV-Cath,可改善杆状病毒表达系统对分泌蛋白和膜结合蛋白的表达。  相似文献   

15.
Park YS  Min HJ  Ryang SH  Oh KJ  Cha JS  Kim HY  Cho TJ 《Plant cell reports》2003,21(10):1027-1034
Salicylic acid is a messenger molecule in the activation of defense responses in plants. In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp. pekinensis) by subtractive hybridization. Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein. The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein. The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase. Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain. The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences. These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole. Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv. tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage. None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.  相似文献   

16.
17.
Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers.  相似文献   

18.
The plsC gene of Escherichia coli encoding sn-1-acylglycerol-3-phosphate acyltransferase was modified by inserting an endoplasmic reticulum retrieval signal to its 3 end and introduced into rapeseed (Brassica napus L.) plants under the control of a napin promotor. In developing seeds from transgenic plants an sn-1-acylglycerol-3-phosphate acyltransferase activity was detectable which showed substrate specificities typical of the E. coli enzyme. Moreover, seed oil from the transformants unlike that from untransformed plants contained substantial amounts of triacylglycerol species esterified with very-long-chain fatty acids at each glycerol position. Analysis of fatty acids at the sn-2 position of triacylglycerol showed hardly any very-long-chain fatty acids in untransformed plants, but in certain transformants these fatty acids were present, namely about 4% erucic acid and 9% eicosenoic acid. These data demonstrate that the bacterial acyltransferase can function in developing rapeseed and alters the stereochemical composition of transgenic rape seed oil by directing very-long-chain fatty acids, especially cis-11 eicosenoic acid, to its sn-2 position.  相似文献   

19.
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