转Cry1Ac活性杀虫蛋白及慈菇蛋白酶抑制剂B基因的棉花

根据植物基因的结构特征。合成了Cry1Ac活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K。构建了含Bt29K基因及慈菇蛋白酶抑制剂B（API-B）基因表达框的双抗虫基因植物表达载体。通过根癌土壤杆菌（Agrobacterium trmefaciens(Smith et TOwnsend)ConnLBA4404)介导转化了棉花（Gossypium hirsu-tunL.)的两个生产品种（系）。根据抗棉铃虫（Heliothis armigera)试验及农艺性状的观察调查结果。经6代筛选,获得了抗棉铃虫90.0%_99.7%且农艺性状优良的9个双价抗虫棉纯合品系。分子生物学分析结果表明,两个抗虫基因在棉花基因组中的插入拷贝数为1个或2个,活性Cry1Ac和API-B蛋白在转基因抗虫棉株系中的表达量分别约占总可溶性蛋白的0.17%和0.09%。对双抗纯合系植株及仅转Bt基因的棉花纯合系抗虫性检测结果表明前者的抗虫性明显高于后者,因此推断本研究采用的双抗虫基因表达载体构建策略是合理的。     

转新型双抗虫基因棉花的遗传分析

首次用含合成的BtCrylAc活性杀虫蛋白嵌合基因及慈菇蛋白酶抑制剂B(API-B)基因表达框的双抗虫基因植物表达载体,通过土壤根癌杆菌介导转化棉花品种冀合321,获得一批抗虫的转化再生棉花植株。利用叶片涂抹卡那霉素、叶片离体养虫和PCR扩增等检测方法对6个不同转双抗虫基因株系的抗虫性进行遗传分析。结果显示,转化株系自交的T1抗虫性状遗传较为复杂;农杆菌介导获得的转基因抗虫棉在早期世代不易选到纯合系,但是随着对抗虫性状进行单向选择,到T4和T5就能获得抗虫纯合系。利用抗虫性稳定的转化后代材料和转化受体进行田间杂交,发现F2抗虫性分离完全符合一对或两对显性基因的分离规律,并证明了DR248和DR193两个材料为外源基因双拷贝插入。转化株系的Southern杂交也证明了上述结果。     

盐碱土壤转Bt基因棉花外源蛋白表达量时空变化及对抗虫性的影响

盐碱地是潜在的可利用耕地资源,但土壤盐碱化严重制约了农业生产的可持续发展。基于棉花机械化程度低、劳动力成本和生产资料投入剧增、比较效益下降和实施粮食生产安全战略等因素影响,我国长江流域和黄河流域棉花面积锐减,种植区域向内陆盐碱旱地或滨海盐碱地转移,但目前针对盐碱地转Bt基因棉种植可能带来的生态安全性问题研究甚少,正成为国内外研究的焦点和热点。伴随着棉花向盐碱地大面积转移种植趋势,检测盐胁迫是否影响转基因抗虫棉抗虫性,明确其影响程度,直接关系到转基因抗虫棉种植的安全性,也是目前抗虫棉扩大生产中迫切需要解决的问题。以非转基因棉花为对照,分别在低盐、中盐和高盐土壤种植的棉花的苗期、蕾期和花铃期采样,室内测定了转Bt基因棉花叶片对棉铃虫幼虫校正死亡率和外源蛋白表达量。研究结果发现盐分胁迫下转Bt基因棉花苗期叶片对棉铃虫幼虫校正死亡率下降了9.22%—47.46%,蕾期下降了31.61%—45.42%,花铃期下降了3.59%—18.52%;土壤盐分显著降低了转Bt基因棉花叶片中外源蛋白的表达量,苗期功能叶外源蛋白表达量下降了7.66%—29.86%;蕾期下降了3.77%—36.85%;花铃期下降了18.13%—41.02%;相关性分析表明,盐分胁迫条件下转Bt基因棉花叶片中外源蛋白表达量与其对棉铃虫抗性程度存在正相关关系。结果表明,盐碱土壤显著降低了转Bt基因棉花叶片外源杀虫蛋白表达量,从而导致转Bt基因棉花叶片对棉铃虫的抗虫性下降。研究土壤盐分对转Bt基因棉花对棉铃虫的影响及其作用机制,可为建立盐碱地转Bt基因棉花田害虫综合防控技术体系、转Bt基因棉花环境安全评价及转Bt基因棉安全管理提供依据。     

转cry1Ab基因抗虫水稻的田间试验

采用农杆菌介导的遗传转化技术, 将Bt基因cry1Ab导入三系恢复系明恢63获得了一批抗虫性及农艺性状较好的转基因纯合株系。研究中, 进一步检测了这些纯合株系的拷贝数、Bt蛋白含量、抗虫性及主要农艺性状。结果显示: 5个转基因纯合株系均为单拷贝; 纯合株系T_1Ab-10及其杂种的Bt蛋白含量最高; 人工接虫和自然感虫试验中, T_1Ab-10对二化螟、三化螟和稻纵卷叶螟均表现高度抗性; 喷药试验中, 阳性植株与阴性植株、对照植株的主要农艺性状均没有显著差异。这说明转cry1Ab基因纯合株系T_1Ab-10具商品化潜力, 可作基因聚合材料。     

转基因抗虫棉Bt基因不同剂量的聚合与抗虫性表现

通过有性杂交手段培育出聚有不同数目Bt基因的植株,在不同生育期进行抗虫性测定和Bt毒蛋白表达的ELISA检测,旨在揭示聚合不同数目Bt基因的植株抗虫性的互作表达机理。聚合有1－4个Bt基因的植株在整个生育期的抗虫性、毒蛋白表达特性和单价抗虫棉时空表达一致,生育前期抗虫性好、毒蛋白表达量高;生育中、后期抗虫性有所下降,毒蛋白表达量降低。聚合有4个Bt基因的纯合材料并未因Bt基因的增加而起到抗性增强的效果,相反还因同源抑制而有所降低。不同来源的Bt基因处于杂合状态时其抗虫性和Bt毒蛋白量均得到充分表达。     

不同抗虫遗传背景对棉花经济性状的影响

为培育高产、优质、抗病虫的棉花新品种,本实验以一组转基因抗虫棉为材料,对不同类型抗虫棉的经济性状,农艺性状,早熟性,抗红铃虫和抗黄萎病进行了研究。结果表明,转(Bt CpTI)基因抗虫杂交棉新组合667表现为高产,纤维品质优良,高抗红铃虫、耐黄萎病,综合性状好。在参试材料中,双价抗虫棉优于单价(Bt)抗虫棉;杂交抗虫棉优于常规抗虫棉。利用外源抗虫基因转导的棉花新材料为杂交亲本,可以培育出丰产优质的高抗虫的棉花新品种。     

转双抗虫基因杂种741毛白杨的研究

用部分改造后的苏云金芽孢杆菌 (Bt)杀虫蛋白基因和慈菇蛋白酶抑制剂 (API)基因A构建了植物表达载体。然后通过根癌土壤杆菌 (Agrobacteriumtumefaciens (SmithetTownsend)Conn .)介导将此表达载体上的双抗虫基因转入杂种 741毛白杨 [PopulusalbaL .× (P .davidianaDode P .simoniiCarr.)×P .tomentosaCarr.]获得了一批抗卡那霉素的转化再生植株。用杨扇舟蛾 (Closteraanachoreta (Fabricius) )进行虫试的结果表明有 3株抗虫杨树 ,其中有1株杨树的叶片可使试虫在 6天内的死亡率达 90 %以上 ,而且存活幼虫的生长发育受到了明显的抑制。PCR检测及基因组DNASouthern杂交分析的结果都表明Bt杀虫蛋白基因和API基因已整合到以上 3株抗虫杨树的基因组中 ,而且表现为单拷贝整合。用Bt毒蛋白抗血清进行滤膜免疫反应及ELISA检测结果表明 3株转基因杨树都有Bt杀虫蛋白的表达 ,表达量约占叶总可溶性蛋白的 0 .0 15 %。这是国内外首次报道用双抗虫基因获得的抗虫 741毛白杨植株。     

盐胁迫对Bt棉棉蕾杀虫蛋白表达的影响

为探讨土壤盐分对转Bt基因抗虫棉棉蕾抗虫性的影响程度,采用盆栽试验,以2个Bt棉品种‘新棉33B’(盐敏感)和‘中07’(耐盐)为试验材料,设置5个土壤盐分水平(0、0.15%、0.30%、0.45%和0.60%), 研究盐分对蕾期Bt棉棉蕾中杀虫蛋白含量、Bt基因相对表达量及氮代谢相关酶和物质代谢的影响.结果表明: 棉蕾中Bt杀虫蛋白含量随土壤盐分的升高而降低,与对照相比(0%),2个不同类型棉花品种在土壤盐分0.30%及以上时,棉蕾中杀虫蛋白含量显著下降,且随土壤盐分水平的升高,Bt 杀虫蛋白含量下降幅度增大.土壤盐分胁迫提高了棉蕾中Bt基因的相对表达量.在相同土壤盐分水平下,盐敏感品种棉蕾中Bt杀虫蛋白含量受土壤盐分影响较大.杀虫蛋白表达量下降幅度大的品种,其可溶性蛋白含量、谷氨酸丙酮酸转氨酶(GPT)和谷氨酸草酰乙酸转氨酶(GOT)活性下降幅度较大,游离氨基酸含量、蛋白酶和肽酶活性上升幅度也较高.说明土壤盐分胁迫下,Bt棉棉蕾蛋白质合成能力下降,分解能力增强,可能是导致杀虫蛋白表达量下降的主要原因.     

转Bt/CpTI基因棉花抗虫性鉴定与筛选

以国内育成和国外引进的52个转Bt/CpTI基因棉花品种(系)为材料,在河北农业大学棉花育种圃种植,采用室内生物鉴定方法,研究供试转基因棉花的棉铃虫抗性和甜菜夜蛾抗性,并进行抗虫性筛选.结果表明:国内育成的品种(系)在棉田二代棉铃虫危害期对棉铃虫的抗性不低于国外引进的品种(系);在三代棉铃虫危害期对棉铃虫的抗性,国内育成的品种(系)还略高于国外引进品种.新棉33B对甜菜夜蛾表现高抗,其他品种抗性一般.转Bt和CpTI的双价抗虫棉对甜菜夜蛾幼虫的抗性不高.根据供试品种(系)的抗虫性鉴定结果,筛选出SGK321等15个在二代、三代棉铃虫危害期对棉铃虫表现高抗的品种(系)和兼抗棉铃虫、甜菜夜蛾的新棉33B.     

双价抗虫基因叶绿体共转化植株抗虫性及其后代表型分析

利用基因枪法将含有水稻巯基蛋白酶抑制剂(Oryzacystatin,OC)基因烟草叶绿体表达载体和含有苏云金芽孢杆菌晶体毒蛋白基因(Bt cry IAc)烟草叶绿体表达载体,共转化烟草叶绿体,获得壮观霉素抗性植株。转基因植株抗棉铃虫试验表明,转双价抗虫基因植株比转单一抗虫基因植株具有更强的杀虫活性。转基因植株后代Southern检测及其遗传学分析试验证明,双价抗虫基因可以稳定地遗传给后代,且表现为叶绿体特有的母系遗传规律。 Abstract:The Bt gene and OC gene were cotransformed to tobacco chloroplast with particle bombardment method and spectinomycin resistance tobacco seedlings were obtained.Bioassays showed that the transgenic tobacco containing both genes had enhanced toxicity to the larvae of cotton bollworm (helicoverpa zea) by comparison with the plants containing only Bt or OC gene.Southern-blotting analysis and genetic analysis of progenies showed that the Bt and OC gene expressed and was inherited maternally to the progenies.     

转基因烟草中Bt毒蛋白基因的表达行为

Bt toxin genes were the insecticidal genes most widely used in genetic engineering of pest resistant plant, were of important significance to study their expression behavior in transgenic plants. In this work, a plant expression vector, pBinMoBc, was constructed. It contained the Cry IA(c) gene under control of chimeric OM promoter and the Ω factor. The vector was transferred into tobacco (Nicotiana tabacum L.) plant via Agrobacterium-mediated transformation. ELISA assay showed that the expression levels of the Cry IA(c) gene in transgenic tobacco plants were significantly higher than that in wild-type tobacco plants. The highest could be up to 0.255% of total soluble proteins; the expression level of CryIA(c) gene in transgenic tobacco plant was changeable during the development stages of tobacco plant. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal activity than the wild-type tobacco plants. The above results indicated that pBinMoBc was an effective pest-resistent plant expression vector. This study would be very helpful in screening transgenic cotton with high resistance to cotton bollworm (Heliothis armigeva Hubner).     

转单、双Bt基因741杨外源基因表达和抗虫性比较

【目的】研究联合使用两种或两种以上的抗虫基因的抗虫效果, 同时鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫有较强抗性的株系。【方法】选取转三基因（Cry3Aa+Cry1Ac+API）741杨8个株系、 转双基因（Cry1Ac+API）741杨1个株系和转单基因（Cry3Aa）741杨3个株系为试材, 从外源基因PCR检测、 毒蛋白表达和抗虫性三方面对转基因株系进行对比分析。【结果】经PCR扩增后各转基因株系出现了预期的电泳条带。ELISA蛋白检测显示转基因株系中都有与所含基因相应的Bt杀虫蛋白表达。用转基因株系新鲜叶片进行柳蓝叶甲Plagiodera versicolora和美国白蛾Hyphantria cunea室内饲虫实验表明： 转入不同抗虫基因的杨树对昆虫的抗性具有选择性, 对非靶标昆虫没有毒杀作用。转双Bt基因741杨具有双抗性, 不同转基因株系表现出高中低的抗性水平： 在对柳蓝叶甲的抗性上, 筛选出的其中5个高抗株系（pCCA1, pCCA2, pCCA5, pCCA6和pCCA9）的抗性水平明显比含Cry3Aa单Bt基因的3个高抗株系（pCC11, pCC53和pCC84）高; 在对美国白蛾的抗性上, 有7个株系（pCCA2~pCCA7和pCCA9）的抗性水平与含Cry1Ac单Bt基因株系（pB29）表现一致, 只有1个株系（pCCA1）对美国白蛾表现出了极低的抗性。【结论】多个抗虫基因在741杨上的联合使用, 不仅扩大了抗虫谱, 其中的高抗株系还具有了更高的抗虫能力, 有效地发挥了基因的叠加效应。     

Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker)

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.     

Seasonal expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the Yangtze River valley of China

Seasonal levels of Bacillus thuringiensis (Bt) insecticidal protein and its control efficacy against Helicoverpa armigera (Hübner) in Bt transgenic cotton GK19 (carrying a Cry1Ac/Cry1Ab fused gene) and BG1560 (carrying a Cry1Ac gene) were investigated in Tianmen County, Hubei Province, located in the Yangtze River valley of China, in 2001 and 2002. The results showed that the toxin content in Bt cotton changed significantly over time, and that the structure, growth stage, and variety were significant sources of variability. Generally, insecticidal protein levels were high during the early stages of cotton growth; they declined in mid-season, and rebounded in late season. On most dates sampled, the toxin contents in leaf, square, petal, and stamens (including nonovule pistil tissue) were much higher than those in ovule and boll. Compared with BG1560, the expression of Cry1Ac/Cry1Ab protein in GK19 was more variable during the whole growth period of cotton. The field evaluation on larval population dynamics of H. armigera in Bt and conventional cotton showed that the larval densities in BG1560 and GK19 fields decreased, respectively, 92.04 and 81.85% in 2001, and 96.84 and 91.80% in 2002.     

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for pest resistance management in China.     

Transgenic sugarcane plants expressing high levels of modified cry1Ac provide effective control against stem borers in field trials

To improve transgene expression level, we synthesized a truncated insecticidal gene m-cry1Ac by increasing its GC content from 37.4 to 54.8%, based on the codon usage pattern of sugarcane genes, and transferred it into two sugarcane cultivars (ROC16 and YT79-177) by microprojectile bombardment. The integration sites and expression pattern of the transgene were determined, respectively, by Southern, northern and western blot analyses. The transgenic sugarcane lines produced up to 50?ng Cry1Ac protein per mg soluble proteins, which was about fivefold higher than that produced by the partially modified s-cry1Ac (GC%?=?47.5%). In greenhouse plant assay, about 62% of the transgenic lines exhibited excellent resistance to heavy infestation by stem borers. In field trials, the m-cry1Ac transgenic sugarcane lines expressing high levels of Cry1Ac were immune from insect attack. In contrast, expression of s-cry1Ac in transgenic sugarcane plants resulted in moderately decreased damages in internodes (0.4-1.7%) and stalks (13.3-26.7%) in comparison with the untransformed sugarcane controls, which showed about 4 and 26-40% damaged internodes and stalks, respectively. Significantly, these transgenic sugarcane lines with high levels of insect resistance showed similar agronomic and industrial traits as untransformed control plants. Taken together, the findings from this study indicate a promising potential of engineering an insect-resistant gene to tailor its protein expression levels in transgenic sugarcane to combat insect infestations.     

Similar genetic basis of resistance to Bt toxin Cry1Ac in Boll-selected and diet-selected strains of pink bollworm

Genetically engineered cotton and corn plants producing insecticidal Bacillus thuringiensis (Bt) toxins kill some key insect pests. Yet, evolution of resistance by pests threatens long-term insect control by these transgenic Bt crops. We compared the genetic basis of resistance to Bt toxin Cry1Ac in two independently derived, laboratory-selected strains of a major cotton pest, the pink bollworm (Pectinophora gossypiella [Saunders]). The Arizona pooled resistant strain (AZP-R) was started with pink bollworm from 10 field populations and selected with Cry1Ac in diet. The Bt4R resistant strain was started with a long-term susceptible laboratory strain and selected first with Bt cotton bolls and later with Cry1Ac in diet. Previous work showed that AZP-R had three recessive mutations (r1, r2, and r3) in the pink bollworm cadherin gene (PgCad1) linked with resistance to Cry1Ac and Bt cotton producing Cry1Ac. Here we report that inheritance of resistance to a diagnostic concentration of Cry1Ac was recessive in Bt4R. In interstrain complementation tests for allelism, F(1) progeny from crosses between AZP-R and Bt4R were resistant to Cry1Ac, indicating a shared resistance locus in the two strains. Molecular analysis of the Bt4R cadherin gene identified a novel 15-bp deletion (r4) predicted to cause the loss of five amino acids upstream of the Cry1Ac-binding region of the cadherin protein. Four recessive mutations in PgCad1 are now implicated in resistance in five different strains, showing that mutations in cadherin are the primary mechanism of resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona.