Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying CrylAc, Crylle or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of CrylAc and Crylle proteins were 0.173% and 0.131% of the total proteins, respectively. CrylAc protein content was 0.182 % and Cry1 le protein content was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm （Helicoverpa armigera）. The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and CrylAc-resistant cotton bollworm than those carrying CrylAc or Crylle alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resistance to transgenic crops.     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

Insecticidal activity of residual Bt protein at the second trophic level

Since the first commercial release of transgenic crop expressing genes from Bacillus thuringiensis (Bt), there have been concerns about its potential impact on the environment. Research has focused on the ecological effects from second exposure to Bt prot…     

Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development ofHelicoverpa armigera

The resistance ratio ofHelicoverpa armigera to Cry1 Ac insecticidal protein fromBacillus thuringiensis (Bt) is 13.1- and 3.02-fold after 18 generations of selection by transgenic tobacco expressing Bt or two (Bt and CpTI) insecticidal protein genes, in which the average corrected mortality for each selection treatments is about 60%. The mortality of selected population by transgenic Bt gene tobacco is significantly lower than the control strain when fed on transgenic tobacco plants. The mortaltty of the selected population by transgenic two genes tobacco was not significantly different from the control strain. This is the first experiment under laboratory condition which has proved that transgenic two genes tobacco could significantly delay resistance development ofH. armigera compared with one gene.     

Commercial production of transgenic Bt insect-resistant cotton varieties and the resistance management for bollworm (Helicoverpa armigera Hubner)

There are currently three kinds of transgenic Bt insect-resistant cotton germplasm lines, Shanxi 94-24, Zhongxin 94 and R19, in China. They showed high resistance to the neonate larvae of bollworm (Helicoverpa armigera). Transgenic Bt insect-resistant cotton varieties or hybrids have been bred using the three kinds of germplasm lines as parents. Our researches reveal that there exist different expressions in resistant level at different developmental stages in the three categories of germplasm lines. When neonate larvae are fed with leaves of cotton plant at the seeding stage with less than 10 leaves on the main stem, the mortality of the neonate larvae is 100%, but the resistance level will decline at later season. When Bt gene has been transferred to the cotton genome, it can be steadily transferred to the progeny, the level of resistance to bollworm keeps fundamentally uniform. Such insects as tobacco budworm (Heliothis virencens) in laboratory directive selection are very apt to produce resistance to the Bt insecticidal crystal protein. From the present crop system of cotton region in the Yangtze and Yellow River Valleys, and the expression characteristic of transgenic Bt resistant cotton, we suggest that the resistance to toxin protein in bollworm is not apt to be produced if the transgenic Bt insect-resistant cotton varieties are released and grown in the regions except in the Xinjiang cotton region. The managing strategies to delay or retard the resistance are discussed.     

Expression of two insect-resistant genes crylA ( b&c )/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesized Bacillus thuringiensis insecticidal protein gene crylA(b&c) and the synthesized gene GNA, (the mannose specific lectin from snowdrop ( Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes of Nico-tiana tabacum var. K326 have been transformed with Agrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreign crylA and GNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show that GNA has a functional activity. Leaf disc bioassays against cotton bollworm ( H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays against Myzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%-70% that on untransformed tobacco plants. ELISA analysis of CrylA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays against H. armigera and M. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

转Bt棉不同生长期及不同器官杀虫蛋白表达量的免疫学方法测定

转苏云金杆菌(Bacillus thuringensis)杀虫蛋白基因棉花(下称Bt棉)在不同生长期及不同器官上对棉铃虫杀虫活性存在明显差异。目前国内普遍使用生物测试法检测Bt棉对棉铃虫的杀虫活性,但无法定量检测Bt棉中杀虫蛋白的表达量。本实验室建立的酶联免疫学测定方法(ELISA),可定量检测棉器官内杀虫蛋白的含量。它具有检测周期短,操作简便的优点。用ELISA方法检测Bt棉杀虫蛋白的含量,结果表明,棉叶和花瓣杀虫蛋白含量最高,铃和蕾次之。在棉株生长的后期,各器官杀虫蛋白的含量则有大幅度的下降。     

Expression of two insect-resistant genescryIA (b&c)/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesizedBacillus thuringiensis insecticidal protein gene cryIA(b&c) and the synthesized geneGNA, (the mannose specific lectin from snowdrop (Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes ofNicotiana tabacum var. K326 have been transformed withAgrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreigncryIA andGNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show thatGNA has a functional activity. Leaf disc bioassays against cotton bollworm (H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays againstMyzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%–70% that on untransformed tobacco plants. ELISA analysis of ClyIA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays againstH. armigera andM. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

苏云金芽孢杆菌猝倒亚种Cry1Aa13基因的克隆及序列分析

根据Gen Bank中Cry1A类基因序列设计一对特异性引物，以苏云金芽孢杆茵猝倒亚种质粒DNA为模板，应用PCR扩增技术得到一大小约为3．6kb的DNA片段(Cry1Aa13)．通过引物步行法测定该片段长3598bp，其中开放阅读框(ORF)长3540bp，编码1180个氨基酸，分子量为133．49kD，等电点pI=5．0．序列比较表明该基因与Cry1Aa类基因高度同源(达95％以上)，该基因已在GenBank中登录，登录号为AF510713，并被Btδ-内毒素基因国际命名委员会正式命名为Cry1Aa13。     

Variation induced by DNA rearrangement in a transgenicBt+CpTI cotton strain

In the development of transgenicBt + CpTI cotton cultivars, one male and female sterile mutant has been found in a homozygous T₄ strain in our laboratory. The mutant plant, as well as its leaves, buds and flowers, is only 1/2–1/3 as large as that of the wild transgenicBt + CpTI bivalant cotton plants. Cytological observation found that the chromosome number of the mutant is 2n = 52; however, there are 4–8 univalents observed in meiosis I of pollen mother cells. Laboratory bioassay indicated that the mutant was highly resistant to bollworm as the wild plants. PCR amplification revealed thatBt andCpTI genes in the mutant were still intactly inserted. However, small deletion of flanked area had been observed in the mutant by Southern blotting analysis. So it is proposed that the mutant phenotype might result from either the DNA deletion or T-DNA transferring in plant genome. No such report has been presented that the rearrangement of chromosome structure in a homozygous transgenic line occurred. Further analysis is ongoing.     

Cry1A型杀虫晶体蛋白活性区的空间结构比较分析

对9种代表性Cry1A型杀虫晶体蛋白成员进行活性区空间结构的同源模建与比较分析.分析结果表明:Domain Ⅰ和Domain Ⅲ相对保守,其中,Domain Ⅰ整体走向及结构基本重叠,只有Cry1Aa和Cry1Af多了一个螺旋;Domain Ⅱ的主要差异体现在loop上;将Domain Ⅲ结构一致的成员Cry1Ab,Cry1Ad,Cry1Ae和Cry1Af归为一个亚型,其他5种成员归为另一个亚型.研究确定了影响Cry1A型杀虫晶体蛋白结构差异的关键氨基酸及关键结构片段.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.)

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutin1 gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors (pUUOAH). The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of Cry1Ah protein in the construct containing the ubi1 intron (pUUOAH) was 20% higher than that of the intronless construct (pUOAH). Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubi1 intron was higher than that of the intronless construct. These results indicated that the maize ubi1 intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

桃蚜关键抗性基因挖掘及抗蚜Cry蛋白预测

为了挖掘桃蚜(Myzus persicae)的关键抗性基因并构建抗性调控网络,通过加权基因共表达网络分析(WGCNA)和差异表达基因分析(DEGs)对桃蚜抗性研究的GEO数据库进行分析,筛选出2 426个枢纽基因和2 263个差异表达基因探针.将关键抗性基因在String数据库中进行蛋白质相互作用(PPI)分析,获得154个桃蚜关键抗性基因,并绘制桃蚜的抗性调控网络.结果表明:acpp基因的上调表达可能是大多数Cry蛋白不能有效杀死桃蚜的原因之一;参考抗蚜Cry1Cb2蛋白和11个靶标蛋白的对接规则,通过同源建模和分子对接等生物信息学技术,预测了10个新的Cry蛋白可能对桃蚜具有杀虫活性.     

Inheritance of resistance to Helicoverpa armigera of 3 kinds of transgenic Bt strains available in upland cotton in China

There are 3 kinds of transgenic Bt strains, Shanxi 94-24, Zhongxin 94, and R19, in upland cotton in China. Their transgenic Bt insect-resistance cultivars or hybrids have been developed and grown by farmers. Genetic studies indicate that the resistance of the 3 transgenic Bt cotton strains to Helicoverpa armigera is controlled by one pair of non-allelic dominant genes. Linkage relationship between the resistant genes of R19 and Shanxi 94-24 transgenic Bt strains shows that they may be inserted in the same chromosome. F₁ hybrids crossed among the 3 strains show that high levels of protection from feeding damage are the same as that of their parents. Therefore, there is no co-suppression phenomenon in many transgenic plants. The results presented here afford a fundamental reliance in developing transgenic Bt insect-resistant cultivars and exploiting the heterosis of hybrids in upland cotton.     

Cloning and functional analysis of chloroplast division gene NtFtsZ2-1 in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2. On the basis of the research on ftsZ1 family, we analyzed the function of NtFtsZ2-1 gene in Nicotiana tabacum. Microscopic analysis of the sense and antisense NtFtsZ2-1 transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-1 gene is involved in plant chloroplast division.     

The Recent Advances in Plant Protection Researches of China

There are eight examples briefly given in this paper, namely, (1) Polymyxa graminis and the cereal viruses it transmits; (2) the geographical types and facultative migration of cotton bollworm as well as the safety of Bt transgenic cotton; (3) development of crop near-isogenic lines with resistance to diseases; (4) molecular-biological researches induced resistance of rice by infection of blast fungus;(5) to use cytological and molecular-biological techniques for breeding wheat varieties resistant to barley yellow dwarf virus; (6) mass rearing and field releasing of Microplitis mediator for cotton bollworm control; (7) identification and recombination of insecticidal crystal genes of Bacillus thuringiensis; and (8) interplanting of diverse resistance rice varieties for sustainable control of blast disease; which reflect the general situation of recent advances in plant protection researches of China.     

Subcellular localization and functional analyses of structural domains of COP1 in transgenic tobacco

Plants have evolved an extremely exquisite light signal regulatory network to adapt to the changing ambient light conditions, in which COP1 plays a critical role of the light signal transduction. Based on the cloned pea COP1 cDNA sequence and its protein structure, four individual gene fragments encoding different structural domains of the COP1 were designed to fuse to the GFP gene. The plant expression vectors containing these fusion genes as well as the COP1GFP fusion gene were constructed and used to transform tobacco by Agribacterium as confirmed by Southern analyses. Antibodies were raised against the recombinant GFP-COP1 overproduced in Escherichia coli. Immunoblotting results demonstrated that all of the fusion genes were constitutively expressed in transgenic tobacco plants. We systematically investigated the different subcellular localization of these fusion proteins and the resulting phenotypic characteristics of these transgenic plants under light and dark conditions. Our data show that (1) the molecular mass of the tobacco endogenous COP1 protein is 76 kD. It is constitutively expressed in all of the tested tissues and the total cellular content of COP1 protein is not noticeably affected by light conditions. (2) The nuclear localization signal of COP1 plays a critical role in regulation of its nuclear-cytoplasmic partitioning. The subcellular localization of the COP1 protein containing nuclear localization signal is regulated by light in the epidermal cells of leaves, but, it is located in nucleus constitutively in root cells. (3) The coiled-coil domain is very critical to the function of COP1 protein, while the zinc binding RING finger domain only plays a supportive role. (4) The WD-40 repeats domain is essential to the COP1 function, but this domain alone does not affect photomorphogenesis. (5) Overexpression of COP1 protein not only inhibits the photomorphogenesis of the stems and leaves of the transgenic tobacco, but also results in the generation of short and clustered roots. In contrast, overexpression of COP1 protein without WD-40 repeats domain promotes the photomorphogenesis process in the stems and leaves and lead to root elongation and lack of lateral roots. The COP1-COP1 interaction happens not only in the nucleus, but also in cytoplasm.     

The role of β18-β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

The β18-β19 loop in domain Ⅲ of Cry1Ac toxin is unique among Bacillus thuringlensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 revealed that a charged amino acid in this position would cause very unfavorable influence on insecticidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18-β19 loop in insecticidal activity of the Cry1Ac toxin.