Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.

A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from Bacillus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in approximately 15% of Bacillus strains analyzed. The sequence of the vip3A(b) gene, a homologue of vip3A(a) isolated from B. thuringiensis strain AB424 is also reported. Vip3A(a) and (b) proteins confer upon Escherichia coli insecticidal activity against the lepidopteran insect larvae mentioned above. The sequence of the gene predicts a 791-amino acid (88.5 kDa) protein that contains no homology with known proteins. Vip3A insecticidal proteins are secreted without N-terminal processing. Unlike the B. thuringiensis 5-endotoxins, whose expression is restricted to sporulation, Vip3A insecticidal proteins are expressed in the vegetative stage of growth starting at mid-log phase as well as during sporulation. Vip3A represents a novel class of proteins insecticidal to lepidopteran insect larvae.

棉花抗虫基因工程研究

本文通过根农杆菌（Agrobacterium tumfaciens）介导法分别将Signal和KDEL修饰的豇豆胰蛋白酶抑制剂（Cowpea trypsin inhibitor, CpTI）基因、豌豆外源凝集素（Pea lectin, P-Lec）和大豆Kunitz型胰蛋白酶抑制剂（Soybean Kunitz typsin inhibitor, SKTI）双价抗虫基因、雪花莲外源凝集素（Galanthus nivals agglutinin, GNA）基因以及高效复合启动子OM控制的苏云金杆菌（Bacillus thuringiensis, B.t.）杀虫毒蛋白基因导入了陆地棉（Gossypium hirsutum L.）栽培品种新陆早1号、新陆中2号、晋棉7号、冀合321、辽9和晋棉12号，并获得了大批转基因再生植株。 实验中对影响棉花转化和再生的一些条件进行了研究，从根农杆菌培养、棉花无菌苗的制备、转化操作和共培养等方面对转化条件进行了探讨;从激素配化、植物表达载体、外植体类型、基因型等方面对抗性愈伤组织的诱导进行了摸索;从激素、从碳源、培养容器、pH值、抗褐化剂及固化剂的选择等方面对影响植株再生的条件进行了优化。 本文开创性地采用嫁接代替移栽，从而极大地提高了转化植株定植成活率，缩短了缓苗时间并增加了转化植株当代的繁殖系数。 在建立了一套较为高效的陆地棉转化及再生系统基础上，本文还进行了其它转化方式和转化体系的初步探讨。利用棉花幼嫩种子无菌苗下胚轴作为外植体，通过改变愈伤组织诱导培养基配方面提高胚性愈伤组织的诱导频率，进而得到更多的体细胞胚状和再生植株，缩短再生周期;尝试用胚性愈伤组织作为外植体的根农杆菌介导法转化，确定了一些与转化有关的条件;建立了一套棉花茎尖培养程序，为运用基因枪法轰击棉花茎尖分生组织或用根农杆菌直接转化茎尖分生组织，以克服根农杆菌转化棉花时体胚发生的基因型局限开辟了一条新途径。 本文还建立了一种快速鉴定转化植株后代的方法。这一简便方法还有助于进行转基因棉纯合系的筛选以及外源基因的遗传稳定性研究。 转基因植株经Npt-II ELISA、PCR、PCR Southern 检测证明外源抗虫基因CpTI、SKTI、P-lec、GNA以及B.t.基因已存在于转化植株基因组内。修饰的CpTI转基因植株抗棉铃虫（Heliothis armigera Hubner）试验结果表明，其杀虫效果显著优于前期未修饰的CpTI转化植株。P－lec和SKTI双价转基因植株抗棉铃虫试验结果表明，转基因植株对棉铃虫幼虫具有较强的杀虫活力。 目前，已获得转以上抗虫基因棉花T1代植株。为今后进一步将植物基因工程技术应用于棉花遗传改良打下了基础。

Vertical transmission efficiency and insecticidal properties of Spodoptera exigua Nucleopolyhedrovirus (SeMNPV) genotypes for their use in biological control

El nucleopoliedrovirus de Spodoptera exigua (SeMNPV) es un patógeno natural de las poblaciones larvarias de S. exigua que constituye la base de un bioinsecticida comercializado en España para el control biológico de esta plaga en pimiento. Recientes estudios han demostrado que la transmisión del virus a la descendencia (transmisión vertical) se da con frecuencia y podría ser una característica deseable para su uso en aplicaciones de campo. En el presente trabajo se discute la conveniencia de utilizar una mezcla de dos genotipos SeAl1 (transmisión vertical) y SeG25 (transmisión horizontal) en determinadas proporciones para mejorar las características que cada uno de ellos presenta por separado y así explotar cada una de las vías de transmisión. La patogenicidad (CL50) del genotipo SeG25, y de cualquiera de las mezclas que contienen un 25, 50 o 75 % del mismo, fue más alta que la del aislado SeAl1. Sin embargo, en términos de virulencia (TMM) y productividad (OBs/larva) no se observaron diferencias significativas entre genotipos ni entre sus mezclas. Además se evaluó la capacidad de producir infecciones encubiertas de cada genotipo y sus mezclas sometiendo larvas de S. exigua a infecciones subletales del virus. Se encontraron transcritos del virus para el gen temprano ie0 mediante RT-PCR en los adultos supervivientes a infecciones provocadas por el genotipo SeG25 y todas las mezclas. También se testaron otros dos genes virales que se expresan de manera temprana y tardía en la infección de baculovirus (DNA-polimerasa y polihedrina) para los que en ningún caso se detectaron transcritos.

Expression of a new Bacillus thuringiensis Cry1Ia gene in Escherichia coli with strong activity against cotton pests

The control of cotton pests may be accomplished using Bacillus thuringiensis Cry proteins. For this purpose, the objective of this work was to evaluate the insecticidal activity of a new Cry1Ia protein against neonatal larvae of Spodoptera frugiperda and Anthonomus grandis. The complete cry1Ia gene, previously obtained by PCR with oligonucleotide primers based on the sequenced gene, was cloned into the vector pET28a(+), introduced into Escherichia coli BL21(DE3) and expressed by induction with IPTG. The expression of the Cry1Ia protein was confirmed with molecular weight of approximately 81 kDa. The results demonstrated the efficiency of the bacterial system for the expression of B. thuringiensis Cry1Ia protein, which was subsequently used in quantitative bioassays against S. frugiperda and A. grandis larvae, resulting in an extremely toxic protein for both species. This characteristic is exceptionally important for obtaining transgenic cotton plants resistant to these pests.

Identificação de genes cry1 em isolados de Bacillus thuringiensis e expressão do gene cry1la em Escherichia coli visando o controle de pragas da cultura algodoeira

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização do gene vip3A e toxicidade da proteína Vip3Aa50 à lagarta-do-cartucho e à lagarta-da-soja

O objetivo deste trabalho foi caracterizar o gene vip3A de Bacillus thuringiensis e verificar a toxicidade da proteína Vip3Aa50 a larvas da lagarta-do-cartucho (Spodoptera frugiperda) e da lagarta-da-soja (Anticarsia gemmatalis). O gene vip3A foi amplificado por PCR, com iniciadores específicos, e gerou um fragmento de 2.370 pb. Esse fragmento foi clonado em vetor pGEM-T Easy e, em seguida, sequenciado, subclonado em vetor de expressão pET-28a (+) e inserido em células de Escherichia coli BL21 (DE3). A expressão da proteína Vip3Aa50 foi induzida por isopropil-β-D-1-tiogalactopiranosídeo (IPTG), visualizada em SDS-PAGE e detectada por Western blot. Os ensaios de toxicidade revelaram alta atividade da proteína Vip3Aa50 contra as larvas neonatas da lagarta-da-soja e da lagarta-do-cartucho, com CL50 de 20,3 e 79,6 ng cm-2, respectivamente. O gene vip3Aa50 é um novo gene da classe vip3A.

Comprehensive Analysis of Gene Expression Profiles of the Beet Armyworm Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Vip3Aa Toxin

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Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants. (c) 2012 Elsevier Inc. All rights reserved.

Premature Polyadenylation at Multiple Sites within a Bacillus thuringiensis Toxin Gene-Coding Region1

Some foreign genes introduced into plants are poorly expressed, even when transcription is controlled by a strong promoter. Perhaps the best examples of this problem are the cry genes of Bacillus thuringiensis (B.t.), which encode the insecticidal proteins commonly referred to as B.t. toxins. As a step toward overcoming such problems most effectively, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, cryIA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacum) cells. Most cell lines transformed with the cryIA(c) B.t.-toxin gene accumulate short, polyadenylated transcripts. The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabilize many unstable transcripts. Using a series of hybridizations, reverse-transcriptase polymerase chain reactions, and RNase-H-digestion experiments, poly(A+) addition sites were identified in the B.t.-toxin-coding region corresponding to the short transcripts. A fourth polyadenylation site was identified using a chimeric gene. These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants. Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.

No evidence for association of ataxia-telangiectasia mutated gene T2119C and C3161G amino acid substitution variants with risk of breast cancer

Background There is evidence that certain mutations in the double-strand break repair pathway ataxia-telangiectasia mutated gene act in a dominant-negative manner to increase the risk of breast cancer. There are also some reports to suggest that the amino acid substitution variants T2119C Ser707Pro and C3161G Pro1054Arg may be associated with breast cancer risk. We investigate the breast cancer risk associated with these two nonconservative amino acid substitution variants using a large Australian population-based case–control study. Methods The polymorphisms were genotyped in more than 1300 cases and 600 controls using 5' exonuclease assays. Case–control analyses and genotype distributions were compared by logistic regression. Results The 2119C variant was rare, occurring at frequencies of 1.4 and 1.3% in cases and controls, respectively (P = 0.8). There was no difference in genotype distribution between cases and controls (P = 0.8), and the TC genotype was not associated with increased risk of breast cancer (adjusted odds ratio = 1.08, 95% confidence interval = 0.59–1.97, P = 0.8). Similarly, the 3161G variant was no more common in cases than in controls (2.9% versus 2.2%, P = 0.2), there was no difference in genotype distribution between cases and controls (P = 0.1), and the CG genotype was not associated with an increased risk of breast cancer (adjusted odds ratio = 1.30, 95% confidence interval = 0.85–1.98, P = 0.2). This lack of evidence for an association persisted within groups defined by the family history of breast cancer or by age. Conclusion The 2119C and 3161G amino acid substitution variants are not associated with moderate or high risks of breast cancer in Australian women.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.