A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis ␦-endotoxin nomenclature committee.Crystal proteins from the gram-positive spore-forming bacterium Bacillus thuringiensis are toxic to a wide variety of insects that are economically important as pests. Many different genes encoding the B. thuringiensis endotoxin have been isolated and characterized. The genes have been classified as cry1 to cry40, cyt1, and cyt2 and are ranked according to their homology (10, 21; see also the B. thuringiensis toxin nomenclature website at http://www.biols.susx.ac.uk/home/Neil_Crickmore /Bt/). Cry1 proteins that are active against lepidopteran insects are produced as crystalline parasporal inclusions during sporulation. Generally, the crystals are composed of protoxins of approximately 130 kDa, but cry1I-type genes are usually silent genes capable of encoding a protein of about 81 kDa in B. thuringiensis strains (9,13,21,24). We decided to screen B. thuringiensis isolates for cry1I genes with the aim of finding novel cry1I genes, which could encode insecticidal proteins toxic to insensitive or resistant insect pests.This screening approach included the development of an analysis protocol based on PCR-restriction fragment length polymorphism (RFLP). PCR-based methods have been developed to detect different cry genes from B. thuringiensis strains (1-8, 11, 13, 15, 17, 23). More than 80 primer pairs have been designed to identify entire groups and individual cry genes (19). Several specific primers and probes for Southern blotting were designed to detect cry1I-type genes. A wide distribution of cry1I-type genes among many different B. thuringiensis strains has been reported (13,22,25). However, the list of cry genes is increasing, and novel PCR primers are needed in order to identify some of the recently described genes (5).The present study establishes a PCR-RFLP method for identify...

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Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on epigenetics and can help us to gain new insight into the time of departure as well as caste trajectory influencing elements at the molecular level.

A novel cry1A was cloned from Bacillus thuringiensis strain BT8 and expressed in the B. thuringiensis acrystalliferous mutant HD73(-). The gene, designated cry1Ah1, encoded a protein with a molecular weight of 134 kDa. Reverse transcriptase-PCR and Western blotting showed that Cry1Ah was expressed in the host strain BT8. The toxin expressed in HD73(-) exhibited high toxicity against lepidopteran larvae of Ostrinia furnacalis, Helicoverpa armigera, Chilo suppressalis, and Plutella xylostella. The 50% lethal concentrations (LC(50)s) were 0.05, 1.48, 0.98 microg g(-1) and 1.52 microg mL(-1), respectively. The LC(50)s of Cry1Ah were significantly lower than that of Cry1Ac for H. armigera, C. suppressalis, and O. furnacalis, and lower than that of Cry1Ab and Cry1Ie for Ostrinia furnacalis. The high toxicity against a range of pest species makes this novel toxin a potential candidate for insect biocontrol.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is an important disease that causes substantial yield losses in wheat (Triticum aestivum) in China and other parts of the world. This foliar disease can be effectively managed by host resistance. The Chinese landrace Hongyanglazi from Shaanxi province is highly resistant to many Bgt isolates at the seedling stage. Genetic analysis using an F2:3 population derived from a cross between Hongyanglazi and susceptible cultivar Zhongzuo 9504 indicated that Hongyanglazi carried a single recessive gene (tentatively designated PmHYLZ) conferring its resistance to Bgt isolate E09. PmHYLZ was flanked by EST marker BE606897 and microsatellite marker Xgwm46 on chromosome 7BS at genetic distances of 1.7 and 3.6 cM, respectively. This gene differed from Pm40, also located on 7BS, by origin, linked markers, and reactions to 13 Bgt isolates. Based on these findings, PmHYLZ was permanently designated as Pm47.

Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation and crystallization of large amounts of insecticidal Cry proteins. This process normally coincides with sporulation and is regulated by various factors operating at the transcriptional, post-transcriptional, metabolic and post-translational levels.

A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding a coleopteran-specific insecticidal protein was constructed and introduced into wild Bacillus thuringiensis subsp. aizawai G03, which contained cry1Aa, cry1Ac, cry1Ca, and cry2Ab genes and was highly toxic to lepidopteran insect pests. The genetically engineered strain were named G033A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry3Aa7 gene was expressed normally and produced a 67 kDa protein in G033A, and the flat rectangular crystals of Cry3Aa7 toxin protein was observed under scanning electron microscope. The recombinant plasmid was maintained in bacteria cultured for 180 generations in culture media containing no antibiotics. Synthesis of the Cry3Aa7 toxin conferred high and broad toxicity to the recombinant strain G033A against coleopteran order, elm leaf beetle (Pyrrhalta aenescens) (LC(50) 0.35 mg/ml), for which the parental strain G03 was not toxic. Both the parental strain G03 and recombinant strain G033A showed strong insecticidal activity to lepidopteran pests, beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera), respectively. The lethal concentration 50% (LC(50)) of G033A against S. exigua, P. xylostella, and H. amigera was 4.26, 0.86, and 1.76 microg/ml, respectively.