Mapk-like, the signaling transduction related mitogen-activated protein kinase gene, shares a conserve motif with p38 MAPK that involves in cellular defense of Caenorhabditis elegans against Bacillus thuringiensis(Bt) Cry toxins. Little validation work, however, had been done in its important role for defense response of Aedes aegypti to Cry toxins. Therefore, RNaseⅢ-absent Escherichia coli HT115, with the high-efficiency and low-cost, was applied to establish a convenient and economical method for mass production of dsRNA for RNA silencing. Using specific primers, mapk-like gene (GenBank No. AAEL003728-RA) was amplified. Then 361 bp PCR products were cloned into the cloning vector pMD18-T and subcloned into the expression vector pLitmus28i, which contained 2 T7 promoters located in each side of multiple cloning sites with the digestion of restriction endonuclease XbaⅠ/XhoⅠ. The recombinant plasmid pLitmus28i-mapk-like was transformed into the HT115. dsRNAs of 1.18 μg/mL of bacteria liquid with high quality were obtained after 0.5 mmol/L IPTG induction. In addition, nanoparticles were prepared by mixing resulted dsRNA and chitosan and the supernatant was examined by agarose gel electrophoresis. These results indicated a good coagulation effect for chitosan, by which nanoparticles could protect dsRNA from dissociation from the mixture of feed andagarose and the stability was improved. Finally, the relative expression levels of mapk-like gene after the effective dsRNA feeding decreased to 65%. These results provide potential for use in RNAi, screening for more defense related gene, and basic research for RNA silencing to control mosquito transmitted diseases.

The extensive exploitation and usage of lead resources have caused serious environmental pollution problems currently. Bioremediation of Pb2+ contaminated water and soil environments using microbes is regarded as a promising technology due to the advantages of its cost-effective, easy operation and environmental-friendly properties. In order to understand Pb2+ biosorption characterization of microbes, an indigenous lead-resistant bacterium-Arthrobacter sp. 12-1(GenBank No. KM362724) was isolated from lead-zinc mine tailing dam, and the process and mechanism of Pb2+ biosorption by Arthrobacter sp. 12-1 were furtherly investigated in this study. Study on the growth of Arthrobacter sp. 12-1 in LB medium containing different concentration of Pb2+ suggested that the highest Pb2+ tolerant concentration of Arthrobacter sp. 12-1 was 800 mg/L. In water solution, 105 mg/L of Pb2+ could be reduced to 2.17 mg/L by Arthrobacter sp. 12-1 within 24 h with biosorption rate of 97.93%. Microscopic investigation (atomic force microscopy and scanning electronic microscopy) combined with energy dispersive X-ray spectroscopy analysis showed that lead containing mineral was formed on the surface of cell after Pb2+ sorption by Arthrobacter sp. 12-1. Further fourier transform infrared (FT-IR) analysis revealed that carboxyl, amide and phosphate groups of Arthrobacter sp. 12-1 might be involved in Pb2+ biosorption process. The results demonstrated that the Arthrobacter sp. 12-1 isolated from lead-zinc mine tailing dam had strong ability of Pb2+ resistance and biosorption, indicating an attractive prospect of practical applications in bioremediation Pb2+ contaminated water and soil environments. The present work provides much fundamental information for help in constructing feasible strategies for Pb2+ bioremediation in the environment.

Bacillus thuringiensis (Bt) is an insect pathogenic bacteria that has been widely used in biological control of agricultural insect pests. In this study, one Bt strain was isolated from 159 samples collected from coalfields in Tianhu Mountain, Yongchun County, Fujian Province. Bipyramidal parasporal crystal of isolated Bt strain was observed under microscope. PCR results showed that isolated Bt strain carried cry1 type gene using general primers of cry1/7/9, cry2, cry3, cry4/10, cry5, cry6, cry8, cry11 and cry1I genes, then cry1 gene was cloned and sequenced. Homology analysis of nucleotide sequence of cry1 showed that it was of high similarity with the reported cry1Ac genes. Results of SDS-PAGE and 2-dimensional electrophoresis/mass spectrometry (2-DE/MS) showed that isolated Bt strain encoded approximately 130 kD crystal protein, which was then identified as insecticidal protein Cry1Ac by mass spectrometry (MALDI-TOF/TOF-MS). Molecular chaperone GroEL, ATP F0F1 synthase subunit beta, elongation factor Tu and pyruvate dehydrogenase were identified by MALDI-TOF/TOF-MS, respectively. Further understanding of protein functions were conducted through bioinformatics analysis, leading to correct folding of nascent peptides, energy production and conversion, as well as improving the ability of cells to withstand adverse external stimuli. This work provides basic data for discovering new Bt resources, new insecticidal protein and using microbial to control heavy metal pollution.

Production of Luzhou-flavor liquor is based on pits where ample microbial communities are living, and interactions among all sorts of microbes especially Bacilli in it make fermented sapor of Luzhou-flavor liquor very unique. In this study, 23 aerobic Bacilli were isolated from 300-year pit mud of cellar affiliate to Luzhou laojiao, Luzhou, Sichuan Province. Microscopic and colonial morphology analysis showed that the difference between these aerobic Bacilli were not significant. Through PCR amplification of 16S rDNA gene and NCBI blast, a phylogenetic tree was preliminarily constructed. The results showed that the strains of aerobic Bacilli were mainly Bacillus thuringiensis, B. cereus and B. anthracis. The genetic relationship of these strains was inconformity. As a whole, the phylogenetic relationship was close and the genetic polymorphism was low between these Bacilli isolated from 300300-year pit mud. Aged pit mud was always in the surroundings of anaerobic or facultative anaerobic, studying on the survival condition and genetic relationship of the aerobic Bacilli in aged pit mud was helpful to get a insight in the change of surrounding environment during the liquor fermentation, and improve the quality of liquor. The result provides basic data for research on the change of surrounding environment during the liquor fermentation, and improving the quality of liquor.

Many phytopathogens use N-acylhomoserine lactone (AHL) as quorum sensing (QS) signals, inducing the expression of virulence factors to destroy plant. N-acylhomoserine lactonase (AiiA) can degrade AHL, resulting in the disruption of AHL-mediated QS system in gram-negative bacteria. In this study, auto inducer inactivation A(aiiA) gene was firstly inserted into the shuttle expression vector of pPICZαB and introduced to GS115 strain of Pichia pastoris, resulting in a recombinant yeast strain GS115-pPICZαB-aiiA. On the other hand, codon optimized aiiA gene, named as MaiiA, was carried out by using site-directed mutagenesis to adapt to P. pastoris expression system betterly, and was also inserted into the expression vector and introduced into yeast, and constructed GS115-pPICZαB-MaiiA. Both GS115-pPICZαB-aiiA and GS115-pPICZαB-MaiiA were successfully induced to express secreted AiiA proteins by the addition of 1% methanol at 28 ℃. Antimicrobial activity analysis showed that the AiiA protein inhibited the virulence of Erwinia carotovora. The expression of secreted AiiA protein in P. pastoris, broadening the acquisition route of AiiA protein, provides a theoretical basis and new idea for AiiA industrialization. Codon optimization provides a new way of thinking for the future reconstruction and the efficient expression of AiiA protein.

The aim of this study was to determine the diversity of the cultivable Bacillus species population in the rhizosphere of maize (Zea mays). Bacillus species were isolated from 10 soil samples by using the dilution method on NA medium and 69 isolates were obtained through morphological difference, and identified by 16S rRNA. Phylogenetic analysis based on 16S rRNA gene sequencing showed that the 69 isolates were grouped into 3 genera (Bacillus, Lysinibacillus and Psychrobacillus) with 23 distinct species. Most of these species were owned to the genus Bacillus. The number of species isolated from every maize variety was different. The most number of Bacillus species (8 species) occurred in the varieties QB662×QB2219 and QB1013×QB446, and the most colonies occurred in the variety J106×QB572. The dominant populations in the maize rhizosphere were B. aerophilus, B. aryabhattai, B. simplex and B. thuringiensis, other species only occurred in one maize variety or few maize rhizosphere. The highest Shannon-Wiener index was QB662×QB2219, the second was QB948×QB48 but owning the highest evenness index. The lowest Shannon-Wiener index and evenness index was J106×QB572. Strains FJAT-17411, FJAT-17472, FJAT-17430 and FJAT-17442 may be the new species, which had lower similarities with the known Bacillus spcies in GenBank database. In conclusion, the result of this study showed that the population diversity of the cultivable bacteria was abundant and there were some potential novel species in the rhizosphere of maize. The findings provide an important scientific base for the development and utilization of Bacillus resources.

It has important practical significance to isolate and screen antagonistic actinomycetes from marine shrimp shell and study their antimicrobial activity. Streptomyces luteoverticillatus strain T0907-107 was isolated and identified from shrimp shell. In order to furtherly exploit and utilize the bacteria, antimicrobial activity substance was extracted from the thalli of strain T0907-107 which was collected from fermentation broth by 5 different solvents, acetone, ethyl acetate, n-butanol, ethanol and methanol. Antimicrobial activity and stability of the antimicrobial metabolites produced from strain T0907-107 were carried out. The results showed that acetone and methanol were the best solvents for the crude extract, and the inhibition zones were 26.20 and 22.50 mm, respectively. After protease K treatment, the crude extract still had inhibitory effects to the fungus. The crude extracted substance was material of thermostability (60 ℃) and had a good performance of acid and alkali resistance. The thalli of the strain and the crude extracted substance presented a strong antifungal activity against half of tested plant pathogenic fungi such as Colletotrichum nicotianae Sacc., Alternaria alternata(Fries)Keissler, Rhizoctonia solani Kühn, C. gloeosporioides (Penz.) Sacc. and C. higginsianum Sacc..The results showed that the acetone and methanol were good extraction solvents, the crude extracted substance had strong polarity and a strange of thermostability, acid and alkali resistance, which indicated that the bacteria had good development prospect. This study provides basic data for further identification of the active material and the exploitation and utilization of the strain T0907-107.

Spinosad is a kind of bio-insecticide showing advantages such as high-efficiency and low-toxicity, which is wildly used in pesticide, veterinary drug, sanitary field, etc. However, industrial production of spinosad still has not been implement in China, because of relatively late and undeveloped study on spinosad high-yield strain breeding and relative fermentation process. Previously, a spinosad high-yield strain, Saccharopolyspora spinosa ASAGF73, was obtained through multiple rounds mutagenesis. In this study, we focused on analysing spinosyn biosynthetic gene cluster(spn) expression profile and comparing the difference between wide-type strain ATCC49460 and high-yield ASAGF73 during fermentation process, and preliminarily elucidated the rate-limiting step for spinosad synthesis. Bioinformatic analysis revealed that spn gene cluster contained 9 transcripts, and their expressions were detected using RT-PCR with primers designed inside each transcript. The results showed that the high production of ASAGF73 strain could be due to the high expression of some transcripts in spn cluster, the expression of genes coding for polyketide synthase(PKS) and cross-bridging in ASAGF73 was higher than those in ATCC49460. spnG, which coded rhamnosyltransferase, showed high expression in ASAGF73 strain at the beginning of the fermentation but decreased to a quite low level later, which was not ideal for spinosad high production and might be the rate-limiting step for spinosad synthesis. The present study was the comprehensive and comparative expression analysis of spinosad biosynthetic gene cluster and genes for rhamnose biosynthesis in the 2 S. spinosa strains during fermentation, provided some clues to understand the molecular mechanisms which can lead to the increasing of spinosad production yield.

Receptor binding is a key step in the mechanism of Bacillus thuringiensis Cry toxins and mainly determined by 3 loops locate on the apex of β-sheet in domainⅡ. The important role of these 3 loops, function in binding with specific receptors in the midgut of target insect, has currently been widely reported. However, the importance of 3 loops to influence the toxicity of Cry is different. To determine the important role of these 3 loops in insecticidal activity of Cry1A toxins, in this research, overlap PCR was conducted by using a pea aphid (Acyrthosiphon pisum) gut APN (amino peptidase N) receptor binding peptide GBP3.1 to replace 3 loops in domainⅡ. Then 3 plasmids carrying each modified cry1Ab gene were constructed. After that, prokaryotic expression and purification of 3 modified Cry toxins were conducted. Leaf residue bioassay was conducted by feeding each modified Cry toxin to Plutella xylotella lavae to test the impact of loop substitution on insecticidal activity. Bioassay results indicated that substitution of each loop decreased the toxicity against P. xylotella larvae at different degree, with the LC50 of 12.17, 32.25 and 23.00 μg/mL for the substitution of loop 1, 2 and 3, respectively, compared with wild-type toxin (LC50=0.88 μg/mL). It suggested that compared with loop 1 and 3, loop 2 played the most important role in exhibition of insecticidal activity of Cry1Ab toxin. Prediction of 3-dimensional structure of Cry1Ab toxin and 3 modified Cry toxins showed that replacement of each loop by GBP3.1 did not change the main structure of Cry1Ab protoxin, which meaned that the variety of toxicity against P. xylatella was mainly attributed to the substitution of each receptor binding loop. Moreover, analysis of 3-dimensional structure of Cry1Ab protoxin indicated that loop 2 was the most exposed receptor binding loop compared with loop 1 and 3. These results will be helpful for the understanding of mechanism of Cry1A toxins in the midgut of target insects and benefit further research on gene modification in receptor binding regions of Bt Cry toxins.

Rice gall dwarf virus (RGDV) is a member of the genus Phytoreovirus in the family Reoviridae, which is transmitted by leafhopper Recilia dorsalis and Nephotettix cincticeps in a persistent-propagative manner. The viral genome of RGDV encodes 6 structural proteins and 6 nonstructural proteins. The structural protein P8 is a component of viral outer capsid protein, but the function of this protein in viral infection in insect vector is still unknown. Here, RNA interference (RNAi) induced by double-stranded RNA(dsRNA) synthesized in vitro was used to investigate the role of RGDV P8 protein in viral infection in the cultured cells derived from R. dorsalis. Immunofluorescence assay of RGDV infection in cultured cells after the treatment of dsRNAs from P8 gene (dsP8) howed that dsP8 did not only knock down the expression of P8, but also reduced the accumulation of RGDV in the insect vector cells. Then viral dsRNA genome and proteins were detected by SDS-PAGE and Western blot, the results indicated that the treatment of dsP8 significantly decreased the synthesis of viral dsRNA genome and the expression of P8 protein in insect vector cells. Thus, the results suggested that structural protein P8 of RGDV might play a role in viral assembly and multiplication in insect vector cells. This provides important theoretical basis for controling the spreading of rice gall dwarf disease by using RNAi technology.

Insertion sequence (IS) elements of organisms have the ability to move into genes or DNA sequences as a single unit, implying that these elements play an important role in the evolution of the organisms. A new IS (ISRso21) was isolated and characterized from Ralstonia solanacearum strain FJAT-1458. Sequence analysis indicated that the ISRso21 was closely related to the members of ISL3 family. The results of insertion sites polymorphism of ISRso21 showed that the frequency of positive strains isolated from Northern of Fujian Province was higher than that isolated from other areas. There was significant difference in the distribution of ISRso21 among R. solanacearum isolated from different geography. Therefore, the present of ISRso21 in R. solanacearum strains might be associated with geographic origin. Moreover, the different insertion sites of ISRso21 in R. solanacearum genome were associated with the different pathogenicity. The results indicated that genomic rearrangements of phenotype conversion transcriptional regulator A (phcA) which was inserted by ISRso21, might play a major role in the control of pathogenicity. In upstream of phcA gene of R. solanacearum, the inserted detection rate of ISRso21 in all strains was 4.71%. The detection rates in avirulent strains and virulent strains were 28.57% and 0.00%, respectively. The insertion of ISRso21 in diaminopimelate decarboxylase gene might make the R. solanacearum strains adapt betterly to the environment. The different insertion sites of ISRso21 in the R. solanacearum strains need to be furtherly investigated, which can provide an alternative way to reveal the pathogenicity of R. solanacearum.

Anoectochilus roxburghii (Wall.) Lindl is a traditional Chinese medicine, which has been used for diabetes and tumor treatment. The wild A. roxburghii and its endophytes live together to form a balanced symbiotic system. The endophytes produce various secondary metabolites to protect the host from being attacked by fungi and pests. Endophytes are rich bio-resources. Because of a major limitation of isolation methods and cultivation media, the diversity endophytes still remain undiscovered. Metagenomics simultaneous study of endophytes genomes from environmental samples may avoid the limitations of culture-dependent methods. We enriched the microbiota from the root of A. roxburghii and extracted and purified the metagenomic DNA.16S rDNA fragments were amplified by PCR and cloned to Escherichia coli DH5α to sequence. The endophytes were mainly uncultured bacterium, uncultured compost bacterium, Enterobacteriaceae, Paenibacillus sp., Bacillus sp. and Brevibacillus sp., according to 16S rDNA information. This research provided the basis for mining endophytes resources of A. roxburghii. Analyzed the groups of endophytes by partial sequence of 16S rDNA. This work opens further insight into the great potential of A. roxburghii microbiota.

This article mainly compares the similarities and differences between Prima produced by Malaysia Pupuk Alam company and protein bait for Bactrocera dorsalis (Hendel) developed by our research institute. The biological test was used to compare attractive effects, and High Performance Liquid Chromatography with Precolumn Derivatization (AccQ·Tag) was used to analyse the kinds and contents of amino acids. The results showed the attractive effects of Prima and protein bait were similar, and the two protein baits all contained 17 kinds of free amino acids and 18 total amino acids. Prima contained 52.847 6 mg/mL of total amino acids, 5.736 4 mg/mL of free amino acid, and 47.111 2 mg/mL of peptides. The protein bait was consisted of 65.624 3 mg of total amino acids, 6.301 0 mg of free amino acid, 59.323 3 mg of peptides in one milliliter. Besides, the attractive effect of hydrolyzed protein was stronger than before. The results can provide a basis for monitoring protein bait quality on the industrial production.

Vegetative insecticidal protein (Vip) is secreted by Bacillus thuringiensis (Bt) during the period from vegetative growth to logarithmic metaphase. To obtain efficient expression condition of Vip3Aa protein, 3 main factors (concentration of isopropyl-β-d-thiogalactoside(IPTG), inducing time and inducing temperature) were screened for the optimization inducing condition of recombinant Escherichia coli BL21-pCzn1-Vip in producing the Vip3Aa by orthogonal experiment. The highest expression level of target protein could be attained with the condition of 200 μg/mL IPTG inducing for 8 h at 21 ℃. The data were analyzed by direct calculation and one-way ANOVA method. R value (resulting from direct calculation) of inducing temperature was the highest among the 3 factors, followed by the inducing time and the concentration of IPTG, wihch indicated that the inducing temperature was the most important factor to affect expression level of target protein. P value from one-way ANOVA of inducing temperature and inducing time was lower than 0.01 and 0.05, respectively, and the P value of the other factor was higher than 0.05, which were similar as the results of direct calculation. The amount of the expressed target protein at the optimal inducing condition was up to 27.6% of total protein, which was 2.3 times of the production before optimization. It can provide the basis for the Vip3Aa manufacture, in-depth theoretical research and practical application.

Rice gall dwarf virus(RGDV), the genus Phytoreovirus in the family Reoviridae, is transmitted by the leafhopper vector(Recilia dorsalis) in a persistent-propagative manner. Nonstructural protein(Pns12), encoded by segment 12 of RGDV, is one of the components of viroplasm which is the site for viral replication and assembly of progeny virons during viral infection in its insect vector cells. In this study, to investigate the functional role of Pns12 in the formation of viroplasm in its insect vector cells, the polyclonal antibody against Pns12 was prepared and purified, then conjugated to fluorescein isothiocyanate. The immunoglobulin G(IgG) of P8 was purified and conjugated to rhadamine. Immunofluorescence microscopy demonstrated that Pns12 antibodies specifically distributed in the viroplasm matrix during RGDV infection in the vector cells in monolayers (VCMs), while outer capsid protein P8 were accumulated at the periphery of the viroplasm. The viroplasm increased in size over time. Knockdown of Pns12 by RNA interference (RNAi) induced by dsRNAs, targeting Pns12 gene of RGDV, significantly inhibited the formation of viroplasm compared with dsGFP, suggesting that RGDV replication was inhibited. Western blot showed that viral Pns12 and P8 expression reduced in the VCMs treated with dsPns12. Thus, the present study indicated that Pns12 of RGDV played an important role in viroplasm formation and viral replication in its insect vector cells. RNAi induced by dsRNAs derived from the viral genes of viroplasm matrix protein may be an ideal tool for inhibiting the infection and transmission of RGDV by leafhopper vectors.

Vegetative insecticidal proteins(Vips) are one kind of novel toxic proteins, which are generated and excreted by Bacillus thuringiensis(Bt) cells during growth from mid-exponential phase to the stationary phase. Vips may generally be classified into 3 groups of Vip1, Vip2 and Vip3, of which Vip3 is the most intensively studied. It has important research significance because of broad-spectrum insecticidal activities to Lepidoptera pests. The entire coding region of vip3Aa(GenBank No. AAM22456) gene was amplified by PCR with total DNA extracted from BtWB5 as template. PCR product was purified and ligated with pMD18-T to form the recombinant pMD18-T-vip3Aa, and then transformed into Escherichia coli DH5. The recombinant plasmid DNA extracted from the selected positive clone was used for nucleotide sequencing and NdeⅠ/XbaⅠ digested analysis. Using Universal DNA Purification Kit, an approximate 2.4 kb DNA product was purified from NdeⅠ/XbaⅠdigested pMD18-T-vip3Aa, and then inserted into multiple cloning sites of the expression vector pCzn1 to generate the recombinant expression vector pCzn1-vip3Aa. The inserted fragment and its reading frame were confirmed by NdeⅠ/XbaⅠdigestion and nucleotide sequence analysis. pCzn1-vip3Aa was transformed into E. coli ArcticExpressTM (DE3) and induced with isopropyl β-D-1-thiogalactopyranoside(IPTG). SDS-PAGE showed that the relative molecular weight of the expressed protein was about 88 kD in both supernatant and precipitated. In addition, the expressed protein was purified by Ni-NTA affinity chromatography. The success of the subcloned vip3Aa gene and the expression and purification of Vip3Aa provide basic data for furthur research on its action receptor in insect mid gut.

Natural biological control agents such as insecticides that exhibit high efficacy, low toxicity, superior selectivity and environmental friendliness, have maintained an important role in integrated pest management of agriculture. This paper provided an overview of recent promising insecticides from natural sources. They were avermectins, spinosyns, meibemucins, and ivermectin. The researches on their efficacies, biochemical properties, mechanism of actions, as well as recent advances in the molecular genetics of the producing microorganisms at home and abroad would be discussed here.