Geographical Distribution of Bacillus thuringiensis Strains and vip3 Genes in Different Climatic Zones in China

Vegetative Insecticidal Proteins(VIPs),a large family of insecticidal proteins,are produced from Bacillus thuringiensis (Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity.This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical (Hainan Province), subtropical (Guangxi Province) and temperate zones (Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes,while vip3 genes were not expressed from temperate zone in Heilongjiang Province.Restriction Fragment Length Polymorphism(RFLP)was used to identify different types of vip 3 genes that were within the same family and three full-length vip3 genes were isolated.The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E.coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects (Spodoptera exigua and Agrotis ipsilon)were performed.The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay.Taken together,the results indicated that the distribution of Bt strains and vip3 genes had regional preference.Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region.These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources.The new vip 3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.     

高大冷杉温室Strophosoma象甲的微生物防治

In Denmark, the weevils Strophosoma melanogrammum and S.capitatum cause economic damage in Noble fir due to the adult stage feeding on the needles.No chemical treatments of these weevils are allowed in Denmark,so biological control is an attractive solution.We evaluated the potential for microbial control of larvae of Strophosoma spp.based on laboratory bioassays and field applications,taking effect on both target and non-target into consideration,as well as persistence of the applied fungus.In the laboratory Beauveria bassiana,Paecilomyces farinosus and Metarhizium anisopliae were able to infect and cause mycosis in Strophosoma larvae.Among the tested isolates the most virulent isolate was M.anisopliae BIPESCO 5,which resulted in 80 % mortality.In the field experiment M.anisopliae,isolate BIPESCO 5,was applied to the soil as a conidial suspension against larvae of Strophosoma spp.The effect of the fungus on the target population was monitored at a weekly basis by counts of emerging adult weevils during their activity periods.The population of Strophosoma spp.was reduced by up to 60% in treated plots compared to control plots.The non-target effects of M.anisopliae were studied by sampling insects and ticks from both treated and control plots.Seven days after treatment,two sampled insect orders (Hemiptera and Coleoptera) and ticks were found with prevalences of M.anisopliae above 50%,compared to no infection in the insects collected from control plots.Infections in coccinellids were found as long as 277 days after treatment.However,the effect on population level of non-target is still unexplored.The persistence of the fungus was documented by plating a soil suspension onto agar.We documented that conidia of M.anisopliae could persist in the greenery plantation for at least 418 days after application.     

Transgenic cry1Ab/vip3H＋epsps Rice with Insect and Herbicide Resistance Acted No Adverse Impacts on the Population Growth of a Non-Target Herbivore,the White-Backed Planthopper,Under Laboratory and Field Conditions

Numerous Bt rice lines expressing Cry protein derived from Bacillus thuringiensis Berliner （Bt） have been developed since 1989. However, the potential risks posed by Bt rice on non-target organisms still remain debate. The white-backed planthopper （WBPH）, Sogatella furcifera （Horváth）, is one of the most economically important insect pests of rice in Asian countries and also one of the main non-target herbivores of transgenic rice. In the current study, impacts of transgenic cry1Ab/vip3H＋epsps rice （G6H1） with both insect and herbicide resistance on WBPH were evaluated to ascertain whether this transgenic rice line had potential risks for this sap-sucking pest under laboratory and ifeld conditions. The laboratory results showed that no signiifcant difference in egg developmental duration, nymphal survival rate and female fecundity was found for WBPH between G6H1 and its non-transgenic isoline （XS110）. However, the development duration of nymphs was signiifcantly shorter and female longevity signiifcantly longer when WBPH fed on G6H1 by comparison with those on its control. To verify the results found in laboratory, a 3-yr ifeld trial was conducted to monitor WBPH population using both the vacuum-suction machine and beat plate methods. Although the seasonal density of WBPH nymphs and total density of nymphs and adults were not signiifcantly affected by transgenic rice regardless of the sampling methods, the seasonal density of WBPH adults in transgenic rice plots was slightly lower than that in the control when using the vacuum-suction machine. Based on these results both from laboratory and ifeld, it is clear that our tested transgenic rice line will not lead higher population of WBPH. However, long-term ifeld experiments to monitor the population dynamics of WPBH at large scale need to be conducted to conifrm the present conclusions in future.     

Ecological Consequences of Elevated CO2 and Bt Cotton on Soil Collembola

Transgenic cotton was modified to express a gene derived from the bacterium Bacillus thuringiensis （Bt） to combat agriculturally important Lepidopteran pests. Elevated CO2 is expected to further alter the chemical composition of the plant, and this change may affect the role soil fauna plays in decomposition of Bt plants. A 3 months litterbag field study, consisting of four treatments using leaves from Bt cotton and near-isolines of non-Bt cotton grown under ambient and elevated CO2 levels, was conducted to investigate the abundance and community structure of soil Collembola that developed on the decaying leaf material. A total of 4,884 collembolans, including 13 genera of five families, were extracted in the present study. These results suggest that collembolan distribution was relatively uniform among the Bt cotton, elevated concentration of CO2 and control treatments, except for a significant difference in the densities of Onychiurus and Folsomides. No significant effects were detected in the decomposition rate between the two cotton varieties and two CO2 treatments. These findings indicated that transgenic Bt cotton plants and elevated CO2 do not have any adverse effect on the soil collembolans through the decomposition way in soil ecosystem.     

Evaluation of Impact of Pollen Grains from Bt, Bt/CpTI Transgenic Cotton and Bt Corn Plants on the Growth and Development of the Mulberry Silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae)

The δ-endotoxin genes of Bacillus thuringiensis (Bt) and proteinase inhibitor (PI) genes aretwo kinds of genes popularly used for developing transgenic plants resistant to insect pests. To clarify whetherthere is any risk concerning the effects of pollens from these transgenic crops on non-target insects with eco-nomic importance, such as the effects on the growth and development as well as cocoon quality of the silk-worm, Bombyx mori Linnaeus, a series of feeding experiments were conducted, using pollens from transgeniccotton or corn containing crylAc, cry1A+-CpTI or crylAb genes, compared with pollens from non-transgenicnormal cotton and corn as well as the non-pollen treatment. In contrast to the latter ones, pollens from trans-genic plants showed no significant adverse effects on larval mortality, cocoon weight, pupa weight, cocoonshell weight, pupation rate, emergence rate and fecundity of the silkworm after neonates were fed with thepollens for 72 h. In addition, no dosage effects of pollens were found. Though the duration of 1st instar larvaewas prolonged in the case of feeding with transgenic pollens as compared with those of the non-pollen treat-ment, but they were not significantly different from those fed with pollens from non-transgenic cotton or corn.Meanwhile, the body weight of the 3rd instar molters fed with transgenic pollens was obviously different fromthose for non-pollen treatment, and was all significantly heavier than that of the controls. Consequently, it isconsidered that the adverse effect of pollens from transgenic insect-resistant cotton and corn on the growth anddevelopment of the silkworm is negligible.     

Relationship Between Leaf C/N Ratio and Insecticidal Protein Expression in Bt Cotton as Affected by High Temperature and N Rate

Expression of insecticidal protein for transgenic Bacillus thuringiensis （Bt） cotton is unstable and related to nitrogen metabolism. The objective of this study was to investigate the relationship between leaf carbon nitrogen ratio （C/N） and insecticidal efficacy of two Bt cotton cultivars. C/N ratio and Bt protein content were both measured at peak square period and peak boll period respectively under 5-7 d high temperature and different nitrogen fertilizer rates on the Yangzhou University Farm and the Ludong Cotton Farm, China. All plants were grown in field. The results showed that the C/N ratio enhanced slightly and the Bt protein content remained stable at peak square period, but significant increases for the C/N ratio and decreases markedly for the leaf Bt protein concentration were detected at the peak boll period. The similar patterns at the two growth periods were found for the leaf C/N ratio and Bt protein content by different N fertilizer treatments. When nitrogen rate was from 0 to 600 kg ha-l, the C/N ratio was reduced by 0.017 and 0.006 for Sikang 1 and Sikang 3 at peak square period, compared to the 1.350 to 1.143 reduction for Sikang 1 and Sikang 3 at peak boll period, respectively. Correspondingly, the leaf Bt protein contents were bolstered by 2.6-11.8 and 26.9-36.9% at the two different growth periods, respectively. The results suggested that enhanced C/N ratio by high temperature and nitrogen application may result in the reduction of inseetiocidal efficacy in Bt cotton, especially in peak boll period.     

Impacts of Environmental Factors on Degradation of CrylAb Insecticidal Protein in Leaf-Blade Powders of Transgenic Bt Rice

The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of CrylAb protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry lAb were measured using enzyme-linked immunosorbent assay （ELISA）. The degradation rate of CrylAb was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of CrylAb, but the effects of soil pH and temperature were relatively greater. In general, CrylAb degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.     

Construction and Characterization of a cDNA Library from the Pulp of Coconut （Cocos nucifera L.）

To investigate the gene expression profile of endosperm development, a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages. The constructed cDNA library incorporated approximately 1 × 10^7 clones in total, and the size of the insertion fragments ranged from 800 to 2 000 bp. Sequencing results of 100 randomly picked clones showed that the recombination rate was 96%. In subsequent sequence analysis, 41 clones （41%） were homologous to known function proteins, and 23 clones showed high amino acid identity （more than 80%） with the corresponding genes of different plants. Semi-quantitative RT-PCR indicated that oleosin and globulin genes are pulpspecific expression, and have differential expression level in different developmental stage. Clone 29, recognized as homologous to KIAA1239 protein （Homo sapiens）, was observed to occur nine times, indicating that this gene may be over-expressed during the endosperm development stage. However, the homologous protein was found only in mammals, and the detailed function is still unknown. Elucidation of the functional characterization of these genes will be carried out immediately.     

Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences

The coat protein （CP） genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus （CTV） collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations （du） to synonymous mutations （ds） was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV.     

The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Ⅰ on the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase Ⅰ on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato （Lycopersicon esculentum） were cloned, such as the l-aminocyclopropane-1-carboxylic acid oxidase 1 gene （LeAC01）, 1- aminocyclopropane-l-carboxylic acid oxidase 3 gene （LeAC03）, EIN3-binding F-box 1 gene （LeEBF1）, pathogenesis-related protein 1 gene （LePR1）, pathogenesis-related protein 5 gene （LePR5）, and pathogenesis-related protein osmotin precursor gene （LeNP24） by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig （AC＋＋） and the LeAC01 co-suppression tomatoes （V1187 and T4B）, respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carded out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.     

Developing transgenic maize (Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis(Bt) cry gene and epsps(5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.     

Gene Expression Profiles of Response to Water Stress at the Jointing Stage in Wheat

Drought is one of the most adverse environmental factors that impact on plant growth and reduce crop yields. To decipher the molecular mechanism of drought tolerance, we constructed a cDNA library using suppression subtractive hybridization （SSH） and dissected the gene expression profiles in seedling and jointing wheat plants after stress with PEG-6000. A total of 2 046 ESTs from the jointing stage library （J-Lib） were allocated to 961 contigs. Among the ESTs, 265 uni-genes in 12 categories were identified on the basis of sequence similarities and functional classifications. Most were known to be involved in protection, directly or indirectly, against water stress. To determine differences in gene expression profiles for water stress responses at the jointing and seedling stages, data from the J-Lib were compared with those from a 2- leaf seedling library （S-Lib） constructed by the same method. Significant differences were observed between the two libraries for function-known genes; signal transduction genes were far more active at jointing than at the seedling stage.     

Construction of Marker-Free GFP Transgenic Tobacco by Cre/Iox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.     

Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated crylAh Gene via Agrobacterium-Mediated Transformation

A novel insecticidal gene crylAh was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active CrylAh toxin has a toxicity level similar to that of the full-length CrylAh toxin. In this study, plant expression vector pMhGM harboring truncated crylAh gene was transformed into maize （Zea mays L.） immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions （madMARs） were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer （Ostriniafurnacalis）. These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the crylAh gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene crylAh was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T l-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.     

Root Function in Nutrient Uptake and Soil Water Effect on NO3- -N and NH4＋-N Migration

Root function in uptake of nutrients and the effect of soil water on the transfer and distribution of NO3^--N in arable soil were studied using summer maize （Zea mays L. var. Shandan 9） as a testing crop. Results showed that root growth and water supply had a significant effect on NO3^--N transfer and made NO3^--N distributed evenly from bulk soil to rhizosphere soil. Under a natural condition with irrigation, the difference of NO3^--N concentration at different distance points from a maize plant was smaller, while obvious difference of NO3^--N concentration was observed under conditions of limited root growth space without irrigation. Whether root growth space was restricted or not, the content of soil NO3^--N decreased gradually from 10 to 0 cm from the plant, being opposite to the root absorbing area in soils. When root-grown space was limited, changes of NO3^--N concentration at different distances from a plant were similar to that of water content in tendency. Results showed that NO3^--N could be transferred as solute to plant root systems with water uptake by plants. However, the transfer and distribution of NH4^--N were not influenced by root growth and soil water supply, being different to NO3^--N.     

Effects of Chlorination on Soil Chemical Properties and Nitrogen Uptake for Tomato Drip Irrigated with Secondary Sewage Effluent

Chlorination is usually an economical method for treating clogging in drip emitters during sewage application. Appropriate assessment of the responses of soil and crop is essential for determining an optimal chlorination scheme. During 2008 to 2009, field experiments were conducted in a solar-heated greenhouse for tomato drip irrigated with secondary sewage effluent, to investigate the influences of chlorine injection intervals and levels on soil chemical properties and nitrogen uptake. Injection intervals ranging from two to eight weeks and injection concentrations ranging from 2 to 50 mg L-1 were used. A salinity factor and a nutrient factor were extracted from the pool of the nine soil chemical constituents using factor analysis method. The results demonstrated that chlorination practices increased the residual Cl in the soil, resulting in an increased salinity factor, especially for the frequent chlorination at a high injection concentration. Chlorination weakened the accumulation of nutrients factor in the upper soil layer. Nitrogen uptake of the tomato plants also was inhibited by the increased salinity in the upper soil layer caused by high chlorination levels. In order to reduce the unfavorable effect on soil chemical properties and nitrogen uptake, chlorination scheme with concentrations of lower than 20 mg L-1 was recommended.     

High plant density increases seed Bt endotoxin content in Bt transgenic cotton

Plant density is the cultivation practice usually employed to manipulate boll distribution, boll setting and yield in cotton production. In order to determine the effect of plant density on the insecticidal protein content of Bacillus thuringiensis(Bt) cotton plants, a study was conducted in Yangzhou University of China in 2015 and 2016. Five plant densities(PD1–PD5, representing 15 000, 30 000, 45 000, 60 000, and 75 000 plants ha–1) were imposed on two Bt cotton cultivars, Sikang 1(the conventional cultivar, SK-1) and Sikang 3(the hybrid cultivar, SK-3). The boll number per plant, boll weight and boll volume all decreased as plant density increased. As plant density increased from 15 000 to 75 000 plants ha~(–1), seed Bt protein content increased, with increases of 66.5% in SK-1 and 53.4% in SK-3 at 40 days after flowering(DAF) in 2015, and 36.8% in SK-1 and 38.6% in SK-3 in 2016. Nitrogen(N) metabolism was investigated to uncover the potential mechanism. The analysis of N metabolism showed enhanced soluble protein content, glutamic-pyruvic transaminase(GPT) and glutamate oxaloacetate transaminase(GOT) activities, but reduced free amino acid content, and protease and peptidase activities with increasing plant density. At 20 DAF, the seed Bt toxin amount was positively correlated with soluble protein level, with correlation coefficients of 0.825** in SK-1 and 0.926** in SK-3 in 2015, and 0.955** in SK-1 and 0.965** in SK-3 in 2016. In contrast, the seed Bt protein level was negatively correlated with free amino acid content, with correlation coefficients of –0.983** in SK-1 and –0.974** in SK-3 in 2015, and –0.996** in SK-1 and –0.986** in SK-3 in 2016. To further confirm the relationship of Bt protein content and N metabolism, the Bt protein content was found to be positively correlated with the activities of GPT and GOT, but negatively correlated with the activities of protease and peptidase. In conclusion, our present study indicated that high plant density elevated the amount of seed Bt protein, and this increase was associated with decreased boll number per plant, boll weight and boll volume. In addition, altered N metabolism also contributed to the increased Bt protein content under high plant density.     

Expression of Aminopeptidase N1 （APN1）, the Main Receptor Protein for Bacillus thuringiensis CrylA Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 （APN1） gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt.