Improvement in Tol2 transposon for efficient large-cargo capacity transgene applications in cultured cells and zebrafish(Danio rerio)

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.

Impact Evaluation of Insect-Resistant Transgenic Rice on the Feeding and Oviposition Behavior of Its Non-Target Insect, the Brown Planthopper, Nilaparvata lugens (Homptera: Delphacidae)

The feeding and oviposition behavior of the brown planthopper (BPH), Nilaparvata lugens on two transgenic indica rice homogenous genotypes (B1 and B6) with cry1Ab gene from Bacillus thuringiensis and transgenic restored line of hybrid rice (MSA) with SCK gene (a modified CpTI gene) were measured, compared with those on their corresponding non transgenic parental cultivars Jiazao935 and Minghui86 performed by BPH. Under the selection condition of host plants by BPH, loading percentage, oviposition preference and laying egg number of BPH both on transgenic cry1Ab rice and transgenic SCK rice were not significantly different from those on their controls, while their total number of probing wound caused by PBH expect for feeding on B1 plants was markedly more than that on the control. In contrast, under the non selection condition, total number of probing wound caused by BPH on either transgenic cry1Ab rice or transgenic SCK rice was pronouncedly more than those on their controls. Conversely, their honeydew amount excreted by BPH after feeding for 24 h was significantly less than those on the control. As a conclusion, three tested transgenic rice genotypes with insect resistance acted adverse effect on BHP feeding, and no marked effect on BPH oviposition.

Construction of Plant Expresion Vector Carrying Two Insecticidal Genes and Obtain Insect-resistant Transgenic Tobacco Plants

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Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Recombinant adenovirus serotype 5(Ad5)vector has been widely applied in vaccine development targeting infectious diseases,such as Ebola virus disease and coronavirus disease 2019(COVID-19).However,the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines.Thus,there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors.Herein,we address this need by utilizing biocompatible nanoparticles to modulate Ad5–host interactions.We show that positively charged human serum albumin nanoparticles((+)HSAnp),which are capable of forming a complex with Ad5,significantly increase the transgene expression of Ad5 in both coxsackievirus–adenovirus receptor-positive and-negative cells.Furthermore,in charge-and dose-dependent manners,Ad5/(+)HSAnp complexes achieve robust(up to227-fold higher)and long-term(up to 60 days)transgene expression in the lungs of mice following intranasal instillation.Importantly,in the presence of preexisting anti-Ad5 immunity,complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity.These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

See the color,see the seed:GmW1 as a visual reporter for transgene and genome editing in soybean

A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a GmW1 gene encoding a flavonoid 3'5'-hydroxylase from a soybean cultivar ZH42(purple flower).We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds(white flower).All plants with purple flowers were positive,and this trait seems stably inherited in the offspring.We have also obtained the editing plants,which were classified into three types according to the different flower colors appeared.We analyzed the phenotype and the homozygous types of the T_1mutants.We also found that a correspondence between flower color and stem color.This study provides a visible color reporter on soybean transformation.It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings,which substantially reduces the amount of labor and cost.

Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.

Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Study on Insect-resistant Transgenic Cotton Harbouring Double-gene and Its Resistance to Insect Pests

By using the method of pollen tube pathway, the synthesized GFM CryIA gene and modified CpTI gene were transfered into the elite cotton( Gossypium hirsutun L. ) varieties(lines). Through the field and lab identifications, the insect-resistant transgenic plants were obtained. PCR analysis indicated that both the synthesized GFM CryIA gene and modified CpTI gene presented positive reaction. In R1 the boliworm resistance of each transformant was different, and the insect-resistance of R3 of ZGK9708 was stable.

Advances in Insect-resistant Transgenic Rice and Its Developing Tendency

Focusing on several commonly used insect resistance genes,we reviewed the advances in insect-resistant transgenic rice,and analyzed the problems and developing tendency in transgenic rice research in this paper.

Insect-resistant transgenic Brassica crops

Although millions of hectares of cotton and corn carrying Bacillus thuringiensis (Bt) genes are now being grown worldwide, questions remain about the most effective way to delay development of resistant insect pests. In a collaborative project with the Shelton lab at Cornell, we have developed a model system for use in empirical studies of various proposed resistance management strategies. The plant components of the model system are broccoli (Brassica oleracea ssp. italica) lines transformed with cry1Ac or cry1C Bt genes under control of constitutive (35S CaMV) or chemically-inducible promoters. The insect components are susceptible, Cry1A-resistant and Cry1C-resistant populations of the diamondback moth (Plutella xylostella). We identified transgenic lines with high (>1 000 ng/mg total soluble protein) constitutive expression of Cry1Ac or Cry1C proteins and used them in sexual crosses to produce plants expressing both Bt genes. The two-gene plants showed Bt gene expression comparable to the parental lines, i.e. no gene silencing occurred. The cry1Ac+ cry1C plants controlled P. xylostella resistant to Cry1Ac or Cry1C proteins. Chemically inducible broccoli lines that provided insect control after treatment with the crop protectant BTH were also developed. These showed rapid high production of Cry1Ab protein and some signal transduction to untreated or newly formed leaves and heads. Older leaves of these plants produced some Bt protein and retarded insect development even when not induced, so this system may not be suitable for resistance management. Our cry1C broccoli, cauliflower, and Chinese cabbage plants also control other lepidopteran insect pests (Trichoplusia ni, Pieris rapae). A further area of work is production of two types of Bt-transgenic trap crops: glossy leaf collards (B. oleracea ssp. acephala) and Indian mustard (B. juncea). These are more attractive for P. xylostella oviposition than cabbage and also kill all larvae hatched from the eggs laid. Bt-trap crops may offer an attractive combination of biotechnology and biological control, especially when transgenic commercial crops are not accepted. A field test of how well plantings of these materials protect cabbage from insect damage will be conducted in the summer of 2005.

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR(TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35 S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

Indication of the Expression of Transgene in Rice Plant Based on Hyperspectral Remote Sensing Technique Ⅱ——Growth Monitoring of Samples in the Contrast Experiment

Since the complication of monitoring and evaluating the problems about the transgenic expression and its impacts on the receptor in the transgenic crop breeding and other relevant evaluated works,the authors in the present work tried to assess the differences of spectral parameters of the transgenic rice in contrast with its parent group quantitatively and qualitatively,fulfilling the growth monitoring of the transgenic samples.The spectral parameters(spectral morphological characteristics and indices) chosen are highly related to internal or external stresses to the receipts,and thus could be applied as indicators of biophysical or biochemical processes changes of plant.By ASD portable field spectroradiometer with high-density probe,fine foliar spectra of 8 groups were obtained.By analyzing spectral angle and continuum removal,the spectral morphological differences and their locations of sample spectra were found which could be as auxiliary priori knowledge for quantitative analysis.By investigating spectral indices of the samples,the quantitative differences of spectra were revealed about foliar chlorophyll a+b and carotenoid content.In this study both the spectral differences between transgenic and parent groups and among transgenic groups were investigated.The results show that hyperspectral technique is promising and a helpful auxiliary tool in the study of monitoring the transgenic crop and other relevant researches.By this technique,quantitative and qualitative results of sample spectra could be provided as prior knowledge,as certain orientation,for laboratory professional advanced transgenic breeding study.

Minor modifications in obtainable Arabidopsis floral dip method enhances transformation efficiency and production of homozygous transgenic lines harboring a single copy of transgene

Many researchers have developed various methods for in-planta or floral dip transformation of Arabidopsis thaliana, one of the simple protocol and widely used to produce transgenic Arabidopsis. As the efficiency and ease of getting a transformant is very much time consuming effort and less number of the transformants people get, we have developed a little modified transformation protocol to avoid the disparities. Four types of inoculums (inoculum1, inoculum2, inoculum3 and inoculum4) were used to check the transformation efficiency out of which Inoculum3 showed the highest rate of transformation among the four types. 0.07% Twin-20 also acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis. After vacuum infiltration keeping the Agrobacterium infected plants for 7-8 hrs horizontally in low light at 280C temperature condition, considered best to get an increased number of transformed seeds. Modified protocol produced ~12-14% increase in transformants. Selection pots (kanamycin supplemented soil filled pots) in place of selection plates (Kanamycin supplemented Murashige and Skoog agar plates) proved beneficial as no MS medium and no aseptic condition is required for selection of transformed plants. This increase in transformation efficiency consequently increased the percentage of homozygous and single copied stable transgenic lines.

Effect of three insect-resistant maizes expressing Cry1le,Cry1Ab/Cry2Aj and Cry1Ab on the growth and development of armyworm Mythimna separata(Walker)

Three transgenic maize events(IE09S034,Shuangkang 12--5 and C0030.3.5)produced Cry1le,Cry1Ab/Cry2Aj and G10-EPSPS,Cry1Ab and EPSPS,respectively,all of which target the Asian corn borer.The oriental armyworm Mythimna separata(Walker)is the secondary target.In this study,the effects of the three Bt maizes on the development and survival of armyworm were studied.The results showed that IE09S034 had insecticidal activity against 1st instar larvae,and the survival rate of armyworm fed with Bt maize for 10 days was 462%,significantly lower than that of the control.The larvae at 3rd--6th instar were more tolerant of the Bt toxin than the early instar larvae.However,Shuangkang 12-5 had good insecticidal activity against 1st-5th instar larvae.The mortality was nearly 100%when the larvae were fed with Shuangkang 12-5 before 3rd instar,and the toxin had quick acting eficacy.This event significantly ihibited the development of armyworm;that is,the larval duration of the 3rd and 4th instar larvae fed with Shuangkang 12-5 was prolonged by 4.5 and 3.0 days,respectively.The pupal weight and egg number were also significantly lower than those of the control.For C0030.3.5,it could control 1st--5th instar larvae effectively.The mortality rates were all over 50%if 1st-3rd larvae were fed with this event.The pupal weight of 4th-6th instar larvae fed with Bt maize were only 53.9,56.8 and 54.6%,respectively,compared to that of the control.The number of eggs laid was significantly less than the control.The results indicate that all three transgenic maize events exhibit the potential to provide effective control of early instar larvae of armyworm.which can be commercialized in future to control lepidoptera pests such as Asian corn borer and armyworm.

Transgene Flow from Glufosinate-Resistant Rice to Improved and Weedy Rice in China

The development of transgenic rice with novel traits in China can increase rice productivity, but transgene flow to improved or weedy rice has become a major concern. We aimed to evaluate the potential maximum frequencies of transgene flow from glufosinate-resistant rice to improved rice cultivars and weedy rice. Treatments were arranged in randomized complete blocks with three replicates. Experiments were conducted between 2009 and 2010 at the Center for Environmental Safety Supervision and Inspection for Genetically Modified Plants, China National Rice Research Institute, Hangzhou, China. Glufosinate-resistant japonica rice 99-1 was the pollen donor. The pollen recipients were two inbred japonica rice （Chunjiang 016 and Xiushui 09）, two inbred indica rice （Zhongzu 14 and Zhongzao 22）, two indica hybrid rice （Zhongzheyou 1 and Guodao 1）, and one weedy indica rice （Taizhou weedy rice）. The offspring of recipients were planted in the field and sprayed with a commercial dose of glufosinate. Leaf tissues of survivors were analyzed by polymerase chain reaction to detect the presence of the transgene. The frequency of gene flow ranged from 0 to 0.488%. In 2009, the order of gene flow frequency was as follows： weedy rice 〉 Chunjiang 016 〉 Xiushui 09 and Zhongzu 14 〉 Guodao 1, Zhongzheyou 1 and Zhongzao 22. Gene flow frequencies were generally higher in 2009 than in 2010, but did not differ significantly among rice materials. Gene flow frequency was the highest in weedy rice followed by the inbred japonica rice. The risk of gene flow differed significantly between years and year-to-year variance could mask risk differences among pollen recipients. Gene flow was generally lesser in taller pollen recipients than in shorter ones, but plant height only accounted for about 30% of variation in gene flow. When flowering synchrony was maximized, as in this study, low frequencies of gene flow occurred from herbicide-resistant japonica rice to other cultivars and weedy rice. Averaged across years, the risk of gene flow to weedy rice was higher than that of improved rice and hybrids. Greater resources must be dedicated to the management of remnant weedy rice in fields planted with herbicide-resistant rice, and to prevent the evolution of resistant weedy rice populations.

Selection of Homozygous Cotton Lines Transformed with Two Insect-Resistant Genes

A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera , nine transgenic homozygous cotton lines with insect resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.

INFLUENCE OF CHEMOTHERAPEUTANTS AND CYTOKINES ONGROWTH AND TRANSGENE EXPRESSION OF BONE MARROWCELLS FROM MT/P210^(bcr-ab1) TRANSGENIC MICE

Objective: To investigate the influcnce ofchemotherapeutic agents and cytokines on growth ofbone marrow cells from MT/p210 her-ab1 transgenic mice.Methods: The bone marrow cells of transgenic chronicmyelogenous leukemia (CML) model mice carryingmetallothionein (MT) promoter/enhancer, her-abl (p210)cDNA and SV40 splicinglpoly (A) signal sequences werecultured in liquid and soft agar with hydroxyurea (Hu),5-nuorouracil (5-Fu), mouse stem cell factor (mSCF)and mouse interleukin-3 (mIL-3) independently orcollectively. The cells and colonies were counted. Thelevels of transgene expression were detected by reversetranscriptase-polymerase chain reaction (RT-PCR).Results: The cell proliferation, colony formation andtransgene expression of the bone marrow cells werestimulated with mSCF and mIL-3, but there was littlegrowth without any growth factors, or when mSCF,mIL-3 and Hu or 5-Fu were added. Conclusion: Thecombined utilization of chemotherapeutants andcytokines is a potentially effective strategy of clinicaltreatment for CML.

Characterization of Genomic Integration and Transgene Organization in Six Transgenic Rapeseed Events

To characterize the DNA rearrangement of both the T-DNA region and the genomic insertion site during T-DNA insertion, the Genomewalker strategy was used to isolate the junctions between the inserted DNA and the plant genomic DNA in six rapeseed events as well as the genomic DNA at the sites before integration. During transformation in each of the six events, portions of both the right border（RB） and left border（LB） regions of the T-DNA were deleted, ranging from a 7 nucleotide deletion of the LB repeats in event RF1 to a 207 bp deletion of the LB region in event RF2. For the six events, T-DNA integration resulted in a deletion at the target site spanning less than 100 bp. Sequence analysis indicated that the T-DNA was integrated into the coding region of various native rapeseed genes in events RF1 and RF2. Duplications of the genomic DNA target site were observed in events RF2, RF3 and Topas 19/2. And multimerization of transgenes was found in event Topas 19/2, in which, the T-DNA was integrated as a head-to-head（RB-to-RB） concatemer into the recipient genome. In event MS1, chromosomal translocation or a large target-site deletion may have occurred during T-DNA integration, which was identified due to a failure to amplify the presumptive insertion site based on the flanking rapeseed DNA sequences. Our results provide comprehensive data concerning transgene organization and the genomic context of the T-DNA in six rapeseed events, which can aid in the developing of insert fingerprinting and the monitoring of long-term genetic stability and potential unintended effects of transgenic events.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgeniccommon carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of thepMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes fromtwo individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recoveredtransgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5%respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicatingthat most transgenes were faithfully inherited during the four generations of reproduction. The other five classes weredifferent from the original configuration in both molecular weight and restriction map, indicating that a few transgeneshad undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types ofaberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carpβ-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermalkeratin 14, respectively.

Breeding and Identification of Insect-Resistant Rice by Transferring Two Insecticidal Genes,sbk and sck

The plasmid of pCDMARUBA-Hyg, which contained two insect-resistance genes, sbk （modified from CrylA（c）） and sck （modified from CpTI）, was transformed into an Agrobacterium EHA105 for infection of the calli of a super japonica rice Nanjing 45. Primarily, using polymerase chain reaction （PCR） detection with the primers of sbk and sck genes, 42 positive transgenic plants that were marker-free and contained the two target genes were selected from 97 regenerated plants. Results of southern-blotting indicated that 23, 11, 5, 2 and 1 plants had one, two, three, four and five copies of the transformed genes, respectively. Analysis of reverse transcription PCR （RT-PCR） and Bt gene testing paper showed that 28 T3 generation plants derived from four transgenic plants having a single copy were insect-resistant. Feeding experiment with rice stem borer revealed that the insect resistance was greatly increased with the larva mortality ranging from 94% to 100%. In addition, among the transgenic plants, three T3 transgenic plants possessed some desirable characteristics for breeding and production, such as plant height, seed-setting rate, 1000-grain weight and larva mortality. The mechanism of insect resistance of Bt .qene and its application in rice transgenic research were also briefly discussed.