Plant Expression Vector Research Articles

Multiplex-genome-editing based rapid directional improvement of complex traits in rice.

Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.

Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Expression Studies of BmNPV Antiviral Proteins Serine Protease and Lipase in Mulberry Plant

Sericulture is a significant agricultural cottage enterprise. The silkworm Bombyx mori L. is used in the commercial manufacture of silk. Silkworms are prone to fungal, bacterial, viral, and protozoan infections. Bombyx mori nuclear polyhedrosis virus (BmNPV) causes grasserie, a viral illness in silkworm that is a severe economic loss to the sericulture business. The current study focuses on the transformation of antiviral genes, serine protease, and lipase into mulberry leaves by agro infiltration. Antiviral proteins from BmNPV were cloned into the plant expression vector pBI121. The plant expression vector pBI121 was effectively transferred into Agro-bacterium cells for mulberry agroinfiltration investigations. PCR amplification and SDS-PAGE analysis validated the gene integration and production of antiviral proteins, which were discovered to be 885 bp of lipase gene with protein size of 29 Kda and 855 bp of serine protease gene with protein size of 24 Kda. Lipase and serine protease antiviral protein genes were cloned into the bacterial expression vector pET32a for bioassay experiments.

In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV)

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD.

Isolation and Functional Characterization of a Constitutive Promoter in Upland Cotton (Gossypium hirsutum L.).

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a β-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3-10-fold higher compared to those under the CaMV 35S promoter at 10-30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.

A preliminary study of the immunogenic response of plant-derived multi-epitopic peptide vaccine candidate of Mycoplasma gallisepticum in chickens.

Mycoplasma gallisepticum (MG) is responsible for chronic respiratory disease in avian species, characterized by symptoms like respiratory rales and coughing. Existing vaccines for MG have limited efficacy and require multiple doses. Certain MG cytoadherence proteins (GapA, CrmA, PlpA, and Hlp3) play a crucial role in the pathogen's respiratory tract colonization and infection. Plant-based proteins and therapeutics have gained attention due to their safety and efficiency. In this study, we designed a 21.4-kDa multi-epitope peptide vaccine (MEPV) using immunogenic segments from cytoadherence proteins. The MEPV's effectiveness was verified through computational simulations. We then cloned the MEPV, introduced it into the plant expression vector pSiM24-eGFP, and expressed it in Nicotiana benthamiana leaves. The plant-produced MEPV proved to be immunogenic when administered intramuscularly to chickens. It significantly boosted the production of immunoglobulin Y (IgY)-neutralizing antibodies against cytoadherence protein epitopes in immunized chickens compared to that in the control group. This preliminary investigation demonstrates that the plant-derived MEPV is effective in triggering an immune response in chickens. To establish an efficient poultry health management system and ensure the sustainability of the poultry industry, further research is needed to develop avian vaccines using plant biotechnology.

Cloning an RBD-T4-LINKER-C5a sequence encoding the SARS-CoV-2 antigen into a plant expression vector

This study aims to make a plant expression vector with an RBD-T4-Linker-C5a sequence that codes for the SARS-CoV-2 antigen and an A3Dsp signal peptide from the rice 3D amylase gene located before the RBD. Methods of molecular cloning were applied in this study. The plant expression vector pNHL22 harboring the RBD-T4-Linker-C5a sequence was successfully established and conjugated into Agrobacterium tumefaciens LBA4404 by triparental mating. Bacteria A. tumefaciens containing the RBD-T4-Linker-C5a sequence are now ready for genetic transformation into the Nicotiana benthamiana plant for future applications.

Development of an efficient Agrobacterium-mediated transformation method and its application in tryptophan pathway modification in Catharanthus roseus.

The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l-1 kanamycin and 20 mg l-1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.).

Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants.

Control of erucic acid biosynthesis in Camelina (Camelina sativa) by antisense technology.

Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistanceto abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elangase 1 (FAE1) gene encoded the enzyme β-ketoacyl COA synthase (KCS), which is a key factor in the biosynthesis of erucic acid. In this study, isolation and cloning of the FAE1 from the Camelina sativa were performed to prepare an antisense structure. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning FAE1 into the Bluescriprt II SK+ vector and sequencing, these fragments were used for the preparation of antisense structurein the pBI121 plant expression vector. The approved structurewas transferred to the camelina plant via the Agrobacterium-mediated method. Also, the conditions of tissue culture and gene transfer were optimized. Moreover, the erucic acid content of the immature seeds of T0 transgenic plants was analyzed with gas chromatography (GC). Results showed significant changes in erucic acid levels of two control plants (0.88%), while two lines of the RFAE1 transgenic plants showed a decrease of approximately 0% in erucic acid level. It can be concluded that the antisense structure can be effective in reducing erucic acid.

Soybean GmGolS2-2 improves drought resistance of transgenic tobacco

Galactinol synthase (GolS) genes play important roles in plant response to abiotic stress. In this research, the plant expression vector of soybean GmGolS2-2 gene was constructed and transformed into tobacco to study the drought tolerance of transgenic tobacco. A GmGolS2-2 gene with 975 bp coding sequence was cloned from soybean leaves by reverse transcription-polymerase chain reaction (RT-PCR). GmGolS2-2 was linked to the plant expression vector pRI101 by restriction enzyme sites Nde Ⅰ and EcoR Ⅰ, and transformed into tobacco by leaf disc method. Genomic DNA PCR and real-time PCR showed that three GmGolS2-2 transgenic tobacco plants were obtained. The growth status of GmGolS2-2 transgenic tobacco under drought stress was better than that of wild-type tobacco. After drought stress treatment, the electrolyte leakage and malondialdehyde content of transgenic tobacco were lower than those of wild-type tobacco, but the proline content and soluble sugar content were higher than those of wild-type tobacco. The results of real-time PCR showed that the heterologous expression of GmGolS2-2 increased the expression of stress-related genes NtERD10C and NtAQP1 in transgenic tobacco. The above results indicated that GmGolS2-2 improved drought resistance of transgenic tobacco.

Effect of plant produced Anti-hIL-6 receptor antibody blockade on pSTAT3 expression in human peripheral blood mononuclear cells

As a response to invasion by pathogens, the secretion of interleukin 6 (IL-6) which is a cytokine, activates IL-6/JAKs/STAT3 intracellular signaling via., phosphorylation. Over expression of pSTAT3 induces IL-6 positive feedback loop causing cytokine release syndrome or cytokine storm. Plants have gained momentum as an alternative expression system. Hence, this study aims to produce mAb targeting human IL-6 receptor (hIL-6R) in Nicotiana benthamiana for down regulating its cellular signaling thus, decreasing the expression of pSTAT3. The variable regions of heavy and light chains of anti-hIL-6R mAb were constructed in pBYK2e geminiviral plant expression vector and transiently co-expressed in N. benthamiana. The results demonstrate the proper protein assembly of anti-hIL-6R mAb with highest expression level of 2.24 mg/g FW at 5 dpi, with a yield of 21.4 µg/g FW after purification. The purity and N-glycosylation of plant produced antibody was analyzed, including its specificity to human IL-6 receptor by ELISA. Additionally, we investigated the effect to pSTAT3 expression in human PBMC’s by flow cytometry wherein, the results confirmed lower expression of pSTAT3 with increasing concentrations of plant produced anti-hIL-6R mAb. Although, further in vivo studies are key to unveil the absolute functionality of anti-hIL-6R, we hereby show the potential of the plant platform and its suitability for the production of this therapeutic antibody.

Production of Plant-Derived Japanese Encephalitis Virus Multi-Epitope Peptide in Nicotiana benthamiana and Immunological Response in Mice.

The current production of the Japanese encephalitis virus (JEV) vaccine is based on animal cells, where various risk factors for human health should be resolved. This study used a transient expression system to express the chimeric protein composed of antigenic epitopes from the JEV envelope (E) protein in Nicotiana benthamiana. JEV multi-epitope peptide (MEP) sequences fused with FLAG-tag or 6× His-tag at the C- or N-terminus for the purification were introduced into plant expression vectors and used for transient expression. Among the constructs, vector pSK480, which expresses MEP fused with a FLAG-tag at the C-terminus, showed the highest level of expression and yield in purification. Optimization of transient expression procedures further improved the target protein yield. The purified MEP protein was applied to an ICR mouse and successfully induced an antibody against JEV, which demonstrates the potential of the plant-produced JEV MEP as an alternative vaccine candidate.

Comparison of Post-transcriptional Gene Silencing (PTGS) Strategies for Developing Transgenic Plants Resistant to Tomato Leaf Curl Virus (ToLCV)

Tomato (Solanum lycopersicum L.), an economically important crop is host to many whitefly transmitted geminiviruses including tomato leaf curl virus (ToLCV). Genetically engineering resistance of pathogen through Post-transcriptional gene silencing (PTGS/RNAi) is a powerful strategy that can provide alternative to existing methods of producing virus resistant plants. We cloned and characterized ToLCV-replicase (TRP) gene from a local Dharwad, Karnataka, India ToLCV isolate for development of transgenic tomato plants. Plant expression vectors carrying viral replicase (rep) gene in sense, antisense, ihp (intron spaced hairpin) and HUTR (inverted repeats of heterologous 3΄-untranslated region) were constructed. Transgenic tomato plants carrying rep gene in different strategies when challenged with whiteflies carrying ToLCV showed varied degrees of resistance. Such plants were confirmed through PCR, GUS, Dot blot, Southern blot and semiquantitative PCR analysis. High degree of resistance was observed in the construct carrying both sense and antisense strand interrupted by intron (ihp). Our results demonstrate that, transgenic plants with simultaneous expression of sense and antisense strands are more efficient in gene silencing of ToLCV than those expressing either sense or antisense strand alone.

Construction of an Agroinfectious Clone of a Korean Isolate of Sweet Potato Symptomless Virus 1 and Comparison of Its Infectivity According to Agrobacterium tumefaciens Strains in Nicotiana benthamiana

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus <i>Mastrevirus</i> (family <i>Geminiviridae</i>) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into <i>Nicotiana benthamiana</i> using three <i>Agrobacterium tumefaciens</i> strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The <i>A. tumefaciens</i> strain LBA4404 was the most effective at transferring the SPSMV-1 genome to <i>N. benthamiana</i>. We confirmed the viral replication in <i>N. benthamiana</i> samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

Functional Characterization of a Novel Synthetic Herbicide Resistance Gene in a Model Plant

As crop losses can occur owing to the abrupt growth of uncontrollable weeds in the field, research is currently being conducted in Pakistan to eradicate herbs. To carry out our proposed research domain, we have designed a novel synthetically modified EPSPS gene that has a potent role in herbicide-resistance development. The novel codon-optimized synthesized mEPSPS sequence was inserted into the pXCSG-mYFP plant expression vector. Results of colony PCR (1400 bp) confirmed the integration of genes into bacteria. For functional validation of pXCSG-mYFP-EPSPS, transient expression in tobacco (Nicotiana benthamiana) in comparison with PBSF-16 was done. Benth infiltration results showed that transient expression was successfully confirmed through ELISA and western blot analysis via anti-YFP antibody in tobacco leaves. For further validation of transient expression, the stable transformation results of the pXCSG-mYFP-EPSPS vector showed that successful transformation was done via two days of co-cultivation followed by selection and regeneration of transformed tobacco plants. The regenerated tobacco plants were then confirmed through gene-specific-based PCR. After PCR-based confirmation, western blot further validates the 26 kDa anti-YFP antibodies expression in transformed tobacco plants. Another important finding of the study was the bar and PAT gene real-time expression elucidating that the bar gene was 4.9-fold more expressive under the 35S promoter than the PAT gene under the nos promoter.

AtCIPK16, a CBL-interacting protein kinase gene, confers salinity tolerance in transgenic wheat.

Globally, wheat is the major source of staple food, protein, and basic calories for most of the human population. Strategies must be adopted for sustainable wheat crop production to fill the ever-increasing food demand. Salinity is one of the major abiotic stresses involved in plant growth retardation and grain yield reduction. In plants, calcineurin-B-like proteins form a complicated network with the target kinase CBL-interacting protein kinases (CIPKs) in response to intracellular calcium signaling as a consequence of abiotic stresses. The AtCIPK16 gene has been identified in Arabidopsis thaliana and found to be significantly upregulated under salinity stress. In this study, the AtCIPK16 gene was cloned in two different plant expression vectors, i.e., pTOOL37 having a UBI1 promoter and pMDC32 having a 2XCaMV35S constitutive promoter transformed through the Agrobacterium-mediated transformation protocol, in the local wheat cultivar Faisalabad-2008. Based on their ability to tolerate different levels of salt stress (0, 50, 100, and 200 mM), the transgenic wheat lines OE1, OE2, and OE3 expressing AtCIPK16 under the UBI1 promoter and OE5, OE6, and OE7 expressing the same gene under the 2XCaMV35S promoter performed better at 100 mM of salinity stress as compared with the wild type. The AtCIPK16 overexpressing transgenic wheat lines were further investigated for their K+ retention ability in root tissues by utilizing the microelectrode ion flux estimation technique. It has been demonstrated that after 10min of 100 mM NaCl application, more K+ ions were retained in the AtCIPK16 overexpressing transgenic wheat lines than in the wild type. Moreover, it could be concluded that AtCIPK16 functions as a positive elicitor in sequestering Na+ ions into the cell vacuole and retaining more cellular K+ under salt stress to maintain ionic homeostasis.

Engineering of cry genes “Cry11 and Cry1h” in cotton (Gossypium hirsutum L.) for protection against insect pest attack

Cotton is white gold and contributes significantly to the economy of the countries. The process of evolution of resistance in various cotton insects directly affects the life of people. Among the cotton insect pests, pink bollworm (Pectinophora gossypiella) and army bollworm (Spodoptera litura) have turned out to be destructive. The development of seed using gene stacking approach has tremendously reduced the applications of agrochemicals making the technology greener for society. A gene cassette containing Cry11 and Cry1H genes was constructed. The cassette was cloned into the pCAMBIA2300 plant expression vector using the CAMV3-35S promoter. The construct was transformed into cotton line FBS-222 using the shoot apex-cut Agrobacterium-mediated transformation method. The expression of the Cry genes was 3-fold higher in transgenic than the non-transgenic cotton plants using qRT-PCR. Finally, the insect bioassay with transgenic cotton showed 90% mortality against pink bollworms and 80% against cotton army bollworms. The results demonstrate the development of seeds using the gene pyramiding approach is an effective strategy to control insect pest attacks.

Functional verification of the JmLFY gene associated with the flowering of Juglans mandshurica Maxim.

In this study, a pBI121-JmLFY plant expression vector was constructed on the basis of obtaining the full-length sequence of the JmLFY gene from Juglans mandshurica, which was then used for genetic transformation via Agrobacterium inflorescence infection using wild-type Arabidopsis thaliana and lfy mutants as transgenic receptors. Seeds of positive A. thaliana plants with high expression of JmLFY were collected and sowed till the homozygous T3 regeneration plants were obtained. Then the expression of flowering-related genes (AtAP1, AtSOC1, AtFT and AtPI) in T3 generation plants were analyzed and the results showed that JmLFY gene overexpression promoted the expression of flowering-related genes and resulted in earlier flowering in A. thaliana. The A. thaliana plants of JmLFY-transformed and JmLFY-transformed lfy mutants appeared shorter leaves, longer fruit pods, and fewer cauline leaves than those of wild-type and the lfy mutants plants, respectively. In addition, some secondary branches in the transgenic plants converted into inflorescences, which indicated that the overexpression of JmLFY promoted the transition from vegetative growth to reproductive growth, and compensate the phenotypic defects of lfy mutant partially. The results provides a scientific reference for formulating reasonable genetic improvement strategies such as shortening childhood, improving yield and quality, and breeding desirable varieties, which have important guiding significance in production.