Protein Engineering Of Bt Genes cry1Ab And cry1Ba For The Development Of Chimeric Genes cryAbabba, cryBabaab And cryAbbaab Via Domain Swapping

Bacillus thuringiensis is renowned for its production of insecticidal cry proteins, widely utilized in crop protection to combat insects. However, the risk of insect resistance emerges due to the relatively loose binding of toxins to target sites on larvae's midgut brush boundary membranes. This resistance primarily stems from modifications in binding sites within midgut cells. To address potential threats, the discovery of new Cry proteins is imperative as insects continually evolve resistance against existing ones. Combining Cry toxins with diverse binding sites in larval midguts is proposed as an effective strategy to delay the onset of resistance. In this study, three chimeric B. thuringiensis proteins—CryAbAbBa, CryBaBaAb, and CryAbBaAb—were engineered via domain swapping, utilizing crystal proteins CrylAb and CrylB. Structural validation was conducted, confirming their integrity through Ramachandran Plots. The chimeric proteins can be used as additional resources in crop improvement programmes

Expression and Specificity of EcNAC1 Transcription Factor from Eleusine Coracana in Escherichia Coli

Dehydration stress is an important environmental factor which affects the crop yield. Eleusine coracana (Finger millet) is one of the relatively drought tolerant crop plant and it can also grow with significant levels of salinity. The present work presents cloning, expression and purification of the moisture stress induced transcription factor, EcNAC1. The EcNAC1 gene was cloned into pGEX-4T1 expression vector and transferred into Escherichia coli BL21 (DE3) pLysS strain. The positive clones were grown and induced with IPTG for 4h at 30ºC. The SDS-PAGE analysis of the lysate proteins showed the 66 kDa fusion protein (i.e., GST tag 26 kDa, EcNAC1 approximately 41 kDa). The fusion protein was insoluble and localized as inclusion body. The protein was separated by preparative SDS-PAGE, electroeluted and antiserum was raised in adult healthy rabbits. The antiserum specifically detected EcNAC1 transcription factor from finger millet with no cross reactivity in western blot analysis

In planta transformation strategy to generate transgenic plants in chickpea: proof of concept with a cry gene

The paper presents a non-​tissue culture-​based transformation of chickpea using cry1AcF gene with 5'UTR. The protocol involves raising of plant transformants (T0 plants) directly from Agrobacterium-​infected young seedlings. The apical meristem of the seedling axes were targeted for transformation. The resulting chimeric plants were allowed to grow in the greenhouse and the transgenics were analyzed in the T1 generation. The T1 generation plants were raised in the greenhouse and initial screening was carried out in 109 plants using ELISA for the expression of the cry1AcF protein. On the basis of this, the plants were grouped as non-​transformants and transformants, expressing low and high level of the cry protein. The plants expressing the cry1AcF protein in the range of 2.06-​9.70 μg​/g fr wt were selected for further anal. Bioefficacy of these 44 plants against Helicoverpa armigera allowed identification of 14 plants that not only accumulated good amt. of protein but were also effective against Helicoverpa. Mol. anal. by PCR for the amplification of both the cry1AcF and nptII genes confirmed the transgenic nature of the selected plants. The protocol ensured generation of transgenic chickpea plants with considerable ease in a short time and might be applicable across different genotypes​/cultivars of the crop and offers immense potential as a supplemental or an alternate protocol for generating transgenic plants of difficult-​to-​regenerate crops

Cloning and characterization of phloroglucinol biosynthetic gene phlC from an Indian strain of Pseudomonas fluorescens

Phloroglucinol or 2,4-diacetyl phloroglucinol (DAPG) is a polyketide compound produced by gram negative soil bacteria Pseudomonas. It shows broad spectrum antibacterial and antifungal properties against soil-borne plant pathogens. In Pseudomonas spp., genes for biosynthesis of 2,4-DAPG are localized in phlABCD operon. All the four genes in phlABCD operon are indispensable and DAPG synthesis is attenuated even in the absence of one of the genes. In the present study, we identified and cloned phlC gene from an Indian strain of Pseudomonas and analyzed its sequence. The structural details ofthe PHLC protein was generated by three-dimensional homology modelling. Additionally, stereo-chemical properties of PHLC were analyzed by Ramachandran plot analysis and the generated model was validated by PDBsum. Our results demonstrate that the cloned PHLC protein contains structural features typical of a condensing enzyme involved inpolyketide synthesis

Cloning and structural elucidation of a brassinosteroids biosynthetic gene (Atdwarf4) and genetic transformation of Indian mustard (Brassica juncea L.)

320-330Phytohormones play critical roles in plant growth and development. Brassinosteroids (BRs) are essential group of phytohormones required for optimum growth of plants and their deficiency causes distinctive dwarf phenotypes in plants. Homeostasis of BRs in plants is maintained by DWARF4 enzyme that mediates multiple 22α-hydroxylation steps in brassinosteroid biosynthesis. Arabidopsis plants over-expressing DWARF4 show increase in inflorescence, number of branches and siliques; thereby increased number of seeds/plant. This suggests that engineering DWARF4 biosynthesis in Brassica plant can be strategized to enhance yield in mustard. In the present study (i) we cloned dwarf4 gene from Arabidopsis using gene specific PCR strategy, (ii) elucidated the three-dimensional structure of DWARF4 protein at molecular level where it revealed presence of four beta sheets and 20 alpha-helices, and (iii) transformed mustard cultivar Pusa Jaikisan with an objective to develop transgenic mustard with enhanced number of siliques. We obtained several putative transgenics with an average transformation efficiency of 3.3%. Molecular characterization with nptII specific primers confirmed presence of transgene in six putative transgenic plants

Vacuolar Targeting of Cry1Ac and its Effects on Expression and Stability in Tobacco.

Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes.Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes

Simultaneous expression of regulatory genes associated with specific drought‐adaptive traits improves drought adaptation in peanut

Adaptation of crops to drought-prone rain-fed conditions can be achieved by improving plant traits such as efficient water mining (by superior root characters) and cellular-level tolerance mechanisms. Pyramiding these drought-adaptive traits by simultaneous expression of genes regulating drought-adaptive mechanisms has phenomenal relevance in improving stress tolerance. In this study, we provide evidence that peanut transgenic plants expressing Alfalfa zinc finger 1 (Alfin1), a root growth-associated transcription factor gene, Pennisetum glaucum heat-shock factor (PgHSF4) and Pea DNA helicase (PDH45) involved in protein turnover and protection showed improved tolerance, higher growth and productivity under drought stress conditions. Stable integration of all the transgenes was noticed in transgenic lines. The transgenic lines showed higher root growth, cooler crop canopy air temperature difference (less CCATD) and higher relative water content (RWC) under drought stress. Low proline levels in transgenic lines substantiate the maintenance of higher water status. The survival and recovery of transgenic lines was significantly higher under gradual moisture stress conditions with higher biomass. Transgenic lines also showed significant tolerance to ethrel-induced senescence and methyl viologen-induced oxidative stress. Several stress-responsive genes such as heat-shock proteins (HSPs), RING box protein-1 (RBX1), Aldose reductase, late embryogenesis abundant-5 (LEA5) and proline-rich protein-2 (PRP2), a gene involved in root growth, showed enhanced expression under stress in transgenic lines. Thus, the simultaneous expression of regulatory genes contributing for drought-adaptive traits can improve crop adaptation and productivity under water-limited conditions

Heterologous Expression of Serine Hydroxymethyltransferase-3 From Rice Confers Tolerance to Salinity Stress in E. coli and Arabidopsis

Among abiotic stresses, salt stress adversely affects growth and development in rice. Contrasting salt tolerant (CSR27), and salt sensitive (MI48) rice varieties provided information on an array of genes that may contribute for salt tolerance of rice. Earlier studies on transcriptome and proteome profiling led to the identification of salt stress-induced serine hydroxymethyltransferase-3 (SHMT3) gene. In the present study, the SHMT3 gene was isolated from salt-tolerant (CSR27) rice. OsSHMT3 exhibited salinity-stress induced accentuated and differential expression levels in different tissues of rice. OsSHMT3 was overexpressed in Escherichia coli and assayed for enzymatic activity and modeling protein structure. Further, Arabidopsis transgenic plants overexpressing OsSHMT3 exhibited tolerance toward salt stress. Comparative analyses of OsSHMT3 vis a vis wild type by ionomic, transcriptomic, and metabolic profiling, protein expression and analysis of various traits revealed a pivotal role of OsSHMT3 in conferring tolerance toward salt stress. The gene can further be used in developing gene-based markers for salt stress to be employed in marker assisted breeding programs.HIGHLIGHTS- The study provides information on mechanistic details of serine hydroxymethyl transferase gene for its salt tolerance in rice

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Not AvailableBackground Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. Results In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. Conclusions The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants.Not Availabl

In Vitro Screening and Identification of Putative Sunflower (Helianthus Annus L.) Transformants Expressing ECNAC1 Gene by Salt Stress Method

A simple and effective screening methodology to identify transformants at plant level under NaCl stress has been established. Leaf disc of putative transformants (T1 individual, 6–8 weeks old) and wild type plants were placed on filter paper inthepresence of NaCl (200mM) for 48hr at room temperature. The leaf discs of EcNAC1 gene transformed plants showed salt tolerance by retaining green colour after 48 hours, while leaf discs fromnon-transformed plants turned dark brown or black in colour. Further, molecular analysis of these salt tolerant plants showed PCR positive to EcNAC1 gene specific and HPT II specific primers, and digestion of amplified EcNAC1 gene product with Sac I restriction enzyme showed expected bands size on agarose gel. Our data suggest that in vitro screening strategy at plant level based on the target gene incorporated would result in the initial identification of promising transformants for further analysis