HERBICIDE TOLERANT TRIPLE GENE INSECTICIDAL COTTON AND OTHER PLANTS

20170233758

15/162632

May 24, 2016

The present invention refers to the next-generation three genes cotton (Gossypium hirsutum) expressing chloroplast-targeted herbicide tolerant gene resistant to broad and narrow-leaved weedicide sprays and two B. thuringiensis insecticidal genes δ-endotoxin Cry2A and vegetative insecticidal protein gene VIP3A. Both Cry2A and VIP3A genes have different modes of action in controlling a wide spectrum of lepidopteron insect pests, therefore, likely risk of pest resistance will be minimized which is a prevalent problem with single gene Bt cotton expressing Cry1Ac in Pakistan. The invention comprises novel nucleic acid segments encoding proteins comprising herbicide tolerance, Cry2A and VIP3A insecticidal toxins. The polynucleotide segments are revealed, as are Agro-bacterium-mediated transformation vectors holding the nucleic acid segments, plants transformed with claimed segments, methods for transforming plants, and methods for controlling plant infestation by pests.

1. A plant or a part of plant conferring enhanced resistance to insect feeding on the plant or the part of the plant comprising: a plurality of cassettes having a plurality of polynucleotide sequences assisting a triple gene for encoding insecticidal toxins and a herbicide tolerant protein; each of the plurality of cassettes having each of the triple genes with a 5′ end attached to a promoter operably joint to an intron sequence, a cTP, and a 3′ end attached to a NOS terminator, and wherein the triple gene is encoded so as to provide transgenic plants having chloroplast-targeted higher expression to yield fusion proteins i.e. insecticidal toxins and the herbicide tolerant proteins increasing effectiveness against the insect feeding on the plant by acting in synergist way and decreasing possibility of resistance against the insecticidal toxins and the herbicide tolerant protein.

2. The plant or the part of plant as claimed in claim 1, wherein the plurality of cassettes includes a first cassette, a second cassette, a third cassette, and a fourth cassette are within a T-DNA region of vector flanked by a left border sequence and a right border sequence.

3. The plant or the part of plant as claimed in claim 1, wherein the intron sequences and the cTP have a SEQ ID 13, SEQ ID 14, and SEQ ID 15 for targeted expression of protein coded from the triple gene.

4. The plant or the part of plant as claimed in claim 1, wherein the first cassette coding the herbicide tolerant EPSPS gene having a SEQ ID 17 operably linked to a 35CaMV35S promoter, a second intron of a Petunia Cab gene, and the cTP of the Petunia Cab gene at the 5′ terminal.

5. The plant or the part of plant as claimed in claim 1, wherein the second cassette coding the insecticidal vip3 gene having a SEQ ID 18 operably linked to a 35CaMV35S promoter, a first intron of Brassica rapa Cab gene, and the cTP of Ricin Cab gene at the 5′ terminal.

6. The plant or the part of plant as claim as claimed 1, wherein the third cassette coding the insecticidal Cry2ab gene having a SEQ ID 16 operably linked to a 35CaMV35S promoter, a first intron of G. hirusutum, and the cTP of the Petunia EPSPS gene at the 5′ terminal.

7. The plant or the part of plant as claimed 6, wherein the third cassette coding the insecticidal Cry2ab gene having a SEQ ID 16 may be operably linked to a GhSCFP promoter.

8. The plant or the part of plant as claimed in claim 6, wherein the third cassette coding the insecticidal Cry2ab gene having a SEQ ID 16 may be operably linked to PNZIP promoter and a first intron of pnzip gene.

9. The plant or the part of plant as claimed in claim 6, wherein the fourth cassette includes a 35CaMV35S promoter linked to a gene coding a protein conferring antibiotic resistance and a NOS adenylation sequence.

10. The plant or the part of plant as claimed in claim 9, wherein the antibiotic resistance is to hygromycin or kanamycin.

11. The plant or the part of plant as claimed in claim 1, wherein the plant is a monocotyledonous plant selected from the group consisting of maize, rice, wheat, and sugarcane plant.

12. The plant or the part of plant as claimed in claim 1, wherein the plant is a dicotyledonous plant selected from the group consisting of cotton, tomato, and potato plant.

13. The plant or the part of plant as claimed in claim 12, wherein the dicotyledonous plant is a cotton plant.

14. The plant or the part of plant as claimed in claim 1, wherein the yield of fusion protein is derived by a 4B3G transgenic event.

15. The plant or the part of plant as claimed in claim 1, wherein the yield of fusion protein is derived by a 4BG3 transgenic event.

16. The plant or the part of plant as claimed in claim 1, wherein the fusion protein having a SEQ ID 21, 22 and 23 are present in the plant or the part of plant.

17. A method to achieve expression of a triple gene for conferring enhanced resistance to insect feeding on the plant or the part of the plant, without undesirably affecting the normal phenotype and agronomic characteristics of a transgenic plant comprising the steps of: designing and constructing a plurality of cassettes, wherein each of the plurality of cassettes has each gene of the triple genes having a 5′ end attached to a promoter operably joint to an intron sequence, a cTP, and a 3′ end attached to a NOS terminator are inserted within a T-DNA region of vector flanked by a left border sequence and a right border sequence; transforming plants using an Agrobacterium mediated transformation vector having the T-DNA with the plurality of cassettes expressing the triple gene, so that the transgenic plant expresses the chloroplast-targeted higher expression of fusion proteins.

18. The method as claimed in claim 17, wherein the plurality of cassettes has a first cassette coding the herbicide tolerant EPSPS gene having a SEQ ID 17 operably linked to a 35CaMV35S promoter, a second intron of a Petunia Cab gene, and the cTP of the Petunia Cab gene at the 5′ terminal.

19. The method as claimed in claim 17, wherein the plurality of cassettes has a second cassette coding the insecticidal vip3 gene having a SEQ ID 18 operably linked to a 35CaMV35S promoter, a first intron of Brassica rapa Cab gene, and the cTP of Ricin Cab gene at the 5′ terminal.

20. The method as claimed in claim 17, wherein the plurality of cassettes has a third cassette coding the insecticidal Cry2ab gene having a SEQ ID 16 operably linked to a 35CaMV35S promoter, a first intron of G. hirusutum, and the cTP of the Petunia EPSPS gene at the 5′ terminal.

21. The method as claimed in claim 20, wherein the third cassette coding the insecticidal Cry2ab gene having a SEQ ID 16 may be operably linked to a GhSCFP promoter.

22. The method as claimed in claim 20, wherein the third cassette coding the insecticidal Cry2ab gene having a SEQ ID 16 may be operably linked to PNZIP promoter and a first intron of pnzip gene.

23. The method as claimed in claim 18 or claim 19 or claim 20, wherein the cTP is joint at the N-terminal for the enhanced targeted expression of Cry2A, VIP3A and GTG gene in transgenic cotton plants.

24. The method as claimed in claim 17, wherein, the plurality of cassettes includes a fourth cassette includes a 35CaMV35S promoter linked to a gene coding a protein conferring antibiotic resistance and a NOS adenylation sequence.

25. The method as claimed in claim 24, wherein the antibiotic resistance is to hygromycin or kanamycin.

26. The method as claimed in claim 17 or claim 18 or claim 19 or claim 20, wherein the intron sequences and the cTP have a SEQ ID 13, SEQ ID 14, and SEQ ID 15 for targeted expression of protein coded from the triple gene.

27. The method as claimed in claim 17, wherein the fusion protein having a SEQ ID 21, SEQ ID 22 and SEQ ID 23 are present in the plant or the part of plant.

28. A process for detection of a fusion protein i.e. insecticidal toxins and a herbicide tolerant protein coded by a triple gene conferring enhanced resistance to insect feeding on the plant or the part of the plant, in transgenic, the process consisting of steps of: Producing of antibodies specific to purified EPSPS protein, Cry2Aprotein and VIP3A protein; Purifying of antibodies using purified Protein A affinity resin; Characterizing of antibodies using indirect ELISA; and Extracting of protein.

29. The process of claim 28, wherein antibodies are detected through ELISA by the process consisting of: denaturing the purified proteins to inactivate endogenous alkaline phosphatase; loading denatured samples in microtiter plate; blocking the unbound non-specific sites with BSA/TBS blocking buffer, allowing the protein samples to react with antibodies such as anti-EPSPS, anti-Cry2A and anti-VIP3A respectively; detecting bound antibodies such as anti-EPSPS, anti-Cry2A and anti-VIP3A with AP-conjugated goat antirabbit IgG using BCIP/NBT substrate, after standard washings; stopping the ELISA reaction by adding 1N HCl and estimating the absorbance rate at 430 nm spectrum, using negative control as blank; and plotting a graph with standards between optical densities (OD) of different concentrations of standards, determining the concentrations of GTG, Cry2A and VIP3A by placing their respective OD values on standard graph curve and using the following formula for protein quantification: [mathematical expression included]

30. A process of DNA extraction from parts of transgenic plants and a PCR analysis, the process comprising steps of: plucking fresh leaf samples and immediately keeping in liquid Nitrogen container; grinding finely leaves or other parts of transgenic plants using in pre-chilled sterile mortar & pestle using liquid Nitrogen; taking dry powder and mixing thoroughly with pre-heated DNA extraction buffer (2% CTAB, 1% Mercaptoethanol, 2M NaCl, 200 mM EDTA and 100 mM Tris-HCl, RNase A); incubating at 700C for 30 minutes in mixture of dry powder and pre-heated DNA extraction buffer, adding one volume of Phenol (pH 8), vortexing and spinning for 10 minutes at maximum speed and extracting supernatant with equal volume of Chloroform: Isoamylalcohol (24:1) and spinning again; taking supernatent and adding 0.7 volume of isopropanol and kept at room temperature for one hour, after spinning, washing the DNA pellet twice with 70% freshly prepared ethanol, air dried and resuspending in 50 microL sterile water, and quantifying DNA on 0.8% Agarose gel through electrophoresis.

31. The process of claim 30, wherein in PCR analysis the PCR amplification of EPSPS, Cry2A and VIP3A genes from genomic DNA is carried out using gene-specific primers having SEQ ID NOs: 1-12.

12; 12; 07; 01

edspap.20170233758