In the present study truncated Hvt gene, synthetic Cry1Ac gene without transient peptide, Vip gene and stacked gene construct of Hvt and Vip gene were used with specific cis-element to improve the expression of these genes in plant system and to induced optimum resistance in plants against the herbivore insects. Synthetic Hvt and Cry1Ac gene were already available in plant molecular biology and transformation lab in plant biotechnology division of the National Institute for Biotechnology and Genetic Engineering (NIBGE). The sequence of the gene encoding Hvt was deduced, with codon optimization for expression in plants from the published amino acid sequence of the protein (Fletcher et. al. 1997) and was synthesized commercially (Medigenomics, Germany). The sequence of the Cry1Ac gene was deduced from the known amino acids sequence of the Bt protein (Perlak et. al. 1991) and modified according to the dicot preferred plant triplet codons as described in (Appendix22). The synthetic Cry1Ac gene used in this study was commercially synthesized from Medigenomics, Germany. Vip gene was obtained from ICGEB (International Centre for Genetic Engineering and Biotechnology) New Dehli, India. To facilitate the cloning of Cry1Ac, Hvt, and Vip genes under desirable expression cassette, few restriction sites were added to the flanking regions of the gene at the 5ï‚¢ as well as 3ï‚¢ ends of the gene. Three different plant expression vectors were developed at the Plant Molecular Biology and Transformation Lab at NIBGE, which contained 35S promoter, specific gene (Hvt, Cry1Ac or Vip or Hvt+Vip) or and CaMV or OCS terminator. These vectors were named as pZMCII, pSAKVII, pJSVD.
1. Amplification of Cry1Ac gene by Polymerase Chain Reaction (PCR)
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Based on the Cry1Ac gene sequence a set of primers was designed which was used to amplify Cry1Ac gene from plasmid pSAKIV that was already available in the Plant Molecular Biology and Transformation Labortary at NIBGE Faisalabad Pakistan.
Cry1Ac gene was amplified by using the reverse and forward primers (Appendix4). The PCR reaction (Appendix 5) was carried out in Thermal Cycler (Eppendorf) and incubated at different annealing temperatures for optimization.
1.1. Gel electrophoresis
To analyze the amplified PCR products 0.8% agarose gel (Appendix 1) along with DNA marker (Appendix 26) was used. DNA fragment of 1.8 kb was eluted from the agrose gel using DNA Extraction kit from Fermantas (Appendix 6). Elution was confirmed by running 2μl of eluted DNA on 0.8% agrose gel. Purified Cry1Ac gene fragment was cloned into T/A cloning vector (pTZ57R/T) using the profile as shown in appendix 7.
1.2. Heat shock transformation of ligation product in E. coli strain Top 10 F
Escherichia coli (E. coli) Top10 F strain was used for making heat shock competent cells (Appendix 8) following the procedure modified by Cohen et. al. (1972). Competent cells were stored at -70°C for further experiments. For bacterial transformation the competent cells were allowed to thaw on ice and mixed with 5 ïl of ligation reaction was added. Then heat shock (42°C) was given for one and half minutes (Sambrook et. al., 1989). The cells were immediately transferred to ice and kept for 10 minutes. Then 800 ïl of LB medium (Appendix 11) was added and the culture was incubated at 37°C for one hour. This culture was spread on LB agar plates (Appendix 10) containing the ampicillin (100 µg/ml) and 1 mM IPTG + 1mM X-Gal. On the following day, the appearance of white colonies along with blue colonies confirmed the true transformation. After 24 hours, few white colonies selected randomly from LB agar plates and were grown over night in 3.0 ml of LB broth (Appendix 11) with selection antibiotic ( ampicillin 100 mg/ml). Plasmid minipreparation was done by alkaline lysis method (Appendix 13). The new construct was named as pZMC I.
1.3. Confirmation of Cry1Ac in pZMC I by restriction/digestion
Restriction analysis was done at 370C for 2 hours by using BamHI and HindIII (Appendix 12). To confirm the desired clone it was loaded on agarose gel. After confirmation through digestion midiprep of selected clone (Appendix 14) was done by using Invitrogen (USA) kit and clone was reconfirmed through restriction digestion using same restriction enzymes asGi above.
1.4. Cloning of Cry1Ac gene in expression vector pGREEN-ZSA
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pGreen 0029 is a small binary Ti vector whose size and copy number in E.coli offer increase efficiencies in routine invitro recombination procedure. pGreen can replicate in Agrobacterium tumefaciens only if another plasmid pSoup is co- resident in the same strain. pSoup provide replication function in transformed pGreen. pGreen 0029 was modified in our lab by cloning expression cassette (1.5 kb) containing duplicated CaMV35S promoter and CaMV terminator and named as pGreen-ZSA . Cry1Ac gene was cloned in pGreen-ZSA. Cry1Ac gene was digested by double digestion by using BamHI and HindIII. pGreen-ZSA was also digested by the same enzymes (Appendix 12 ). The digestion products were run on agrose gel from where 1.8kb Cry1Ac and pGreen-ZSA fragments were purified (Appendix 6). Afterwards the Cry1Ac fragment was cloned in pGreen-ZSA. The ligation product was transformed into heat shock competent cells of E.coli Top10 F (Appendix 8). Transformed cells were plated on LB agar plate having kanamycin antibiotic (100 µg/ml) and were incubated at 370C overnight. Colonies were picked and grown in LB liquid media (Appendix 11). The recombinant clone was isolated from this culture by plasmid miniprep method (Appendix 9). The resultant clone was named as pZMCII.
1.5. Confirmation of Cry1Ac in pZMC II through restriction/digestion
The restriction analysis was done by using BamHI and HindIII and was incubated for 2 hours at 370C. Afterwards it was loaded on agrose gel. After confirmation through digestion, midiprep of selected clone (Appendix 14) was done by using Invitrogen (USA) kit and again clone was confirmed through restriction digestion using same restriction enzymes.
2. Cloning of Vip gene in plant expression cassette
2.1. Cloning of Vip gene in T/A cloning vector.
The Vip gene was already available in the Plant Molecular Biology and Transformation Lab at NIBGE Faisalabad Pakistan. In order to clone this gene in a desirable vector, specific oligonucleotides were designed for the PCR amplification of the Vip gene. The plasmid obtained from ICGEB through material transfer agreement was used as a template for the amplification of Vip gene. The amplified fragment was ligated within T/A cloning vector according to the manufacturer's instructions (MBI Fermentas). The resultant vector was named as pJSVA which was then used in further cloning.
2.2. Cloning of Vip gene under 35S promoter
As mentioned above, pJSVA was used in next step of cloning. The sites BamH1 and Cla1 present in pJSVA were digested and a 2.4 kb fragment of Vip gene was released. The pN6 expression vector having single 35S promoter and OCS terminator was digested with same enzymes i.e. BamH1 and Cla1. Restricted pJSVA and pN6 vector were run on gel electrophoresis. Enzymes digested insert (Vip gene fragment) and vector (pN6) vector were eluted (Appendix6) from the agrose gel and ligated as described in Appendix 34. The resultant expression vector was named pJSVB containing Vip gene under 35S promoter and ocs terminator.
2.3. Cloning of Vip gene in pGreen binary Vector
The vector obtained in the previous cloning step named pJSVB having 35S promoter and OCS terminator was digested with NotI restriction enzyme. The enzymes restricted a whole cassette containing the promoter, Vip gene and the terminator. pGreen expression vector was also digested with same enzyme. These two restrictions were eluted from the gel. The resultant Vip gene cassette and the restricted pGreen cloning vector were ligated according to the manufacturer's instructions (MBI Fermentas). The resultant vector was named as pSAKVII.
3. Cloning of Stacked gene construct of Hvt and Vip
Hvt gene consrteuct was already available in the lab in vector pSAKII. In order to clone Vip and Hvt in the same vector, the construct pJSVB and p SAKII were digested with BamHI and SacI (Appendix36) and were placed at 370C for 4 hours. After 4 hours both restrictions were run on the 1% agrose gel, desirable fragments were eluted using DNA Extraction kit from Fermatas (Appendix 6) and were ligated (Appendix 33).
3.1. Heat shock transformation of ligation product in E. coli strain Top 10 F
The ligation product was transformed into heat shock competent cells of E.coli Top10 F (Appendix 8). Transformed cells were plated on LB agar plate having kanamycin antibiotic (100 µg/ml) and were placed at 370C overnight. Colonies were picked and grown in LB liquid media (Appendix 11) recombinant clone was isolated from this culture by miniprep method (Appendix 13) the recombinant clone was confirmed by digestion with a combination of restriction enzymes i.e. BamHI and SacI. The recombinant clone was named as pJSVG.
3.2. Cloning of terminator in pJSVG
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In order to clone terminator in pJSVG the vector pJIT60 which was already available in the lab was digested with BamHI and XhoI. (Appendix 37) The vector pJSVG was also digested with same enzyme i.e BamHI and XhoI. Linearized product of pJSVG and 0.7 kb fragment of ocs termintor from pJIT60 was eluted from Gel and were ligated as shown in (Appendix 8). The ligation product was transformed into heat shock competent cells of E.coli Top10 F (Appendix14). Transformed cells were cultured on LB agar plate having kanamycin antibiotic (100µg/ml) and were placed at 370C overnight. Colonies were picked and grown in lb (Appendix 11) recombinant clone was isolated from this culture by miniprep method (Appendix 13), the recombinant clone was confirmed by digestion with a combination of restriction enzymes i.e. BamHI and XhoI. The recombinant clone was named as pJSVD.
4. Transformation of recombinant binary vectors in Agrobacterium strain Gv
Electro-competent cells of Agrobacterium strain Gv already harboring pSoup plasmid were prepared using the procedures as described in the (Appendix 16). The recombinant vectors pZMCII, pSAKVII, pJSVD were transformed in Agrobacterium strain GV through electroporation method as given in the (Appendix 17). Transformed cells were plated on LB agar plate having 10 μg/ml Tetracycline, 50 μg/ml Kanamycin and 50μg/ml Rifampicin antibiotics and were placed at 280C overnight. Colonies were picked and grown in LB liquid media (Appendix 11). The recombinant clone were confirmed through PCR.
5. Stable transformation of pZMCII, pSAKVII, pJSVD through Agrobacterium mediated transformation in Nicotiana tabacum cv. Samsun
5.1 Materials required
Sterilized Nano pure water for rinsing seeds, Whatman No.1 filter papers (9cm), petri dishes, scalpel holder, forceps and spatula are required.
Single colony of A. tumefaciens having pZMCII was picked and inoculated in 25 ml of liquid LB medium (Appendix 11) having 10 μg/ml tetracycline, 50 μg/ml kanamycin and in 50 μg/ml rifampicin in flask and shaken at 150-250 rpm for 48 hrs in dark at 28°C.
Leaf discs (aproximately 20 discs per plate) were cut from top 2-3 leaves under aseptic conditions and placed on MS0 medium (Appendix 23). The leaf discs were placed upside down. Plates were sealed with Para film and incubated at 16/8 light and dark cycle at 25± 1°C. Explants were left to pre-incubate for 48 hours.
Recombinant A. tumefaciens suspension was poured into sterile petri-plate and all leaf discs were placed in the bacterial suspension for 25-30 minutes with gentle shaking after regular interval. The leaf discs were removed from suspension and blot dried on sterile filter paper and transferred to co-culture medium (Appendix 24) for 48 - 72 hours at 25 ±1°C (depending on the growth of bacteria) under light conditions. Plates were sealed with Para film.
Leaf discs were removed from co-culture medium and placed on selection medium (Appendix 24). Some discs were placed in liquid MS0 medium having cefotaxime (250 mg/l) to remove excess of A. tumefaciens before placing them on the reaction medium. Then these leaf discs were placed with cut edges in contact with medium. 10 -15 leaf discs were placed on regeneration medium per petri plate, plates and incubated under 16 hours photoperiod at 25±1° C for two weeks. Regenerated leaf discs were transferred to fresh selection medium (Appendix 25). They were shifted into magenta containers when shoots became large for the petri dishes,
Shoots were separated from the regenerating tissue at the stage when at least one internode was formed. Shoots were transferred into rooting medium with reduced antibiotic concentration (Appendix 28).
5.4. Transfer to soil
Plants were transferred to sterile soil pots after removing media by washing under tap water. Plants were covered with polyethylene bags to retain humidity and kept at 25± 2°C under 16 hours photoperiod for hardening. After 7-10 days, envelopes were backed off to reduce humidity gradually until plants were acclimatized to ambient humidity and temperature conditions. As a control experiment, few non-transformed leaf discs were placed on two different media (with and without antibiotic) to observe the effect of selection agent (kanamycin). Regenerated leaf discs were transferred to fresh selection medium after 2 weeks (Appendix 25).
6. PCR Analysis of transgenic plants
Putative transgenic plants were screened through PCR analysis. The forward primer and reverse internal primers were designed to confirm the DNA of transgenic plants. The PCR profile used was 94 oC initial denaturation for 5 minutes followed by 40 cycles of 94 oC 1 minute, 60 oC 1 minute (depending upon primers melting temperature), 72 oC 1 minute and a final extension at 72 oC for 10 minutes before holding at 4 oC
Eppendorf thermal cycler was used for PCR and amplified products were analyzed by Gel electrophoresis on 1% agarose gels (Appendix 1) along with DNA marker (Appendix26). Total Genomic DNA extraction was done from 10 transformed tobacco plants using the procedure as given in (Appendix 18).
7. Southern blot analysis of transgenic plants
The transgene insertion in genome for all three types of transgenic plants was also determined by Southern hybridization performed. For this purpose 10 µg of total genomic DNA of selected lines was digested with Not I and incubated at 37 0C overnight. The digested DNA was size fractionated on 1 % agrose gel supplemented with 10 µg ml-1 ethidium bromide at 40 V in TAE buffer for 5 to 6 hours. The DNA image was obtained under UV light in gel documentation apparatus DNA was depurinated by submerging the gel in glass tray contained 250 ml depurination solution 0.25M HCl, twice for 15 minutes on an orbital shaker and rinsed briefly with deionized water. Denaturation of DNA was carried out by floating the gel twice in denaturation solution (1.5 M NaCl and 0.5N NaOH) for 15 minutes and washed thoroughly with deionized water. Finally neutralization was done by soaking the gel twice in neutralization solution (1 M Tris (pH 7.4), 1.5 M NaCl) for 15 minutes. DNA in the gel was transferred on nylon membrane (Hybond-Amersham) by capillary method using 10 X SSC (150 mM sodium citrate and 1.5 M NaCl) as transferred buffer for 20 hour (Sambrook et. al. 1989). The membrane was removed from gel and crosslinked in UV crosslinker (CL-1000 Ultraviolet Crosslinker-UVP) at 120 mJ cm-2 energy. The crosslinked membrane was further washed in a solution containing 0.1X SSC, 0.5 % SDS at 65 ÌŠC for 45 minutes to remove residual agrose. To block the attachment of probe to non-specific nucleic acid binding sites, the membrane was treated with 0.2 ml cm-2 pre-hybridization solution [6X SSC, 5X Denhardt's solution (0.1 % each of BSA, Ficol and PVP), 50 % deionized formimide, 0.5 % SDS and 50 µg ml-1 salmon sperm DNA in a hybridizer for 2-4 hours at 42°C.
Biotin DecaLabel DNA Labeling kit (Fermentas, Germany) was used for labeling the purified DNA products (section 2.4.3) of specific genes following the manufacturer's instructions. The reaction mixture for DNA labeling was prepared in 1.5 mL microcentrifuge tube containing 100 ηg to 1 µg DNA template (usually PCR amplified purified product), 10 µl decanucleotide in 5X reaction buffer and total of 44 µl of volume was made up by nuclease free water. The reaction mixture was mixed briefly, spinned down and DNA was denatured in a boiling water bath for 5-10 minutes. The tube was cooled immediately on ice, added 5 µl biotin labeling mixture and 1 µl Klenow fragment, mixed briefly by vortexing and spinned down quickly. The tube was incubated at 37 °C for 1 to 20 hour and the reaction was stopped by adding 1 µL 0.5 M EDTA (pH 8.0) to reaction. DNA labeled probe was again denatured at 100 ÌŠC for 5 minutes chilled on ice and added to the pre-hybridization solution (25-100 ηg ml-1). After the treatment of pre-hybridization solution for 2-4 hours, the membrane was treated with hybridization solution (60 µl cm-2) in the hybridization tubes and incubated overnight in a hybridizer at 42 °C. The membrane was first washed twice with 2X SSC and 0.1 % SDS for 10 minutes at room temperature then twice with 0.1X SSC and 0.1 % SDS at 65 °C for 20 minutes. To detect the biotin-labeled DNA the membrane was floated in 30 ml Blocking/Washing Buffer (provided by the manufacturer) for 5 minutes at room temperature. To block non-specific binding sites, membrane was treated with 30 ml Blocking Solution for 30 minutes. The membrane was incubated for 30 minutes in Streptavidin-AP conjugate diluted in 20 ml blocking solution. The next washing was of 60 ml Blocking/Washing buffer twice for 15 minutes and incubated for 10 minutes in 20 ml Detection Buffer. Finally 10 ml Substrate Solution was prepared and the membrane was incubated in it at room temperature in the dark until blue-purple precipitate became visible. The reaction was terminated by washing the membrane with distilled water for few seconds after discarding the Substrate Solution.
8. Insect Bioassay
Leaves were detached from the transgenic plants of all three constructs as well as from non-transgenic plants and were placed in the petri plates underneath the leaves blotting paper was placed to keep the leaves moist. H. armigera larvae were caged on detached, mature leaves from either transformed or nontransformed tobacco (N. tabacum) plants. Within 24 h of being placed on leaves of transformed plants the insects showed the effects of toxin ingestion consisting of cessation of feeding, uncontrolled movement followed by paralysis. Typically the larvae died within 48 h when placed on detached leaves of transgenic line T21, while the insects on leaves from non-transformed plants continued feeding throughout the experiment, eventually consuming the whole leaf H. armigera larvae feeding on leaves of the transgenic lines showed significantly higher mortality rates compared to control plants.