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ANALYSIS OF TRANSGENIC CHINESE CABBAGE INDUCED CRYIAC GENE

Nashr ma`lumotlari: // Ўзбекистонда озиқ-овқат хавфсизлигини таъминлашда мева-сабзавот ҳамда узумчилик соҳасининг роли ва аҳамияти // мавзусидахалқаро илмий-амалий анжуман.\\ –Тошкент - 2017
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ANALYSIS OF TRANSGENIC CHINESE CABBAGE INDUCED CRYIAC GENE

H.S.Ahn, Y.D. Park, J.E.Park
KOPIA Uzbekistan center

In this study, we analyzed the stability of the introduced gene in the T2 generation of BT cabbage transformants with CryIAc gene introduced by using these characteristics. If we carry out tests on the transformed BT cabbage seeds and develop the generation and fix the traits, we will be able to breed the insect resistant Chinese cabbage in the future and contribute to the increase of Chinese cabbage yield.
Key-words: Chinese cabbage, transformants, CryIAc, gene, pest resistance

Introduction. In past agrarian society, many problems such as the emergence of pesticides resistant to insecticides, residue of chemical pesticides and soil accumulation, destruction of ecosystem, environmental pollution have been caused by the use of chemical pesticides indiscreetly. To address this problem, molecular breeding methods have been introduced to improve traits such as resistance to insect pests that harm plants.
There are primary environmental factors such as temperature, humidity, and precipitation, and secondary factors such as damage to the pest maximizing as the primary environmental factors are equipped. There are more than 10 species of moth, such as moths and aphids, which are harmful to Chinese cabbages. Among them, B. thuringiensis is an aerobic bacterium with major maternal flagella, which forms an endotoxin protein in sporangia during spore formation, which is activated at the high pH of the insecticidal medium and is toxin And pores are killed by making holes in the plasma membrane. In particular, it has been shown that lepidoptera, diptera, and Coleoptera larvae have high specificity for insect pests, and are also specific for parasitic zygotes and protozoans.
In this study, we examined the translocation of CryIAc and the control cabbage transformants without CryIAc transfection. Then, each of the Chinese cabbage seeds was sowed, DNA was isolated, and the transformant selection was carried out by PCR. Transgenic Chinese cabbage selected by PCR will be used for insect resistance functional test.
Methods and materials. A transformant with BT over-expression vector inserted into Chinese cabbage (Brassica rapa ssp. Pekinensis) was named BT. BT 5 lines (BT-14, 21, 23, 24, 28) and CT001 and CT004 as control lines were seeded on 10 * 5 seedlings by 10 lobes each and germinated in the greenhouse. The seeds were sown for DNA extraction when 6-8 leaves were formed after germination. At the sampling time, greenhouse powder, aphids, and soil attached to the main leaf were removed and then the middle part of 3-5 large leaves except the stem was cut into oblique lines and sealed in a foil and stored in a liquid nitrogen container. At each sampling, the scissors cutting the leaves were disinfected with 70% EtOH to prevent mixing of DNA.
The DNA of the sampled individuals was subjected to grinding to separate them. Prior to grinding, liquid nitrogen was poured into a mortar bowl sterilized with an autoclave to lower the temperature of the bowl itself so as not to dissolve when contacted with the Chinese cabbage sample. Then, the Chinese cabbage sample was poured into a sterilized pestle and continuously poured liquid nitrogen to maintain a low temperature. The above procedure was repeated three times to finely grind the particles. The ground sample was placed in a 2 ml tube which had been cooled down to a liquid nitrogen temperature, and about 0.8 g of the sample was placed using a spoon. The tube was stored in a cryogenic freezer.
DNA was extracted for the transformation test of Chinese cabbage. 2.5 x CTAB DNA extraction buffer was added to 800 ql of sample, vortexed, and placed in a water bath at 65°C for 30 minutes. At this time, invert was performed at intervals of 5 minutes so as to mix well. After cooling for 10 minutes, 800 ql of PCI was added and invert for 10 minutes. After centrifugation using a centrifuge for 15 minutes, the supernatant was added to a 2ml tube, and the pellet was confirmed by invertting with 3M sodium acetate 40 and 880 ql of 100% EtOH. After storing in a freezer at -20°C for 1 hour, the pellet was rescued and washed in 70% EtOH for 15 minutes.
Afterwards, the pellet was rescued and dried for about one day. The dried DNA was dissolved by adding 40 gl of 1 x TE buffer and 2 gl of RNase, tapping, and mixed thoroughly. Then spin down and store at 4 ° C for one day.
PCR reaction was performed in order to identify transformants using BT-F (5'-ATG GAC AAC AAC CCA AAC ATC A-3 ') and BT-R (5'-GAT AGT TAT GCT GTT CAA GAT GTC C-3 ') which specifically amplify the CryIAc gene of the introduced T-DNA(Fig. 1). Each primer was first synthesized with oligo­nucleotide at 100 pM / ul. Therefore, the distilled water was diluted to 10 pM / ul, and a mixture for PCR reaction was prepared. For the prepared mixture, 1 gl of each of Primer F and R, 1 gl of DNA, and 17 gl of distilled water were added to make a total volume of 20 gl. The finished mixture was then dispensed into the pre-mix. The PCR reaction was performed using a pre-mix in which Tag polymerase, MgCl2, and dNTP required for DNA replication and amplification were quantified.
A total of 38 samples were made including 36 DNA samples, BT vector plasmid DNA as a positive control and CT001 gDNA as a negative control. Then, the mixture was taped so that the mixture was evenly mixed and then put into a PCR machine and operated. The PCR program was initially denatured at 95 ° C for 10 minutes, followed by 35 cycles of 95 ° C for 40 seconds, 59 ° C for 30 seconds, and 72 ° C for 1 minute and 10 seconds.
In a 500ml Erlenmeyer flask, add 2 g of agarose and 200 ml of 1 x TBE buffer, mix 10 gl of bluemango, mix well using microwave range, and make 1% agarose. After the solution was poured into a tray, a comb was inserted, and the wells were allowed to cool to prepare gels for electrophoresis. PCR samples were loaded in a 1% agarose well and electrophoresed at 100V for 30 minutes. The electrophoretic gel was photographed under UV to confirm the band presence.
The research results. The concentration and purity of cabbage gDNA in the BT transformants and the control groups CT001 and CT004 were analyzed using a Nanodrop spectrophotometer. Concentrations and purity of isolated gDNA were analyzed by major system (Tables 1, 2, 3, 4, 5, Figs. 3, 4, 5, 6, 7). The DNA concentration was measured as 10.56 ~ 4527.52 ng/^. The A260/A280 ratio was 0.73 ~ 1.87 and the A260/A230 ratio was 0.10 ~ 2.23. The A260 value indicates the amount of nucleotide (ssDNA, dsDNA, RNA), and the value of A280 indicates the amount of protein, phenol and other contaminants. A230 represents the amount of EDTA, carbohydrates, and phenol.
The purity of the DNA was analyzed by the ratio of the absorbance value at 260 nm to the absorbance value at 280 nm or 230 nm. The DNA purity is high when the A260 / A280 ratio is close to 1.8. However, when the A260 / A280 ratio is low, the concentration of DNA itself is very low or the DNA concentration is normal but the ratio is low. Although the DNA concentration is normal but the ratio is low, there is a high level of protein contamination, or the pH of the solution in which the DNA is dissolved is low. On the other hand, high A260 / A280 ratio is due to high DNA concentration or DNA fragmentation.
The A260 / A230 ratio has a high purity when close to 2.0. The A260 / A230 ratio is often low due to pollutants. Especially, when plant DNA is extracted, carbohydrate remains and the ratio is low.
According to the above results, the individuals with good DNA isolation in this experiment were 14-4 BT line which concentration is 2631.09, A260 / A280 ratio is 1.81, A260 / A230 ratio is 2.01, 28-1 BT line which concentration is 2665.68, A260 / A280 ratio is 1.82, A260 / A230 ratio is 2.0 and 28-7 BT line which concentration is 2858.17, A260 / A280 ratio is 1.82 and A260 / A230 ratio is 2.01(Table 2, 3, 4, and 5).
PCR was performed to select the T2 generation of Chinese cabbage transformed with Agrobacterium through CryIAc gene. PCR was performed using the primers BT-F and BT-R, which specifically identified the CryIAc gene, and confirmed by electrophoresis (Fig. 8). As a result, PCR products of 915 bp were confirmed in 25 out of 12 BT lines. Finally, the selected system was BT 14-1-5, BT 14-4-2, BT 14-4-3, BT 21-4-5, BT 21-5-1, BT 24-1-7, BT 28 -1 -1, BT 28-1-6, BT 28-3-3, BT 28-3-5, BT 28-4-2, BT 28-4-4, BT 28-4-6, BT 28- 5-5, BT 28­5-7, BT 28-7-3, BT 28-7-8, BT 28-7-10, BT 28-8-1.
PCR and electrophoresis showed PCR product band in 915 bp which is estimated as a transformant.
Of the total of 12 BT lines, 25 samples were identified as transgenic lines by using BT-F and BT-R
primers.
Since the Chinese cabbage transformants have been identified through PCR, more specific selection will be required through gene expression testing using such as RT-PCR method.
The conclusions, offers and recommendations. In this study, transgenic Chinese cabbage and control group were germinated for selection of transgenic plants by isolation of autologous fertilization in Chinese cabbage T2 generation transformed with insect resistance gene using Bacillus thuringiensis.
  1. Transgenic BT cabbage T2 generation which is induced of insect resistance gene CryIAc and control cabbage were seeded in a greenhouse and grown for 60 days. The leaves were frozen in liquid nitrogen to extract genomic DNA and the purity was measured.
  2. PCR was performed with a primer that specifically select the CryIAc gene among the induced T-DNA extracted genomic DNA, and a Chinese cabbage BT transformant line containing the CryIAc gene was selected by electrophoresis.

The references:
  1. Kim J. B., Kim K. D., Park D. S., “Establishment of a transformation protocol combination particle bombardment with Agrobacterium tumafaciens in different zoysiagrass cultivars”, 2004, Turfgrass and Environment Research Institute, Samsung Everland Inc.
  2. Park Y. Y., Kim J. M., Kim Y. S., “Construction two metal-ion binding sites to Improve the 3’- 5’Exonuclease Activity of Taq DNA Polymerase”, 1998, 471-477, Youngnam University.
  3. Kim Y. M. et al., "Characterization of 5-endotoxin produced by Bacillus thuringiensis BT-1 and BT-2", Journal of Life Science and Biotechnology, 2007, p658-663.
  4. Kim, JH, Lee, JH, Lee, JH, Kwon SB, Hwang MR, Kim IJ and Choi BL, "Studies on the Chinese cabbage moths and thorn moths in Chinese cabbage", Proceedings of Korean Society of Applied Entomology, , 2015., 295-295, Korean Society of Applied Entomology.
  5. "Insecticidal activity of cabbage moths against Bacillus thuringiensis and Neem oil", 2009, p315-324, Pesticide Science.
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