Nghiên cứu chuyển gen cry8db có tính kháng côn trùng vài cây mía

antlet regeneration (ROC22 – 97,43%) mean while axillary buds have the highest regeneration at ROC22 variety (90,24%) and 13,67 shoots/explant. The study showed that the regeneration via embryogenesis is the most effective to use in transgenic sugarcane and. This protocol may be use for further studies in genetic transformation of sugarcane. 5.3 Construction vector for transgenic sugarcane and the efficiency selectable marker hpt We constructed the plant expression vector pCAMBIA1300 containing two expression cassettes, CaMV35S/cry8Db/NOST and CaMV35S/hpt/NOST. The target gene cry8Db was placed under the control of the constitutively expressed double promoter CaMV35S. Consistent with this, the Cry8Db protein content in the leaves of transgenic sugarcane lines ranged from 1.62-11.89 ng/µg. A previous study reported that the expression of cry1Ab regulated by CaMV35S yielded only a maximum of 27.23 ng/mg Cry1A(b) protein in transgenic sugarcane leaves (Arencibia et al., 1998). This suggests that the promoter CAMV35S can significantly enhance the expression of target genes. 129 The hpt gene is a high efficiency selectable marker in plant genetic transformation. It has been widely used in many plant species (Vasil et al., 1992) including sugarcane (Manickavasagam et al., 2004) due to its advantage in screening putative transformants. Sugarcane is more sensitive to hygromycin than kanamycin (Luo et al., 2002). The screening concentrations of hygromycine for the sugarcane cultivar ROC22 in tissue culture was 50 -100 mg/L hygromycin for in vitro genetic protocol and 2000 mg/L hygromycin for ex vitro genetic protocol. Putative transformants surviving in tissue culture could be re-screened by spray treatment with hygromycine at the seedling stage after soil transplantation. This procedure allows rapid and cost effective identification of transformants because the leaves of transgenic plants will remain green but leaves from non-transgenic leaves turn yellow, wither and die. Additionally, the hpt gene (hypoxanthine phosphoribosyl transferase) which is resistant to hygromycine. This is also an important agronomic character for sugarcane with respect to against pets of sugarcane. Thus, transgenic sugarcanes with the cry8Db gene have great potential for commercial use. According to Brukhin et al. (2000), the appropriate selective concentration threshold is achieved when 90% of non-transgenic crops are removed. Thus, in this study, hygromycin 50 mg /L was suitable for selection transgenic sugarcane shoots (approximately 90% removal). This concentration is two times higher than the selective concentration of transgenic sugarcane in vitro. In vitro gene transfer experiments use 50 mg /L hygromycin to select transgenic sugarcane (Joyce et al., 2010). Mean while others use concentration hygromycin were lower, Arencibia et al. (1998) used 25 mg/L hygromycin supplementation medium for selection of Ja60-5 sugarcane cultivar scar tissue and 20 mg/L for transgenic plants (Arencibia et al., 1998). The cause of this phenomenon is that ex-vitro sugarcane are able to grow and tolerate stress much better when in the natural environment, under artificial conditions, in vitro plants are less resistant, so the selection condition will be lower. 5.4 Cry8Db protein expression level in transgenic sugarcane The first transgenic insect resistance sugarcane plant using electroporation was 130 reported in 1997. A truncated cry1Ab gene encoding the active region of the Bt insecticidal crystal protein was expressed in transgenic plants under the control of CaMV35S promoter. Although the expression level of Cry1Ab was low (0.5–1.4 ng/mg), several selected transgenic plants showed significant larvicidal activity (Arencibia et al., 1997). The subsequent field trial showed that elite transgenic lines reduced the incident of internode inoculation by sugarcane stem borers (Arencibia et al., 1999). Expression of cry8Db transgenic sugarcane mediated-Agrobacterium in ex vitro condition The δ-endotoxin toxin, encoded by genes from Bt, is widely used in insect- resistant genetically modified (GM) crops, but the degree to which insect protection is effective against insect pests.Very low due to low level of protein expression. The cause of this phenomenon is that if the Bt gene is used directly after isolation without modification such as increasing the G-C rate in the gene, modifying the code to target host variety, these two factors determine The level of expression of the toxin gene when it is introduced into the plant. Our cry8Db gene was synthesized, modified based on the cry8Db gene sequence isolated from the Bt51 strain and the full nucleotide sequence of this gene on Genbank with code AB303980.1 (not show in this study). The results showed that recombinant recombinant protein concentrations in the transgenic lines were very high. This is suitable with the results of Weng et al. (2011), when the team made cry1Ac gene modification by increasing the G-C content of the gene and modifying the encoded trio. The trio suit is in the sugar cane. Concentrations obtained were highest in the 79mt11 bagasse, the Cry1Ac toxicity analyzed in this line was 50.5 ng/mg total soluble proteins in leaves (Weng et al., 2011), which was lower with the result of we obtained in ES2 sugar cane, the Cry8Db toxin protein obtained at 11 89 ng/μg soluble proteins in leaves. The recombinant protein concentrations we obtained in transgenic sugarcane flocks were similar to the results of Wu et al. (2015), recombinant protein concentrations ranged from 9.5-122.5 ng/20 μg total protein Thus, modifying the genetic code of the target gene is a very important factor in determining the viability of the transgenic plant. In addition, the expression of the 131 foreign gene expression depends on the localization of the transgene in the genome of the sugarcane. Because the sugarcane genome is one of the most complex chromosomes in plants, we are continuing to investigate the effect of localization of target genes on the plant genome to the extent that gene expression of transgene. Our study showed that, vector pCAMBIA1300/CaMV35S/cry8Db/NOST was transfected in sugarcane via A. tumefaciens. Among the 9 PCR positive plants, all of them were determined to be positive using protein analysis (ELISA) and contained a range of 1.62-11.89 ng/µg total soluble proteins Cry8Db in leaves, so the expression of cry8Db gene was detected in all these lines. While the remaining 4 lines (ES2; ES3, ES6, ES7) had a high protein expression 6.4-11.89 ng/µg total soluble proteins in leaves. The recombinant Cry8Db protein content in transgenic sugarcane of our study was higher than that of Weng et al., 2011 that the transgenic sugarcane lines produced up to 50 ng Cry1Ac per mg soluble proteins. Accelerating the production of transgenic sugarcane plants not only saves time and effort but will likely also minimize somaclonal variation. Expression of Cry8Db transgenic sugarcane Agrobacterium -mediated in vitro condition ELISA A protocol is described that supports the production of transgenic sugarcane plants ready for transfer to soil within 3 months from culture initiation. cry8Db gene transfer into embryogenesis via A. tumefaciens resulted in the stable genetic transformation of the commercially important sugarcane cultivar ROC22. Among the 4 PCR positive plants, 2 lines IS2, IS3 were determined to be positive using protein analysis (ELISA) and contained a range of 11.87-21.73 ng/µg total soluble proteins Cry8Db in leaves. Thus, although the number of gene transfer lines was lower, the target protein concentration obtained by using the in vitro gene transfer method was approximately two times higher than that of the ex vitro procedure. Therefore, this protocol has great potential for the generation of commercial transgenic sugarcane events. 132 Western blot and insect bioassay Western blot assays were performed to examine the integrity of the Cry8Db protein produced by the transgenic plants (Figure 3.33). This experiment gave nearly similar results for all the transgenic lines. A well-defined band was observed at 73 kDa, a molecular size value similar to the one predicted from the DNA sequence of the corresponding gene. However, some weaker bands of lower molecular size were also noted, which may indicate that the Cry8Db protein undergoes proteolytic processing. Arencibia et al.,1997 also published this problem on the transgenic sugarcane. Transgenic plants had significantly less dead at the seedling stage and there was a negative correlation between protein expression and the dead rate. The differences in resistance to resistant L. signata Fabricius may be due to two factors: the level concentration of Cry8Db protein expression and resistance to different transgenic sugarcane lines. The second point was the difference suspicion of sugarcane in the greenhouse environment. The results demonstrated that higher Cry8Db protein expression results in reduced damage. IS3 transgenic sugarcane line, with the highest protein expression in leaves (21,73 ng/µg total soluble proteins) had the lowest percentage of damaged (12.91%).This result was similar to some of the bioassays of cry transgenic sugarcane published by Gao et al. (2016); Weng et al. (2011). Specifically, when the concentration of Cry1Ac was low (1.8 ng/mg total solube protein), the percentage of the damage to the transgenic lines ranged from 23.33% to 36.67%. In contrast, transgenic sugarcane expressing high Cry1Ac concentration (70 92 μg / Wg that lead to the percentage of damage which was only 13.33% (non- transgenic control damage level up to 76.67% ) (Gao et al., 2016). Conclusions In conclusion, the data presented herein reveal the potential Bt insecticidal action of Cry8Db against L. signata Fabricius in Vietnam. In this reseach, an efficient in planta Agrobacterium-mediated genetic transformation was developed for sugarcane using 2 protocol: in vitro condition using embryogenesis and ex vitro using setts as explant. The results showed that the ex vitro transgene is more 133 effective than the in vitro protocol. When the buds were pricked with a fine needle, sonicated for 6 minnutes in A. tumefaciens strain EHA105 harbouring pCAMBIA 1300/cry8Db plasmid suspension containing 5% sucrose, 0.1% Silwett L-77, and 100 µM of acetosyringone, and vacuum infiltered at 500 mm Hg for 2 minutes in Agrobacterium suspension recorded a maximum transformation efficiency of 29.6 % (with var. ROC22). Among the 9 PCR positive plants, all of them were determined to be positive using protein analysis (ELISA) and contained Cry8Db a range of 1.62-11.89 ng/µg total soluble proteins in leaves, so the expression of cry8Db gene was detected in all these lines. Mean while the remaining 4 lines transgenic sugarcane had a high protein expression 6.4-11.89 ng/µg total soluble proteins in leaves. This is very high protein expression concerntration toxic protein to achieve.This is the first report on in planta Agrobacterium-mediated genetic cry8Db transformation of sugarcane using setts as explant in Vietnam. In protocol for in vitro transgenic sugarcane, the embryonic calli infected were co-cultured with A. tumefaciens onto MS medium supplemented 100 μmol/L AS in dark for 4 days, and then transferred to selective medium. After 4 weeks, non-transformed calli became brown and died gradually. However, the resistant calli, with the characters of white-yellow color, lustrous, dense and grainlike tissues were appeared on the surface of transformed embryonic calli. The survival shoots in selective medium were planted in rooting medium with 30 mg/L hygromycin. After 3 months in green house, the suvival shoots were tested by PCR and ELISA. Among the 4 PCR positive plants, 2 lines IS2 and IS3 were determined to be positive using protein analysis (ELISA) and contained a range of 11.87-21.73 ng/µg total soluble protein Cry8Db in leaves. Thus, although the number of gene transfer lines was lower, the target protein concentration obtained by using the in vitro gene transfer method was approximately two times higher than that of the ex vitro procedure. Therefore, this protocol has great potential for the generation of commercial transgenic sugarcane events. Western blot and insect bioassay showed that IS3 line was the best resistance to L. signata Fabricius. IS3 line has the lowest rate of damage (12.91%) by bioassay and lead to the larval weight which was reduced approximately 2.85 times after 14 days. 134 TÀI LIỆU THAM KHẢO 1. Aditya P, Jitendra K (2014). Alien Gene Transfer in Crop Plants. Spinger, NY. 2. 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