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Integration and expression of Bluetongue VP2 gene in somatic embryos of peanut through particle bombardment method

机译：粒子轰击法在花生体细胞胚中整合蓝舌VP2基因并表达

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摘要

After pre-culture and treatment of osmosis, zygotic embryos of peanut (Arachis hypogaea L.) were transformed via particle bombardment with a plasmid containing a Bluetongue VP2 gene (BTVP2) comprising neutralizing epitopes. Selection for Kanamycin resistant calluses and somatic embryos was initiated at 12th day post-bombardment on medium containing 25mg/L Kanamycin. Under continuous selection, 12.38% Kanamycin resistant plantlets were regenerated from bombarded somatic embryos. The presence and integration of BTVP2 DNA in regenerated Kanamycin resistant plants were confirmed by southern hybridization assay using non-radioactive Digoxiginin BTVP2 probe. beta-Glucuronidase (GUS) enzyme activity was detected in transgenic somatic embryos but not from control, non-transformed embryos. The expression of the BTVP2 protein was confirmed through RT-PCR (reverse transcription polymerase chain reaction) using the RNA isolated from the transgenic callus employing BTVP2-specific primers. The production of transgenic peanut was mainly focused on evaluating a newly improved somatic embryogenesis regeneration system as well as the gene transfer method and to produce the Bluetongue outer coat protein that comprises the neutralizing epitopes.
机译：在预培养和渗透处理之后，用含有包含中和表位的蓝舌VP2基因（BTVP2）的质粒通过粒子轰击来转化花生的合子胚（花生）。在含有25mg / L卡那霉素的培养基上轰击后第12天，开始选择抗卡那霉素的老茧和体细胞胚。在连续选择下，从轰击的体细胞胚中再生出12.38％的抗卡那霉素苗。通过使用非放射性洋地黄毒苷BTVP2探针进行的Southern杂交测定，证实了再生的卡那霉素抗性植物中BTVP2 DNA的存在和整合。在转基因体细胞胚胎中检测到β-葡萄糖醛酸苷酶（GUS）酶活性，但未从对照，未转化的胚胎中检测到。 BTVP2蛋白的表达通过RT-PCR（逆转录聚合酶链反应）使用从BTVP2特异性引物分离自转基因愈伤组织的RNA进行确认。转基因花生的生产主要集中在评估新改良的体细胞胚发生再生系统以及基因转移方法上，并生产包含中和表位的蓝舌外衣蛋白。

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