转基因甘蔗BtG-2的T-DNA侧翼序列分析及其转化事件特异性检测

转基因甘蔗BtG-2是利用农杆菌介导法把Cry1Ac-2A-gna融合抗虫基因导入‘新台糖22号’的转基因甘蔗株系,具有良好的抗虫特性和农艺性状。为了明确转基因甘蔗BtG-2的分子特征及其检测方法,推进其生物安全性评价工作,以BtG-2的T2代为研究材料,利用Southern杂交检测外源基因在转基因甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立了该转化体高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BtG-2株系;经过3次的热不对称巢式PCR扩增,获得外源基因T-DNA左边侧翼序列984 bp和右边侧翼序列705 bp;以这2个序列和相应的T-DNA的左右端序列分别设计3对检测引物对,建立了BtG-2株系的转化事件特异性PCR检测方法,扩增效率最高的引物对LS011/LA451和RS160/RA588分别扩增到440 bp和428 bp的特异片段。其中T-DNA左侧设计的LS011/LA451检测引物对扩增的灵敏度高、特异性强,能够在甘蔗BtG-2基因组DNA相对含量为0.1%的模板中检测出转基因目的成分,相当于9个单倍体基因组拷贝数。本研究完成了转基因株系BtG-2的分子特征及其转化事件特异性检测,为该转基因甘蔗及其衍生产品的检测和身份识别提供技术依据。

Analysis of the T-DNA Flanking Sequence and Event-specific Detection for Insect-resistant Transgenic Sugarcane BtG-2

Sugarcane BtG-2 is an insect resistance transgenic sugarcane strain, developed by introducing the Cry1Ac- 2A-gna fusion gene into ‘ROC22’ with the Agrobacterium-mediated method. It has strong insect resistance and excellent agronomic traits. In order to clarify the molecular characteristics and detection of transgenic sugarcane BtG-2, and promote biological safety evaluation, the T2 generation of BtG-2 was selected, and the copy number of foreign genes in the transgenic sugarcane genome was detected by Southern hybridization. The flanking sequence of the insertion site of the foreign gene was isolated using the chromosome walking technology, and an efficient specific PCR detection method of the strain was established. The results showed that the foreign T-DNA insertion of BtG-2 strain was a single copy. After three times amplifications of thermal asymmetric interlaced PCR, 984 bp of the left flanking sequence and 705 bp of the right flanking sequence of the foreign gene T-DNA were obtained. According to the flanking sequences, three pairs of detection primers were designed respectively, then the event-specific PCR detection for transgenic sugarcane BtG-2 was established. The primer pairs with the highest amplification efficiency were LS011/LA451 and RS160/RA588, with 440 bp and 428 bp specific amplified fragments respectively. Among them, the pair of primers LS011/LA451 designed on the left side of T-DNA had high sensitivity and specificity for detection, and this method could detect the genetically modified ingredients in samples containing 0.1% genomic DNA of sugarcane BtG-2. This study completed the molecular characteristics and event-specific detection of the transgenic strain BtG-2, which provided a technical basis for the detection and identification of the transgenic sugarcane and its derivatives.