摘要
为研究不同转基因作物T-DNA插入位点侧翼序列分离中所用的通用简并引物对TAIL-PCR的影响,本研究使用5个通用简并引物分别对8个转基因大豆、30个转基因水稻和2个转基因油菜株系进行了TAIL-PCR分析,结果表明,5个简并引物在TAIL-PCR中的扩增效果存在显著差异,同一简并引物在不同转基因物种的扩增效果显著不同。AD1引物在转基因水稻和转基因油菜中扩增效果较好,AD2引物在转基因水稻、油菜和转基因大豆中扩增效果均较好,AD3引物在转基因水稻中扩增出1个株系,在转基因油菜中扩增出1个株系,但AD3引物与pFGC5941 T-DNA RB上游有匹配,不适用于以pFGC5941为载体的转基因材料分析。AD4和AD5 2个引物在3个转基因物种的扩增均较差。对AD1和AD2两个引物的TAIL-PCR扩增产物进行测序,结果表明,扩增产物均对应转基因材料的侧翼序列。研究表明,不同通用引物在不同转基因事件中侧翼序列的分离效果差异较大,通用引物AD1和AD2TAIL-PCR成功率最高。不同通用引物的应用效果不仅与不同物种有关,也与外源基因的插入位点有关,而且还与载体序列匹配程度相关。为提高TAIL-PCR中T-DNA插入位点侧翼序列分离成功率,建议同时采用多个通用引物平行扩增。
To study the effect of arbitrary degenerate primers of TAIL-PCR on the flanking sequence isolation, eight independent transgenic soybean lines, 30 independent transgenic rice lines and two independent transgenic rapeseed lines were selected for TAIL PCR analysis with the 5 broadly used arbitrary degenerate primers AD1 to AD5. The results indicated that different amplification effects existed among the 5 degenerate primers in TAIL-PCR. Even in using the same degenerate primer, amplification effects were significantly different for different crops. AD1 amplified better in transgenic rice and rapeseed than what it did in transgenic soybean. AD2 generated good amplification effects in all the three crops. AD3 worked well only in one line of transgenic rice, and one line of transgenic rapeseed. However, the AD3 primer has one anchor point in upstream of pFGC5941 T-DNA RB, so it is not suitable for TAIL PCR analysis of pFGC5941 transformed crops. AD4 and AD5 arbitrary degenerate primers didn＇t generate ideal amplification result in all the three crops. The TAIL-PCR＇ s products of AD1 and AD2 were sequenced and analyzed by the software DNAMAN. The results indicated that all the products contained the flanking sequences of the transgenic material. This research discovered that different arbitrary primers are suitable for different crops. AD1 and AD2 works better than other primers in all the three crops. The amplification effects depend on the species analyzed, the flanking sequences of T- DNA inserted, and the primer matching points on the vectors. In order to successfully isolate the T-DNA flanking sequences, we suggest multiple arbitrary primers be used in parallel amplification.
出处
《核农学报》
CAS
CSCD
北大核心
2013年第7期922-928,共7页
Journal of Nuclear Agricultural Sciences
基金
浙江省重大项目(2008C12074)
浙江省农业科学院中科院合作项目(2011R19Y03E01
2012R19Y01E01)
