3种筛选NPT-Ⅱ标记转基因油菜方法研究

Study on Three Methods Screening Transgenic Rapeseed Tagging NPT-Ⅱ

为获得一种简便的筛选含有NPT-Ⅱ标记基因的转基因油菜种子的方法,对卡那霉素溶液叶片涂抹法、种子浸泡法以及卡那霉素MS培养基筛选3种方法进行研究。结果表明,3种筛选方法的最佳卡那霉素选择压为200mg/L;涂抹叶片法4～5天可以明显识别植株对卡那霉素抗性与否,此法筛选到的4株植株PCR检测全部呈阳性,可靠性达100%;浸泡种子法2～3天可以明显识别植株对卡那霉素抗性与否,此法筛选到的5株植株PCR检测全部呈阳性,可靠性100%;MS培养基筛选法一般10天可以明显识别植株对卡那霉素抗性与否,此法筛选到20株植株,移栽后存活5株,PCR检测3株呈阳性,2株是假阳性植株,可靠性为60%。因此,可以认为卡那霉素涂抹叶片和浸泡种子两种方法是转基因油菜进行大规模、快速的筛选及后代的遗传分析的理想方法。

In order to obtain the simple method of sieving the seeds of transgenic rapeseed tagging NPT-Ⅱ. Using three methods such as Kanamycin daubing vane, dipping seeds and MS culture medium containing Kanamycin to study. The results indicated that the best Kanamycin choice press was 200 mg/L. The method of Kanamycin daubing vane can distinguish to Km- resistance or not for 4-5 days. Four plants were chosen in this method and its PCR detection was positive. The reliability reached 100%. The method of dipping seeds can distinguish to Km- resistance or not for 2-3 days. Five plants were chosen in this method and its PCR was positive. The reliability reached 100%. The method of Kanamycin MS culture medium can distinguish to Km- resistance or not commonly for 2-3 days. Twenty plants were chosen in this method but only five saved in transplanting. The result of PCR detection is that there plants were positive and two were negative. The reliability reached 60%. So, it could be thought Kanamycin daubing vane and dipping seeds were ideal methods for fast screening transgenic rapeseed and descendant＇s heredity analysis.