转基因水稻抗性基因漂移频率检测的抽样技术研究

Research on sampling technique for verifying gene flow frequency of transgenic rice

[目的]本文旨在为转基因水稻抗性基因漂移频率的检测提供科学的抽样技术,包括抽样方法和最佳抽样量,以达到既能真实反映田间实际发生的基因漂移频率,又能节省检测时间和经济成本。[方法]把同种常规水稻的红墨水染色种子与非红墨水染色种子按照混杂比为2.5%、1.25%、0.8%、0.625%、0.5%、0.4%、0.3%、0.2%、0.1%、0.05%、0.025%共11个比例充分混合,通过模拟转基因水稻向花粉受体(非转基因栽培水稻、普通野生稻和杂草稻)抗性基因漂移后形成的杂交种混杂在收获的花粉受体种子中的空间分布型,确定抽样方法;以抽样得到的混杂比与实际混杂比的偏离系数小于0.05为标准,研究抗性基因漂移频率为0.025%~0.5%时的最佳抽样量。[结果]抗性基因漂移后形成的杂交种混杂在收获的花粉受体种子中的空间分布型均符合随机分布;在混杂比为0.025%时,最佳检测量为40000粒种子;在混杂比为0.05%和0.1%时,最佳检测量为30000粒种子;在混杂比为0.2%、0.3%和0.5%时,最佳检测量为20000粒种子。[结论]在检测转基因水稻抗性基因漂移频率时,应采取随机抽样法抽样。根据本研究获得的不同混杂比下的最佳抽样量,建议在检测抗性基因漂移频率时,可先检测20000粒花粉受体种子,若得到的漂移频率大于0.2%,则不需要增加花粉受体种子的检测量;若得到的抗性基因漂移频率小于0.2%,则需再检测10000粒花粉受体种子,这时若得到的抗性基因漂移频率大于0.05%,则不需要增加检测量;若抗性基因漂移频率小于0.05%,还需再检测10000粒花粉受体种子。

[Objectives]This paper aimed to develop a robust,affordable and rapid sampling technique for verifying gene flow frequency from transgenic rice to compatible crop relatives(nontransgenic rice,wild rice and weedy rice)using seed harvested from transgenic rice pollen recipients,including sampling methods and appropriate sample size,in order to achieve a true response to the actual occurrence of gene flow frequency in the field.[Methods]Conventional rice was used as experimental material.Redink dyed rice seed was thoroughly mixed with nondyed rice seed at 11 weight ratios(2.5%,1.25%,0.8%,0.625%,0.5%,0.4%,0.3%,0.2%,0.1%,0.05%,0.025%).Sampling method based on the obtained distribution patterns was adopted.To determine the appropriate sample size,red-ink dyed rice seeds and non-dyed rice seeds were mixed using the same procedure at ratios(2.5%,1.25%,0.8%,0.625%,0.5%,0.4%,0.3%,0.2%,0.1%,0.05%,0.025%).The appropriate sampling size was determined(aiming to obtain a deviation coefficient lower than 0.05).[Results]Hybrid seeds produced by gene flow from transgenic rice to relatives distributed randomly in the sample of pollen receptor seeds at 11 weight ratios.When the gene flow frequency was 0.025%,0.05%and 0.1%,0.2%and 0.5%,sizes corresponding to 40000,30000 and 20000 seeds,respectively,were required for proper detection.[Conclusions]Random sampling methods should be adopted for verifying gene flow frequency of transgenic rice.According to the appropriate sample size under different mixed ratios obtained in this study,it is suggested that 20000 pollen receptor seeds should be verified first.If the gene flow frequency is more than 0.2%,no more pollen receptor seeds need to be verified.However,if the gene flow frequency is less than 0.2%,an additional 10000 pollen receptor seeds should be verified.At this time,if the gene flow frequency is more than 0.05%,no further verification is required.However,if the gene flow frequency is less than 0.05%,another extra 10000 pollen receptor seeds need to be verified.