小麦花粉管途径转化及高效筛选体系的建立

Wheat Transformation by Pollen-Tube Pathway and High Effective Screening System

本文报道了一种从大量花粉管途径转化处理后代中快速有效地筛选到转基因植株的方法。转化质粒为含bar筛选标记基因的天麻抗真菌蛋白(GAFP)与兔防御素(NP-1)双价抗病基因植物表达载体(pBI35S-gafp-NP1-bar),受体小麦品种为扬麦158和Alondra。对花粉管途径转化处理获得的种子分3步进行筛选鉴定:首先,转化处理种子密播于塑料盘中,待小苗长到3叶期时,用浓度为120mg/L的Basta除草剂弥雾喷施筛选,约10d后绝大部分小苗变黄枯死,只有约1%的高抗Basta除草剂的T0小苗存活;然后对其Basta抗性植株用gafp基因引物进行PCR检测,其中62.5%的Basta抗性植株为PCR阳性;最后将T0代PCR阳性植株后代种子(T1)分别发芽并按株系混合取样提取DNA,分别用gafp和bar基因引物进行PCR检测验证,结果均为阳性。表明外源基因已整合入小麦基因组并由T0代植株稳定遗传到T1代,同时证明该筛选方法是可靠有效的。

A screening method can obtain quickly and effectively transgenic wheat plants from a great deal of progenies by pollen-tube pathway transformation is reported in this experiment. The plasmid vector pBI35S-gafp -NP1-bar containing selective marker gene bar?disease- resistance genes GAFP (Gastrodia Antifugal protein) and NP-1(Rabbit Defensin) genes, and the acceptors is wheat varieties Alondra and Yangmai158. The screening to the transformation seeds is separated as 3 steps: at first, sowing densely T0 seeds in a plastic box, and when the seedlings are in 3-leaves stage, spraying 120mg/L Basta solution on these seedlings to screen them. After 10 days, the most of them turn yellow and died, and only 1% Basta-resistance seedlings survive. Then, the PCR analysis with gafp primer to the T0-plants shows that 62.5% Basta-resistance plants are positive. Finally, germinating T1-seeds derive from PCR positive T0-plants, we take mixture samples according T0-lines and extract their DNA. PCR analysis is performed with gafp and bar genes primers again, and the PCR result is still positive. It confirms that the gafp and bar genes have integrated into the wheat, and inherited stability from T0-plants to T1-plants,and shows that the screening method is effective and reliable.