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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。
This paper reports a rapid and effective screening method for transgenic plants from a large number of pollen tube transformation treatments. The transformation plasmids were pBI35S-gafp-NP1-bar, a plant expression vector containing gastrodin antifungal protein (GAFP) and rabbit defensin (NP-1) 158 and Alondra. Pollen tube pathway transformation treatment of seed obtained in three steps for screening identification: First, the transformation treatment of seeds close to the plastic tray until the seedlings grow to 3 leaf stage, with a concentration of 120mg / L Basta herbicide mist spray Screening, most of the seedlings became yellowish after about 10 days, and only about 1% of T0 seedlings with high anti-Basta herbicide survived. Then Basta-resistant plants were detected by PCR with gafp gene primers, of which 62.5% The positive plants were PCR positive. Finally, the progenies of T0 generation PCR-positive plants were germinated and mixed with strains to extract DNA. The PCR products were verified by gafp and bar primers respectively. The results were positive. This indicated that the foreign gene has been integrated into the wheat genome and inherited from the T0 generation to the T1 generation. At the same time, the screening method is proved to be reliable and effective.