论文部分内容阅读
采用花粉管通道技术,用雪花莲凝集素基因(Galanthusnivalisagglutinin,GNA)转化吉林省主推品种吉林20号、吉林30号、吉林45号品种大豆。通过接蚜鉴定和PCR鉴定,从所获得的种子苗中筛选出转基因植株。对转基因植株的后代进行分子生物学鉴定:(1)PCR分析,转基因植株97TGR1和97TGR2的T2代表现阳性,第5代表现阳性纯合;97TGR1、97TGR2和98FD1￣98FD20的T3代Westernblotting检测结果证明,GNA基因在蛋白质水平有表达,最高表达量占总可溶性蛋白的0.7%;97TGR1、98TGR2和99JI45TGR2的Southernblotting检测结果显示,GNA基因已插入大豆基因组;(2)遗传学分析,97TRG1的T2代呈孟德尔3:1分离,97TGR2的T3代出现种皮颜色不规则分离。经过抗蚜性鉴定和连续的筛选,获得抗性纯系;(3)抗蚜性鉴定,转基因株的T1、T2世代转基因植株可抑制蚜虫繁殖量50%￣90%;(4)品系鉴定,转基因大豆的抗蚜性达到农学标准抗(R)和高抗(HR)水平;大面积环境释放试验自然感蚜鉴定,转基因系蚜虫发生的高峰比对照延迟,高峰期过后群体蚜量的下降速度也比对照快。本研究认为,大豆花粉管通道技术可以利用于大豆的转基因研究和应用中,GNA基因在改良大豆的抗蚜性上是可取的。
Pollen tube pathway technology was used to transform the main varieties Jilin Jilin 20, Jilin 30 and Jilin 45 soybean with Galanthus ningisagglutinin (GNA). Aphids identification and PCR identification, obtained from the seedlings screened transgenic plants. The progeny of the transgenic plants were identified by molecular biology: (1) PCR analysis showed that the T2 plants of 97TGR1 and 97TGR2 transgenic plants were positive and the 5th generation plants were positive homozygous; the Western blotting results of T3 generation of 97TGR1, 97TGR2 and 98FD1 ~ 98FD20 proved , GNA gene was expressed at the protein level, and the highest expression level accounted for 0.7% of the total soluble protein. Southern blotting analysis of 97TGR1, 98TGR2 and 99JI45TGR2 showed that GNA gene was inserted into soybean genome. (2) Genetic analysis showed 97TG1 T2 generation was Mendel 3: 1 separation, 97TGR2 the T3 generation of seed coat irregular color separation. (3) Identification of resistance to aphids, T1, T2 generation transgenic plants can inhibit aphid reproduction 50% ~ 90%; (4) Identification of lines, The resistance to aphids of transgenic soybean reached the agronomic standard resistance (R) and high resistance (HR) level. The large-area environmental release test was used to identify the natural aphids. The peak of the aphids in the transgenic lines was delayed compared with the control. The decline of population aphids after the peak period Also faster than the control. This study suggests that the soybean pollen tube pathway technology can be used in transgenic research and application of soybean, GNA gene is desirable to improve aphid resistance of soybean.