大豆花粉管通道技术转化雪花莲凝集素(GNA)基因

采用花粉管通道技术，用雪花莲凝集素基因（Galanthus nivalis agglutinin，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Western blotting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99JI45 TGR2的Southern blotting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。

Transforming the Snowdrop Lectin (Galanthus nivalis agglutinin, GNA) Gene to Soybean by Pollen Tube Pathway Technique

Snowdrop lectin (Galanthus nivalis agglutinin, GNA) gene was introduced into 3 soybean cultivars (Jilin20, Jilin30 and Jilin45) via pollen tube pathway technique, which were widely grown in Jilin province. The transformed plants were selected out by the analysis of resistance to aphids and PCR. The progeny of some transgenic plants were identified by molecular biological methods. (1) PCR results revealed that T2 generation of the transgenic plants 97TGR1 and 98TGR2 was positive and T5 generation of those plants was positive and homozygous. Western blot analysis showed that GNA gene had expressed in T3 generation of 97TGR1, 98TGR2 and 98FD1~ 98FD20 on protein level and the highest expression level was over 0.7% of total soluble protein. Southern blot analysis showed that GNA gene had integrated into the soybean genome of 97TGR1, 98TGR2 and 99JI45TGR3. (2) Genetics analysis showed that the inheritance of GNA gene in T2 generation of 97TRG1 complied with Mendel segregation ratio of 3:1, while in T3 generation of 97TGR2 displayed an irregular segregation in the color of seed capsule. The purely resistant lines were obtained by field appraisal of resistance to aphids and continuous selection. (3) Bioassay results showed that in earlier generations of transgenic plants the propagation quantity of aphids could be reduced as much as 50%~90%. (4) The resistance to aphids of the transgenic lines was up to R and HR level by agronomy standard. The investigation of the large-scale natural occurrence of aphids of the pure lines approved to be released to the environment showed that the damage peak of aphids for the transgenic lines was delayed, the quantity of aphid declined very quickly after the peak and the holistic damage was lightened. The results indicated that the pollen tube pathway technique is an effective, feasible method for soybean transgene. The results indicated that as an extraneous gene, GNA gene had an obvious resistance to aphids and could be applied in soybean breeding.