论文标题:大豆利用花粉管通道技术导入抗病虫目的基因的研究
The Study on the Technique of Pollen Tube Path Way Used to Introduce Insect-resistant and Disease-resistant Gene into Soybeans
论文作者 崔岩
论文导师 杨庆凯,论文学位 硕士,论文专业 作物遗传育种
论文单位 东北农业大学,点击次数 109,论文页数 35页File Size1875k
2000-05-01论文网 http://www.lw23.com/lunwen_993630052/ 花粉管通道技术,目的基因,转基因,植物基因工程
Pollen tube path way,Target-gene,Transgene,Genetic,engineering of plant
本试验对利用花粉管通道技术将抗虫、抗病目的基因导入大豆的适宜时期、成活率、转化率进行了研究，并运用特异PCR技术从分子水平上对转基因D1代、D2代进行了检测，验证了此项技术在大豆上应用的可行性。研究结果表明：1、用液滴法将几丁质酶基因、Bt基因导入27个大豆品种，导入花朵数为1667朵，收获荚数为712个，平均成活率为42.7％。2、将转基因D1代27个品种中的15个品种第二年种下，总粒数为1060粒，成株数为676株，平均成株率为63.8％。3、不同的基因型大豆之间、不同的导入基因之间的成活率和成株率没有明显差异，其影响主要因子是操作技术本身的问题。4、用x—Gluc溶液检测转Bt基因D1代植株。检测了132株，没有发现阳性反应。5、对转入Bt基因的132株植株的幼叶提DNA，作PCR检测。D1代获得5株阳性转化植株，分别是90035的4株，85—593的1株，转化率为3.8％。6、将D1代获得5株阳性转化植株的种子在温箱中发芽，提取DNA检测，得到D2代稳定遗传的阳性转化植株2株。7、应用荧光镜制片法观察大豆自花授粉后花粉管在花柱中延伸情况。结果表明：1）根据大豆自花授粉后花蕾形态特征来选择花粉管通道法转基因用的花具有可行性。2）用花粉管通道法向大豆体内转基因，转化率低的原因之一是：与其他用花粉管通道法转基因成功的植物（如棉花、黄瓜、番茄等）相比，大豆子房较小，花粉管非常细，DNA溶液很难渗入。 本试验研究结果表明，花粉管通道法在大豆上应用是完全可行的，从而为创造新的大豆抗病虫种质资源提供了一条切实可行的基因转化途径。
In order to study the optimum introducing period, survival rate and inversion rate, thetechnique of pollen tube path way was used to introduce insect-resistant and disease-resistant gene into soybeans. PCR was also done to test the Dland D2 generations of transgenic plants. All these have proved that the technique of pollen tube path is feasible to study soybean.The results showed that:1. The way of drop of liquid was used to introduce chitinase and Bt gene into 27 soybean varieties. The totaliniroduced flower counts was 1667 and the pods obtained was 712. The mean value of survival rate was 42.7%.2.ln the following year, 15 transgenic varieties among 27 of Dl generation were planted. The total number of seeds was 1060 and the number of maturity plants was 676. The mean rate of maturity plants was 63.8%.3 .The suviral rate and matarity rate of different genes soybeans and different transmiting genes have no obvious differnce. The main effect factor exist in the operation technique.4. X-Gluc was used to test the plants of Dl generation of Bt-transgenic plants. 132 were done and no positive reaction was found.5. The DNA of the young leaves of 132 Bt transgenic plants was extracted to do PCR Five positive inversion plants were obtained and the inversion rate was 3.8%.6. The DNA of fire positive inversion plants obtained in Dl generation was extracted and tested. Two stable positive inversion plants were obtained in D2 generations.7 The way of flourescent mirror flat was used to observe the pollen tube extension circumstance in the flower post after self-pollination. 1) According to flower bud, we can choose the optimum ones to transfer gene by pollen tube path way 2) When the tube is used in genetic lrunsformation, the reason of the low inversion rate is that soybeans seed capsule is smell and the pouen is slim,comparing with other plants (such as cotton,cucumber and tomato etc),so the DNAis difficult to permeate into. The experiment shows that pollen-tube pathway method is fully feasible in soybeans plants,and it provides a practical and feasible pathway of genetic transformation to create varieties of new soybeans insect-resistantand disease resistan. Postgraduate: Cui Yan Supervisor: Yang QingkaiMajor: Crop genetic and breeding
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