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论文题名(中文):

 农杆菌介导PVX cp基因转化马铃薯获无标记转基因植株的研究

作者:

 白冬梅

论文语种:

 chi

学科名称:

 作物遗传育种

学位:

 农学硕士

学校:

 山西农业大学

院系:

 农学院

专业:

 作物遗传育种学

研究方向:

 细胞工程和染色体工程

第一导师姓名:

 黄晋玲

论文完成日期:

 2006-06-01

论文题名(外文):

 Research on Pvxcp Gene into Potato to Obtain Marker-free Transgenic Plants by Agrobacterium-mediated

关键词(中文):

 马铃薯 pvxcp基因 转基因 无标记 农杆菌

关键词(外文):

 potato pvxcp gene transgene have no mark farming bacillus LBA4404

论文文摘(中文):

马铃薯(solanum tuberosum L.)是世界上最重要的作物之一。马铃薯粮菜兼用,营养丰富,生育期短,单位面积产量高,适应性强,生产潜力巨大,是解决粮食问题的理想作物;也是纺织、医疗造纸等工业生产的重要原料,因此它的需求和种植面积将进一步扩大。但病害常给马铃薯生产造成巨大损失,马铃薯X病毒(potato viros x,pvx)是马铃薯上分布最广泛的病毒之一 ,培育抗病毒品种是一条经济有效的途径。目前已知有多种马铃薯抗病毒育种途径,而转cp基因途径被认为是最有应用潜力的途径之一。

本试验克隆了pvxcp基因,通过特异重组酶转化系统构建了无选择标记pvxcp基因的表达载体pFMpvxcpIR。采用冻融法将pFMpvxcpIR载体导入农杆菌LBA4404,然后用农杆菌介导两个基因型不同的品种东北白和晋薯7号,获得无标记转基因植株。试验转化过程中,对外植体的选择、农杆菌侵染浓度、预培养时间、共培养时间等进行了系统的研究,结果如下:

1)本实验室克隆的pvxcp基因与其它株系具有较高的序列同源性;(2构建了pvxcp基因的无选择标记基因的植物表达载体pFMpvxcpIR3)马铃薯不同外植体的愈伤诱导及分化能力差异较大,东北白茎段和叶片分化效果较好,可做外植体;而晋薯7号的茎段愈伤诱导能力很差,几乎不能成活,不能做为外植体;但其叶片愈伤诱导及分化效果好;(4)试验确定了农杆菌OD6000.3-0.5、预培养2d、侵染10min、共培养4d的最佳转化条件;(5)对东北白所得的560个再生芽植株进行PCR检测, 174株扩增得到了与阳性对照一致的目的条带,初步确定pvxcp基因已经整合到该马铃薯受体基因组中,转化率约31%;晋薯7号所得的320个再生芽植株, 60扩增得到了与阳性对照一致的目的条带,初步确定pvxcp基因已经整合到该马铃薯受体基因组中,转化率18.8%;(6)部分转基因植株 dot blot 杂交有杂交信号产生,表明外源基因已经整合到再生植株苗基因组中。

    本试验探索了一条高效、安全的马铃薯抗病毒转基因育种途径,为培育高效表达并稳定遗传的马铃薯抗病毒品种奠定了基础。

文摘(外文):

Potato is one of the most important crops in the world whose demand and planting area will be enlarged further. But diseases frequently cause huge loss to potato production. Potato X virus (potato virus x, pvx) is one of the viruses that spread most extensively on potato. It is an effective and economic method to cultivate virus-resistant breed. Nowadays, there are various methods to resist potato viruses. The method of transgene of cp is considered as one of the most potential ways. In this research pvxcp gene was cloned. Mark-free pvxcp gene expression vector pFMPVXcpIR was constructed through Site Specific Recombination. PFMPVXcpIR was transferred into Agrobacterium LBA4404 through freezing and melting. Then the mark-free transgenic plants were obtained by Agrobacterium-mediated into two different species: Northeast White and Jin No.7. In this test the option of explants, the bacterium density, the time of pre-culture and co-culture were researched systematically. The results showed:

(1) The cloned pvxcp gene in this test showed high homologous with other breed. (2)Mark-free PVXcp gene expression vector pFMPVXcpIR was constructed through Site Specific Recombination. (3) The induction and differentiation of injury were different among various explants. The Northeast White’s stem and leaf disc could be used as explants. Whereas Jin No.7 was not suitable for explants because of its low efficiency of stem induction. (4) This experiment determined that the best transformation condition could be gained by training Agrobacterium 2 days beforehand in OD600 0.3-0.5, transfer training for 10 minutes and culturing for 4 days. (5) 174 plants of 560 regenerative buds of Northeast White got the consistent strip with the positive plants detected by PCR. Therefore it was preliminarily confirmed that pvxcp gene had been transformed into potato accepted body genome and the rate of transfer was 31%; 60plants of 320regenerative buds of Jin 17 got the consistent strip with the positive plants detected by PCR and it was preliminarily confirmed that pvxcp gene had been transformed into potato accepted body genome and the rate of transfer was 18.8%.(6) The transgenic plants were detected by dot blot and definited pvxcp gene which had been transformed into potato accepted body genome.

This test explored a high efficient and safe transgenic method to resist potato viruses, which would pave the way for the breeding of virus-resistant potato species that have high efficient expression and stable heredity.

开放日期:

 2007-06-01

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