论文标题:农杆菌介导MSI-99基因转化马铃薯及其植株再生研究
Agrobacterium-mediated Transformation of Potato (Solanum Tuberosum) with MSI-99 Gene and Plant Regeneration
论文作者 赵文锋
论文导师 杨清,论文学位 硕士,论文专业 生物化学与分子生物学
论文单位 南京农业大学,点击次数 146,论文页数 71页File Size9212k
2004-05-01论文网 http://www.lw23.com/lunwen_31030692/ 马铃薯;根癌农杆菌;MSI-99;再生体系;转化
potato(Solanum tuberosum); Agrobacterium tumefaciens; MSI-99;regeneration system; transformation
马铃薯(Solanum tuberosum)是粮菜兼用型作物，栽培分布广泛，在世界上，其产量仅次于小麦、玉米、水稻而位居第四。但在生产上，各种病害严重，常常造成巨大的经济损失。因此，培育广谱抗病新品种是育种学家的首选目标。MSI-99为一广谱抗菌肽，对多种植物病原细菌、真菌及病毒均有强烈抑制作用。将MSI-99基因导入马铃薯栽培种，可以改良品种的抗病性。试验从筛选马铃薯高效再生体系入手，并在此基础上，以农杆菌为媒介将抗微生肽基因MSI-99导入马铃薯栽培种。 1．马铃薯高效再生体系的筛选 以Superior、Shepody和Favorita三种基因型的叶片、茎段和块茎为材料，通过对7种再生体系培养基配方进行比较，筛选出三种基因型材料再生的最佳培养基配方：①适于叶片和茎段再生的培养基：MS基本培养基+2.25mg／L6-BA+0.18mg／LNAA(愈伤组织诱导培养基)，MS基本培养基+2.25mg／L6-BA+5.0mg／L GA3(芽分化培养基)；②适于块茎片再生的培养基配方为：MS基本培养基+0.9mg／L IAA+1.8mg／L ZT(愈伤组织诱导与芽分化培养基)。 2．农杆菌介导MSI-99基因转化马铃薯 用电转化仪将携有目的基因的双元载体质粒pBIN-MSI99导入农杆菌LBA4404菌株，再用其分别浸染Superior、Shepody和Favorita三个马铃薯品种的叶片、茎段和块茎片外植体，诱导再生后，获得抗Kan的再生苗36株，其中有2株不能生根。经PCR检测有13株呈阳性，Southern杂交有7株有杂交信号，表明外源基因已经整合到再生苗基因组。移入盆栽后，转基因植株比对照植株叶片小，颜色深，叶数多；转基因小薯外形也有变异，表现为细长和椭圆两种。
Potato is a world-cultured crop and ranked 4th in tube production following wheat, maize and rice. It can be used as food and vegetable. However, in potato culture, diseases often cause huge loses. Creating a new broad-spectrum resistant variety has become the goal of potato breeders. MSI-99, a analog of antimicrobial peptide magainin II, is a broad-spectrum antibacterial peptide. It can effectively inhibit the growth of many species of bacteria, fungi and viruses which infect plants. The introduction of MSI-99 gene into potato cultivars could improve the resistance of the cultivars to diseases. This study includes the screening of high effective regeneration systems and the transformation of potato with MSI-99 gene by Agrobacterium.1. Screening of high effective regeneration systemsThe leaf discs, stem segments and tuber pieces of 3 potato cultivars, Superior, Shepody and Favorita, were used as the experimental materials. The high effective regeneration systems were obtained by the comparison of 7 different regeneration systems:(1)The system used for leaf discs and stem segments culture: MS base medium complemented with 2.25mg /L 6-BA + 0.18mg /L NAA (for callus induction); MS base medium complements with 2.25mg /L 6-BA+5.0mg /L GA3 (for shoots generation);(2)The system used for tuber pieces culture: MS base medium complements with 0.9mg /LIAA + 1.8mg /L ZT (for callus and shoots induction)2. Transfer of gene MSI-99 to potatoes mediated by AgrobacteriumThe binary vector pBIN-MSI99 carrying MSI-99 gene was transferred to Agrobacterium LBA4404 by electroporation apparatus. Then the Agrobacterium LRA4404/pBIN-MSI99 was cocultured with the leaf discs, stem segments and tuber pieces cut from Superior, Shepody and Favorita respectively. The callus and shoots were induced on the regeneration system and 36 adventitious shoots were gained. In these adventitious shoots, 2 shoots couldn"t root. PCR analysis gave 13 positive regeneration plants and Southern blot analysisconfirmed the MSI-99 gene has been integrated into the genome of 7 regenerated plants. After being transplanted to flowerpot, the transgenic plants have smaller and more leaves than the control and the color of the leaves is darker also. The shape of oval transgenic microtubers revealed thinner and longer than the control.
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