论文标题:花生花粉管通道法转基因育种的基础研究
Basic Studies on the Peanut Transgenic Breeding with Pollen Tube Pathway Technique
论文作者 李庆云
论文导师 庄东红,论文学位 硕士,论文专业 生物化学与分子生物学
论文单位 汕头大学,点击次数 102,论文页数 88页File Size5153k
2003-04-01论文网 http://www.lw23.com/lunwen_108378907/ 花生;RAPD;花粉管通道法;转基因
Peanut;RAPD;Pollen Tube Pathway;Transgenic
分子育种就是定向、有目的地对农作物品种的遗传进行改良和修饰，更有效地培育植物新品种。在多种抗植物虫害、耐除草剂基因中，豇豆蛋白酶抑制剂基因(cowpea trypsin inhibitor，CpTI)和耐除草剂基因(bar)因其抗虫和耐除草剂的广谱性而成为效果最为理想的外源基因，被广泛地应用于农作物抗虫、耐除草剂基因的改良上。本论文研究了用花粉管通道法将CpTI和bar基因转入花生品种“汕油523”和“汕油21”的实验方法，初步探讨了花粉管通道法转化花生的机理，并分析了花生品种间生化成分的变化及和RAPD聚类的遗传距离的关系，以期为花生分子育种提供依据。 本实验进行了两次RAPD分析，每次选用19个花生品种。结果表明，花生具有丰富的多态性，第一次RAPD分析中的多态性条带达到90.5％，19个花生品种共分为5大类，后代品种和亲代品种基本聚在一类；第二次分析中的多态性条带达到83.5％，19个品种共分为5大类，汕油系列基本聚在一类，后代品种和亲代品种基本也是在一类。两次分析说明聚类的树状图和品种间亲缘关系的一致性。花生品种间的遗传差异比较小，两次分析中品种间的平均遗传距离分别为0.313和0.261。利用RAPD技术的多态性可以为鉴定花生品种提供依据，也可以对花生转基因后代进行初步的检测。本实验根据RAPD分析结果和品种株型特征为汕头市农业科技园鉴定了三个不确定亲缘关系的花生品种：“五菲四”和“粤选58/1”、“粤选58/14”，确定了“粤选58/1”是“五菲四”的亲代品种。 但是在两次RAPD分析中，有4个品种重复，两次聚类矩阵中，相同品种之间的遗传距离差别较大，如“湛油30”和“NCAC17090”先后两次的距离分别是0.3617和0.2561，“汕油523”和“狮头企”分别是0.2319和0.1847；两次聚类的树状图中，这4个品种的归类也不相同。这说明在本实验中RAPD分析的重复性较差。 第一次聚类的19个花生品种中，“金花1012”和距离最远的“天津豆”在蛋白质、粗脂肪和主要脂肪酸的含量之间有显著性差异，但是和距离最近的“粤油256”之间只有粗脂肪的含量接近；第二次聚类的19个花生品种中“汕油27”和距离最远的“NCAC17090”在蛋白质、油酸之间差异汕头大学硕士论文不显著，和距离最近的“狮油15”之间只有粗脂肪接近。所以RAPD聚类结果中，花生品种间遗传距离的远近和生化成份的差异性没有完全一致的关系。 为了提高花粉管通道法的转化效率，探索分析了“汕油523”和“汕油21”两个品种的开花规律。两个品种的盛花期平均都是8一10d，盛花期平均每天每株开花3朵以上。春季开花数量波动较大，秋季开花数量波动较小。秋季开花数量比春季明显增多，而且同一个季节，“汕油 523”比“汕油21”的单株开花数要多。由此可见，秋季利用“汕油523”进行花粉管通道法转化操作比较合适。 用改良的注射法和涂抹法分别在3个不同季节进行了花粉管通道法转基因操作，共处理了3998朵花，得到了861株D。代植株。对这些植株进行PCR检测，结果共有4株呈阳性反应，品种上“汕油523”和“汕油21”各两株，方法上注射法和涂抹法各两株;三次转基因操作的转化率分别是0.49%、0.42%和 0.48%。DI代一共得到35株植株，PCR检测结果，只有1株D。代转基因植株在D;代中检测到2株有目的基因片断的遗传。 本实验结果还表明，转基因操作对花生单株果针数和结果数的影响较大，都比对照明显减少:座果率和对照相比较，没有明显的变化。可见花粉管通道法操作对果针的发生和种子形成的影响比较一致。单株结果数和座果率的统计中，方差总体上都比对照要大，说明花粉管通道法操作对花生性状的整齐性影响较大。
Molecular breeding is to breed new plant variety via improving or modifying genetic character of crop. The cowpea trypsin inhibitor (CpTI) gene and the bar gene discovered in recent years are found to be used for insect-resistant and anti -herbicide respectively because of their broad spectrum and are used in gene improvement of many crops widely now. Using the peanut (Arachis hypogaea L. cv. Shanyou 523 and Arachis hypogaea L. cv. Shanyou 21) in this report, method of transforming the CpTI and bar gene to peanut with pollen tube pathway technique was studied, the mechanism of the technique in the peanut was discussed; biochemic ingredient changing among the peanut cultivars was analyzed, and genetic distance was studied with RAPD analysis, we hope that these study will be useful to crop breeding.In the two analyses different 19 peanut cultivars were analyzed by RAPD technique respectively. The result shows there are plenty of polymorphism among the peanut cultivars. In the first analysis, rate of polymorphic fragments is 90.5%, 19 peanut cultivars is classified in 5 categories, the parent and filial generation cultivars is classified in the same categories approximately; In the second analysis, rate of polymorphic fragments is 83.5%, 19 peanut cultivars is classified in 5 categories approximately, the series of cv. Shanyou are classified in a category, the parent and filial generation cultivars is classified in the same category approximately too. The two analyses show the UPGMA tree is accord with the relationship of the cultivars. The differences among the peanut cultivars are small. The mean genetic distance of the two analyses is 0.313 and 0.261 respectively. It may provide the identification of the peanut cultivars or transgenic plant with proof via RAPD polymorphism. In this report we identified three questionable peanut cultivars via RAPD analysis, the result shows that the cv.Yuexuan58/l is parent of the cv.Wufeisi.But in two analyses, there is 4 repeat cultivars. In the two RAPD distancematrix, between the same pairs the difference of distance is significant, for example, the distance between the pair of the cv.Zhanyou 30 and cv.NCAC 17090 is 0.3617 and 0.2561 respectively, between cv.Shanyou 523 and cv. Shitouqi is 0.2319 and 0.1847 respectively; in the two RAPD trees ,the four cultivars lies in different categories. It shows that repeat of the RAPD analysis is poor.Biochemic ingredient data and morphological data of the cv.Zhanyou 30 and its parents cv.Shanyou 523 and cv.Yueyou 223 was analyzed, the mean value difference was tested by Ttest. The result shows that protein, raw fat and morphological data between the cv.Zhanyou 30 and its one of two parents always is different significantly except the fatty acid data. These indicate that biochemic ingredient and morphological character can be inherited steadily via crossbreeding.Comparing the result of the first RAPD analysis with the result of the biochemic analysis ,the difference of the pair(cv.Jinhual012 and cv.Tianjindou) which distance is longest is significant in the protein ,raw fat and the main fatty acid ,but only in the raw fat the difference between the cv.Jinhua!012 and cv.Yueyou 256(distance between the pair is very close) is not significant; In the second RAPD analysis, the difference of the pair(cv.Shanyou27 and cv. NCAC 17090)which distance is longest is not significant in the protein and oleic acid, only in the raw fat the difference between the cv.Shanyou27 and ev.Shiyou!5 (distance between the pair is very close) is not significant .From all above ,we thought that the long distance is not consonant with the biochemic ingredient difference.In order to increase the translation efficiency of the pollen tube pathway technique, comparing the shanyou523 with shanyou21 in the blossom rule. The result shows that the two cultivars" full-blossom stage is about 8-10 days respectively, in this stage every peanut blossom out more than 3 flowers. In the spring, the flower number per peanut plant is fluctuant widely; but in the
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