论文标题:用花粉管通道法将豌豆DNA导入小麦及其后代的分子验证
Molecular Test of Variant Wheat after Injected Pea DNA by Pollen Tube Pathway
论文作者 张立
论文导师 王宪泽,论文学位 硕士,论文专业 生物化学与分子生物学
论文单位 山东农业大学,点击次数 116,论文页数 43页File Size2599k
2002-06-01论文网 http://www.lw23.com/lunwen_801545272/ 小麦;豌豆DNA;花粉管通道法;过氧化物酶;RAPD;蛋白质含量
Wheat quality,Pollen tube pathway,Exogenous DNA,Variation,Gliadin,Peroxidase,RAPD,Electrophoresis,Protein content
在我国小麦产量已处于较高水平的前提下，改善和提高小麦品质就成为育种工作者一项迫切的任务。本研究通过花粉管通道法将豌豆基因组DNA导入高产低蛋白小麦品种鲁麦22号，以期提高其蛋白质含量，并探讨该方法在改良小麦品质方面的作用。以筛选的变异体T220X为材料，对其醇溶蛋白、同工酶表达和DNA水平上的差异以及蛋白质含量的变化等方面进行了研究，获得了如下结果： 1．种子醇溶蛋白电泳结果显示，变异体T220X酶带与受体鲁麦22号相比，既有新的酶带增加，也有酶带缺失的情况。说明外源DNA的导入影响到醇溶蛋白组分的变化，根据醇溶蛋白基因定位，初步认定外源DNA影响到小麦的第一或者第六号染色体。 2．小麦幼苗期过氧化物酶活性的测定显示，变异体T220X的酶活性高于受体鲁22；过氧化物酶电泳显示，T220X和鲁22过氧化物酶在酶带深浅和数目方面都存在差异，说明外源遗传物质已影响到过氧化物酶的变化。 3．RAPD分析结果显示，变异体T220X和受体鲁22在DNA水平上存在较大差异。经引物S103扩增，在变异体T2201中出现850bp的特异片段；引物S74在变异体T2202中扩增出1500bp和400bp的两条新带；引物S64在变异体T2203中扩增出两条新带，大小分别为980bp和550bp；引物S118在变异体T2204扩增出一条480bp的新带。这可能是外源DNA片段整合进受体的基因组中，改变基因的顺序或者引起基因碱基的缺失、插入，从而在基因水平上发生突变。 4．蛋白质含量分析结果显示，变异体T220X的蛋白质含量较之受体鲁22有较大的提高，平均提高6.4个百分点。这证明利用花粉管通道法提高蛋白质含量是行之有效的。 本实验结果表明：利用花粉管通道法将外源DNA导入小麦中获得目的性状的植株，从而改良小麦品质，在实践上是可行的，在生产中具有极大的潜力。
Under the conditions of high wheat yield in china, it is a insistent task for breeders to improve wheat quality. In the experiment pea genomic DNA were intrAuced into wheat Lu22 through the pollen tube pathway in order to enhance wheat protein content and improve wheat quality. Then the variant wheat T220X were studied from gliadin, isoperoxidase, the difference in DNA and protein content. The main results were as follows.1. The results of seed gliadin electrophoresis showed that there were difference in zymotype between Lu22 and T220X, which verified that the injection of exogenous DNA influenced the component of gliadin. It was affirmed preliminarily that exogenous DNA had made an effect on the wheat 1 Chr. or6Chr.2. The peroxidase activity of variant T220X was obviously higher than acceptor Lu22 at seedling stage. The result of peroxidase electrophoresis indicated that there were difference both in the depth and in the amount of zymogram between Lu22 and T220X.. So it was concluded that the variety of peroxidase was the result of change of heriditary substance.3. The result of RAPD analysis showed there were many differences in DNA between variant T220X and acceptor Lu22. After the amplication of primer S103, there was a specific fragment about 850bp in T2201; after the amplication of primer S74, there were two new fragment about ISOObp and 400bp in variant T2202. The amplified prAuct of SI 18 had a 480bp new fragment in variant T2204. In variant T2203 there were two specific fragmentsabout 980bp and 550bp after ampliation of primer S64. It is possible that exogenous DNA fragment integerated into acceptor genome, changing gene base sequence or base deletion or base insertion, inducing to mutant at gene level.4. The detecting of protein content indicated that there was a obviously enhandcement of protein content in variant T220X than in acceptor Lu22. The result affirmed that it is available to improve protein content through pollen tube pathway.These results indicated that it is practicable inducing exogenous DNA into wheat through pollen tube pathway to obtain taget plant. There are great potential power to improve wheat quality by this way.
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