【作者】 周会；

【导师】 黄玉碧；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 品种真实性和纯度是种子质量的重要指标,对作物产量及品质具有直接的影响。因品种真实性和纯度问题造成大幅减产的事件时有发生,严重影响了农业生产。转基因作物是应用生物技术对农作物基因组进行改造获得的。虽然各国均对转基因农作物商业化种植持有相对谨慎的态度,但转基因作物的种植面积仍然快速增加和推广的转基因品系不断增多。随着对转基因产品进行风险评估及相关法规相继出台,转基因成分的检测和鉴定显得越来越重要。我国目前种子经营主体数量多,市场监管技术和手段落后,假冒伪劣种子禁而不绝,套牌侵权问题突出,种子市场秩序比较混乱。《国务院关于加快推进现代农作物种业发展的意见》明确提出,建立手段先进、监管有力的种子管理体系,严厉打击套牌侵权、生产经营假劣种子行为,切实维护公平竞争的市场秩序。分子检测技术为解决这一问题提供了有效的途径,可以破解管理技术瓶颈。因此,加快分子检测技术的研发和应用,是当前种子质量管理工作的一项重要而迫切的任务。许多研究表明,利用SSR分子标记技术鉴定品种真实性和纯度的方法是可行的,检测结果是准确的。但大多数都是从理论的角度进行研究,本实验结合种子监管工作实际,对实际应用中存在的问题进行研究和验证。主要结果如下：1.在品种纯度检测方面,对市场上主推的23个杂交水稻品种筛选出了特定的引物,并通过海南异地田间小区种植鉴定和当地当季田间小区种植鉴定对这些引物的SSR分子标记鉴定结果进行验证。用所筛选的引物对包含这23个品种的226份样品,代表种子数量近400万千克的“两系”杂交水稻种子进行SSR分子标记检测。检测结果显示：SSR分子标记鉴定与田间正季纯度鉴定结果基本一致,最大相差11.2%,其平均差距为0.9%;而海南异地小区种植纯度鉴定结果与田间正季纯度鉴定结果差异较大,最大相差44.0%,其平均差距为3.6%。另外,我们选取7个有代表性样品分单株同时进行SSR分子标记和田间正季鉴定,结果显示每个单株用这两种方法鉴定的结果吻合度达99%。实验结果表明,筛选的SSR分子标记引物能准确、快速地鉴定“两系”杂交水稻纯度,且用一对具有双亲互补带型的引物即可分辨出不育株。另外,第一次提出SSR分子标记鉴定“两系”杂交水稻品种纯度结果比海南异地田间小区种植鉴定结果准确可靠的观点。检测中,通过利用双亲作为对照,准确的将杂株分为母本型、父本型及其他杂株类型,利于分析杂株的来源,很好的解决了在分子检测中难以分辨杂株类型的问题。2.在品种真实性鉴定方面,检测的样品纯度可能较低,样品中的杂株可能对电泳分析结果产生影响。在对全国农作物种子检验机构能力验证4份样品的SSR品种真实性鉴定中,通过分单株进行DNA提取检测,发现有27%的单株与对照在24个位点中未检测出差异,有73%的单株与对照在24个位点中检测出差异。用混合样提取的DNA,其扩增谱带表现为深浅带,深带与对照样品一致,浅带与对照样品不一致,不易于准确读带。本研究通过提取单株DNA进行PCR扩增,有效避免了杂株对鉴定结果的影响。3.在品种真实性和纯度鉴定中,人们一直认为SSR分子标记鉴定结果比田间鉴定结果更严格,利用SSR分子标记鉴定品种真实性和纯度,其结果对企业有失公平。本研究通过大量的SSR分子标记检测结果与田间鉴定结果比较分析显示,6%的聚丙烯酰胺凝胶电泳结果更接近田间鉴定结果。这一研究结果为下一步SSR品种真实性和纯度检测标准制定中电泳方法选择提供了有力科学依据。本研究用中华人民共和国农业行业标准NY/T1433-2007上公布的24对引物,对代表种子批77680千克的11份杂交水稻种子送检样品进行田间小区种植鉴定和SSR分子标记鉴定,通过6%非变性聚丙烯酰胺凝胶电泳分析结果显示：SSR分子标记鉴定结果与田间小区种植鉴定结果一致,两种鉴定结果都低于国家质量标准值96.0%,判定为不合格。SSR分子鉴定结果略低于田间小区种植鉴定结果,前者鉴定结果平均值为86.6%,后者鉴定结果平均值为87.3%,两者相差0.7%。对226份材料的纯度检测中,3%的琼脂糖凝胶电泳结果与田间鉴定结果平均相差0.9%。由此可见,用6%非变性聚丙烯酰胺凝胶电泳分析的结果更接近田间鉴定结果。用中华人民共和国农业行业标准NY/T1433-2007上公布的24对引物,以农业部提供的标准样品作为对照,对448份样品进行品种真实性鉴定,通过6%非变性聚丙烯酰胺进行电泳,共检测出37份样品真实性不合格。将SSR分子标记鉴定结果与田间品种真实性鉴定结果进行比较,有13个没有标准样品,未进行SSR检测,有6个样品田间没有对照样品,无法进行田间鉴定,其余429个样品的SSR鉴定结果与田间鉴定结果一致。由此可见,利用SSR分子标记检测品种真实性和纯度,通过6%非变性聚丙烯酰胺电泳,其结果准确可靠。在转基因成分的检测中,国际上还没有统一的方法和标准。随着转基因农作物检测技术的不断发展,PCR技术已经成为最主要的检测方法之一。但是常规的PCR每次只能扩增一个基因,而单独的多重PCR和常规电泳相结合的技术由于受多重PCR技术自身限制,每次扩增的基因数目也很有限,因此不能满足当前转基因农作物检测的要求。基因芯片是一种点有若干寡核苷酸探针的基片,每个点都含有序列特异的探针和目标基因互补结合。因此,基因芯片可以和多重PCR技术相结合同时检测多个目标基因。本研究通过对现有玉米转基因商业化信息收集,利用已公布的基因序列信息设计11对引物和探针,以CryIAb, Nos-T, neo, Camv35S-P, EPSPS,18S的引物组合和bar,pat, CrylAc, hpt和IVR的引物组合分别进行多重PCR扩增,扩增产物与固定有相应目标外源基因特异性探针的基因芯片进行杂交,通过芯片扫描仪对杂交芯片进行扫描,最终达到在同一个芯片中同时检测9个外源目标基因的目的。在实验中,通过对多重PCR扩增和杂交条件的优化,最终达到利用生物芯片技术准确、快速检测出市场上主要的Bt176、Bt11、MON810、 GA21、NK603、MON863、1507、3272、MIR604及59122共10个玉米转基因品系的转基因成分。更多还原

【Abstract】 The identity and purity of variety have an signicant effects on the yield and quality, which are important evaluation index for seed quality. And the problem of varietal identity and purity can lead to dramatical yield declining, such events often happened in the past years, and which seriously affected the increase of the agricultural production. Genetically modified crops are obtained via the genetic transformation of crop genome. Although all countries hold a relatively cautious attitude to commercialization of the transgenic crops, but the acreage and varieties of genetically modified crops still are rapid increased. With the enaction of the relevant laws and regulations for the risk assessment and detection of genetically modified products, the detection and identification of genetically modified component have become increasingly important.Now there are a great many seed business entities in China, and market supervision technology and means are lagged. Fake and shoddy seeds could not be forbidden with absolutely, and infringement problems of sets licensing outstand issues, so the order of the seed market is chaotic, Which seriously infringe the legitimate rights and interests of farmers and legitimate enterprises."The view of the State Council on accelerating the development of modern crop seed industry" clearly pointed out that establishing the seed management system with advanced means and effective supervision, to crack down the behavior of infringement of licensing and counterfeit seed production and management, and feasible safeguarding the market order of fair competition. Molecular detection techniques provide effective measures to solve these problems mentioned above. It can break the bottlenecks of management technology. Therefore, It is an important and urgent task for current seed quality management to accelerate the development and application of molecular detection technology.Many studies have shown that the method is feasible, and that the technique of SSR molecular markers can be used to accurately appraise the varietal identity and purity. But most of these reports focused on the theoretical point of research. So in present work, we combined the practical supervision of seedand theoretical research together, to try to solve the existing problems in the practical application. The main results were as follows:1. Variety purity testing:screened out specific primers for23rice hybrids which are good sold variety on the market, checked the result of SSR molecular identification through field planting in growing season and field planting in Hainan province. By SSR molecular marker, use the primers screened to check226samples, which can stand for nearly4million kilograms "two-line" rice hybrids and contained23rice hybrids sold well on the market. The results showed:the identification results of SSR marker and the field planting in growing season were basically the same, the biggest difference was11.2%, the average gap was0.9%; the identification results of field planting in growing season and field planting in Hainan province were quite different, the biggest difference was44.0%, the average gap was3.6%.In addition, we selected20representative samples and identified per plant of the samples by SSR marker and the field planting in growing season. The result showed the goodness of fit of each individual plant with these two identification methods was99%. In conclusion, the purity of the two-line rice hybrids could be accurate and rapid identification with the primers screened, and the sterile lines can be distinguished out only with a pair of primers according the parents’ complementary types.In addition, through analyze the experimental data, at first to raise a viewpoint that the identification results of SSR marker were more accurate and reliable than field planting in Hainan province, when identify the purity of two-line rice hybrids. When purity testing, off-types could easy divided into female parent type and paternal type when parents as standard control, which conducive to analyze the sources of the off-types, being a good solution of the problem that difficult to distinguish off-types during the actual molecular detection.2. Varietal identity:when identify lower purity sample, electrophoretic analysis often appeared the bands that were not parents complementary bands, which brings difficulties to read tape, it often easy causes miscarriage of justice when identify the varietal identity. In this study, a good solution for the problems above was that use per plant to identify the varietal identity. Samples in the institutional capacity of national agricultural seed testing to verify the varietal identity of the SSR markers by DNA extraction of per plant, whose differences could not be detected about27%per plant checked in the24sites, whose differences were detected about73%per plant checked in the24sites. DNA extracted with a mixed sample, the amplification bands manifest as deep-undertone bands, deep band was the same with the check, shallow tape was inconsistent with the check, and this was not easy to accurately read the bands. By the method of extracting the DNA of per plant could be more accurately distinguish the differences of the electrophoretic bands, and could avoid the interference of the off-types.3.In making test of the varietal identity and purity, people always thought that the results of SSR Markers were more stringent than the results of field planting test, and the results were unfair to the enterprise when use SSR Marker to identify the varietal identity and purity. After making analysis on a large number of the results of SSR molecular marker test and the results of field planting test,this study showed that the results by6%polyacrylamide electrophoresis were closer to the results of field planting test,.The results in this study provided a strong scientific basis for chosen electrophoresis method, which was the setting standard use for varietal identity and purity. In this study,24pairs of primers which announced in agricultural industry standard NY/T1433-2007of China were used to identify11rice hybrid samples on behalf of the seed lots of77,680kg by SSR molecular marker test and the field planting inspection. The analysis by electrophoresing by6%non-denaturing polyacrylamide showed that:The result of SSR molecular marker test was86.6%,87.3%by field planting test, and the slightly difference of two methods was0.7%. The results of two identifications were all below the grade of96.0%that was the national quality standard, so the samples were unqualified. In addition, the average difference was0.9%between the results of3%agarose gel electrophoresis and the results of field planting test. The average difference was0.7%between the results of6%non-denaturing polyacrylamide gel electrophoresis and field planting test. It clearly showed that the results of6%non-denaturing polyacrylamide gel electrophoresis much more close to the results of field planting test. Use24pairs of primers which published on agricultural industry standard NY/T1433-2007of the People’s Republic of China to identify the varietal identity of448samples with standard samples which provided by the Ministry of Agriculture, and37samples was unqualified tested with6%non-denaturing polyacrylamide electrophoresis. Comparing the results of SSR molecular marker test and the results of field planting test show that13samples with no standard sample were not made SSR detection,6samples with no field control samples could not be carried out field test, and the remaining429samples SSR identification results consistentted with the field test results. This shows that the results of the SSR molecular marker identification of varietal identity and purity by6%non-denaturing polyacrylamide gel electrophoresis were accurate and reliable.The international community still was no uniform methods and standards to identify genetically modified component. With the development of identification technology for genetically modified crops, the technology of PCR has become one of the most important identification methods. But regular PCR only can amplify a gene one time, due to the limitations of multiplex PCR technology, therefore, the technology combined the individual multiplex PCR and regular electrophoresis could not meet the current requirement of transgenic crops test. And the number of gene amplification was very limited at one time. Gene chip was a basic chip which had a number of oligonucleotide probes, each point containing complementary linked with the sequence-specific probe and the target gene. Therefore, the gene chips could be combined with multiplex PCR technology, this could be use to detect many target genes simultaneously. This study applied the hybridization between the amplified product of multiple asymmetric PCR and the gene chip which fixed target exogenous gene-specific probes, in order to achieve the purpose to detect more target genes at one time. In this work, by collecting the commercial information on existing transgenic corn, designed11pairs of primers and probes by using the gene sequence information that had published, and optimized the conditions of asymmetric PCR amplification and hybridization, Finally the technology of bio-chip was used to accurate and rapid detect10genetically modified corn varieties sold well in the market.更多还原