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我的毕业论文摘要，请诸位议一议
摘 要
大豆是人类主要的植物性蛋白质、油脂的重要来源。人类消费的油脂中，植物油正逐渐代替动物油，高油大豆市场需求量大。提高大豆中油分含量一直是科研工作者研究的课题。植物转基因技术将会比常规育种更有效地改造栽培品种的某些缺点。理论上，植物转基因技术可根据品种的要改造的缺点有针对性地转移单个或少数几个基因，避免了大量不良基因的干扰，所需的群体小，周期短，因此具有较高的效率。生物技术的发展，为通过基因工程手段提高大豆含油量的育种工作奠定了坚实的基础。
反义技术根据核酸杂交原理设计针对特定靶序列的反义核酸，通过向细胞内导入反义核酸或其表达载体，就可特异地阻止相应基因的表达。PEP 是高等植物中普遍存在的胞质酶。从植物生理学研究认为丙酮酸羧化酶（PEPCase）活性的抑制能够提高籽粒中油脂含量。有关研究也证实了利用反义基因技术抑制PEP表达是提高籽粒含油量的有效途径。
本文根据Genebank发表的大豆PEP基因cDNA序列,设计了一对引物用RT-PCR方法,扩增出了PEP基因片段。将扩出的的基因序列,插入T载体，重组质粒转化大肠杆菌，穿刺培养的菌株送交联合基因测序，测序结果为，克隆的序列片段长度符合预先设计，为968bp ，碱基的准确率为99.99%。将此片段插入SK（-）载体的XmaI，EcoRI位点，构建了中间载体Sp，再利用重组质粒上的Kpn和SaI位点将PEP基因反向连入有35S启动子的表达载体中，构建了含有大豆PEP羧化酶的反义基因双元载体质粒Dantip。根据内含子能提高基因表达，发夹式序列结构能够使调控的基因表达适度的原理，构建了反义基因的发夹结构的质粒载体Dpip，一个中间带有内含子，基因的上游有35S启动子。用反复冻融法将质粒载体转入农杆菌EH105。利用携带双元载体质粒p3301（含有GUS报告基因和卡那霉素抗性基因NPTⅡ）的根癌农杆菌菌株LBA4404，用百脉根子叶为外植体，以GUS基因瞬时表达效果为指标，优化了百脉根的农杆菌侵染条件。以建立的较优化的转化体系，将农杆菌EH105携带的反义PEP羧化酶基因采用共培养法，导入百脉根，在已获得的109株再生植株中，经PCR检测和卡那抗性检测，获得了转化植株10株，转化率为9.1%。本论文内容初步证实了所构建的植物转化载体Dantip能将反义PEP羧化酶基因导入植物体。为PEP羧化酶反义基因进一步导入大豆植株并稳定表达和遗传，最终提高大豆籽粒油脂含量，改良大豆品质奠定了实验基础。
Abstract
Soybean is the main source of plant protein and lipid for mankind. With the improvement of standard of living, People are paying more attention to consumption of plant lipid rather than animal ones.
How to increase the lipid content of soybean, however is a great challenge for the soybean breeders. Now biotech, which can combine the favorite characters of different species into one variety, provides an effective means to reach the goal. It has mainly three advantages. Firstly, it can not only shorten the cycle of routine of breeding, but also be practicable; secondly, it can transfer the foreign gene into plant acceptor, integrate it into genome of acceptor and eventually express the foreign gene in the plant; and thirdly, it can use gene of different species without restriction of species.
Phosphoenolpyruvate carboxylase (PEPCase) is critical enzymes of pyruvic acid metabolism. Studies had confirmed that the prevention of PEP activity can transfer the reaction of substrate into fatty metabolism. This fact presents great possiblity to manipulate fatty acid metabolism in plant seeds by gene engineering .
Using antisense RNA , which based on nucleic acid hybridization theory, we designs antisense sequence versus special target gene, so the expression of target gene is refrained from expression by the interaction of partnership.
In this dissertation, primers are designed according the sequence of PEP registed in Genebank. A DNA fragment of was amplified with polymerase chain reaction (PCR) using soybean totle RNA as template. The gene was sequenced and the results showed that the sequence contained 968 nucleotides and only five different from the PEP gene. We ligase the gene into T-vector and then digist out it at XmaI and EcoRI sites and insert the vector SK(-). In order to make the gene express in plant, the PEP gene fragment was inserted in an antisense orientation using mosaic virus 35S promoter to drive transcription and the nopaline synthase terminator.
Crowtoe (Lotus corniculation L.) is the acceptor plant of this antisense gene in this experiment. Plasmid Dantip was mobilized into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method, using that plasmid cloned in E.coli.DH5α.The antisense PEP gene was transferred into crowtoes via the Agrobacterium mediated method.
The antisense PEP was integrated into the acceptor which confirmed by PCR. Among 109 regenerated plants there are 9 contained the transgene.
This work has laid a preparation for engineering such gene into soybean in the future. We hope to increase the fatty content of soybean seed by this way finally. |
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发表于 2006-11-15 09:20:53
