【作者】 李清；

【导师】 傅永福；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 MFT(MOTHER OF FT AND TFL1)基因属于磷脂酰乙醇胺结合蛋白(PEBP)基因家族，广泛存在于多细胞陆生植物中。最初的研究认为MFT基因可能是个开花调控因子，但是后来的一些关于MFT基因的转化和表达分析研究表明MFT基因更可能在种子发育或萌发过程中起作用。对MFT基因进行研究，明确其在种子发育或萌发过程中的作用，将为植物种质的分子改良提供一定的理论参考。本研究从大豆中克隆到GmMFT基因，对它进行了生物信息学分析，用实时荧光定量PCR(RT-qPCR)分析了它在不同组织器官和发育过程的时空表达模式，并在拟南芥中过表达该基因以研究其功能。与此同时，因为合适内参基因的选择对于获得正确的RT-qPCR结果至关重要，所以为了更好地研究GmMFT基因在种子发育和萌发过程的表达模式，本研究还评价了18个大豆候选内参基因在34个不同的种子样本中的表达稳定性。主要研究结果如下：1.在大豆种子发育过程中最适合的内参基因是TUA5和UKN2，在种子萌发过程最适合的内参基因是Glyma05g37470和Glyma08g28550，在总样本中则需要用Glyma05g37470、Glyma08g28550、Glyma18g04130和UKN24个内参基因做标准化分析。2. GmMFT基因主要在大豆种子中表达，并且在种子发育过程中表达量逐渐升高，在种子萌发过程中表达量逐渐降低。GmMFT基因的这种表达模式暗示其很可能调控大豆种子发育和萌发。3. GmMFT蛋白同拟南芥AtMFT和小麦TaMFT蛋白一样定位在细胞核和细胞质中，暗示其可能是一个转录因子。4.转基因拟南芥表型分析结果表明GmMFT基因可能抑制种子萌发，并不参与开花时间或种子发育的调控。分析GmMFT转基因和野生型种子中一些与萌发相关的基因的表达情况，我们发现GmMFT基因可能从两方面来抑制种子萌发：一方面通过促进ABA合成基因的表达，同时抑制ABA降解基因的表达来促进ABA在种子中的积累以抑制种子萌发；另一方面通过抑制GA合成基因的表达，同时促进GA降解基因的表达来抑制GA在种子中的积累进而抑制萌发。更多还原

【Abstract】 MFT (MOTHER OF FT AND TFL1) is a member of phosphatidylethanolamine-binding protein(PEBP)gene family and evolutionarily conserved in a wide range of multicellular land plants. MFT wasinitially identified as a floral regulator in some research. However, later studies using expressionanalysis and transformation analysis of MFT genes indicated that MFT genes may function in seeddevelopment or germination rather than in flowering. An analysis of MFT genes aimed to clarify theirdefinite roles in seed development or germination may be helpful to improve the quality of seeds at themolecular level. In this study, GmMFT gene was cloned and bioinfomatically analyzed. Itsspatio-temporal expression profiles across different developmental stages and in various tissues/organswere performed using real-time quantitative PCR (RT-qPCR). Furthermore, functional analysis ofGmMFT gene was performed in transgenic Arabidopsis. At the same time, due to selection of properinternal reference genes is critical for the reliable RT-qPCR results, we evaluated the expressionstability of18candidate reference genes in34different seed samples in order to study the expressionpattern of GmMFT gene during seed development and germination. The main results were as follows:1. The reference genes identified as optimums for soybean seed development were TUA5andUKN2, whereas for seed germination were Glyma05g37470and Glyma08g28550. Furthermore, fortotal samples it was necessary to combine four genes of Glyma05g37470, Glyma08g28550,Glyma18g04130, and UKN2for normalization.2. In soybean, GmMFT gene mainly expressed in seeds and the expression level was increasingduring seed development, whereas decreasing during seed germination. This expression pattern ofGmMFT gene indicated that GmMFT may function in seed development or germination of soybean.3. GmMFT protein was localized in both nuclear and cytoplasm as Arabidopsis AtMFT and wheatTaMFT, indicating it may be a transcription factor.4. The phenotype analysis of transgenic Arabidopsis indicated that GmMFT gene may suppressesseed germination rather than regulate flowering or seed development. In comparison of the expressionof germination-related genes between GmMFT transgenic and wild-type seeds, We found that GmMFTgene may inhibit seed germination in two ways: On one hand, GmMFT promotes the accumulation ofABA in seeds by increasing the expression of ABA biosynthetic genes and decreasing the expression ofABA degradation genes; On the other hand, GmMFT inhibits the accumulation of GA in seeds bydecreasing the expression of GA biosynthetic genes and increasing the expression of GA degradationgenes.更多还原