【作者】 谢彬；

【导师】 黄玉碧；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2010， 硕士

【摘要】 玉米是重要的粮饲作物,也是农作物中最主要的淀粉来源。许多学者对小麦、水稻等作物籽粒的淀粉合成和积累已做了研究,然而关于玉米淀粉生物合成机理的研究却很少。参与淀粉合成的酶类主要有：腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、淀粉合成酶(starch synthases,SS)、淀粉分支酶(SBE)和淀粉去分支酶(DBE)。腺苷二磷酸葡萄糖(Adenosine diphosphate glucose, ADPG)是植物体内淀粉合成的直接前体,而它则由AGPase以ATP和1-磷酸葡萄糖为底物代谢合成。已分离到的低淀粉突变体伴随有AGPase活力的下降,可以看出它在淀粉的生物合成中有着重要作用,是高等植物淀粉生物合成的限速酶。AGPase由四个亚基组成,高等植物中AGPase是由大小亚基组成的异源四聚体蛋白。AGPase两种大小亚基都由不同的基因编码。在玉米中编码AGPase的基因有3个大亚基和3个小亚基。因此,研究AGPase基因的时空表达规律有助于我们了解其与淀粉生物合成的关系,从而达到通过调节其含量和活性来提高淀粉含量以及改造淀粉品质的目的。本试验通过RT-PCR检测了玉米6个AGPase基因在玉米不同组织和不同时段的时空表达规律。试验结果表明：1.玉米6个AGPase基因的表达具有组织特异性和发育阶段特异性。2.授粉后10-16天,随着细胞质型AGPase基因表达水平的升高,AGPase酶活性也在同时显著增强。当AGPase酶积累到一定浓度,淀粉积累速率也开始显著增长。3. AGPase基因对日夜周期变化的不同反应与其所在组织有关。日夜周期变化对6个AGPase基因表达量影响较小的组织有老叶、幼叶和叶鞘。与此相反,日夜周期变化对AGPase基因表达量影响较大的组织是成熟叶片,在成熟叶片中,agpl1、agpl2、agpl3、agps1和agps2基因在夜间的表达量远远大于在白天的表达量。4.不同的AGPase基因对糖和植物激素的反应存在显著差异。在本研究中,我们通过序列对比发现agps1和agps2存在很高的同源性,二者对激素的反应也很相似。另一方面,这两个基因也存在组织特异性表达的特性,对糖处理有不同的反应。这说明它们存在功能上的差异,糖也许是其差异的影响因素之一。5.葡萄糖对玉米AGPase基因表达水平的影响大于蔗糖、PEG6000和甘露醇的影响。各基因中,糖处理对agps2基因表达的影响相对较小,各糖或激素处理均能降低agps2基因的表达。而在籽粒灌浆过程中,其内源性蔗糖含量在授粉后13d左右达到最大值,而后降低,而AGPase基因相对表达量最高值并不是出现在授粉后第13d。这样看来似乎AGPase基因表达水平受内源性蔗糖的影响规律与受外源性蔗糖的影响规律不符。但ABA和蔗糖共同处理对AGPase基因表达的影响远远大于单独蔗糖处理的影响,因此最恰当的解释是,淀粉新陈代谢的复杂性决定了它由许多重要因素共同影响。更多还原

【Abstract】 Maize is one of the most important feed crops and is the major source of starch. Starch is the most significant carbon reserve in higher plant. Many researches about the biosynthesis and accumulation of starch of wheat, rice grain have been done, however, further research to address the starch synthesis net-work is necessary. The production of starch in higher plant is orchestrated by four major types of enzymes, named ADP-glucose pyrophosphorylase (AGPase), starch synthase (SSs), starch branching enzymes (SBEs) and starch-debranching enzymes (DBEs). The regulatory and rate-limiting step of starch biosynthesis is the synthesis of the ADP-glucose, catalysised by ADP-glucose pyrophosphorylase, which could affect the final starch content directly in higher plant for that low AGPase activity was observed in AGPase genes deficient mutant. This enzyme in higher plant is a heterotetrameric (α2β2), consisting of two "regulatory" subunits (AGPL) and two slightly smaller "catalytic" subunits (AGPS). In higher plant, each subunit of AGPase is encoded by distinct genes. In maize, six gene encoding AGPase can be found in the database, three of which encode for large subunit, AGPL1, AGPL2 and AGPL3; and the others of which encode for small subunit, AGPS1, AGPS2 and AGPS3. Therefore, study on the expression patterns of maize AGPase genes can help us better understand the relationship between AGPase genes and starch biosynthesis, and also can improve starch content quality.The expression patterns of six maize AGPase genes in different organ and different developmental stage of maize were studied by using Quantitative Real-time RT-PCR in this work. The results showed that:1).The expression profillings of AGPase genes in maize were tissue-specific and developmental stage-specific.2).As the expression level of cytoplasmic AGPase gene increased in 10d to 16d after pollination, AGPase activity was also significantly enhanced. When the AGPase enzyme accumulated to a certain level, starch accumulation rate also began to grow significantly and had the same change trends.3).The different influence of day and night cycles on the exppression of AGPase genes may relate to the different tissues. The influence of day and night cycles on the exppression of AGPase genes in old leaf, young leaf and sheath were less than the influence of day and night cycles on the exppression of AGPase genes in developed leaf. In developed leaf, the expression level of agpl1, agpl2, agpl3, agps1 and agps2 gene in the night was far more than in the day.4).Our data showed significant difference in response to sugar or hormone among differenct AGPase isoforms. In this work, we presented that both agps1 and agps2 are nearly identical and similarly induced by hormone. On the other hand, tissue-specific expression profilling and different response to sugar of the two genes were observed. These facts support the functional divergence between the two genes and that sugar may be one of the impact factors.5).Glucose had greater effect on the expression of maize AGPase gene than sucrose, PEG6000 and manitol. However, minor changes were observed for trancripts of agps2 was observed. The amount of endogenous surcose in developing grain reached max at approximately 13 days after pollination and then decreased, but the expression level of AGPase genes did not appear 13 days after pollination. It seemed that the data of the endogenous sucrose, expression level of AGPase genes and exdogenous sucrose did not consist with each other. The most suitable explanation for this discrepancy was that many signal factors effect the complicated starch metabolism together for that more obvious changs of transcript for AGPase genes in ABA and sucrose treatment than sucrose alone treatment were observed.更多还原