有机肥对烟草淀粉生物合成的影响及NtGBSSI基因的过表达调控研究

发布时间：2018-06-03 02:42

本文选题：烟草 + 有机肥　；参考：《河南农业大学》2016年硕士论文

【摘要】：淀粉的含量和组分是影响烟叶质量和特色的主要因素之一。为探索烟叶成熟过程中淀粉组分的变化和积累,探讨直链淀粉对烟叶品质的影响,本研究设计比较不同有机肥处理对烟叶淀粉合成代谢的影响;在此基础上,克隆烟草NtGBSS1基因,通过农杆菌介导法获得转基因阳性植株,并分析了NtGBSS1基因过表达对植株形态、淀粉合成酶、淀粉含量和淀粉组分的影响,为研究烟草淀粉合成的分子调控机制奠定了基础。主要研究结果如下:1添加有机肥有利于叶片细胞中淀粉粒的积累,随着有机肥施用量的增加,叶绿体中淀粉粒的数目随之增多、体积明显变大;定量检测发现:总淀粉和支链淀粉含量相应增加,直链淀粉含量反而减少,淀粉组分显著改变;淀粉合成关键酶基因的表达变化与淀粉含量变化一致,ADPG焦磷酸化酶基因(AGPase)的表达量变化并不明显,但颗粒结合型淀粉合成酶基因(GBSS1)的表达量随有机肥的施用量增加而减弱,可溶性淀粉合成酶基因(SS1)的表达量随有机肥的施用量增加而增强。2 NtGBSS1基因在烟草中过表达:克隆烟草NtGBSS1基因插入载体,成功构建了植物表达载体pCAMBIA1305.1-35S-NtGBSS1-GUS;利用农杆菌介导法转化普通烟草K326,在添加潮霉素筛选压的MS+6-BAl.5+NAA0.15分化培养基上获得幼芽,在添加同样筛选压的生根培养基MS+NAA0.1上进行培养,两周后形成幼苗。RT-PCR和GUS组织化学染色检测得到12株阳性植株。3转基因植株的比较分析:将NtGBSS1组成型过表达的阳性株系进行大田种植,观察其形态变化,对淀粉合成酶活性、直链淀粉、总淀粉含量进行分析。结果显示,在烟草中过表达NtGBSS1基因并未影响植株表型;NtGBSS1转基因株系的淀粉总含量不变,但淀粉组分发生改变,直链淀粉含量显著增加。
[Abstract]:The content and composition of starch is one of the main factors affecting the quality and characteristics of tobacco leaves. In order to explore the changes and accumulation of starch components during tobacco maturation and the effect of amylose on the quality of tobacco leaves, this study was designed to compare the effects of different organic fertilizer treatments on starch synthesis and metabolism in tobacco leaves, and to clone the NtGBSS1 gene of tobacco. Transgenic plants were obtained by Agrobacterium tumefaciens and the effects of overexpression of NtGBSS1 gene on plant morphology, starch synthase, starch content and starch composition were analyzed, which laid a foundation for the study of molecular regulation mechanism of starch synthesis in tobacco. The main results were as follows: (1) the addition of organic fertilizer with 1 / 1 was beneficial to the accumulation of starch granules in leaf cells. With the increase of application amount of organic fertilizer, the number of starch granules in chloroplasts increased and the volume of starch granules in chloroplasts increased obviously. The quantitative analysis showed that the content of total starch and amylopectin increased correspondingly, the amylose content decreased and the starch component changed significantly. The change of the expression of the key enzyme gene of starch synthesis was consistent with the change of starch content. The expression of ADPG pyrophosphorylase gene (AGPase1) was not obvious, but the expression of granular-bound starch synthase gene (GBSS1) decreased with the application of organic fertilizer. The expression of soluble starch synthase gene (SS1) enhanced the overexpression of 2. 2 NtGBSS1 gene in tobacco with the increase of the amount of organic fertilizer. The NtGBSS1 gene was cloned into the vector. The plant expression vector pCAMBIA1305.1-35S-NtGBSS1-GUSwas successfully constructed and transformed into common tobacco K326 by Agrobacterium tumefaciens. Shoots were obtained on MS 6-BAl.5 NAA0.15 differentiation medium supplemented with hygromycin, and cultured on MS NAA0.1, a rooting medium with the same screening pressure. A comparative analysis of 12 transgenic plants was obtained by RT-PCR and GUS histochemical staining after two weeks. The NtGBSS1 component positive lines were planted in the field to observe the morphological changes and the activity of starch synthase. Amylose, total starch content was analyzed. The results showed that the over-expression of NtGBSS1 gene in tobacco did not affect the total starch content, but the amylose content increased significantly.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S572

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