【作者】 李祥；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 发育生物学， 2008， 博士

【摘要】 玉米(Zea mays L. ssp. Mays)是食品和饲料的主要来源之一,其淀粉产量和品质决定了它在人类生产和生活中的地位。为深入了解玉米淀粉合成的分子调控机理,本研究首先以B73、ae/wx和sh1授粉后15天的胚乳为材料,利用18K Affymetrix玉米基因组芯片平台,研究了突变体材料中基因表达谱的变化,发现了淀粉合成受阻后糖的积累对其它物质代谢和基因表达调控的影响;在此基础上,对可能参与淀粉合成的基因以多个自交系为材料,分析它们在叶片和胚乳发育过程中的表达模式,研究了不同成员对胚乳淀粉合成的贡献。同时,为满足工业上对高直链淀粉的需求,利用RNAi策略敲除ZmSBEⅡb或ZmSBEⅠ&ZmSBEⅡb。主要结果如下:1.胚乳基因表达谱分析以B73、sh1、ae/wx授粉后15天的胚乳为材料进行寡聚核苷酸表达谱芯片分析。与B73相比,sh1突变体中2706个探针对发生显著变化,ae/wx突变体中1966个探针对发生显著变化(FDR<0.1%;q value<0.01)(玉米探针总量17,622个,检测13,495个基因)。为减少玉米自交系背景差异对基因表达的影响,分析了B73和Mo17授粉后13天和19天的胚乳中基因表达的差异,分别找到差异基因609个和889个(FDR<5%),认为是由遗传背景差异造成的,从ae/wx vs.B73中扣除了351个,从sh1 vs.B73中扣除390个,分别剩下1429(命名为Dae/wx)和1557(命名为Dsh1)个差异表达基因进行后续的分析,其中共同变化的835个。qRT-PCR结果表明,90.6%(29/32)的差异基因是可靠的。2.差异基因的GO分析Dae/wx中按p-value(<0.1)由小到大依次为脂类转运(6/12,指该芯片中参与脂类转运的12个成员中6个成员表达变化,下同)、丙酮酸脱羧酶(4/6)、糖类运输(4/7)和细胞壁结构成分(3/6),说明对碳水化合物代谢和脂类转运影响显著;Dsh1中按p-value(<0.1)由小到大依次为糖类运输(6/7)、高尔基体(4/5)、蔗糖合成酶(4/6)、半胱氨酸蛋白酶抑制剂(5/9)、脂类运输(5/12)、细胞壁成分(6/18)和丙酮酸脱羧酶(3/6),说明除碳水化合物代谢和脂类转运影响显著外,半胱氨酸蛋白酶抑制剂的表达降低反应了胁迫和细胞发育的变化,说明该突变体中可能有更多基因表达变化和细胞学改变。3.差异基因的功能注释和分类约72%的差异基因得到注释并按照代谢途径和功能分为14类,碳水化合物和能量代谢、基因的转录和转录后修饰、蛋白质合成、蛋白调控和分子伴侣、胁迫/防御/衰老及物质转运是受影响很大的代谢和调控过程,占共同变化基因的46.5%,占ae/wx单独变化基因的53.7%,占sh1单独变化基因的47.1%。进一步分析发现,与淀粉生物合成相关的基因变化不多,而与糖中间代谢和能量相关的基因变化较多;TPS表达的降低反应了淀粉合成基因sh2翻译后AGPase活性下调。启动子分析发现,参与糖代谢的多数基因的启动子中有糖响应元件。Pul和TPS可能是直接受高糖抑制表达的基因,而且SREATMSD (TTATCC)、TATCCAOSAMY (TATCCA)和TATCCAY MOTIFOSRAMY3D (TATCCAY) 3个糖抑制元件可能是下调基因表达的糖抑制位点。两个突变体中基因表达的不同变化反应了淀粉积累和细胞结构的差异。突变体中糖的积累,可能形成了糖信号和胁迫信号,改变了信号转导,引起激素响应和细胞组成蛋白的变化,改变了细胞周期。另外,还发现母性遗传基因和种子成熟相关基因的变化。4.碳水化合物代谢基因和细胞周期基因随胚乳发育的变化以B73、ae/wx、sh1授粉后7天种子、15天和25天胚乳为材料,分析18个糖代谢差异基因和9个细胞周期调节相关的差异基因的表达模式,研究发现sh1突变体中Pul、SBEⅡa、UGPase、SUS3和SSⅡa基因表达模式发生改变,ae/wx突变体中Sh2-1、SUS3和SSⅡa基因表达模式发生改变,揭示了突变体对这些基因的巨大影响,反映了它们可能存在相互作用。糖转运、信号调节相关基因只改变胚乳发育过程中的表达量而不改变表达模式,可能是随着糖的积累、细胞的发育等过程表达水平发生了改变。细胞周期调节基因表达水平发生了较明显改变,但是它们的表达模式没有明显变化,表明虽然突变体中可能促进了细胞分裂但并未打乱细胞周期,只是延长了细胞分裂时间,延缓了胚乳细胞发育进程。徒手切片显示突变体7天胚乳没有明显的分层,ae/wx突变体细胞核明显变大而细胞大小差异不明显,sh1突变体细胞核明显变小,细胞数目增加,说明突变体胚乳细胞结构明显改变。5.淀粉生物合成基因的不同贡献以Q319、C7-2、q404、ZN-A和S8自交系的幼叶、成熟叶、成熟子房、授粉后1d、5d、10d的种子和15d、20d、25d的胚乳为材料,研究了参与淀粉合成的44个基因的表达模式,研究发现ZmGBSSⅠ、ZmSSⅢ、ZmSBEⅡb、ZmBT2-2、ZmSh2-2、ZmSh2-3、ZmBT1在胚乳发育中后期特异性表达,ZmSUS1、ZmSus1L、ZmSUS3、ZmSSⅠ、ZmSSⅡa、ZmSBEⅠ、ZmISO1、ZmPul的表达在淀粉合成期显著上调,ZmUGP3和ZmSUT2在所有组织中高水平表达,说明这些基因可能主要负责胚乳淀粉的生物合成。尽管ZmBT2-1和ZmSh2-1随着胚乳发育表达下调,但是一直有较高水平。亚细胞定位发现,ZmSUS1、ZmSUS1L、ZmBT2-1、ZmSH2-1、ZmBT1、ZmSS、ZmSBE、ZmISO1、ZmISO2、ZmPUL、ZmGPT和ZmPPT定位于质体,ZmSUS2、ZmSUS3、ZmUGPase、ZmBT2-2、ZmSH2-2、ZmSH2-3、ZmPGI和ZmPPM定位于细胞质中,而ZmSUT定位于细胞质膜。这些结果为胚乳淀粉生物合成研究提供了新资料。6. RNAi技术创造高直链淀粉玉米为培育高直链淀粉玉米材料,根据淀粉合成相关基因的表达模式及已发表资料,利用RNAi技术使转基因玉米SBEⅡb和SBEⅠ&SBEⅡb转录后沉默,以创造高直链淀粉玉米新品系。克隆了玉米Sh2基因,在第495位和496位两个酪氨酸中插入酪氨酸和丝氨酸各一个(Sh2YS),降低Pi的负调节作用,以提高ADP葡萄糖焦磷酸化酶的活性,最终提高淀粉的合成效率。采用基因枪和农杆菌浸染法已经获得相应的转基因植株。更多还原

【Abstract】 Maize (Zea mays L. ssp. Mays) provides us food and feed for many years due to its starch content and quality. To study the molecular mechanisms of starch biosynthesis in maize, we employed 18K Affymetrix? maize Genomic Genechip? to reveal the differences of gene expression profiling of the 15 days endosperm after pollination (DAP) in ae/wx and sh1 mutants compared to B73. We found the relationship among starch metabolism, metabolisms of other components and gene expressing profiling due to the accumulation of sugar. Then, genes expression patterns of starch biosynthesis in leaves and developing endosperms of 5 maize inbreds were studied for the contribution of different genes to starch biosynthesis in endosperm. In addition, RNA interference was employed to knock down ZmSBEⅡb or ZmSBEⅠ&ZmSBEⅡb for the requirement of high-amylose starch in industry. The main results were as follows:1. DNA microarrayThe 15 DAP endosperms of B73、sh1、ae/wx were employed to investigate the differenc of their gene expression profiling. Compared to B73, total 2706 and 1966 probe sets were different significantly (FDR<0.1%; q value<0.01) in sh1 and aw/wx mutants (total 17,622 probe sets detecting 13,495 genes in chip). To reduce the effect of gene expression in different genetic backgrounds, differentially expressed genes were analyzed in endosperm of B73 and Mo17 at 13 DAP and 19 DAP, from which 609 and 889 differentially expressed genes were identified (FDR<5%) as the ones caused by different backgrounds. After deducting 351 probe sets from ae/wx vs.B73 and 390 probe sets from sh1 vs.B73, there were 1429 (named Dae/wx) and 1557 (named Dsh1) probe sets surplus for further analysis, among which 835 probe sets kept the same tendency in both mutants. 90.6% (29/32) differentially expressed genes were confirmed by qRT-PCR.2. GO analysis of differentially expressed genesAccording to p-value, lipid transport (6/12, which means 6 differentially expressed genes of 12 members on chip, and the same as follows), pyruvate decarboxylase activity (4/6), sugar:hydrogen symporter activity (4/7) and structural constituent of cell wall (3/6) were revealed in Dae/wx, suggesting that carbohydrate metabolism and lipid transportation were affected significanty. Whereas, sugar:hydrogen symporter activity (6/7), Golgi apparatus (4/5), sucrose synthase activity (4/6), cysteine protease inhibitor activity (5/9), lipid transport (5/12), cell wall (6/18) and pyruvate decarboxylase activity (3/6) were displayed in Dsh1, suggesting that except for carbohydrate metabolism and lipid transportation, down-regulated expression of cysteine protease inhibitor suggests changes in stress responses and in cell biology.3. The functional annotation and classification of differentially expressed genesAbout 72% differentially expressed genes were annotated and classified into 14 classes, among which carbohydrate and energy metabolism, transcription and posttranscriptional processing, protein synthesis, posttranslational processing, stress/defence/senescence and transportation were affected significantly. All together, the genes in these processes accounted for 46.5% of the same tendent differences, 53.7% of the genes changed only in Dae/wx (Oae/wx) and 47.1% of the genes changed only in Dsh1 (Osh1). Further analysis suggests that there were a few genes in starch biosynthesis but more genes in sugar and energy metabolism expressed differentially. The down-regulation of trehalose-phosphate synthase (TPS) suggests the down-regulated activity of AGPase after posttranslational redox activation between Sh2 subunits. The analysis of promoters of sugar metabolism genes indicates several sugar response elements in most promoters. Pul and TPS may be repressed directly by high-level of sugar in which SREATMSD (TTATCC), TATCCAOSAMY (TATCCA) and TATCCAY MOTIFOSRAMY3D (TATCCAY) in their promoters should be the sugar repressive elements due to their down-regulated expressions.The differentially expressed genes reflect the difference of the starch accumulation and cell structure in two mutants. Due to the accumulation of sugar, signal transductions, especially sugar signal and stress signal, were changed significantly, which might alter the expression of hormone-related genes, cell organizational proteins genes, and cell cycle-related genes finally. In addition, there were maternally expressed genes (MEG) and seed maturation proteins up-regulated and embryo-specific proteins down-regulated.4. Expression patterns of genes differentially expressed in starch metabolism and cell cycle during endosperm development7 DAP kernals, 15 DAP endosperms and 25 DAP endosperms of B73, ae/wx and sh1 were used to study the expression patterns of 18 differentially expressed genes in sugar metabolism and 9 differentially expressed genes in cell cycle. We found that the expression patterns of Pul, SBEⅡa, UGPase, SUS3, SSⅡa in sh1 and Sh2-1, SUS3, SSⅡa in ae/wx were altered, revealing that these genes may interact with each other. In contrast, only the expression level of genes in sugar transport and signal were altered, suggesting that these genes are regulated indirectly by sugar level, cell development. Similarly, although the expression levels of the genes in cell cycle were changed significantly, the expreeion patterns were not changed distinctly, suggesting that the cell cycle in endosperm cells is not disturbed the cell division. As a result, endosperm cell development is delayed. Compared to B73, layered structure was not found in 7 DAP endosperm of ae/wx and sh1 mutants, and bigger nucleoli in ae/wx mutants and smaller nucleoli and more cell number in sh1 mutants were found, suggesting the significant alternation of the cytoarchitecture in the endosperms of ae/wx and sh1 mutants.5. Different contribution of the genes to starch biosynthesisLeaves and developing endosperms of Q319, C7-2, q404, ZN-A and S8 were used to study the expression patterns of 44 genes participating in starch synthesis. We found that ZmGBSSⅠ、ZmSSⅢ、ZmSBEⅡb、ZmBT2-2、ZmSh2-2、ZmSh2-3、ZmBT1 were expressed exclusively in the metaphase and anaphase of developing endosperm, and the expression patterns of ZmSUS1、ZmSus1L、ZmSUS3、ZmSSⅠ、ZmSSⅡa、ZmSBEⅠ、ZmISO1、ZmPul were consistent with the process of starch synthesis in the endosperm, while ZmUGP3 and ZmSUT2 were highly-expressed in all samples, suggesting that these genes perhaps devoted to starch synthesis in endosperm. Although their expression patterns were down-regulated during the endosperm development, ZmBT2-1 and ZmSh2-1 were still expressed. ZmSUS1、 ZmSUS1L、ZmBT2-1、ZmSH2-1、ZmBT1、ZmSSs、ZmSBEs、ZmISO1、ZmISO2、ZmPUL、ZmGPTs and ZmPPTs were localized to plastids, ZmSUS2、ZmSUS3、ZmUGPs、ZmBT2-2、ZmSH2-2、ZmSH2-3、ZmPGI和ZmPPMs were localized to cytoplasm. Whereas, ZmSUTs were localized on cytoplasm membrane. These data provides more evidence about the different contribution of the genes to starch biosynthesis.6. High-amylose maize inbreds engineered by RNA interference (RNAi)To meet the requirement of high-amylose starch in industry, based on the expression profiles of starch biosynthesis-related genes and published data, SBEⅡb and SBEⅠ&SBEⅡb were selected to knock down in maize by RNA interference approach, to create new high-amylose maize inbreds. Sh2 was cloned and modified by insertion of a tyrosine and a Serine between 495 and 496 amino acids, to enhance the activity of AGPase and to increase the starch biosynthesis. Transgenic plants were recovered via biolistic and Agrobacterium-mediated transformations.更多还原