高赖氨酸转基因水稻的营养评价及代谢关联研究
发布时间：2018-07-17 00:32
【摘要】：稻米富含淀粉和优质蛋白,是人类主要的能量与蛋白来源。但稻米中缺乏赖氨酸,被称为第一限制性必需氨基酸,影响稻米的营养价值。植物中赖氨酸的代谢调控机制较为复杂,其积累不仅受本身反馈抑制,还与降解代谢有关,而且与其它众多代谢通路相关联。其中天冬氨酸激酶(AK)和二氢吡啶二羧酸合酶(DHPS)是赖氨酸合成途径中的两个关键酶,赖氨酸酮戊二酸还原酶／酵母氨酸脱氢酶(LKR/SDH)是其降解途径中最重要的酶。在前期研究中,为通过基因工程技术调控水稻胚乳中赖氨酸的代谢,创新高赖氨酸水稻种质,本实验室培育了大量的转基因水稻材料,包括在水稻中过表达对赖氨酸反馈抑制不敏感的大肠杆菌AK和DHPS基因、抑制水稻内源LKR/SDH基因表达、同时过表达AK和DHPS基因及抑制LKR/SDH表达等。不同转基因水稻事件中对于赖氨酸含量的提高幅度存在明显差异。此外,对于赖氨酸含量稻米的营养价值、田间表型以及代谢关联效应等也不十分明了。本研究重点以粳稻品种武香粳9号及其来源的5个转基因水稻新品系为材料,从分子鉴定、品质分析、田间表现、营养评价、代谢组与转录组比较、以及代谢关联等多个方面开展了研究,以期进一步对高赖氨酸转基因水稻开展系统的评价分析,并为优化代谢调控途径以培育营养改良型作物提供依据。所用的5个转基因水稻分别为：(1)35S-15转基因系,含有由CaMV 35S启动子驱动的大肠杆菌AK和DHPS基因(简称为35S转基因事件)；(2)GR-14和GR-65转基因系,含有分别由水稻谷蛋白基因GluB-1和Gtl启动子(胚乳特异性表达)驱动的大肠杆菌AK和DHPS基因、以及由水稻Gtl启动子驱动的水稻LKR基因的RNAi结构(简称为GR转基因事件)；(3)HFL1和HFL2转基因系,由GR-14或GR-65分别与35S-15杂交并经自交产生的聚合转基因系,均聚合有35S和GR两类转基因事件。所有转基因水稻中都已去除了抗生素选择标记基因。开展的研究工作及取得的主要研究结果如下。1、高赖氨酸转基因水稻的分子鉴定、品质分析与田间表现从分子特征、理化与营养品质、田间表现等方面对5份转基因水稻新品系及其未转化对照(WT)进行了较为详细的比较分析,特别在2个聚合转基因系(HFL1和HFL2)与其双亲(35S和GR)间的效应作了系统比较。定量RT-PCR和Western blot分析显示,转基因水稻中外源AK和DHPS基因能高效表达,水稻内源LKR基因的表达则被有效抑制,但对水稻内源AK和DHPS基因的表达并未产生显著影响。氨基酸测定结果表明,在35S-15转基因水稻成熟籽粒中游离赖氨酸含量与野生型WT相比提高较少,只提升了35%；在GR-14和GR-65转基因成熟籽粒中游离赖氨酸含量显著提高,与WT相比分别提高了10倍和8倍；可喜的是,在聚合转基因系HFL1和HFL2成熟籽粒中游离赖氨酸含量进一步提高,与亲本WT相比分别提高了25倍和20倍。在游离赖氨酸提高的同时,HFL1和HFL2转基因成熟种子中的总游离氨基酸含量、总赖氨酸含量和总氨基酸含量也显著增加,蛋白质含量也有所提高。说明,聚合35S和GR两个转基因事件,可进一步促进赖氨酸在水稻籽粒中的累积。对稻米主要理化品质性状进行测定,结果显示含有较高赖氨酸的聚合转基因系HFL1和HFL2种子中的直链淀粉含量略有下降、胶稠度趋软,但对淀粉品质并未产生显著的影响。田间试验表明,除35S-15的株高稍低于野生型WT外,各转基因水稻的主要农艺性状与WT相比并无显著差异,产量也与WT相当。但随着赖氨酸在水稻籽粒中富集,两个HFL转基因水稻的部分籽粒呈现棕褐色表型。种子萌发试验显示,高赖氨酸转基因成熟种子萌发势要稍快于野生型,这完全不同于拟南芥和烟草等双子叶植物。在种子萌发早期高赖氨酸种子中淀粉水解速度要快于亲本对照,相应的淀粉酶活性也增强；但随着种子萌发和幼苗形态建,后期转基因水稻种子中淀粉水解、还原性糖积累以及相关水解酶活性与WT相比并没有显著的差异。2、高赖氨酸转基因稻米的营养评价为了进一步评价在水稻籽粒中提高赖氨酸含量后的营养价值,本研究以上述培育的2个聚合转基因系HFL1和HFL2及其未转化亲本WT大米为材料配制饲料,通过60天饲喂SD大鼠试验进行分析评估。在试验中还设置了野生型WT稻米添加不同剂量合成赖氨酸的饲料,用以比较并验证添加外源赖氨酸对提高稻米营养价值的作用。添加外源赖氨酸的剂量分别为在WT稻米原赖氨酸含量的基础上提高10%、20%、40%和60%,饲喂的4组SD鼠分别称为WT+10%Lys组、WT+20%Lys组、WT+40%Lys组和WT+60%Lys组。在60天喂养试验中,各组大鼠均表现正常,生命力活跃,无非正常死亡现象。食用含高赖氨酸转基因稻米的HFL1组和HFL2组SD大鼠在体重增长、食物利用率等生物性能上均显著优于食用含非转基因对照大米的WT组,与添加相似赖氨酸剂量的WT+20%Lys组相当。氮平衡试验结果显示,食用高赖氨酸转基因稻米的SD大鼠组的食物表观消化率、蛋白质功效比和赖氨酸利用效率都显著高于非转基因对照WT组,而与WT+20%Lys组相似。由此表明,转基因稻米中提高赖氨酸含量可显著提升其营养价值,且与外添相同剂量的野生型WT+20%Lys组具有相同的效应。试验结果同时还表明,在大米中添加赖氨酸的剂量并不是越多越好,以添加20-40%的剂量对提升稻米营养效率最为合适。此外,与非转基因WT组相比,食用转基因稻米组SD鼠的心、肝、脾等脏器重与体重的比值(脏体比)并未出现明显的差异,初步说明食用高赖氨酸转基因稻米并未对大鼠产生其它不良效应。3、高赖氨酸转基因水稻的代谢组与转录组分析植物中赖氨酸代谢与其它多个代谢途径相关联。根据上述研究,在转基因水稻中累积赖氨酸后会产生籽粒色泽变化等非预期效应。为此,本研究借助整合了正离子LC-MS/MS(液-质联用)、负离子LC-MS/MS和GC-MS(气-质联用)三种分析模式的高通量代谢分析技术,对上述5份不同转基因水稻叶片、发育籽粒及成熟种子的代谢谱进行分析；同时,通过RNA-Seq技术对叶片和发育籽粒中的转录谱进行比较分析,以期从物质代谢和分子水平进一步了解转基因调控赖氨酸代谢后可能的关联效应。对代谢组数据进行分析显示,转基因调控赖氨酸代谢关键酶表达对抽穗期叶片和发育籽粒中代谢物质种类与含量的影响极小,但不同转基因事件的效应有所差异。在由胚乳特异性启动子驱动的GR转基因系中,与亲本对照WT相比仅有1-2种差异代谢物质(p0.05)；在组成型表达外源AK和DHPS基因的35S-15转基因叶片和发育籽粒中,差异代谢物的数量较GR中的稍多。在转基因水稻成熟种子中,与亲本对照WT相比有显著含量差异(p0.05)的代谢物数量较多,尤其是赖氨酸积累多的聚合转基因系HFL1和HFL2。这些差异代谢物质主要为氨基酸类,其次是多肽类、脂类、核苷酸类、碳水化合物等。取各转基因水稻开花后10天或15天发育籽粒,对其总RNA进行RNA-Seq分析。结果显示,与未转化对照WT相比,GR-65和HFL2两个转基因水稻中差异表达基因数量较多,而35S-15、GR-14和HFL1三个转基因系中的差异表达基因相对较少,这可能是由于GR-65与GR-14不同独立转基因事件间差异引起的。通过GO富集和KEGG通路分析,发现虽然不同转基因事件间差异表达基因数量不同,但这些差异表达基因主要在细胞代谢过程、初级代谢过程、生物合成和小分子代谢过程等活动中显著富集。以KEGG数据库作为参考,将差异表达基因归入到不同的代谢通路中,显示它们主要集中在氨基酸代谢、植物胁迫响应、脂类代谢、碳水化合物代谢等途径中。此外,分析结果还显示转基因水稻花后15天发育籽粒中差异表达基因的数量要明显少于花后10天发育籽粒；而灌浆后期种子中的差异表达基因多数与胁迫响应有关。说明来自于天冬氨酸家族通路的赖氨酸代谢通路与多种代谢通路相关联,在植物代谢中具有多重角色。4、高赖氨酸转基因水稻褐色籽粒形成的代谢关联解析在赖氨酸富集的两个HFL转基因水稻籽粒中往往会出现棕褐色的表型。为深入探析其形成的原因,本研究对相关的代谢物及其关联途径进行详细的分析与验证。对不同发育时期籽粒中赖氨酸含量及籽粒色泽进行分析,表明褐色表型与赖氨酸的积累高度相关。综合代谢组和转录组的数据分析,发现在含有棕褐色表型的种子中,赖氨酸代谢、TCA循环和糖酵解、芳香族氨基酸及相关次生代谢、糖水化合物代谢、嘌呤和嘧啶代谢、以及脂类代谢等通路上发生或多或少的更改。结果显示,棕褐色种子中特异性地积累了5-羟色胺和色胺等色氨酸代谢途径中的相关代谢物。从转基因水稻HFL籽粒中提取褐色物质进行HPLC-MS分析,发现其中含有大量的色胺和5-羟色胺；RT-PCR分析也证实色氨酸代谢相关基因TDC(编码色氨酸脱羧酶)和T5H(编码5-羟色胺合酶)表达显著上调。为进一步证实褐色物质是否是由5-羟色胺等引起的,在转基因水稻中过表达TDC1和TDC3基因,结果在转基因的愈伤组织及种子中都有大量色胺和5-羟色胺积累,并同样可以引起褐色表型。由此说明,高赖氨转基因水稻种子的褐色成分主要是由5-羟色胺和色胺等引起的。在高等植物中,色氨酸与赖氨酸两个代谢通路间相距较远,其间的关联还不清楚。为此,经对含褐色成分的种子与正常色泽种-子等进进代谢谱的特异性比较、并结合转录酱比较,显示茉莉酸等大量与植物胁迫响应有关的代谢物及其基因表达水平增加,推测在高赖氨酸转基因水稻中由于赖氨酸累积,增强了茉莉酸途径并诱导了TDC等基因的表因,从而造成5-羟色胺和色胺的积累。进一步用定量RT-PCR等进一步证实了上述推测。此外,又通过在灌浆期设置不同温度处理,显示棕褐色表型受低温诱导,高温条件下充实的种子中几乎没有棕褐色表型。综合上述分析,推断调控赖氨酸代谢通路使籽粒中赖氨酸有效累积,从而提高了植物肋迫响应相关途径的活性,进而诱导色氨酸代谢并引起5-羟色胺和色胺积累,最终产生褐色表型。
[Abstract]:Rice is rich in starch and high quality protein, which is the main source of human energy and protein. However, the lack of lysine in rice is known as the first restrictive essential amino acid, which affects the nutritional value of rice. The metabolic regulation mechanism of lysine in plants is complex, and its accumulation is not only inhibited by its own feedback, but also related to degradation metabolism, but also with others. Many metabolic pathways are associated. Among them, aspartic kinase (AK) and two hydropyridine two carboxylic synthase (DHPS) are the two key enzymes in the lysine synthesis pathway. Lysine diacid reductase / yeast paramaric dehydrogenase (LKR/SDH) is the most important enzyme in its degradation pathway. In the previous study, rice embryos were regulated by genetic engineering technology. The metabolism of lysine in milk, innovation of high lysine rice germplasm, a large number of transgenic rice materials have been cultivated in our laboratory, including overexpressing the genes of Escherichia coli AK and DHPS insensitive to lysine feedback inhibition, inhibiting the expression of LKR/SDH gene in rice endogenous, and overexpressing AK and DHPS genes and inhibiting the expression of LKR/SDH. There are obvious differences in the increase of lysine content in the transgenic rice events. In addition, the nutritional value of lysine content, the field phenotype and the metabolic association effect are not very clear. This study focuses on the molecular identification of the japonica rice variety Wuxiang japonica 9 and the 5 new transgenic rice lines from its source. Quality analysis, field performance, nutritional evaluation, comparison of metabolic and transcriptional groups, and metabolic associations were studied in order to further evaluate the system of transgenic rice with high lysine, and to provide the basis for optimizing the metabolic regulation way to cultivate the nutritive crops. 5 transgenic rice were used respectively. (1) the 35S-15 transgenic line contains the Escherichia coli AK and DHPS gene driven by the CaMV 35S promoter (the 35S transgenic event); (2) the GR-14 and GR-65 transgenic lines contain the AK and DHPS genes, which are driven by the rice glutenin gene GluB-1 and Gtl promoters (the endosperm specific expression), and are driven by the rice promoter. The RNAi structure of rice LKR gene (referred to as GR transgenic event); (3) HFL1 and HFL2 transgenic lines, both GR-14 or GR-65 were hybridized with 35S-15 and produced by self crossbred transgenic lines, all of which were polymerized with 35S and GR two types of transgenic events. All transgenic rice had been carried out in addition to the antibiotic selection marker gene. The main results were as follows:.1, molecular identification of transgenic rice with high lysine, quality analysis and field performance from molecular characteristics, physicochemical and nutritional quality, field performance and other aspects of 5 transgenic rice lines and their unconverted control (WT), especially in 2 polymerized transgenic lines (HFL1 and HF). L2) compared with their parents (35S and GR), quantitative RT-PCR and Western blot analysis showed that the expression of exogenous AK and DHPS gene in transgenic rice could be highly expressed, and the expression of endogenous LKR gene in rice was effectively suppressed, but the expression of endogenous AK and DHPS genes in rice was not significantly affected. The results of amino acid determination showed that in 3 The content of free lysine in the mature grain of 5S-15 transgenic rice was less than that of wild type WT, only increased by 35%. The content of free lysine in GR-14 and GR-65 transgenic mature grains increased by 10 times and 8 times respectively compared with WT, and the gratifying lysine was free lysine in the mature grains of HFL1 and HFL2 of the polymerized transgenic line. The content of total free amino acids in HFL1 and HFL2 transgenic seeds, total lysine content and total amino acid content also increased significantly, and protein content was also higher in HFL1 and HFL2 transgenic seeds. Two transgenic events of polymerization of 35S and GR were available. The accumulation of lysine in rice grain was further promoted. The main physicochemical properties of rice were measured. The results showed that the amylose content in the HFL1 and HFL2 seeds containing high lysine decreased slightly, the gel consistency was softer, but the starch quality was not significantly affected. Field experiments showed that 35S-15 The main agronomic traits of the transgenic rice were not significantly different from that of the WT, and the yield was similar to that of WT, but with the enrichment of the lysine in the rice grain, some grains of the two HFL transgenic rice were brown phenotypes. The seed germination test showed that the seed germination of the transgenic rice with high lysine was slightly more than that of the WT. It is faster than the wild type, which is completely different from the dicotyledonous plants such as Arabidopsis and tobacco. In the early seed germination, the hydrolysis rate of starch in the high lysine seeds is faster than the parent control, and the activity of the amylase is also enhanced. But with the seed germination and the seedling morphology, the starch hydrolysis, the reduction sugar accumulation and the phase in the later transgenic rice seeds are in the form of the seed germination and the seedling morphology. There was no significant difference between the activity of the hydrolytic enzyme and the WT.2. The nutritional evaluation of the high lysine transgenic rice was used to further evaluate the nutritional value of the lysine content in the rice grain. This study was made up of 2 polymerized transgenic lines HFL1 and HFL2 and their unconverted parent WT rice for 60 days. The experiment of feeding SD rats was analyzed and evaluated. In the experiment, the feed of wild type WT rice added with different doses of lysine was added to compare and verify the effect of adding exogenous lysine on the nutritional value of rice. The dosage of exogenous lysine was increased by 10%, 20%, 40%, respectively, on the basis of the content of prolysine in rice. And 60%, the 4 groups of SD rats were called group WT+10%Lys, group WT+20%Lys, group WT+40%Lys and WT+60%Lys. In the 60 day feeding test, the rats were all normal, active and no normal death. HFL1 and HFL2 group SD rats with high lysine genetically modified rice and SD rats of HFL2 group were on the biological properties of body weight, food utilization and so on. The WT group with non GM control rice was significantly better than the WT+20%Lys group adding similar lysine dosage. The results of nitrogen balance test showed that the food apparent digestibility, protein efficiency ratio and lysine utilization efficiency of the SD rats with high lysine transgenic rice were significantly higher than those of the non transgenic control WT group, but with WT+ The 20%Lys group was similar. It showed that the increase of lysine content in transgenic rice could significantly increase its nutritional value, and the same effect was found in the wild type WT+20%Lys group with the same dosage. The results also showed that the dosage of lysine added in rice was not the more the better, and the dosage of 20-40% was added to the rice battalion. In addition, compared with the non transgenic WT group, there was no significant difference in the ratio of body weight to body weight (dirty body ratio) in the heart, liver, spleen and other organs of SD mice of the transgenic rice group. It was preliminarily indicated that the transgenic rice rice with high lysine transgenic rice did not produce other adverse effects.3, and the metabolic group of high lysine transgenic rice was the same as that of the transgenic rice. The transcriptional analysis of lysine metabolism in plants is associated with many other metabolic pathways. According to the above studies, the accumulation of lysine in transgenic rice produces non expected effects on grain color changes. For this reason, three analytical models for the integration of positive ion LC-MS/MS (liquid chromatograph), negative ion LC-MS/MS and GC-MS (gas mass combination) The metabolic profiles of 5 different transgenic rice leaves, developmental grains and mature seeds were analyzed by high throughput metabolic analysis. At the same time, the transcriptional spectrum of leaves and developmental grains was compared by RNA-Seq technology, in order to further understand the metabolism of lysine after metabolism and molecular water level. A possible correlation effect. Analysis of the metabolic data showed that the expression of key enzymes of lysine metabolism had little effect on the types and contents of metabolic substances in the leaves and grains at the heading stage, but the effects of different transgenic events were different. In the GR transgenic line driven by the endosperm specific promoter, it was with the parent. There were only 1-2 different metabolic substances (P0.05) compared to WT; the number of differential metabolites was a little more than that in GR in the 35S-15 transgenic leaves and developing grains expressing the exogenous AK and DHPS genes. In the mature seeds of transgenic rice, there were more significant amounts of metabolites than those of parental control WT (P0.05). HFL1 and HFL2., which have accumulated more amino acids, are mainly amino acids, followed by polypeptides, lipids, nucleotides, carbohydrates, etc., and take 10 days or 15 days after the flowering of the transgenic rice to develop a grain for RNA-Seq analysis of its total RNA. The results show that GR-65 and HFL2 two compared with unconverted WT. There are a large number of differentially expressed genes in transgenic rice, while the differentially expressed genes in the three transgenic lines of 35S-15, GR-14 and HFL1 are relatively small. This may be due to the difference between the different transgene events of GR-65 and GR-14. Through GO enrichment and KEGG pathway analysis, the number of differentially expressed genes between different transgenic events is found. However, these differentially expressed genes are mainly enriched in cellular metabolic processes, primary metabolic processes, biosynthesis and small molecular metabolic processes. Using KEGG database as a reference, differentially expressed genes are classified into different metabolic pathways, showing that they are mainly concentrated in amino acid metabolism, plant stress response, lipid generation. In addition, the results also showed that the number of differentially expressed genes in the 15 days after the transgenic rice flower was significantly less than that in the 10 day after the flower, and most of the differentially expressed genes in the late grain filling were related to the stress response. Metabolic pathways are associated with a variety of metabolic pathways and have multiple roles in plant metabolism,.4, and the metabolic Association of brown grain formation in high lysine transgenic rice tends to appear brown in the two HFL transgenic rice grains enriched with lysine. The contents of lysine and grain color of grains in different developmental periods were analyzed to show that the brown phenotype was closely related to the accumulation of lysine. The data analysis of the comprehensive metabolic and transcriptional groups showed that lysine metabolism, TCA cycle and glycolysis in the seeds containing brown phenotypes were found. Solutions, aromatic amino acids and related secondary metabolism, metabolism of sugar water compounds, purine and pyrimidine metabolism, and lipid metabolism are more or less altered. The results show that the metabolites of 5- hydroxytryptamine and tryptamine are specifically accumulated in brown seeds. Extract from the HFL grain of transgenic rice. HPLC-MS analysis showed a large number of tryptamine and 5- serotonin; RT-PCR analysis also confirmed that the expression of tryptophan metabolism related gene TDC (coded Tryptophan Decarboxylase) and T5H (coded 5- serotonin synthase) was significantly up-regulated. It was further confirmed whether the brown substance was caused by 5- serotonin and so on. In transgenic rice Overexpression of TDC1 and TDC3 genes results in the accumulation of a large number of tryptamine and 5- serotonin in the transgenic callus and seeds, and also can cause Brown phenotypes. Thus, the brown components of the seeds of high lysine transgenic rice are mainly caused by 5- serotonin and tryptamine. In higher plants, two generations of tryptophan and lysine are used in higher plants. The relationship between the metabolic pathways is far away and the correlation between them is not clear. For this reason, by comparing the specificity of the brown components with the normal color seed and the seeds, the metabolites related to the plant stress response, such as jasmonic acid and the expression level of the plant stress response, are increased. The accumulation of lysine in rice enhanced jasmonic acid pathway and induced the appearance of TDC and other genes, resulting in the accumulation of 5- serotonin and tryptamine.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511
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本文编号：2128235
[Abstract]:Rice is rich in starch and high quality protein, which is the main source of human energy and protein. However, the lack of lysine in rice is known as the first restrictive essential amino acid, which affects the nutritional value of rice. The metabolic regulation mechanism of lysine in plants is complex, and its accumulation is not only inhibited by its own feedback, but also related to degradation metabolism, but also with others. Many metabolic pathways are associated. Among them, aspartic kinase (AK) and two hydropyridine two carboxylic synthase (DHPS) are the two key enzymes in the lysine synthesis pathway. Lysine diacid reductase / yeast paramaric dehydrogenase (LKR/SDH) is the most important enzyme in its degradation pathway. In the previous study, rice embryos were regulated by genetic engineering technology. The metabolism of lysine in milk, innovation of high lysine rice germplasm, a large number of transgenic rice materials have been cultivated in our laboratory, including overexpressing the genes of Escherichia coli AK and DHPS insensitive to lysine feedback inhibition, inhibiting the expression of LKR/SDH gene in rice endogenous, and overexpressing AK and DHPS genes and inhibiting the expression of LKR/SDH. There are obvious differences in the increase of lysine content in the transgenic rice events. In addition, the nutritional value of lysine content, the field phenotype and the metabolic association effect are not very clear. This study focuses on the molecular identification of the japonica rice variety Wuxiang japonica 9 and the 5 new transgenic rice lines from its source. Quality analysis, field performance, nutritional evaluation, comparison of metabolic and transcriptional groups, and metabolic associations were studied in order to further evaluate the system of transgenic rice with high lysine, and to provide the basis for optimizing the metabolic regulation way to cultivate the nutritive crops. 5 transgenic rice were used respectively. (1) the 35S-15 transgenic line contains the Escherichia coli AK and DHPS gene driven by the CaMV 35S promoter (the 35S transgenic event); (2) the GR-14 and GR-65 transgenic lines contain the AK and DHPS genes, which are driven by the rice glutenin gene GluB-1 and Gtl promoters (the endosperm specific expression), and are driven by the rice promoter. The RNAi structure of rice LKR gene (referred to as GR transgenic event); (3) HFL1 and HFL2 transgenic lines, both GR-14 or GR-65 were hybridized with 35S-15 and produced by self crossbred transgenic lines, all of which were polymerized with 35S and GR two types of transgenic events. All transgenic rice had been carried out in addition to the antibiotic selection marker gene. The main results were as follows:.1, molecular identification of transgenic rice with high lysine, quality analysis and field performance from molecular characteristics, physicochemical and nutritional quality, field performance and other aspects of 5 transgenic rice lines and their unconverted control (WT), especially in 2 polymerized transgenic lines (HFL1 and HF). L2) compared with their parents (35S and GR), quantitative RT-PCR and Western blot analysis showed that the expression of exogenous AK and DHPS gene in transgenic rice could be highly expressed, and the expression of endogenous LKR gene in rice was effectively suppressed, but the expression of endogenous AK and DHPS genes in rice was not significantly affected. The results of amino acid determination showed that in 3 The content of free lysine in the mature grain of 5S-15 transgenic rice was less than that of wild type WT, only increased by 35%. The content of free lysine in GR-14 and GR-65 transgenic mature grains increased by 10 times and 8 times respectively compared with WT, and the gratifying lysine was free lysine in the mature grains of HFL1 and HFL2 of the polymerized transgenic line. The content of total free amino acids in HFL1 and HFL2 transgenic seeds, total lysine content and total amino acid content also increased significantly, and protein content was also higher in HFL1 and HFL2 transgenic seeds. Two transgenic events of polymerization of 35S and GR were available. The accumulation of lysine in rice grain was further promoted. The main physicochemical properties of rice were measured. The results showed that the amylose content in the HFL1 and HFL2 seeds containing high lysine decreased slightly, the gel consistency was softer, but the starch quality was not significantly affected. Field experiments showed that 35S-15 The main agronomic traits of the transgenic rice were not significantly different from that of the WT, and the yield was similar to that of WT, but with the enrichment of the lysine in the rice grain, some grains of the two HFL transgenic rice were brown phenotypes. The seed germination test showed that the seed germination of the transgenic rice with high lysine was slightly more than that of the WT. It is faster than the wild type, which is completely different from the dicotyledonous plants such as Arabidopsis and tobacco. In the early seed germination, the hydrolysis rate of starch in the high lysine seeds is faster than the parent control, and the activity of the amylase is also enhanced. But with the seed germination and the seedling morphology, the starch hydrolysis, the reduction sugar accumulation and the phase in the later transgenic rice seeds are in the form of the seed germination and the seedling morphology. There was no significant difference between the activity of the hydrolytic enzyme and the WT.2. The nutritional evaluation of the high lysine transgenic rice was used to further evaluate the nutritional value of the lysine content in the rice grain. This study was made up of 2 polymerized transgenic lines HFL1 and HFL2 and their unconverted parent WT rice for 60 days. The experiment of feeding SD rats was analyzed and evaluated. In the experiment, the feed of wild type WT rice added with different doses of lysine was added to compare and verify the effect of adding exogenous lysine on the nutritional value of rice. The dosage of exogenous lysine was increased by 10%, 20%, 40%, respectively, on the basis of the content of prolysine in rice. And 60%, the 4 groups of SD rats were called group WT+10%Lys, group WT+20%Lys, group WT+40%Lys and WT+60%Lys. In the 60 day feeding test, the rats were all normal, active and no normal death. HFL1 and HFL2 group SD rats with high lysine genetically modified rice and SD rats of HFL2 group were on the biological properties of body weight, food utilization and so on. The WT group with non GM control rice was significantly better than the WT+20%Lys group adding similar lysine dosage. The results of nitrogen balance test showed that the food apparent digestibility, protein efficiency ratio and lysine utilization efficiency of the SD rats with high lysine transgenic rice were significantly higher than those of the non transgenic control WT group, but with WT+ The 20%Lys group was similar. It showed that the increase of lysine content in transgenic rice could significantly increase its nutritional value, and the same effect was found in the wild type WT+20%Lys group with the same dosage. The results also showed that the dosage of lysine added in rice was not the more the better, and the dosage of 20-40% was added to the rice battalion. In addition, compared with the non transgenic WT group, there was no significant difference in the ratio of body weight to body weight (dirty body ratio) in the heart, liver, spleen and other organs of SD mice of the transgenic rice group. It was preliminarily indicated that the transgenic rice rice with high lysine transgenic rice did not produce other adverse effects.3, and the metabolic group of high lysine transgenic rice was the same as that of the transgenic rice. The transcriptional analysis of lysine metabolism in plants is associated with many other metabolic pathways. According to the above studies, the accumulation of lysine in transgenic rice produces non expected effects on grain color changes. For this reason, three analytical models for the integration of positive ion LC-MS/MS (liquid chromatograph), negative ion LC-MS/MS and GC-MS (gas mass combination) The metabolic profiles of 5 different transgenic rice leaves, developmental grains and mature seeds were analyzed by high throughput metabolic analysis. At the same time, the transcriptional spectrum of leaves and developmental grains was compared by RNA-Seq technology, in order to further understand the metabolism of lysine after metabolism and molecular water level. A possible correlation effect. Analysis of the metabolic data showed that the expression of key enzymes of lysine metabolism had little effect on the types and contents of metabolic substances in the leaves and grains at the heading stage, but the effects of different transgenic events were different. In the GR transgenic line driven by the endosperm specific promoter, it was with the parent. There were only 1-2 different metabolic substances (P0.05) compared to WT; the number of differential metabolites was a little more than that in GR in the 35S-15 transgenic leaves and developing grains expressing the exogenous AK and DHPS genes. In the mature seeds of transgenic rice, there were more significant amounts of metabolites than those of parental control WT (P0.05). HFL1 and HFL2., which have accumulated more amino acids, are mainly amino acids, followed by polypeptides, lipids, nucleotides, carbohydrates, etc., and take 10 days or 15 days after the flowering of the transgenic rice to develop a grain for RNA-Seq analysis of its total RNA. The results show that GR-65 and HFL2 two compared with unconverted WT. There are a large number of differentially expressed genes in transgenic rice, while the differentially expressed genes in the three transgenic lines of 35S-15, GR-14 and HFL1 are relatively small. This may be due to the difference between the different transgene events of GR-65 and GR-14. Through GO enrichment and KEGG pathway analysis, the number of differentially expressed genes between different transgenic events is found. However, these differentially expressed genes are mainly enriched in cellular metabolic processes, primary metabolic processes, biosynthesis and small molecular metabolic processes. Using KEGG database as a reference, differentially expressed genes are classified into different metabolic pathways, showing that they are mainly concentrated in amino acid metabolism, plant stress response, lipid generation. In addition, the results also showed that the number of differentially expressed genes in the 15 days after the transgenic rice flower was significantly less than that in the 10 day after the flower, and most of the differentially expressed genes in the late grain filling were related to the stress response. Metabolic pathways are associated with a variety of metabolic pathways and have multiple roles in plant metabolism,.4, and the metabolic Association of brown grain formation in high lysine transgenic rice tends to appear brown in the two HFL transgenic rice grains enriched with lysine. The contents of lysine and grain color of grains in different developmental periods were analyzed to show that the brown phenotype was closely related to the accumulation of lysine. The data analysis of the comprehensive metabolic and transcriptional groups showed that lysine metabolism, TCA cycle and glycolysis in the seeds containing brown phenotypes were found. Solutions, aromatic amino acids and related secondary metabolism, metabolism of sugar water compounds, purine and pyrimidine metabolism, and lipid metabolism are more or less altered. The results show that the metabolites of 5- hydroxytryptamine and tryptamine are specifically accumulated in brown seeds. Extract from the HFL grain of transgenic rice. HPLC-MS analysis showed a large number of tryptamine and 5- serotonin; RT-PCR analysis also confirmed that the expression of tryptophan metabolism related gene TDC (coded Tryptophan Decarboxylase) and T5H (coded 5- serotonin synthase) was significantly up-regulated. It was further confirmed whether the brown substance was caused by 5- serotonin and so on. In transgenic rice Overexpression of TDC1 and TDC3 genes results in the accumulation of a large number of tryptamine and 5- serotonin in the transgenic callus and seeds, and also can cause Brown phenotypes. Thus, the brown components of the seeds of high lysine transgenic rice are mainly caused by 5- serotonin and tryptamine. In higher plants, two generations of tryptophan and lysine are used in higher plants. The relationship between the metabolic pathways is far away and the correlation between them is not clear. For this reason, by comparing the specificity of the brown components with the normal color seed and the seeds, the metabolites related to the plant stress response, such as jasmonic acid and the expression level of the plant stress response, are increased. The accumulation of lysine in rice enhanced jasmonic acid pathway and induced the appearance of TDC and other genes, resulting in the accumulation of 5- serotonin and tryptamine.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511
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本文编号：2128235
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