高賴氨酸轉基因水稻的營養(yǎng)評價及代謝關聯(lián)研究
發(fā)布時間：2018-07-17 00:32
【摘要】：稻米富含淀粉和優(yōu)質蛋白,是人類主要的能量與蛋白來源。但稻米中缺乏賴氨酸,被稱為第一限制性必需氨基酸,影響稻米的營養(yǎng)價值。植物中賴氨酸的代謝調控機制較為復雜,其積累不僅受本身反饋抑制,還與降解代謝有關,而且與其它眾多代謝通路相關聯(lián)。其中天冬氨酸激酶(AK)和二氫吡啶二羧酸合酶(DHPS)是賴氨酸合成途徑中的兩個關鍵酶,賴氨酸酮戊二酸還原酶／酵母氨酸脫氫酶(LKR/SDH)是其降解途徑中最重要的酶。在前期研究中,為通過基因工程技術調控水稻胚乳中賴氨酸的代謝,創(chuàng)新高賴氨酸水稻種質,本實驗室培育了大量的轉基因水稻材料,包括在水稻中過表達對賴氨酸反饋抑制不敏感的大腸桿菌AK和DHPS基因、抑制水稻內源LKR/SDH基因表達、同時過表達AK和DHPS基因及抑制LKR/SDH表達等。不同轉基因水稻事件中對于賴氨酸含量的提高幅度存在明顯差異。此外,對于賴氨酸含量稻米的營養(yǎng)價值、田間表型以及代謝關聯(lián)效應等也不十分明了。本研究重點以粳稻品種武香粳9號及其來源的5個轉基因水稻新品系為材料,從分子鑒定、品質分析、田間表現(xiàn)、營養(yǎng)評價、代謝組與轉錄組比較、以及代謝關聯(lián)等多個方面開展了研究,以期進一步對高賴氨酸轉基因水稻開展系統(tǒng)的評價分析,并為優(yōu)化代謝調控途徑以培育營養(yǎng)改良型作物提供依據(jù)。所用的5個轉基因水稻分別為：(1)35S-15轉基因系,含有由CaMV 35S啟動子驅動的大腸桿菌AK和DHPS基因(簡稱為35S轉基因事件)；(2)GR-14和GR-65轉基因系,含有分別由水稻谷蛋白基因GluB-1和Gtl啟動子(胚乳特異性表達)驅動的大腸桿菌AK和DHPS基因、以及由水稻Gtl啟動子驅動的水稻LKR基因的RNAi結構(簡稱為GR轉基因事件)；(3)HFL1和HFL2轉基因系,由GR-14或GR-65分別與35S-15雜交并經自交產生的聚合轉基因系,均聚合有35S和GR兩類轉基因事件。所有轉基因水稻中都已去除了抗生素選擇標記基因。開展的研究工作及取得的主要研究結果如下。1、高賴氨酸轉基因水稻的分子鑒定、品質分析與田間表現(xiàn)從分子特征、理化與營養(yǎng)品質、田間表現(xiàn)等方面對5份轉基因水稻新品系及其未轉化對照(WT)進行了較為詳細的比較分析,特別在2個聚合轉基因系(HFL1和HFL2)與其雙親(35S和GR)間的效應作了系統(tǒng)比較。定量RT-PCR和Western blot分析顯示,轉基因水稻中外源AK和DHPS基因能高效表達,水稻內源LKR基因的表達則被有效抑制,但對水稻內源AK和DHPS基因的表達并未產生顯著影響。氨基酸測定結果表明,在35S-15轉基因水稻成熟籽粒中游離賴氨酸含量與野生型WT相比提高較少,只提升了35%；在GR-14和GR-65轉基因成熟籽粒中游離賴氨酸含量顯著提高,與WT相比分別提高了10倍和8倍；可喜的是,在聚合轉基因系HFL1和HFL2成熟籽粒中游離賴氨酸含量進一步提高,與親本W(wǎng)T相比分別提高了25倍和20倍。在游離賴氨酸提高的同時,HFL1和HFL2轉基因成熟種子中的總游離氨基酸含量、總賴氨酸含量和總氨基酸含量也顯著增加,蛋白質含量也有所提高。說明,聚合35S和GR兩個轉基因事件,可進一步促進賴氨酸在水稻籽粒中的累積。對稻米主要理化品質性狀進行測定,結果顯示含有較高賴氨酸的聚合轉基因系HFL1和HFL2種子中的直鏈淀粉含量略有下降、膠稠度趨軟,但對淀粉品質并未產生顯著的影響。田間試驗表明,除35S-15的株高稍低于野生型WT外,各轉基因水稻的主要農藝性狀與WT相比并無顯著差異,產量也與WT相當。但隨著賴氨酸在水稻籽粒中富集,兩個HFL轉基因水稻的部分籽粒呈現(xiàn)棕褐色表型。種子萌發(fā)試驗顯示,高賴氨酸轉基因成熟種子萌發(fā)勢要稍快于野生型,這完全不同于擬南芥和煙草等雙子葉植物。在種子萌發(fā)早期高賴氨酸種子中淀粉水解速度要快于親本對照,相應的淀粉酶活性也增強；但隨著種子萌發(fā)和幼苗形態(tài)建,后期轉基因水稻種子中淀粉水解、還原性糖積累以及相關水解酶活性與WT相比并沒有顯著的差異。2、高賴氨酸轉基因稻米的營養(yǎng)評價為了進一步評價在水稻籽粒中提高賴氨酸含量后的營養(yǎng)價值,本研究以上述培育的2個聚合轉基因系HFL1和HFL2及其未轉化親本W(wǎng)T大米為材料配制飼料,通過60天飼喂SD大鼠試驗進行分析評估。在試驗中還設置了野生型WT稻米添加不同劑量合成賴氨酸的飼料,用以比較并驗證添加外源賴氨酸對提高稻米營養(yǎng)價值的作用。添加外源賴氨酸的劑量分別為在WT稻米原賴氨酸含量的基礎上提高10%、20%、40%和60%,飼喂的4組SD鼠分別稱為WT+10%Lys組、WT+20%Lys組、WT+40%Lys組和WT+60%Lys組。在60天喂養(yǎng)試驗中,各組大鼠均表現(xiàn)正常,生命力活躍,無非正常死亡現(xiàn)象。食用含高賴氨酸轉基因稻米的HFL1組和HFL2組SD大鼠在體重增長、食物利用率等生物性能上均顯著優(yōu)于食用含非轉基因對照大米的WT組,與添加相似賴氨酸劑量的WT+20%Lys組相當。氮平衡試驗結果顯示,食用高賴氨酸轉基因稻米的SD大鼠組的食物表觀消化率、蛋白質功效比和賴氨酸利用效率都顯著高于非轉基因對照WT組,而與WT+20%Lys組相似。由此表明,轉基因稻米中提高賴氨酸含量可顯著提升其營養(yǎng)價值,且與外添相同劑量的野生型WT+20%Lys組具有相同的效應。試驗結果同時還表明,在大米中添加賴氨酸的劑量并不是越多越好,以添加20-40%的劑量對提升稻米營養(yǎng)效率最為合適。此外,與非轉基因WT組相比,食用轉基因稻米組SD鼠的心、肝、脾等臟器重與體重的比值(臟體比)并未出現(xiàn)明顯的差異,初步說明食用高賴氨酸轉基因稻米并未對大鼠產生其它不良效應。3、高賴氨酸轉基因水稻的代謝組與轉錄組分析植物中賴氨酸代謝與其它多個代謝途徑相關聯(lián)。根據(jù)上述研究,在轉基因水稻中累積賴氨酸后會產生籽粒色澤變化等非預期效應。為此,本研究借助整合了正離子LC-MS/MS(液-質聯(lián)用)、負離子LC-MS/MS和GC-MS(氣-質聯(lián)用)三種分析模式的高通量代謝分析技術,對上述5份不同轉基因水稻葉片、發(fā)育籽粒及成熟種子的代謝譜進行分析；同時,通過RNA-Seq技術對葉片和發(fā)育籽粒中的轉錄譜進行比較分析,以期從物質代謝和分子水平進一步了解轉基因調控賴氨酸代謝后可能的關聯(lián)效應。對代謝組數(shù)據(jù)進行分析顯示,轉基因調控賴氨酸代謝關鍵酶表達對抽穗期葉片和發(fā)育籽粒中代謝物質種類與含量的影響極小,但不同轉基因事件的效應有所差異。在由胚乳特異性啟動子驅動的GR轉基因系中,與親本對照WT相比僅有1-2種差異代謝物質(p0.05)；在組成型表達外源AK和DHPS基因的35S-15轉基因葉片和發(fā)育籽粒中,差異代謝物的數(shù)量較GR中的稍多。在轉基因水稻成熟種子中,與親本對照WT相比有顯著含量差異(p0.05)的代謝物數(shù)量較多,尤其是賴氨酸積累多的聚合轉基因系HFL1和HFL2。這些差異代謝物質主要為氨基酸類,其次是多肽類、脂類、核苷酸類、碳水化合物等。取各轉基因水稻開花后10天或15天發(fā)育籽粒,對其總RNA進行RNA-Seq分析。結果顯示,與未轉化對照WT相比,GR-65和HFL2兩個轉基因水稻中差異表達基因數(shù)量較多,而35S-15、GR-14和HFL1三個轉基因系中的差異表達基因相對較少,這可能是由于GR-65與GR-14不同獨立轉基因事件間差異引起的。通過GO富集和KEGG通路分析,發(fā)現(xiàn)雖然不同轉基因事件間差異表達基因數(shù)量不同,但這些差異表達基因主要在細胞代謝過程、初級代謝過程、生物合成和小分子代謝過程等活動中顯著富集。以KEGG數(shù)據(jù)庫作為參考,將差異表達基因歸入到不同的代謝通路中,顯示它們主要集中在氨基酸代謝、植物脅迫響應、脂類代謝、碳水化合物代謝等途徑中。此外,分析結果還顯示轉基因水稻花后15天發(fā)育籽粒中差異表達基因的數(shù)量要明顯少于花后10天發(fā)育籽粒；而灌漿后期種子中的差異表達基因多數(shù)與脅迫響應有關。說明來自于天冬氨酸家族通路的賴氨酸代謝通路與多種代謝通路相關聯(lián),在植物代謝中具有多重角色。4、高賴氨酸轉基因水稻褐色籽粒形成的代謝關聯(lián)解析在賴氨酸富集的兩個HFL轉基因水稻籽粒中往往會出現(xiàn)棕褐色的表型。為深入探析其形成的原因,本研究對相關的代謝物及其關聯(lián)途徑進行詳細的分析與驗證。對不同發(fā)育時期籽粒中賴氨酸含量及籽粒色澤進行分析,表明褐色表型與賴氨酸的積累高度相關。綜合代謝組和轉錄組的數(shù)據(jù)分析,發(fā)現(xiàn)在含有棕褐色表型的種子中,賴氨酸代謝、TCA循環(huán)和糖酵解、芳香族氨基酸及相關次生代謝、糖水化合物代謝、嘌呤和嘧啶代謝、以及脂類代謝等通路上發(fā)生或多或少的更改。結果顯示,棕褐色種子中特異性地積累了5-羥色胺和色胺等色氨酸代謝途徑中的相關代謝物。從轉基因水稻HFL籽粒中提取褐色物質進行HPLC-MS分析,發(fā)現(xiàn)其中含有大量的色胺和5-羥色胺；RT-PCR分析也證實色氨酸代謝相關基因TDC(編碼色氨酸脫羧酶)和T5H(編碼5-羥色胺合酶)表達顯著上調。為進一步證實褐色物質是否是由5-羥色胺等引起的,在轉基因水稻中過表達TDC1和TDC3基因,結果在轉基因的愈傷組織及種子中都有大量色胺和5-羥色胺積累,并同樣可以引起褐色表型。由此說明,高賴氨轉基因水稻種子的褐色成分主要是由5-羥色胺和色胺等引起的。在高等植物中,色氨酸與賴氨酸兩個代謝通路間相距較遠,其間的關聯(lián)還不清楚。為此,經對含褐色成分的種子與正常色澤種-子等進進代謝譜的特異性比較、并結合轉錄醬比較,顯示茉莉酸等大量與植物脅迫響應有關的代謝物及其基因表達水平增加,推測在高賴氨酸轉基因水稻中由于賴氨酸累積,增強了茉莉酸途徑并誘導了TDC等基因的表因,從而造成5-羥色胺和色胺的積累。進一步用定量RT-PCR等進一步證實了上述推測。此外,又通過在灌漿期設置不同溫度處理,顯示棕褐色表型受低溫誘導,高溫條件下充實的種子中幾乎沒有棕褐色表型。綜合上述分析,推斷調控賴氨酸代謝通路使籽粒中賴氨酸有效累積,從而提高了植物肋迫響應相關途徑的活性,進而誘導色氨酸代謝并引起5-羥色胺和色胺積累,最終產生褐色表型。
[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.
【學位授予單位】：揚州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S511
，
本文編號：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.
【學位授予單位】：揚州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S511
，
本文編號：2128235
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