家蚕吡哆醛激酶和磷酸吡哆醇氧化酶基因的RNA干扰研究
发布时间：2018-10-05 07:40
【摘要】：PLP是主要辅酶形式,参与体内氨基酸、糖原、神经递质、鞘磷脂、血红素和核酸的合成与代谢。由于PLP的缺乏会引起严重的神经系统疾病,PLP浓度过高又会产生毒性作用,导致感觉神经和运动出现问题,因此PLP的合成调节及平衡供给是VB6营养核心问题。酶的调节一般从结构水平和合成水平两个方面进行研究,目前PLP合成酶结构水平研究已经透彻,但是合成调节如转录翻译等方面研究有待深入。有研究发现过表PLP依赖酶,PLP含量随之也增加,这提示PLP的合成酶和PLP依赖酶之间有联动调节关系。家蚕是大型泌丝性昆虫,蛋白质代谢旺盛,PLP作为转氨酶的辅酶,PLP需求量增加。为此本研究以家蚕为实验材料,用RNAi的方法对PLK和PNPO基因进行干扰,用荧光定量的方法检测PLK基因、PNPO基因及转氨酶基因的转录水平表达变化,并用高通量测序的方法对RNAi处理后的家蚕丝腺组织进行基因转录组学分析,探讨涉及PLP合成酶基因调控网络。实验结果如下:1.家蚕PLK和PNPO基因RNAi(1)PLK和PNPO基因在注射干扰片段si RNA 48 h后干扰效率最佳。(2)PLK最佳干扰片段是k-siRNA1下降了80%;PNPO最佳干扰片段是o-si RNA2下降了65%。(3)PLK和PNPO基因RNAi处理后,干扰效果最好的组织都是中肠组织,基因表达量分别下降了55%和52%。脂肪体中几乎没有干扰效率。2.家蚕PLK和PNPO基因RNAi后转氨酶的表达量研究PLK基因RNAi处理后,丝腺中磷酸丝氨酸转氨酶(phosphoserine aminotransferase,Ser B)和天门冬氨酸氨基转移酶(asparate aminotransferase,AST)表达量分别下降了90%和29%;PNPO基因RNAi处理后,丝腺中SerB和AST基因表达量分别下降了28%和26%。3.家蚕PLK和PNPO基因RNAi后丝腺组织转录组学分析PLK基因RNAi处理后,筛选得到差异基因365个,其中基因下调177个,基因上调188个。对PLK基因处理组的差异基因进行GO富集,其中,有显著的GOterms有150个(P≤0.05),包括39个分子功能(Molecular funtion),99个生物过程(Biological process)及12个细胞成分(Cell component)。对KEGG代谢通路分析发现有9条通路有富集现象,差异基因主要富集在内质网蛋白加工、次生物质合成和累积及蛋白输出通路中。PNPO基因RNAi处理后,筛选得到差异基因381个,其中基因下调基因260个,上调基因121个。对PNPO基因RNAi处理组的差异基因进行GO富集,分析发现具有显著性富集的分析发现具有显著性的Goterms有280个(P≤0.05),包括140个生物学过程、68个细胞组分和72个分子功能。对其KEGG代谢通路通路分析发现有10条通路有富集现象,差异基因主要富集在内质网蛋白加工、蛋白质输出、核糖体及氨酰生物合成通路中。本研究证明了家蚕PLP合成酶基因和转氨酶基因存在联动调节,PLP合成酶RNAi干扰后影响到家蚕丝蛋白合成,为进一步分析PLP的动态平衡及VB6的代谢工作奠定基础,为研究人类VB6营养问题提供依据。
[Abstract]:PLP is the main coenzyme form, involved in the synthesis and metabolism of amino acids, glycogen, neurotransmitters, sphingomyelin, heme and nucleic acid. The deficiency of PLP can lead to serious nervous system diseases, such as high concentration of PLP and toxic effect, resulting in sensory nerve and motor problems. Therefore, the synthesis regulation and balanced supply of PLP are the core problems of VB6 nutrition. The regulation of enzyme is generally studied from two aspects: structural level and synthesis level. At present, the structural level of PLP synthase has been thoroughly studied, but the study of synthetic regulation such as transcriptional translation needs to be further studied. It has been found that the content of PLP dependent enzymes increases, which suggests that there is a relationship between PLP synthase and PLP dependent enzymes. Silkworm (Bombyx mori) is a large filamentous insect, and the demand of PLP as a coenzyme for aminotransferase is increasing. In this study, silkworm (Bombyx mori) was used as experimental material, PLK and PNPO genes were interfered by RNAi method, and the transcriptional levels of PLK gene and transaminase gene were detected by fluorescence quantitative method. High throughput sequencing method was used to analyze the gene transcriptome of silkworm silk gland treated with RNAi, and to explore the gene regulatory network of PLP synthase. The results of the experiment are as follows: 1. The interference efficiency of PLK and PNPO RNAi (1) PLK and PNPO gene in Bombyx mori was the best after 48 h of si RNA injection. (2) the best interference fragment of PLK was that k-siRNA1 decreased 80% PNPO best interference fragment was o-si RNA2 decreased 65%. (3) PLK and PNPO gene RNAi treatment. The most effective tissues were midgut tissues, where gene expression decreased by 55% and 52%, respectively. The fat body has little interference with efficiency. 2. Expression of transaminase in silkworm PLK and PNPO after RNAi treatment with PLK gene RNAi, the expression of phosphoserine aminotransferase,Ser B and asparate aminotransferase,AST in silk gland decreased 90% and 29% after RNAi treatment, respectively. The expression of SerB and AST in the silk gland decreased by 28% and 26. 3%, respectively. Transcriptome analysis of silk gland after RNAi of PLK and PNPO genes in silkworm, 365 differentially expressed genes were screened after RNAi treatment with PLK gene, of which 177 genes were down-regulated and 188 genes were up-regulated. The differential genes of PLK gene treatment group were enriched by GO. Among them, there were 150 significant GOterms (P 鈮，
本文编号：2252561
[Abstract]:PLP is the main coenzyme form, involved in the synthesis and metabolism of amino acids, glycogen, neurotransmitters, sphingomyelin, heme and nucleic acid. The deficiency of PLP can lead to serious nervous system diseases, such as high concentration of PLP and toxic effect, resulting in sensory nerve and motor problems. Therefore, the synthesis regulation and balanced supply of PLP are the core problems of VB6 nutrition. The regulation of enzyme is generally studied from two aspects: structural level and synthesis level. At present, the structural level of PLP synthase has been thoroughly studied, but the study of synthetic regulation such as transcriptional translation needs to be further studied. It has been found that the content of PLP dependent enzymes increases, which suggests that there is a relationship between PLP synthase and PLP dependent enzymes. Silkworm (Bombyx mori) is a large filamentous insect, and the demand of PLP as a coenzyme for aminotransferase is increasing. In this study, silkworm (Bombyx mori) was used as experimental material, PLK and PNPO genes were interfered by RNAi method, and the transcriptional levels of PLK gene and transaminase gene were detected by fluorescence quantitative method. High throughput sequencing method was used to analyze the gene transcriptome of silkworm silk gland treated with RNAi, and to explore the gene regulatory network of PLP synthase. The results of the experiment are as follows: 1. The interference efficiency of PLK and PNPO RNAi (1) PLK and PNPO gene in Bombyx mori was the best after 48 h of si RNA injection. (2) the best interference fragment of PLK was that k-siRNA1 decreased 80% PNPO best interference fragment was o-si RNA2 decreased 65%. (3) PLK and PNPO gene RNAi treatment. The most effective tissues were midgut tissues, where gene expression decreased by 55% and 52%, respectively. The fat body has little interference with efficiency. 2. Expression of transaminase in silkworm PLK and PNPO after RNAi treatment with PLK gene RNAi, the expression of phosphoserine aminotransferase,Ser B and asparate aminotransferase,AST in silk gland decreased 90% and 29% after RNAi treatment, respectively. The expression of SerB and AST in the silk gland decreased by 28% and 26. 3%, respectively. Transcriptome analysis of silk gland after RNAi of PLK and PNPO genes in silkworm, 365 differentially expressed genes were screened after RNAi treatment with PLK gene, of which 177 genes were down-regulated and 188 genes were up-regulated. The differential genes of PLK gene treatment group were enriched by GO. Among them, there were 150 significant GOterms (P 鈮，
本文编号：2252561
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