家蠶吡哆醛激酶和磷酸吡哆醇氧化酶基因的RNA干擾研究
發(fā)布時間：2018-10-05 07:40
【摘要】：PLP是主要輔酶形式,參與體內(nèi)氨基酸、糖原、神經(jīng)遞質(zhì)、鞘磷脂、血紅素和核酸的合成與代謝。由于PLP的缺乏會引起嚴(yán)重的神經(jīng)系統(tǒng)疾病,PLP濃度過高又會產(chǎn)生毒性作用,導(dǎo)致感覺神經(jīng)和運動出現(xiàn)問題,因此PLP的合成調(diào)節(jié)及平衡供給是VB6營養(yǎng)核心問題。酶的調(diào)節(jié)一般從結(jié)構(gòu)水平和合成水平兩個方面進行研究,目前PLP合成酶結(jié)構(gòu)水平研究已經(jīng)透徹,但是合成調(diào)節(jié)如轉(zhuǎn)錄翻譯等方面研究有待深入。有研究發(fā)現(xiàn)過表PLP依賴酶,PLP含量隨之也增加,這提示PLP的合成酶和PLP依賴酶之間有聯(lián)動調(diào)節(jié)關(guān)系。家蠶是大型泌絲性昆蟲,蛋白質(zhì)代謝旺盛,PLP作為轉(zhuǎn)氨酶的輔酶,PLP需求量增加。為此本研究以家蠶為實驗材料,用RNAi的方法對PLK和PNPO基因進行干擾,用熒光定量的方法檢測PLK基因、PNPO基因及轉(zhuǎn)氨酶基因的轉(zhuǎn)錄水平表達變化,并用高通量測序的方法對RNAi處理后的家蠶絲腺組織進行基因轉(zhuǎn)錄組學(xué)分析,探討涉及PLP合成酶基因調(diào)控網(wǎng)絡(luò)。實驗結(jié)果如下:1.家蠶PLK和PNPO基因RNAi(1)PLK和PNPO基因在注射干擾片段si RNA 48 h后干擾效率最佳。(2)PLK最佳干擾片段是k-siRNA1下降了80%;PNPO最佳干擾片段是o-si RNA2下降了65%。(3)PLK和PNPO基因RNAi處理后,干擾效果最好的組織都是中腸組織,基因表達量分別下降了55%和52%。脂肪體中幾乎沒有干擾效率。2.家蠶PLK和PNPO基因RNAi后轉(zhuǎn)氨酶的表達量研究PLK基因RNAi處理后,絲腺中磷酸絲氨酸轉(zhuǎn)氨酶(phosphoserine aminotransferase,Ser B)和天門冬氨酸氨基轉(zhuǎn)移酶(asparate aminotransferase,AST)表達量分別下降了90%和29%;PNPO基因RNAi處理后,絲腺中SerB和AST基因表達量分別下降了28%和26%。3.家蠶PLK和PNPO基因RNAi后絲腺組織轉(zhuǎn)錄組學(xué)分析PLK基因RNAi處理后,篩選得到差異基因365個,其中基因下調(diào)177個,基因上調(diào)188個。對PLK基因處理組的差異基因進行GO富集,其中,有顯著的GOterms有150個(P≤0.05),包括39個分子功能(Molecular funtion),99個生物過程(Biological process)及12個細(xì)胞成分(Cell component)。對KEGG代謝通路分析發(fā)現(xiàn)有9條通路有富集現(xiàn)象,差異基因主要富集在內(nèi)質(zhì)網(wǎng)蛋白加工、次生物質(zhì)合成和累積及蛋白輸出通路中。PNPO基因RNAi處理后,篩選得到差異基因381個,其中基因下調(diào)基因260個,上調(diào)基因121個。對PNPO基因RNAi處理組的差異基因進行GO富集,分析發(fā)現(xiàn)具有顯著性富集的分析發(fā)現(xiàn)具有顯著性的Goterms有280個(P≤0.05),包括140個生物學(xué)過程、68個細(xì)胞組分和72個分子功能。對其KEGG代謝通路通路分析發(fā)現(xiàn)有10條通路有富集現(xiàn)象,差異基因主要富集在內(nèi)質(zhì)網(wǎng)蛋白加工、蛋白質(zhì)輸出、核糖體及氨酰生物合成通路中。本研究證明了家蠶PLP合成酶基因和轉(zhuǎn)氨酶基因存在聯(lián)動調(diào)節(jié),PLP合成酶RNAi干擾后影響到家蠶絲蛋白合成,為進一步分析PLP的動態(tài)平衡及VB6的代謝工作奠定基礎(chǔ),為研究人類VB6營養(yǎng)問題提供依據(jù)。
[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，
本文編號：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，
本文編號：2252561
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