{"title":"纳豆芽孢杆菌富蛋氨酸与赖氨酸融合基因的构建与表达","authors":"Zhang Shuang , Luo Chao-chao , Wu Cai-xia , Gao Xue-jun","doi":"10.1016/S1006-8104(15)30028-3","DOIUrl":null,"url":null,"abstract":"<div><h3>Abstract</h3><p>Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. <em>Bacillus natto</em> has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene (<em>zein</em>) from maize endosperm protein with lysine-rich gene (<em>Cflr</em>) from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43<em>/zein-Cflr</em> was constructed. The recombinant plasmid was transferred into <em>Bacillus natto</em>, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined <em>Bacillus natto</em> as feed additive.</p></div>","PeriodicalId":58038,"journal":{"name":"Journal of Northeast Agricultural UniversityEnglish Edition","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2015-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1006-8104(15)30028-3","citationCount":"0","resultStr":"{\"title\":\"Construction and Expression of Methionine-rich and Lysine-rich Fusion Gene in Bacillus natto\",\"authors\":\"Zhang Shuang , Luo Chao-chao , Wu Cai-xia , Gao Xue-jun\",\"doi\":\"10.1016/S1006-8104(15)30028-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Abstract</h3><p>Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. <em>Bacillus natto</em> has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene (<em>zein</em>) from maize endosperm protein with lysine-rich gene (<em>Cflr</em>) from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43<em>/zein-Cflr</em> was constructed. The recombinant plasmid was transferred into <em>Bacillus natto</em>, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined <em>Bacillus natto</em> as feed additive.</p></div>\",\"PeriodicalId\":58038,\"journal\":{\"name\":\"Journal of Northeast Agricultural UniversityEnglish Edition\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2015-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/S1006-8104(15)30028-3\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Northeast Agricultural UniversityEnglish Edition\",\"FirstCategoryId\":\"91\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1006810415300283\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Northeast Agricultural UniversityEnglish Edition","FirstCategoryId":"91","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1006810415300283","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Construction and Expression of Methionine-rich and Lysine-rich Fusion Gene in Bacillus natto
Abstract
Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene (zein) from maize endosperm protein with lysine-rich gene (Cflr) from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.