小花棘豆Embellisia内生真菌酵母氨酸还原酶基因缺失突变株的构建及筛选鉴定
发布时间：2019-02-28 19:35
【摘要】：小花棘豆是广泛分布于内蒙古荒漠化草原的毒草之一,牲畜采食导致中毒。致其中毒的有毒物质为苦马豆素,该物质使牲畜细胞内的甘露糖苷酶失去活性,导致神经细胞空泡化,症状严重时造成死亡,给草原畜牧业带来严重的经济损失。苦马豆素是小花棘豆体内寄生的Embellisia内生真菌合成的次生代谢物,为了更好地治理和利用苦马豆素,探索其在该内生真菌的生化合成途径成为了必要。2012年,美国的学者以其他产苦马豆素的内生真菌为材料,获得酵母氨酸还原酶基因缺失突变株,测定苦马豆素及其前体在其体内的含量变化,推测出苦马豆素部分合成途径。本研究首次探索小花棘豆Embellisia内生真菌原生质体制备的最适条件;利用酵母氨酸还原酶基因敲除载体上的打靶目的片段转化原生质体,在含有潮霉素的原生质体再生培养基上筛选出转化子;根据酵母氨酸还原酶基因上游和下游序列和潮霉素磷酸转移酶基因序列,设计两对引物,分别扩增野生株和转化子基因组DNA,鉴定酵母氨酸还原酶基因缺失突变株。为下一步研究酵母氨酸还原酶基因的功能及其在苦马豆素合成代谢中的作用奠定坚实基础。主要研究结果如下:1.探索了酶浓度、酶解温度、酶解时间、渗透压稳定剂和真菌菌龄对小花棘豆Embellisia内生真菌原生质体制备的影响。选择用1.2mol/L KCl稳渗剂配制3%Driselase、1%Lysing enzymes和0.001%Chitinase三种酶的混合液,选择10天菌龄的菌丝,于30℃,转速为80rpm在恒温振荡器中振荡酶解3h,收集原生质体可达4.42?105个/ml;将原生质稀释后培养到原生质体再生培养基中,于25℃培养10天,再生率达到52%。2.用PEG介导法,将酵母氨酸还原酶基因敲除载体上的目的片段转化到原生质体中,在含有潮霉素B的再生培养基上,25℃恒温培养10天左右,长出转化子。野生株在含潮霉素B培养基上不生长。转化子菌落与野生株相比,菌丝体较致密、菌丝体生长较快。3.用设计的两对引物分别对野生株和转化子基因组DNA进行目的DNA扩增,两次PCR反应中,野生株基因组DNA未扩增出条带,转化子基因组DNA均扩增出目的条带,故鉴定获得的转化子为酵母氨酸还原酶基因缺失突变株。
[Abstract]:Oxytropis vulgaris is one of the poisonous grass widely distributed in the desertification grassland of Inner Mongolia. The toxic substance is matrine, which causes mannosidase activity in livestock cells to be lost, leading to vacuolization of nerve cells, death when symptoms are serious, and serious economic losses to grassland animal husbandry. Sophorin is a secondary metabolite synthesized by endophytic fungi of Embellisia parasitized in Oxytropis vulgaris. In order to better control and utilize it, it is necessary to explore its biochemical synthesis pathway in this endophytic fungus. In 2012, it is necessary to explore the biochemical synthesis pathway of the endophytic fungus in order to better control and utilize it. Other endophytic fungi producing matrine were used as materials to obtain yeast reductase gene deletion mutants. The content changes of matrine and its precursors were determined in vivo, and the partial synthesis pathway of matrine was speculated. In this study, the optimum conditions for the preparation of protoplasts from endophytic fungi of Oxytropis alternata Embellisia were studied for the first time. The target fragment of yeast reductase gene knockout vector was used to transform protoplasts, and the transformants were screened on protoplast regeneration medium containing hygromycin. Based on the upstream and downstream sequences of the yeast reductase gene and the hygromycin phosphatase gene sequence, two pairs of primers were designed to amplify the genomic DNA, of the wild strain and the transformant to identify the yeast reductase gene deletion mutants. It lays a solid foundation for the further study of the function of yeast reductase gene and its role in the synthesis and metabolism of matrine. The main findings are as follows: 1. The effects of enzyme concentration, enzymolysis temperature, hydrolysis time, osmotic stabilizer and fungal age on protoplast preparation of endophytic fungi in Oxytropis minor bean Embellisia were investigated. The mixture of 3% Driselastic, 1% lysing enzymes and 0.001%Chitinase was prepared by using 1.2mol/L KCl stabilizer. The mycelium of 10 days old was selected and the enzymatic hydrolysis of 80rpm in a constant temperature oscillator for 3 hours was performed at 30 鈩，
本文编号：2432108
[Abstract]:Oxytropis vulgaris is one of the poisonous grass widely distributed in the desertification grassland of Inner Mongolia. The toxic substance is matrine, which causes mannosidase activity in livestock cells to be lost, leading to vacuolization of nerve cells, death when symptoms are serious, and serious economic losses to grassland animal husbandry. Sophorin is a secondary metabolite synthesized by endophytic fungi of Embellisia parasitized in Oxytropis vulgaris. In order to better control and utilize it, it is necessary to explore its biochemical synthesis pathway in this endophytic fungus. In 2012, it is necessary to explore the biochemical synthesis pathway of the endophytic fungus in order to better control and utilize it. Other endophytic fungi producing matrine were used as materials to obtain yeast reductase gene deletion mutants. The content changes of matrine and its precursors were determined in vivo, and the partial synthesis pathway of matrine was speculated. In this study, the optimum conditions for the preparation of protoplasts from endophytic fungi of Oxytropis alternata Embellisia were studied for the first time. The target fragment of yeast reductase gene knockout vector was used to transform protoplasts, and the transformants were screened on protoplast regeneration medium containing hygromycin. Based on the upstream and downstream sequences of the yeast reductase gene and the hygromycin phosphatase gene sequence, two pairs of primers were designed to amplify the genomic DNA, of the wild strain and the transformant to identify the yeast reductase gene deletion mutants. It lays a solid foundation for the further study of the function of yeast reductase gene and its role in the synthesis and metabolism of matrine. The main findings are as follows: 1. The effects of enzyme concentration, enzymolysis temperature, hydrolysis time, osmotic stabilizer and fungal age on protoplast preparation of endophytic fungi in Oxytropis minor bean Embellisia were investigated. The mixture of 3% Driselastic, 1% lysing enzymes and 0.001%Chitinase was prepared by using 1.2mol/L KCl stabilizer. The mycelium of 10 days old was selected and the enzymatic hydrolysis of 80rpm in a constant temperature oscillator for 3 hours was performed at 30 鈩，
本文编号：2432108
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