【摘要】：小花棘豆是廣泛分布于內(nèi)蒙古荒漠化草原的毒草之一,牲畜采食導(dǎo)致中毒。致其中毒的有毒物質(zhì)為苦馬豆素,該物質(zhì)使牲畜細(xì)胞內(nèi)的甘露糖苷酶失去活性,導(dǎo)致神經(jīng)細(xì)胞空泡化,癥狀嚴(yán)重時(shí)造成死亡,給草原畜牧業(yè)帶來嚴(yán)重的經(jīng)濟(jì)損失�？囫R豆素是小花棘豆體內(nèi)寄生的Embellisia內(nèi)生真菌合成的次生代謝物,為了更好地治理和利用苦馬豆素,探索其在該內(nèi)生真菌的生化合成途徑成為了必要。2012年,美國(guó)的學(xué)者以其他產(chǎn)苦馬豆素的內(nèi)生真菌為材料,獲得酵母氨酸還原酶基因缺失突變株,測(cè)定苦馬豆素及其前體在其體內(nèi)的含量變化,推測(cè)出苦馬豆素部分合成途徑。本研究首次探索小花棘豆Embellisia內(nèi)生真菌原生質(zhì)體制備的最適條件;利用酵母氨酸還原酶基因敲除載體上的打靶目的片段轉(zhuǎn)化原生質(zhì)體,在含有潮霉素的原生質(zhì)體再生培養(yǎng)基上篩選出轉(zhuǎn)化子;根據(jù)酵母氨酸還原酶基因上游和下游序列和潮霉素磷酸轉(zhuǎn)移酶基因序列,設(shè)計(jì)兩對(duì)引物,分別擴(kuò)增野生株和轉(zhuǎn)化子基因組DNA,鑒定酵母氨酸還原酶基因缺失突變株。為下一步研究酵母氨酸還原酶基因的功能及其在苦馬豆素合成代謝中的作用奠定堅(jiān)實(shí)基礎(chǔ)。主要研究結(jié)果如下:1.探索了酶濃度、酶解溫度、酶解時(shí)間、滲透壓穩(wěn)定劑和真菌菌齡對(duì)小花棘豆Embellisia內(nèi)生真菌原生質(zhì)體制備的影響。選擇用1.2mol/L KCl穩(wěn)滲劑配制3%Driselase、1%Lysing enzymes和0.001%Chitinase三種酶的混合液,選擇10天菌齡的菌絲,于30℃,轉(zhuǎn)速為80rpm在恒溫振蕩器中振蕩酶解3h,收集原生質(zhì)體可達(dá)4.42?105個(gè)/ml;將原生質(zhì)稀釋后培養(yǎng)到原生質(zhì)體再生培養(yǎng)基中,于25℃培養(yǎng)10天,再生率達(dá)到52%。2.用PEG介導(dǎo)法,將酵母氨酸還原酶基因敲除載體上的目的片段轉(zhuǎn)化到原生質(zhì)體中,在含有潮霉素B的再生培養(yǎng)基上,25℃恒溫培養(yǎng)10天左右,長(zhǎng)出轉(zhuǎn)化子。野生株在含潮霉素B培養(yǎng)基上不生長(zhǎng)。轉(zhuǎn)化子菌落與野生株相比,菌絲體較致密、菌絲體生長(zhǎng)較快。3.用設(shè)計(jì)的兩對(duì)引物分別對(duì)野生株和轉(zhuǎn)化子基因組DNA進(jìn)行目的DNA擴(kuò)增,兩次PCR反應(yīng)中,野生株基因組DNA未擴(kuò)增出條帶,轉(zhuǎn)化子基因組DNA均擴(kuò)增出目的條帶,故鑒定獲得的轉(zhuǎn)化子為酵母氨酸還原酶基因缺失突變株。
[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，

本文編號(hào)：2432108