洛伐他汀产生菌原生质体融合育种.docx

广东药学院硕士研究生学位论文中文摘要中文摘要 洛伐他汀（ lavostatin）是 HMG- CoA（3-羟基-3-甲基-戊二酰辅酶 A）还原酶抑制剂（HMG- CoA -RI）类微生物药物，它可以有效的降低冠心病和心肌梗塞的发病率和死亡率近年来虽然有很多相关洛伐他汀（lavostatin）基因工程育种研究的报道，，但是由于洛伐他汀生产菌土曲霉和红曲霉的遗传背景不清楚，在实际中都还难以应用而原生质体融合技术有具有重组频率高、可省去遗传标记、避免基因分离、纯化、酶切和拼接操作等优点，这对遗传背景不清楚的土曲霉和红曲霉的育种来说显得格外重要不同代谢产物产生菌的融合子，有可能将它们的部分生物基因整合在一起产生新的杂合代谢产物，达到与用基因工程方法实现组合生物合成的相同目的 洛伐他汀主要用土曲霉（ aspergillus terreus）和红曲霉（ monascus rubber）经深层发酵制取，红曲霉（ monascus rubber）不仅产生洛伐他汀（也称莫那可林 K，monacolin K），而且产生据推测为洛伐他汀生物合成的中间产物的聚酮类物质莫那可林 J、L、X，这些物质都是 HMG-CoA-RI 类物质,具有很强的降低胆固醇、调节血脂的功能。

而且红曲霉在我国有着广泛的应用，在酿造红酒、制腐乳、肉制品着色及制备中药方面的应用已有悠久的历史，其调节血脂的功能在许多国家都得到认同由于其发酵产品可直接用作食品添加剂，因此有着更广的应用面，并且易被人们接受同时土曲霉是现在国内生产洛伐他汀的原始出发菌，它具有优良的发酵性能、较高的洛伐他汀产生能力、成熟的发酵工艺本项目将通过土曲霉和红曲霉原生质体融合技术，将土曲霉生产菌优良的发酵性能、较高的洛伐他汀产生能力、成熟的发酵工艺和红曲霉他汀类化合物的结构多样性、色素产生能力以及长期实践证明的安全性结合起来，选育他汀类化合物和色素高产菌株，以及全新他汀类物质的产生菌在理论上探讨用原生质体融合技术实现组合生物合成的可能性与途径，为实际生产选育目的化合物稳定、高产菌株在本论文中研究考察了各种细胞裂解酶和渗透压稳定剂等条件对红曲霉和土曲霉的原生质体制备、灭活及融合的影响，同时采用不同的方法对红曲霉和土曲霉的 ＤＮＡ 进行提取纯化，探讨了提取液中不同的 ＥＤＴＡ 浓度对 ＤＮＡ 提取和 ＲＡＰＤ－ＰＣＲ的影响，同时采用 ２７ 条随机引物分别对土曲霉、红曲霉和融合子的 ＤＮＡ 进行ＲＡＰＤ－ＰＣＲ，扩增产物用 ５％的聚丙烯酰胺电泳的进行分离，并采用紫外分光光度I广东药学院硕士研究生学位论文法对土曲霉、红曲霉和融合子 ２ 的发酵液进行 Lovastatin 含量测定。

结果表明：土曲霉和红曲霉在加石英砂混合酶体系中获得的原生质体数最多，而最适宜的渗透压稳定剂为 0.6 mol/L 的 NaCL红曲霉和土曲霉的原生质体分别在紫外灯（ 18W）下 15cm 照射 50 min 和 60oC 水浴下保持 40 min 灭活率达到 100%最适合红曲霉和土曲霉的原生质体融合的条件为 20% PEG 在 28oC 下处理 15-20 min采用氯化苄的方法能提到完整的 DNA，２０ｍＭ 的 ＥＤＴＡ 浓度能得到较好的 ＤＮＡ 模板和稳定的 ＲＡＰＤ－ＰＣＲ 条带，ＲＡＰＤ－ＰＣＲ 结果显示融合子 ２ 号在红曲霉和土曲霉的原生质体融合时发生基因重组，并能稳定遗传Ｌｏｖａｓｔａｔｉｎ 在 ２４６ｎｍ 波长处有最大的吸收，融合子 ２ 产洛伐他汀的能力不及由原始出发菌土曲霉但是比红曲霉的能力由了一定的提高从而为构建一株高产量的菌株做好基础关键词：Lovastatin；红曲霉；土曲霉；融合子；RAPDII广东药学院硕士研究生学位论文The Studies on Protoplast fusion and breeding in Lovastatin-producing FungusLIN Xiaobo Biological Pathogens Supervisor: LI Haoming HUAN Shulin ProfessorLovastatin is the HMG- CoA microbe medicine,it is a very valid to coronary arterydisease and myocardial infarction.There are many reports about the lovastatin of thegene engineering breeding in this years,but because the genetic background of aspergillusterreus and monascus rubber is not clear,So it can’t be apply in the practice. The technologyof protoplast fusion has many merits such as to have hypsi-ecombination frequency, to omitgenetic mark and to avoid gene isolation,purification,digested and splicing, it is very important tothe aspergillus terreus and monascus rubber that the genetic background is not clear.Thedifferent metabolic product of fusant can produce a new heterozygosis metabolic product to utilizegene integration, With the same useing of genetic engineering methods to achieve the samebiosynthetic purpose.The lovastatin of preparation use the aspergillus terreus and the monascus rubber ofsubmerged fermentation,The monascus rubber is not only to produce the lovastatin(monacolin K) but also to produce the monacolin J、L、X that is the intermediateproduct of the biosynthetic lovastatin,this material all is the HMG-CoA-RI materialthat have the Strong function to lower cholesterol and regulate blood lipid. Themonascus rubber have general application in china,it has a long history in the brewingred wine,making Chinese cheese, coloring meat and preparing of traditional chinesemedicine,the function of regulating blood fat is to been accept by manycountries.Because the Fermentation products can been directly used for foodadditives,so it can been widely applied and accept by people. The aspergillus terreusis the original starting strain to produce the lovastatin in china,it has many goodqualitiy such as the good fermentation performance,the high producing Lovastatincapacity and the maturity zymotechnique.The experiment use the technology ofprotoplast fusion to Combine the merits of the aspergillus terreus and the monascusi广东药学院硕士研究生学位论文rubber, In theory it explore the possibility and means of biosynthesis to use thetechnique of protoplast fusion for breedingthe stable compounds and high- yieldstrains.The article study and research various cell lysase and osmotic pressure stab to theinfluence of the Monascus rubber and Aspergillus terreus that of the protoplastformation、inactivation and fusion. Use the different method to extract and pur ify theDNA of the Monascus rubber and Aspergillus terreus,study the different EDTAconcentration to the DNA extraction and RAPD-PCR of influence.Furthermore,use 27random primers to the DNA of Monascus rubber、Aspergillus terreus and protoplastfusion progeny for RAPD-PCR,the PCR products were separated by 5%polyacrylamide electrophoresis.Use UVspectrophotometry to determinthefermentation broth of Aspergillus terreus、the Monascus rubber and the protoplastfusion progeny 2 for Lovastatin.The result showed: the Monascus rubber and Aspergillus terreus in mixture enzymesystem with grit have the most protoplast number, The most suitable of osmoticpressure stab is 0.6 mol/l NaCL.The protoplast of the Monascus rubber andAspergillus terreus in the ultraviolet lamp(18W, 15cm, 50min) and 60oCwaterbath(40min) all are inactivation.The most suitable of protoplast fusion conditionis that 20% PEG handle 15-20 mi。