【作者】 谢宝贵；

【导师】 郑金贵；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2004， 博士

【摘要】 银耳(Tremella fuciformis Berk．)又称白木耳，是一种已大面积人工栽培、被人们普遍接受的滋补食品。本研究应用基因工程技术，遗传转化高赖氨酸蛋白基因、人乳铁蛋白基因、人胰岛素基因和蜜蜂抗菌肽基因，以期进一步提高银耳的营养保健品质。 文献检索结果，从1976年至今，未见银耳遗传转化方面的研究报道。 1 建立了银耳遗传转化系统 研究出银耳遗传转化的标记基因：研究了银耳、大肠杆菌和农杆菌对13种抗生素的敏感性，筛选出银耳芽孢敏感的两种抗菌素——潮霉素、卡那霉素，确定潮霉素磷酸转移酶基因(hpt)、新霉素磷酸转移酶基因(nptII)作为银耳遗传转化的筛选标记基因；测定了银耳的β-半乳糖苷酶(lac z)和β-葡萄糖苷酸酶(GUS)的活性，确定GUS作为银耳遗传转化的报告基因。通过诱变，获得二个营养缺陷型菌株，其中一个是肌醇缺陷型，可应用于营养缺陷型标记的研究。 建立银耳原生质体制备与再生系统：应用正交设计优化银耳原生质体的制备条件，原生质体产量可达到2.75×10~7个／mL；对原生质体的再生条件进行研究，再生率最高可达32.3％。原生质体的浓度与再生率均达到作为遗传转化材料的要求。 研究出2种用原生质体转化的方法：即PEG介导和REMI转化，用20～30μg质粒DNA进行转化，这两种方法的转化率分别为1593和2140个转化子／μgDNA，用微量的DNA(50 ng)进行转化，其转化率分别达到266000和30000个转化子／μgDNA。 研究出3种用芽孢直接转化的方法：即LiAc转化法、超声波转化法和物化结合转化法，不需要制备原生质体，用带壁的细胞进行转化。7.5～30μg质粒DNA进行转化，上述三种方法的转化率分别为304、750和2688个转化子／μgDNA。用微量的质粒DNA(60 ng)进行超声波转化，转化率最高可达17000个转化子／μg DNA；本研究的最高转化率，与香菇(转化率为6个转化子／μg DNA)相比，提高了2833倍。 2 遗传转化三个品质基因 高赖氨酸蛋白基因(lys)：赖氨酸是人体必需氨基酸，也是以大米为主食人群的第一限制性氨基酸，具有重要的营养价值。应用REMI介导，高赖氨酸蛋白基因转化银耳，获得大量的抗性菌株，对26抗性菌株进行GUS活性检测，22个阳性、4个阴性；对17福建农林大学博士学位论文谢宝贵:摘要个抗性菌株的潮霉素抗性检测，在Zoou岁mL的浓度下都能生长:对8个抗性菌株进行PcR检测(包括lys基因、355启动子序列、GUs基因、hPt基因和Tnos终止子序列)，均为阳性.测定了7个菌株的氨基酸组成，与受体菌株(T认us 11)相比，赖氨酸含量提高16%~16.6%，有3个菌株的赖氨酸含量增幅超过巧%，其中几usll一16的增幅最高，达到16.6%。 人胰岛紊基因(BC减):胰岛素是糖尿病的特效药，目前主要从动物胰腺中提取，基因工程重组人胰岛素药物已开发成功，并在临床上应用，但生产成本较高.本研究合成了植物密码子偏好的人胰岛素基因(刀C减)，并从中克隆出人胰岛素A链、B链基因，构建了银耳表达载体，分别用REMI法、电击法和PEG法把上述三个基因导入银耳芽抱，都得到大量的抗性菌株，GUS检测了BCAZI株(18株阳性，3株阴性)、A15株(12株阳性，3株阴性)、B6株(5株阳性，1株阴性)，PcR检测(包括目的基因、Gus基因和Thos终止子序列)了BCA10株、A7株、B6株，均为阳性。 蜜蜂抗菌肚基因认尸):抗菌肤具有广谱抗菌活性、不会使病菌产生抗药性，可提高农产品的卫生品质，是一类很有前景的肤类抗生素，已引起科学家们的重视.本研究合成了蜜蜂抗菌肤基因，应用LIAc法和PEG法把该基因导入银耳芽抱，获得大量抗性菌株，GUS活性检测了19个抗性菌株，其中只有1个阴性，其它都为阳性。PCR检测(包括目的基因、GUS基因和Thos终止子序列)10个抗性菌株，均为阳性。测定了4个菌株对金黄色葡萄球菌的抑菌活性，AP一39菌体提取液的抑菌率为25.4%、培养液的抑菌率达到53.8%。 银耳的双型性(芽抱和菌丝体)赋予它具有原核生物和酵母菌的生长特点，又有高等生物分化发育过程.本论文研究出银耳的高效遗传转化技术，转化效率很高。因此，银耳可作为一种新的模式生物应用于生命科学研究。银耳是一种食药兼用的真菌，即可进行大规模人工栽培生产子实体，也可应用发酵技术培养芽抱营养体，不论是子实体还是芽抱都可以食用.银耳菌种生产是无性繁殖，后代不会发生分离。如果作为外源基因表达的生物反应器，基因产品的生产方式有两种选择，即种植子实体或发酵生产，它将促进基因工程产品的研发与生产。更多还原

【Abstract】 Tremella fuciformis Berk, was call as White Jelly Fungus or White Wood Ear. It was cultivated in large scales and was known as tonic food or health food. In order to increase nutrient and health value of T.fuciformis, three genes of high lysine protein and human insulin and honeybee (Apis mettiferd) antibiotic peptide were transformated respectively applying of genetic engineering technology.The result of retrieval, there have not been reported on transformation of this fungus since 1976.1 Establishing the Genetic Transformation System of T.fuciformis CanyOlmOlliaillBf BOneSOf BMMtemHlMIBfllin; Antibiotic sensitivity of T.fuciformis and E. coli and A. tumefaciens was studies. Hygromycin and kanamycin were the only two sensitivity antibiotic to T.fuciformis among 13 antibiotic tested. Hygromycin and neomycin phosphotransferase gene were established as select marker in genetic transformation of this fungus. Based on results of the activity of P -galactosidase(lac Z) and P -glucosidase(GUS) of T. fuciformis tested, GUS gene was established as report gene for genetic transformation. Two auxotroph were obtained by induction of mutation. One of them was inositol auxotrph, it could be developped and applied as auxotrophic marker after further research.Established a system of protoplast prepare and regeneration: The protoplast preparecondition was optimized applying the experiment schedule of orthogonal design, the yield of protoplast have got up to 2.75 X 107 /mL in the optimized condition. The regeneration condition of protoplast were studies, the highest regeneration rate was 32.3%. Protoplast yield and regeneration rate above were reach the requirement of protoplast genetic transformation.Research and development 2 methods fer T. fuciformis protoplast genetictransformation. These methods are PEG-mediated and REMI-mediated. The rate of transformation of PEG and REMI were 1593 and 2140 transformants/ug DNA respectively when used 20~30 ug plasmid DNA, the rate could be reach 266000 and 30000 transformants /ug DNA when used race plasmid DNA(50ng) respectively.Resoarch and development 3 nettads ftr T. fuciformis Intact cell oenetictransformation. These method were LiAc-mediated and ultrasonic-mediated and physics-chemical-mediated. These methods does not need to prepare protoplast, intact ceil can be transformated. The rate of the three methods above were 304 and 750 and 2688 transformants/ug DNA respectively when used 7.5 ~30 ug plasmid DNA, the rate could be reach 17000 transformants/ug DNA when used race plasmid DNA(60ng) by means of ultrasonic-mediated . This transformation rate was 2833 times to it of Lentinus edodes (6 transformants/ug DNA).2 Transformation three quality genes respectivelyHigh lyslnt CCntaln pratBin gene MysV. Lysine is essential amino acid for human body. It is the first limit amino acid of people who take rice as major food. Lysine have important nutrient value. Large amount of antibiotic resistant colony were obtained applying REMI-mediated to transformation lys gene. GUS activity of 26 antibiotic resistant strains were tested, 22 strains shown positive and 4 strains shown negative. The tolerance of antibiotic of 17 strains were tested, all of them could grow in the medium containing 200 ug/mL hygromycin; 8 strains were verified by PCR of lys gene, GUS gene, hpt gene, 35S promoter and Tnos sequence. The results show that all of them were positive. Amino acid contain of 7 trangene strains were determined, the increase of lysine contain ranged 3.6%~16.6% comparing to the host strain TauSl 1, there were 3 strains that lysine contain increase more than 15%, the strain of Tau811-1 was the highest increment rate, that was 16.6%.HdBian Insulin Ul|ne \BCA\\ Insulin is the specific medicine for diabetes. The major source for drug production was pancreas of animal now. It was success to production recombined drug by means of gene engineering, and this drug was bring to clinic, but the production cost was higher. Insulin gene (BCA) was synthesized with plant codon bias in this studies,更多还原