【作者】 付桂明；

【导师】 许杨；

【作者基本信息】 南昌大学 ， 食品科学， 2007， 博士

【摘要】 本文采用PCR分析方法对7种14株产桔霉素的红曲菌中合成桔霉素基因——pksCT的保守性进行了研究；运用DNA重组技术构建了红曲菌pksCT gene的置换型打靶载体pHD116；通过原生质体PEG介导和电击转化，利用pHD116敲除了橙色红曲菌(M．aurantiacus)AS3．4384中的pksCT gene，并对该基因缺失株的产毒、产色素等功能进行了分析研究。本文还对运用RecET重组系统对快速构建长同源臂置换型打靶载体的方法进行了初步探讨，为红曲菌基因打靶研究提供了一个快速构建长同源臂载体的途径。本文的主要研究结果如下：1、采用PCR分析方法，从14株红曲菌的基因组DNA中均扩增到pksCT基因的翻译起始区部分序列(668bp)和终止密码子区部分序列(591bp)，扩增产物的测序结果通过NCBI进行BLAST同源性分析，结果表明，本文中选用14个扩增产物的序列之间具有高度同源性，且分别与Genbank中紫色红曲菌中的pksCT gene启动子和终止密码子区部分序列一致。结果显示pksCT在14株产桔霉素红曲菌菌株中具有高度地保守性。2、以红曲菌中合成桔霉素基因——pksCT为靶基因，以高产桔霉素的橙色红曲菌(M．aurantiacus)AS3．4384的基因组DNA为模板，扩增靶基因pksCT的翻译起始区部分序列(65-733)A和终止密码子区部分序列(8349-8940)B为靶基因的同源臂序列。通过酶切、连接和转化等DNA重组技术，成功地构建了一个两端含pksCT gene同源臂序列、中间为潮霉素抗性基因的置换型打靶载体——pHD116。该载体中靶基因pksCT gene的同源臂序列，能靶向性精确地界定并敲除基因组上的靶基因，并通过潮霉素B抗性筛选靶基因缺失株。3、优化了红曲菌原生质体PEG介导和电击转化体系中原生质体的浓度以及打靶载体质粒DNA量的条件，并利用原生质体PEG介导转化和电击转化将置换型打靶载体pHD116转入橙色红曲菌(M．aurantiacus)AS3．4384中，敲除了靶基因pksCT gene，采用潮霉素抗性筛选和基因组DNA的PCR扩增法筛选，获得一株pksCT gene缺失株PHDS26，其桔霉素表达量比原始菌株降低了97.2％，同时红色色素和黄色色素的表达量分别提高了49.4％和28.8％。4、合成一对引物，分别在它们的5′端设计与pHD116上潮霉素B抗性基因和上pUC18载体同源的55bp短序列；以橙色红曲菌的基因组DNA为模板，PCR扩增pksCT gene两端外侧同源臂E、F为线性打靶DNA，电击转化到含RecET重组系统的大肠杆菌宿主菌YZ2005中，在RecET重组酶介导下，经过两次同源重组，分别以E和F(长度分别为2437、2694bp)置换pHD116中的同源臂A和B(长度分别为668、591bp)，快速构建了具有长同源臂E、F的打靶载体pHF08。更多还原

【Abstract】 In this paper, PCR analysis was used to research the conservatism of citrininbiosynthesis gene—pksCT in different Monascus species. Fourteen producingcitrinin Monascus spp. coming from seven Monascus species were used inexperiment. A pksCT-replacement vector pHD 116 was constructed to disrupt pksCTgene by the genetic engineering technique, and was transformed into M. aurantiacusAS 3.4384 by protoplast PEG transformation and electroporation transformation.Transformants were screened with hygromycin B resistance and PCR analysis, thenanalysis of the expression of the red pigments and citrinin were used to gain thepksCT-disrupted strain. A method to construct the replacement vector by RecETrecombination system was found, and it provided a fast way to construct thereplacement vector of gene-knockout in Monascus.The major findings were expounded as follows:1. Two DNA fragments from the transcriptional start region and the stop codonregion of pksCT, which are 669bp and 591bp, were gained by genomic PCR fromfourteen Monascus spp effectively. Results of NCBI BLAST showed that PCRproducts exhibited high identity with each other, and were in accordance with thepartial sequence of pksCT genein M. purpureus which was announced in Genbank. Itis suggested that pksCT gene is highly conserved in the fourteen citrinin-producingMonascus spp.2. PksCT was the targeting gene which is responsible for citrinin biosynthesis inMonascus. Homologous arm sequence fragments A and B from the transcriptionalstart region (65-733) and the stop codon region (8349-8940) of pksCT were gainedby genomic PCR from M.aurantiacus AS3.4384, which is a citrinin high-producingstrain. A pksCT-replacement vector (pHD116) was constructed by the DNArecombinant technique such as restriction enzyme cutting and ligase ligation andtransformation. A hygromycin resistance gene in the middle of homologous armsequence fragments A and B was used for the selection marker. The vector couldaim at the targeted gene pksCT on chromosome of Monascus accurately with Homologous arm sequence fragments. A hygromycin resistance gene was used toscreen target gene disruptants.3. The conditions of protoplast-PEG transformation and protoplast-electroporationwere optimized. The pksCT-replacement vector pHD116 was transformed intoprotoplast of M.aurantiacus AS3.4384 to disrupt pksCT. Then the methods ofhygromycin resistance and genomic-PCR were used to screen disruptants. ApksCT-disrupted strain PHDS26 was gained, whose capacity of producing citrininwas decreased by about 97.2％compared with the original strain. At same time, itscapacities of producing red pigments and yellow pigments were increased by 49.4％and 28.8％4. Two short homologous sequences (55bp), which were homologous to hph geneand pUC18 vector respectively, were designed on the 5’end of primers. PCRfragments E and F from exterior sequences of pksCT, were transformed intoEscherichia coli YZ2005 by electroporation in turn. When homologousrecombinations happened twice with the catalysis of homologous recombinationenzyme RecE and RecT, fragments E (2437bp) and F (2694bp) were replaced thehomologous fragments A(668bp ) and B (591bp) in pHD116. A newpksCT-replacement vector pHF08 with longer homologous sequences E and F wasreconstructed.更多还原