(论文 页数:20 字数:10965)摘要:淡紫拟青霉(Paecilomyces lilacinus)是一种重要的植物线虫寄生真菌，目前已在数个国家开发为生物杀线菌剂，广泛应用于根结线虫、胞囊线虫等植物寄生线虫的防治工作。在淡紫拟青霉菌剂的大规模高密度的发酵中，供氧成为提高产品效价的主要限制因素之一。透明颤菌血红蛋白（VHb）能提高细胞内氧传递效率，为解决供氧限制问题提供了一条新的思路。本研究经人工设计，构建出一种适合于淡紫拟青霉高效表达的透明颤菌血红蛋白（VHb）基因——vgbpl基因。拟借助VHb的作用，使淡紫拟青霉的发酵性能得到改善，在供氧不足时可以获得更高的生长速率，以提高发酵的速度和产品效价。
从Genebank中查找透明颤菌血红蛋白（VHb）的氨基酸序列；根据密码子的简并性以及淡紫拟青霉的密码子偏好性原则，人工设计出vgbpl基因的核甘酸序列；根据重叠PCR的引物设计原则，将核甘酸序列分成6条寡聚核苷酸片段；经重叠PCR，将合成的寡聚核苷酸片段组装成vgbpl基因。经酶切、连接后，将vgbpl基因克隆至真核表达载体pPIC9K中，而构建成重组质粒pPIC9K/vgbpl。重组质粒转化大肠杆菌(Escherichia coli )DH5α，采用菌落PCR法对重组子进行筛选，阳性重组子送上海英俊公司进行序列分析。序列分析和比对结果表明，我们获得了序列和阅读框正确的重组质粒pPIC9K/vgbpl。
总之，本研究根据透明颤菌血红蛋白（VHb）的氨基酸序列，人工设计出适合于淡紫拟青霉高效表达的vgbpl基因，通过重叠PCR成功构建出vgbpl基因，并构建了真核表达载体pPIC9K/vgbpl。这些工作为vgbpl基因转入淡紫拟青霉并实现VHb稳定而高效的表达奠定了重要基础。
关键词：淡紫拟青霉,透明颤菌血红蛋白,基因,人工设计,构建
Abstract
Paecilomyces lilacinus is an important fungal parasite of plant nematode eggs. Presently, these nematode-trapping fungi have been developed in a number of countries into biological control agents, which are widely used in root-knot nematode, cyst nematode, and other harmful plant nematodes prevention. In large-scale, high-density fermentation of Paecilomyces, oxygen supply had become one of the major limiting factors for production enhancement. Vitreoscilla hemoglobin (VHb) which can increase intracellular oxygen transfer efficiency has provided a new solution for the oxygen supply constraints. This study, by artificial design, constructs Vitreoscilla hemoglobin (VHb) gene --vgbpl Gene that is suitable for efficient expressing in Paecilomyces. VHb can be used to improve the fermentation performance of Paecilomyces, so that, in the absence of sufficient oxygen, a higher growth rate of Paecilomyces as well as a speeding up and product titer improvement of Paecilomyces fermentation can be observed.
The amino acid sequence of Vitreoscilla hemoglobin (VHb) was obtained from Genebank. The nucleotide sequence of vgbpl gene was artificially designed in accordance with the codon degeneracy and the codon preference of Paecilomyces. The nucleotide sequence was divided into six oligonucleotide fragments according to primer design principles of the overlapping PCR. vgbpl gene was assembled by overlapping PCR from the synthetic oligonucleotide fragments. After endonuclease digestion and ligase connection, vgbpl gene was cloned to the eukaryotic expression vector pPIC9K, forming a recombinant plasmid pPIC9K/vgbpl. Recombinant plasmid was transformed into Escherichia coli (E. coli) DH5α. Colony PCR was adopted to screen the positive recombinants, which was then sequenced by Invitrogen Biotechnology Co., Ltd (Shanghai). Sequence analysis and comparison of the sequencing results confirmed that we have acquired recombinant plasmid pPIC9K/vgbpl of the identical sequence with correct reading frame.
To conclude, in this study, according to the amino acid sequence of Vitreoscilla hemoglobin (VHb), vgbpl gene which is suitable for efficient expressing in Paecilomyces was artificially designed. vgbpl gene was then successfully constructed by overlapping PCR, and a eukaryotic expression vector pPIC9K/vgbpl was constructed. This work has laid a solid foundation for the transformation of vgbpl gene into Paecilomyces and for the stable and effective expression of vgbpl gene in Paecilomyces.
Keywords：Paecilomyces lilacinus；Vitreoscilla hemoglobin（VHb）；Gene；Artificial design；Construction
目录
中文摘要………………………………………………………………………………………………1
英文摘要………………………………………………………………………………………………1
前言……………………………………………………………………………………………………3
淡紫拟青霉及其在植物线虫防治的应用…………………………………………………………3
淡紫拟青霉菌剂的规模化生产与供氧问题………………………………………………………3
透明颤菌血红蛋白（VHb）和透明颤菌血红蛋白基因（vgb）……………………………………4
本课题研究目的和意义……………………………………………………………………………5
材料和方法……………………………………………………………………………………………6
材料…………………………………………………………………………………………………6
细菌菌株及质粒…………………………………………………………………………………6
主要试剂及耗材…………………………………………………………………………………6
主要仪器…………………………………………………………………………………………6
方法…………………………………………………………………………………………………7
vgbpl 基因的人工设计………………………………………………………………………… 7
vgbpl 基因的PCR构建…………………………………………………………………………7
PCR扩增vgbpl 基因……………………………………………………………………………9
重组质粒pPIC9K/vgbpl的构建与转化…………………………………………………………9
重组子的菌落PCR筛选与序列分析……………………………………………………………11
结果与分析……………………………………………………………………………………………12
vgbpl 基因的设计…………………………………………………………………………………12
vgbpl 基因的组装与扩增…………………………………………………………………………13
重组质粒pPIC9K/vgbpl 的构建、菌落PCR筛选与序列分析…………………………………14
讨论……………………………………………………………………………………………………15
参考文献………………………………………………………………………………………………17
1 前言
1.1 淡紫拟青霉及其在植物线虫防治的应用
淡紫拟青霉(Paecilomyces lilacinus (Thom.) Samson) 属半知菌纲、丝孢菌目、丝孢菌科、拟青霉属，是一种土壤及多种植物根围的习居菌。1974年Samson在秘鲁首先从根结线虫Meloidogyne sp.卵囊中分离得到该菌[1]，Jatala博士1979年首次发现该菌对南方根结线虫(Meloidogyne incognita (Kofoid and White) Chitwood) 卵的寄生率高达60%~70%[2]。现已认为淡紫拟青霉是一种非常重要的植物病原线虫天敌真菌，目前已广泛应用于根结线虫、孢囊线虫等有害植物线虫的防治实践中[3]。由于化学杀线虫剂不仅会对环境造成污染，并且在使用过程中对人、畜也不安全，而淡紫拟青霉作为一种生物试剂，在杀线过程中具有高效、安全、寄主广、持效长的特点[4][5]，许多试验表明其杀线活性与化学杀线剂相比较无明显差别，甚至超过化学杀线剂[6]，而且还能促进植物的生长[7]，达到增产的目的。
近年来，国内外纷纷筛选淡紫拟青霉菌优良菌株并进行开发利用。淡紫拟青霉商品菌剂已在菲律宾〔商品名“Biocon”〕、秘鲁、美国〔商品名为 “Melocon”〕、巴基斯坦、澳大利亚和德国等国家用于防治植物线虫病，效果显著。我国采用液体发酵技术工业化生产商品制剂，在黑龙江、云南、南京等地防治大豆抱囊线虫和南方根结线虫等均取得较好的效果。
|
