摘要：近年来，血红素加氧酶（heme oxygenase, HO）的生理作用得到了越来越广泛的关注。本文通过克隆灵芝的HO基因，并对其进行生物学特性分析，研究其功能。首先利用RNAi技术获得了HO基因的沉默载体HO-PET-32a，进一步进行转化子筛选获得HO基因沉默的灵芝突变菌株。通过比较相关表型，分析HO基因对灵芝酸合成的影响，发现在热胁迫条件下，HO基因沉默的灵芝菌株中三萜含量大幅度升高。此外，通过体外原核表达HO基因，并纯化得到了HO蛋白，有利于进一步探究它的作用机制。本研究发现HO基因在热激后显著影响灵芝酸的合成，对提高灵芝营养经济价值具有重要意义。82399

毕业论文关键词：血红素加氧酶；基因克隆；原核表达；灵芝；热胁迫

Cloning and functional analysis of heme oxygenase gene in Ganoderma lucidum

Abstract：In recent years, more and more attentions have been paid to heme oxygenase (HO)。 In this research, the biological characteristics of HO gene are cloned and analysed in Ganoderma lucidum。 Firstly, HO-PET-32a silent vector is constructed by using RNA interference, and HO gene silencing mutant of Ganoderma lucidum is obtained by screening transformants。Then, we analyse the effects of HO gene is analysed on the synthesis of ganoderic acid。 It was found that the content of triterpenoids in the mutants of Ganoderma lucidum was significantly increased under heat stress。 Finally, HO protein is expressed in prokaryotic expression in vitro and purified to further study the functions of HO gene in Ganoderma lucidum。 In this study, it is found that HO gene significantly affects the synthesis of ganoderic acid under heat shock。 It is of great significance to improve the nutritional value of Ganoderma lucidum。

Key words: heme oxygenase; gene clone; prokaryotic expression; Ganoderma lucidum; hot stress

目  录

摘要 1

关键词 1

Abstract 1

Key words 1

1  材料与方法 2

1．1  菌株和质粒 2

1．2  菌株的培养 3

1。2。1  培养基配制配方 3

1。2。2  种子培养 3

1。3  常用溶液 3

1。4  主要仪器 3

1。5  实验方法 3

1。5。1  碱裂解法提取质粒 3

1。5。2  HO基因沉默片段的PCR扩增 4

1。5。3  HO基因沉默片段、沉默载体的双酶切与回收 4

1。5。4  片段与载体的连接 4

1。5。5  大肠杆菌的转化 4

1。5。6  RNA提取 5

1。5。7  RNA中DNA的消化 5

1。5。8  灵芝cDNA的制备 5

1。5。9  实时荧光定量PCR 5

1。5。10  蛋白分离 6

1。5。11  热胁迫体系 6

1。5。12  三萜含量测定 6

2  结果与分析 7

2。1灵芝HO基因的克隆