论文总字数：34338字

摘 要

有“生命之糖”称号的海藻糖，能赋予低等动物、植物和微生物等抵抗营养缺乏、高温、低温、干燥、高渗透压、有毒物质等恶劣环境的能力，在严酷环境下保护生物体的生物膜、脂质体、蛋白质、核酸等结构和功能的特殊功效，还能使细胞内保持湿润，防止细胞内各种养分的损失。本文对于如何从自然界中筛选出一株高产海藻糖酵母菌进行探讨，选择最利于海藻糖积累的培养基进行培养；实验通过诱变育种的方法，初筛，初步选出耐高渗透压的菌株，复筛，进行海藻糖含量测定，选出高产海藻糖的酵母菌；实验所用的是酿酒酵母菌，并通过分子鉴定的方法确定酵母菌的种类；之后，再对选出的这一株高产海藻糖酵母菌在高渗透压、PH、乙醇浓度以及温度等优化胁迫条件下进行培养。通过培养基成分优化、菌种优化以及培养条件优化三个方面，从而确定了如何筛选培育出一株高产海藻糖酵母菌的方法。

关键词：海藻糖 酵母 诱变 筛选 培养

Abstract

As we all know, trehalose can also be called " the sugar of life " , can give the normal animals, plants and microbial the ability of resistance to lack of nutrition, such as high temperature, low temperature, dry, high osmotic pressure, toxic substances and other bad environment, protect organisms from a harsh environment of biofilm, liposomes, such as protein, nucleic acid structure and function of special effect, still can keep moist inside the cell, prevent all kinds of nutrient losses in the cell.This article do researches on how to select a high-yielding trehalose yeast strains from the nature, select the most favorable medium to culture a high-yielding trehalose yeast; experiment through mutation breeding methods, screening, shortlisted strains which can be resistant to high osmotic pressure, rescreening, a high-yielding trehalose yeast strains can be selected; experiments use saccharomyces yeast, and yeast species can be identified by the methods of molecular identification; after then this selected strain of yeast trehalose yield can be cultured under conditions,such as high osmotic pressure, PH, different ethanol concentration and temperature. Through the medium component optimization, optimization, and optimization of culture conditions spawn three aspects to determine how to select and cultivate a high yield of trehalose yeast method.

Key words: trehalose yeast mutation select cultivation

目录

摘要..............................................................................................................................................................2

Abstract........................................................................................................................................................3

1. 文献综述........................................................................................................................................6

1.1 研究背景...................................................................................................................................6

1.1.1 研究意义........................................................................................................................6

1.1.2 作用机理........................................................................................................................7

1.1.3 国内外成果....................................................................................................................7

1.2 研究现状.................................................................................................................................11

1.2.1 制备工艺......................................................................................................................11

1.2.2 提取原理......................................................................................................................11

1.2.3 研究发现......................................................................................................................12

1.3 前景展望.................................................................................................................................12

1.3.1 关键科学问题..............................................................................................................12

1.3.2 研究目标......................................................................................................................13

第二章 高产海藻糖菌株的筛选............................................................................................................14

2.1 实验材料 .............................................................................................................................14

2.1.1 菌株与培养基................................................................................................................14

2.1.2 实验仪器 ......................................................................................................................14

2.1.3 实验试剂 ......................................................................................................................15

2.2实验方法 ................................................................................................................................15

2.2.1 初筛 ..............................................................................................................................15

2.2.2 复筛 ..............................................................................................................................15

2.3 酵母菌的BIOLOG鉴定 ......................................................................................................16

2.4 酵母菌的18s rDNA基因序列鉴定......................................................................................16

2.4.1 基因工程操作方法...................................................................................................... 16

2.4.2 酵母全基因组的提取.................................................................................................. 16

2.4.3 引物的设计与合成 .....................................................................................................16

2.4.4 PCR扩增反应...................................................................................................................17

2.5 结果讨论.....................................................................................................................................17

2.5.1 不同培养基对海藻糖积累的影响..................................................................................17

2.5.2 BIOLOG鉴定结果 .........................................................................................................17

2.5.3 18S rDNA菌株鉴定结果 ...............................................................................................18

2.5.4 培养条件的优化..............................................................................................................19

1. 热酿酒酵母NR11.........................................................................................................................21

3.1 前言.............................................................................................................................................21

3.2实验材料......................................................................................................................................22

3.2.1 菌株与培养基 ................................................................................................................22

3.2.2 实验仪器..........................................................................................................................22

3.2.3 主要试剂..........................................................................................................................23

3.2.4 工具酶、分子量标准和试剂盒....................................................................................23

3.2.5 引物序列_,和样品信息.....................................................................................................24

3.3 实验方法.....................................................................................................................................25

3.3.1 细胞培养方法 ................................................................................................................25

3.3.2 酵母胞内海藻糖的提取与含量测定 ............................................................................25

3.3.3 酵母RNA的提取..............................................................................................................25

3.3.4 RNA浓度及质量检测.......................................................................................................25

3.3.5 反转录..............................................................................................................................26

3.3.6荧光定量PCR检测...........................................................................................................26

3.4 结果与讨论...............................................................................................................................27

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