论文总字数：18927字

摘 要

黑酵母是一类可以产生黑色素的酵母类真菌，其通常生长缓慢，对高盐、辐射、重金属等逆境的耐受性较高。本课题从中国新疆核爆区采集的土壤样品中分离出一种新的黑色酵母样菌株。在菌落形态和表型性质分析的基础上，结合ITS和LSU rDNA序列的比对，鉴定该分离菌株为出芽短梗（Aureobasidium）属的一个新型物种，并命名为A. pullulans sp. nov. F134.通过对其进行生物信息学分析，发现基因组中含有海藻糖代谢的关键酶基因--海藻糖-6-磷酸合酶基因tps1和酸性海藻糖酶基因ath1，进一步构建了工程菌株APtps1, AP∆ath1 和 APT∆A。实验结果显示，该原始菌株及其工程菌株对γ射线、紫外光和重金属离子（尤其是铅离子）显示出优异的抗性。γ射线辐射量达到15 kGyr时，F134及其工程菌株依然有存活，而大肠杆菌在辐射量还未达到5 kGyr时就已基本无存活；在紫外照射5 min后大肠杆菌基本无存活，而F134及其工程菌在照射20 min后仍然有存活；此外，野生型F134对Pb² 的耐受浓度达到1200 mg/L，突变菌株高达1500 mg/L。最后，对A. pullulans sp. nov. F134菌株中海藻糖积累的保护作用和机制进行了调查研究，为放射抗性微生物的应用研究提供新的视角。

关键词：耐辐射黑酵母 重金属 海藻糖 特性研究

Study on the response of black - yeast to heavy metal lead ion resistance

ABSTRACT

Black-yeast is the general term for a type of yeast fungus that produces melanin, which usually grows slowly and has a high resistance to high salinity, radiation, heavy metals and so on. In this study, a new black yeast-like strain was separated from the radiation-polluted soil which was obtained in Xinjiang, Uygur Autonomous Region of China. According to the analysis of colony morphology and phenotypic properties , associating with an comparison between the ITS and LSU rDNA sequences, this study reveals that the isolated strain represents a new species of the genus Aureobasidium, named as A. pullulans sp. nov. F134. Through the analysis of bioinformatics, the key genes of enzymes to synthesize and degrade trehalose were found--the trehalose-6-phosphate synthase gene tps1 and the acid trehalose gene ath1, and the engineering strains, APtps1, AP∆ath1 and APT∆A, were further built. The isolated strain, as well as its engineering strains, show excellent resistance to gamma irradiation, UV light, and heavy metal ions, especially to lead ion. Based on the experiments, when the dose of γ was 15 kGyr, F134 and its engineering strains still survived and E.coli was almost dead with the dose of 5 kGyr. After 2.5 min of UV exposure, E.coli was nearly no survival but F134 and its engineering strains still survived with UV exposure for 20 min. For the tolerance of Pb² , wild strain can grow with the concentration of 1200 mg/L and the engineering strains were up to 1500 mg/L. Finally, we investigated the protective effects and mechanism of intracellular trehalose accumulation in A. pullulans sp. nov. F134，which provided new viewpoints on the adaptations of radiation-resistant microorganisms.

Key Worlds: radioresistant black-yeast; heavy metals; trehalose; characterization

目 录

摘要 Ⅰ

ABSTRACT Ⅱ

目 录 Ⅲ

第一章 文献综述 1

1.1选题背景 1

1.2 重金属处理方法 1

1.3 吸附机理 4

1.4耐辐射黑酵母 4

1.5 海藻糖保护机理 5

1.5.1海藻糖简介 5

1.5.2 作用机制 5

1.6本项目的研究意义 6

第二章 耐辐射黑酵母的培养与鉴定 8

2.1实验材料 8

2.1.1菌种 8

2.1.2实验试剂 8

2.1.3实验仪器 9

2.1.4主要溶液与培养基 9

2.2实验方法 10

2.2.1菌株分离 10

2.2.2 形态学分析 10

2.2.3 系统发育树 10

2.2.4 海藻糖代谢途径研究 11

2.2.5 耐辐射测定 12

2.2.6 重金属耐受性研究 12

第三章 结果分析 13

3.1 耐辐射黑酵母的培养与鉴定 13

3.1.1 菌株筛选 13

3.1.2 形态学分析 13

3.1.3 分子学鉴定 15

3.2 菌株的特性探究 16

3.2.1 耐辐射 16

3.2.2 对重金属的耐受性 18

3.2.3 海藻糖的作用 18

第四章 结论与展望 20

4.1 结论 20

4.2 展望 20

参考文献 22

致谢 25

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