论文总字数：32305字

摘 要

目前，全世界的农业生产都存在过度依赖化肥的现象，这进一步导致了土壤和水体的富营养化，从而对生态系统造成严重破坏。因此，开发新型肥料是解决这一系列问题的重要途经，而微生物肥料因其在促进作物生长，抑制病虫害，提高土壤肥力等方面上效果显著，具有广泛的应用前景，但是对其促生机制的研究还不够完善，所以本论文试图通过生物学分析手段进一步挖掘促生机理以及潜在的生物学特性。

本论文所分析的菌株NRCB010是课题实验室初步筛选的对植物具有较好促生效果的菌株，并于南京工业大学生化中心所保藏的施氏假单胞菌。因其良好的促生效果，本课题计划通过对其全基因组测序分析，并且通过KEGG、GO等多个数据库进行基因注释，挖掘该菌株中所含有的潜在的生物学特性。通过与相关数据库对比发现施氏假单胞菌NRCB010具有反硝化代谢的相关基因，铁载体，细菌素等次级代谢产物的基因簇，并具有阳离子抗菌肽（CAMP）和万古霉素在内的多种抗生素的抗性基因，并对一些植物促生相关的特性进行了进一步的试验验证，包括硝酸盐还原特性，铁载体产量,菌株抗旱抗盐性能以及对于不同类型磷源的溶解能力等性能进行了考量。通过实验证明，NRCB010具有较强的硝酸盐还原能力，能够产生铁载体，具有一定的抗旱和抗盐能力，对于无机磷酸钙和磷酸锌具有一定溶解能力，这些特性将有利于菌株NRCB010增强对环境的适应力和植物促生效果，为后续在农业生产的实际应用提供一定的理论依据。

关键词：施氏假单胞菌 全基因组 生物学分析 促生特性

Biological characteristics and genome-wide analysis of Pseudomonas stutzeri NRCB010

Abstract

At present, there is a phenomenon of over-reliance on chemical fertilizers in agricultural production all over the world, resulting in eutrophication of soils and water bodies, which in turn causes serious damage to ecosystems. Therefore, the Development and utilization of new types of fertilizers is an important way to solve these problems, and microbial fertilizers have a broad application prospect because of their remarkable effects in promoting crop growth, restraining pests and diseases, and improving soil fertility, however, the study of growth-promoting mechanism need more exploration, so this paper attempts to further explore the growth-promoting mechanism and potential biological characteristics through biological analysis.

The strain NRCB010 analyzed in this paper is a strain initially selected by laboratory and has a good growth promoting effect on the plant, and it is deposited as Pseudomonas stutzeri in National Engineering Research Center for Biotechnology. Due to its good growth-promoting effect, the project plans to analyze its whole genome and perform gene annotation through multiple databases such as KEGG,GO, etc. to discover its potential biological characteristics. Through comparison with related databases, it was found that Pseudomonas stutzeri NRCB010 has a string of gene clusters of secondary metabolites related to denitrification metabolism, iron carrier, bacteriocin, etc. and resistance genes of various antibiotics including cationic antimicrobial peptides (CAMP) and vancomycin. Some plant growth-promoting characteristics were further tested and verified, including nitrate reduction characteristics, iron carrier yield, drought and salt resistance, and the ability to dissolve different types of phosphorus sources. It is proved by experiments that NRCB010 has a great nitrate reducing ability, can produce iron carriers, has a certain drought and salt resistance and has a certain dissolving ability for inorganic calcium phosphate and zinc phosphate. These characteristics will help the strain NRCB010 to enhance the adaptability of various environment and plant growth promotion effect ,which provide a certain theoretical basis for the subsequent practical application in agricultural production.

Key words: Pseudomonas stutzeri；Whole genome；Biological analysis；Growth promoting properties

目录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1 引言 1

1.2植物促生菌的研究现状 1

1.3施氏假单胞菌的一般生物学特性以及促生机理的研究进展 2

1.3.1 固氮特性 2

1.3.2 铁载体代谢 3

1.3.3 生物降解特性 3

1.3.4 耐药特性 3

1.3.5 耐盐耐旱特性 3

1.4全基因组研究现状与应用 4

第二章 施氏假单胞菌NRCB010全基因组测序及分析 7

2.1实验材料 7

2.1.1测序菌株 7

2.1.2培养基制备 7

2.2实验方法和步骤 7

2.2.1 DNA提取 7

2.2.2 DNA质量检测 7

2.2.3基因组测序 8

2.2.4基因组拼接 8

2.2.5基因预测与注释 8

2.2.6重复序列分析及基因组圈图绘制 8

2.2.7适应性相关功能基因查找 9

2.2.8前噬菌体及其相关功能预测 9

2.2.9细菌次级代谢产物的预测 9

第三章 施氏假单胞菌NRCB010的生物学特性测定 10

3.1铁载体的测定 10

3.2硝酸盐还原实验 10

3.3细菌溶磷效果鉴定 11

3.4菌株耐盐能力的测定 12

3.5细菌的耐旱能力实验 12

第四章 实验结果与分析 13

4.1施氏假单胞菌NRCB010基因组测序质量分析 13

4.1.1 Illumina二代测序数据与数据质控 13

4.1.2 Pacbio三代测序数据质量分析 13

4.2施氏假单胞菌NRCB010基因组组装 14

4.3编码基因预测 14

4.4非编码RNA预测 14

4.5基因功能注释 14

4.5.1 KEGG数据库注释 15

4.5.2 COG数据库注释 17

4.5.3 GO数据库注释 18

4.6重复序列分析以及基因圈图展示 19

4.7适应性相关作用分析结果 21

4.8前噬菌体基因预测结果 24

4.9假单胞菌次级代谢产物合成基因簇预测及比较分析结果 27

4.10铁载体测定的实验结果 28

4.11硝酸盐还原能力测定实验结果 29

4.12溶磷实验的测定结果 29

4.13菌株耐盐平板实验结果 30

4.14菌株耐旱能力测定 31

第五章 实验结论与展望 32

5.1实验结论 32

5.2展望 32

参考文献 33

致 谢 37

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