摘 要

交替氧化酶（AOX）是交替呼吸途径末端氧化酶，广泛存在于高等植物中。研究表明AOX对于植物的生长发育及抗逆性有重要的作用。在逆境条件下，细胞色素呼吸途径受到抑制，植物通过加强交替呼吸来消除过剩的电子从而避免植物受到损伤。木兰科鹅掌楸（Liriodendron chinense）为珍贵用材及园林观赏树种，挖掘鹅掌楸适应性相关基因，对于提高鹅掌楸适应性有重要意义。本文对实验前期已获得的AOX1基因编码蛋白进行生物信息学分析，利用qRT-PCR方法研究鹅掌楸AOX1基因的组织表达模式，并采用双酶切的方法构建鹅掌楸AOX1基因超表达载体。主要研究结果如下：

鹅掌楸AOX1基因cDNA全长1220bp，其中开放阅读框（ORF）长1032bp，编码343个氨基酸。对AOX1基因编码蛋白进行生物信息学分析可知：AOX1蛋白为亲水性蛋白，存在两个跨膜区，亚细胞定位预测该蛋白位于线粒体中，同源性分析发现AOX1基因表达蛋白C端保守性极强，系统发育分析结果表明该蛋白为AOX1亚家族成员，且与番茄和葡萄的亲缘关系较近。

鹅掌楸AOX1基因在不同组织中表达量差异显著。基因表达量由高到低依次为雄蕊、花瓣、花萼、叶片、雌蕊、花芽、叶芽、茎。其中，雄蕊中的表达量最高，是叶片表达量的2.7倍；而茎中的表达量最低，仅有叶片表达量的40%。

以pCAMIA1302为骨架，利用双酶切法构建鹅掌楸AOX1基因超表达载体，通过测序检验，确定重组质粒正向插入且无变异位点，为进一步开展转基因功能验证奠定基础。

关键词：鹅掌楸；AOX1基因；序列分析；定量表达；过表达载体构建

Quantitative expression analysis and construction of over expression vector of AOX1 Gene from Liriodendron chinese

ABSTRACT

Alternative oxidase (AOX) is a terminal oxidase of the alternative respiratory pathway, which is widespread in higher plants. Studies have shown that AOX plays an important role in the growth and development of plants. Cytochrome respiratory pathway was inhibited under the adverse circumstances, while plants will eliminate excess electrons by increasing the amount of respiration in order to avoid damage to them. Magnoliaceae Liriodendron (Liriodendron chinense) is an important and precious timber ornamental species. Researching and utilizing Liriodendron adaptive gene has the important significance to improve the adaptability of Liriodendron. In this paper, we conduct the bioinformatics analysis of AOX1 gene encoding protein which is already obtained in the early stage of the experiment and study on the expression of LcAOX1 by the method of qPCR model. Also, we use the method of double digestion to construct over expression vector construction of LcAOX1.The main results are as follows:

The full length of AOX1 gene is 1220bp with an open reading frame（ORF） of 1032 bp. This gene encoded a protein of 343 amino acids. Basing on the bioinformatics analysis, the protein is hydrophilic and has two transmembrane regions. Basing on the subcellular localization prediction, we predict that the protein is located in mitochondria. Homology analysis showed that C terminal of the protein was highly conserved and the phylogenetic analysis showed that the protein is a member of the AOX1 subfamily which has the closer relationship with tomato and grape.

There are noticeable differences in the expression quantity of AOX1 in diverse tissues of Liriodendron and the order of expression level from high to low is stamens, petals, calyx, leaf, flower bud, leaf bud, stem and pistil. It holds the highest expression level in the stamen which is about 2.7 times of the leaf. Also, it has the lowest expression quantity in the stem which is just about 40% of the leaf.

We use pCAMIA1302 as the skeleton and construct the over expression vector of LcAOX1 by double digestion method. Also, according to sequencing test, we can ensure the positive insertion of the recombinant plasmid without changing the ectopic site which establishes the foundation for further validation of transgenic function.

Key words:Liriodendron chinese, AOX1 gene,sequence analysis, expression quantity analysis, construction of over expression vector

目 录

1文献综述 1

1.1 AOX基因研究概述 1

1.1.1 AOX基因结构 1

1.1.2 AOX基因功能 2

1.2植物表达载体构建方法 3

1.2.1双酶切法 3

1.2.2 Gateway技术 4

1.2.3重组融合PCR法 4

1.3鹅掌楸研究进展 5

1.3.1鹅掌楸生活习性 5

1.3.2鹅掌楸生物学研究 5

1.3.3鹅掌楸属植物功能基因研究概况 5

1.4目的与意义 6

2材料与方法 7

2.1鹅掌楸AOX1基因生物信息学分析 7

2.1.1实验材料 7

2.1.2实验方法 8

2.2鹅掌楸AOX1基因表达模式分析 10

2.2.1实验材料 10

2.2.2实验方法 10

2.3鹅掌楸AOX1基因过表达载体构建 12

2.3.1实验主要仪器 12

2.3.2实验试剂 12

2.3.3实验方法 13

3结果与分析 16

3.1鹅掌楸AOX1基因生物信息学分析 16

3.1.1总RNA提取 16

3.1.2鹅掌楸AOX1基因的序列分析 16

3.1.3鹅掌楸AOX1基因编码蛋白分析 17

3.2鹅掌楸AOX1表达模式分析 21

3.2.1总RNA提取和质量检测 21

3.2.2鹅掌楸AOX1基因组织表达特性分析 22

3.3鹅掌楸AOX1基因超表达载体构建 23

4结论与讨论 25

4.1结论 25

4.2讨论 25

参考文献 27

1文献综述

1.1 AOX基因研究概述

植物交替呼吸是呼吸主链（图1-1）的分支途径，它从细胞色素途径的辅酶Q分岔，氧化辅酶Q获得4个电子，还原氧分子催化生成水，在主呼吸链抑制剂（例如抗霉素A和氰化物）存在时，电子传递过程依然继续进行，电子依次传递给AOX和O₂。AOX氧化过程没有质子移动，还原能不是以ATP方式贮存，而是以散热方式消耗。植物抗氰呼吸平衡植物体碳代谢和电子传递。在产热植物佛焰花序中，开花时花序产热释放出芳香气味吸引昆虫帮助传粉，由此推测AOX基因与果实成熟[^[1]]、抗逆抗氧化胁迫[^[2]]、抗水分胁迫[^[3]]有关。

图1-1线粒体呼吸链示意图 [^[4]]