摘 要

杨树生长快、分布广，是重要的速生工业用材树种之一，在维护生态环境等方面发挥着重要作用。但干旱、盐碱等因素严重制约了杨树产业的发展。近年来，基因工程技术为培育杨树抗逆新品种提供了新途径。本文将本实验室已分别转入抗逆相关基因AtGolS2、AtSRK2C、PtSnRK-1和PtSnRK-2的转基因杨树作为研究对象，对其进行了分子检测和抗逆性分析，为培育杨树抗逆新品种提供依据。主要研究结果如下：

1.开展了转AtGolS2、AtSRK2C、PtSnRK-1和PtSnRK-2基因南林895杨（Populus×euramericana cv.‘Nanlin895’）的分子检测。在利用组培技术快速繁殖转AtGolS2、AtSRK2C、PtSnRK-1和PtSnRK-2基因南林895杨的基础上，对转基因植株进行了PCR检测，结果显示多数植株检测呈阳性，表明该基因已整合到南林895杨基因组中，且经过一年多的继代繁殖，导入的外源目的基因未发生丢失。

2. 对转基因植株进行了盐胁迫处理分析。在分子检测的基础上，选取PCR检测阳性植株及对照株系分别进行了组培苗和温室移植苗的盐胁迫试验，结果表明随着盐胁迫浓度的增加及处理时间的延长，转化植株和对照植株在生长上均受到抑制，但转基因株系受到的影响显著小于对照植株。

3. 对转基因植株进行了盐胁迫处理后相关生理生化指标的检测。在盐胁迫下转基因株系叶片内脯氨酸（Proline）、过氧化物酶（POD）、等含量增高幅度均大于对照植株，而丙二醛（MDA）含量的增高幅度小于对照。转基因株系叶片水分利用效率也明显高于对照植株。

4.分析结果显示分别转AtGolS2、AtSRK2C、PtSnRK-1和PtSnRK-2不同基因的转基因植株及转同一个基因的不同转基因株系对NaCl胁迫的抵抗能力也不同，其中AtGolS2-1、AtGolS2-4、AtGolS2-13、PtSnRK-1-5、PtSnRK-2-10等株系在盐胁迫处理后相关生理生化指标等方面与对照相比差异达显著水平。

关键词 南林895杨 转基因 分子检测 抗逆性 耐盐

Abstract

Poplar is one of important commercial timber and industrial tree species，with fast growth，easy propagation and widely distribution. It has played a key role in maintenance balance of ecology environment. However ,drought and soil salinity have been threatening growth of wood resulted in woods can’t completely exert their roles in the field of arid，and saline-alkali environment. But，the development of genetic engineering provided an effective alternative approach to create the new transgenic poplar germplasm with stress resistance within a short time. In this study molecular detection and evaluation of stress tolerance in greenhouse, and different circumstance of stress tolerance for the transgenic poplar with different salt in tolerance gene AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2.The major results and conclusions are described follows:

1. Molecular detection of salt tolerance in the transgenic poplars with AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2.

The transgenic poplars with AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 were detected by the molecular ways of PCR amplification，and the result showed that the AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 genes could be transcripted in Populus×euramericana cv.‘Nanlin895’and expressed.

2. Evaluation of growth thing of transgenie poplars(Populus×euramericana cv.‘Nanlin895’)with AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 genes.

By taking salt stress on transgenic poplars in greenhouse，the result indicated that with density of NaCl increase and treatment time lasting，growth of transgenie lines and non-transformed were intimidated control under high concentrations of salt treatments, but degree of inhibition of transgenic plant was lower than control plant.

3. Evaluation of in salt tolerance the transgenie poplars(Populus×euramericana cv.‘Nanlin895’)with AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 genes.

The results indicate that the degradation degree of and dandification degree of content of proline 、RWC and POD in leaves in the transgenic poplars with AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 genes were all lower than those in the control poplar, the MDA content of transgenic poplars were significantly lower than control, which indicated that AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 genes actually enhanced the plant NaCl-resistance ability. By means of genetic engineering, AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 genes can be genetically modified to improve the salt resistance of poplar to reduce salt stress on plants.

4. The result indicated that Populus×euramericana cv.‘Nanlin895’with AtGolS2、AtSRK2C、PtSnRK-1 and PtSnRK-2 genes gene had some differences in salt resistance, Populus×euramericana cv.‘Nanlin895’with AtGolS2-1、AtGolS2-4、AtGolS2-13、PtSnRK-1-5、PtSnRK-2-10 gene had slightly higher salt-resistant capacity than the other transgenic samples.

Key words：Populus×euramericana cv.‘Nanlin895’, transgenie; Molecular detection; stress tolerance; Salt-resistance

目 录

1.国内外研究进展 1

1.1. 植物转基因的研究概况 1

1.2. 杨树转基因研究近况 1

1.2.1. 杨树转抗虫基因工程 2

1.2.2. 杨树抗病基因工程 2

1.2.3. 杨树耐盐碱基因工程 3

1.2.4. 杨树抗性基因工程其它方面的研究 3

1.3. 非生物胁迫下编码逆境蛋白的相关基因 3

1.3.1. 渗透调节基因 3

1.3.2. 活性氧清除的相关基因 6

1.3.3. 蛋白激酶基因 6

2. 研究的目的和意义 7

3. 材料和方法 8

3.1. 植物材料 8

3.2. 分子检测 8

3.2.1. PCR扩增 8

3.3. 转基因植株的扩繁与移栽 9

3.3.1. 分化培养 9

3.3.2. 移栽 10

3.4. 对转基因南林895杨组培苗及盆栽苗继续盐胁迫试验 11