摘 要

杨树是世界上重要造林绿化树种之一，也是生态防护林的主要树种。植物生物钟是在与环境长期生存斗争中进化出来的一种适应性机制，它使生物体能够预测环境因素的变化，从而调节生理、代谢及发育过程。生物钟在植物的生长发育过程中发挥重要功能。本研究在从毛果杨中克隆的有关生物钟基因PtPRR5A和PtPRR5B的基础上，通过构建过量表达载体，采用农杆菌介导法转化南林895杨，通过分子检测和生理检测分析克隆基因PtPRR5A和PtPRR5B的表达及功能。主要研究结果如下：

(1)PtPRR5A和PtPRR5B基因过量表达载体的构建。利用pBI121载体重组质粒，经酶切和测序证实正确后，转化大肠杆菌和农杆菌EHA105，菌液PCR检测显示已成功构建了PtPRR5A和PtPRR5B基因过量表达载体。

(2)遗传转化中筛选压的确定。本研究以卡那霉素为筛选标记基因，通过实验选用卡那霉素20mg/L作为分化筛选时的选择压，选用卡那霉素10mg/L作为生根培养基的选择压，培养基中的头孢浓度为200mg/L。遗传转化效率最高为36.2%。

(3)转PtPRR5A和PtPRR5B基因植株的获得和分子检测。采用农杆菌介导法转化南林895杨的叶片，获得转基因植株，并对转基因植株进行PCR检测、RT-PCR检测和荧光实时定量检测，确定阳性植株。经筛选共获得转PtPRR5A基因植株11个株系，转PtPRR5B基因植株9个株系。

(4)在不同的光照条件下处理转基因植株，转PtPRR5A和PtPRR5B基因植株的株高与未转基因植株相比增高明显，而叶绿素含量增加最高达39.2%。表明PtPRR5A和PtPRR5B基因可能通过调控杨树相关生物钟基因，在提高杨树生长量方面有一定作用，为研究杨树生物钟相关基因表达调控及新品种培育提供了依据。

关键词：PtPRR5A，PtPRR5B，生物钟，功能分析，南林895杨

ABSTRACT

Populus is one of the important afforestation tree species and the main tree species of ecological protection around the world. Circadian clock is an adaptive mechanism which evolves in the struggle with long-term survival environment, it enables organisms to predict the change of environmental factors and adjust physiology, metabolism and development.The research is that PtPRR5A and PtPRR5B genes were cloned from Populus tomentosa and construct the over-expression vectors of PtPRRP5A and PtPRRP5B genes. We used Agrobacterium-mediated method to introduce the genes into Nanlin ‘895’. The molecular detection and physiological tests were used to analyze the gene expression and function of PtPRR5A and PtPRR5B genes. Major results of the research were as follows:

(1)The construct of the over-expression vectors of PtPRRP5A and PtPRRP5B genes. We used the pBI121 vector to recombe plasmid ,then they were confirmed by digestion and sequence, thentransformed into E. coli and agrobacterium EHA105, the results of PCR showed that the over-expression vectors of PtPRRP5A and PtPRRP5B genes had been constructed.

(2)Determination of selective pressure in genetic transformation. In this experiment, the kanamycin was used as the selectable marker, through the experiment, 20 mg/L and 10mg/L kanamycin were respectively considered as suitable selective pressure concentration for differentiation and rooting medium, the concentration of cefazolin was 200mg/L in the medium. Genetic transformation efficiency was up to 36.2%.

(3)Obtain and molecular detection of transgenic plants. We used Agrobacterium-mediated method to introduce the genes into the leaves of Nanlin ‘895’,and obtain the transgenic plants.The detection of transgenic plants by PCR、RT-PCR and real-time quantitative PCR to determine the positive plants.wo got some transgenic plants through screening,the number of

PtPRRP5A plants was 11, the number of PtPRRP5B plants was 9.

(4)The transgenic plants were grown under different lighting conditions, the height of transgenic plants with PtPRRP5A and PtPRRP5B genes were obvious higher than the non-transgenic plants, the chlorophyll content increased up 39.2%.The results showed that the PtPRRP5A and PtPRRP5B genes may be through regulating the clock genes to improve the growth of populus. The result could provide experimental basis for researching the related clock genes and cultivating new varieties of populus.

Keywords: PtPRR5A, PtPRR5B, circadian clock, function analysis,Nanlin ‘895’

目 录

1文献综述 - 1 -

1.1 国内外生物钟基因研究进展 - 1 -

1.1.1 植物生物节律及其特点 - 1 -

1.1.2 生物钟分子机制研究进展 - 1 -

1.1.3 PRRs家族 - 3 -

1.1.4 生物钟的组成部分 - 4 -

1.1.5 PRR5与TOC1 - 4 -

1.2 杨树遗传转化的研究进展 - 4 -

1.3 转基因植株的检测方法 - 4 -

1.3.1 PCR检测 - 4 -

1.3.2 RT-PCR检测 - 5 -

1.3.3 Southern杂交 - 5 -

1.3.4蛋白质印迹法(Western blot) - 5 -

1.3.5 酶联免疫吸附法（ELISA） - 5 -

1.4研究目的和意义 - 5 -

1.4.1 研究目的 - 5 -

1.4.2 研究意义 - 5 -

2 杨树 PtPRR5A 和 PtPRR5B 基因克隆及表达载体构建 - 6 -

2.1 实验材料 - 6 -

2.1.1 菌株和质粒 - 6 -

2.1.2 植物材料 - 6 -

2.1.3 抗生素、酶和试剂盒 - 6 -

2.1.4 仪器与设备 - 7 -

2.1.5 培养基 - 7 -

2.2 实验方法 - 7 -

2.2.1 杨树PtPRR5A和PtPRR5B基因克隆 - 7 -

2.2.1.1 毛果杨总RNA的提取 - 7 -

2.2.1.2毛果杨总RNA纯化 - 7 -