摘 要

病程相关蛋白（PR）是普遍存在于高等植物中的一类防御蛋白，在植物抵御病原体、响应外界压力以适应不良环境过程中发挥重要作用。其中第5家族(PR-5)是一组很独特的蛋白，具有β-1,3葡聚糖酶、蛋白酶、抗真菌、抑制α淀粉酶以及抑制反转录酶等多种生物学活性。

本研究在杨树全基因组序列信息的基础上，克隆获得了PR-5家族的两个类甜蛋白基因PeTLP1和PeTLP2，分别构建过量表达载体，以南林895杨（Populus×euramericana cv ′Nanlin895′）为受体材料，利用农杆菌介导法开展杨树的遗传转化研究，为杨树转基因抗病新品种培育提供依据。主要研究结果如下：

（1）目的基因PeTLP1和PeTLP2的克隆与植物表达载体的构建，为杨树抗病相关基因的遗传转化提供了合适的表达载体。

（2）克隆基因PeTLP1和PeTLP2的遗传转化研究。利用较合适的遗传转化条件，经初步筛选分别获得卡那抗性植株59株和72株。

（3）转基因植株的分子检测。PCR反应的结果显示，相关目的片段已经成功导入部分植株中；实时定量RT-PCR检测结果显示表明，PeTLP1和PeTLP2基因已经成功整合到南林895杨中，分别获得转基因植株14个株系和18个株系。

（4）转基因植株的表达分析。结果表明目的基因成功在南林895杨中过量表达。

（5）转基因植株的抑菌分析。分析结果表明，转基因植株在体外表现出一定的抑菌效果，基因的表达量越高抑菌效果越明显。

关键词：PeTLP1；PeTLP2；过量表达；杨树；抗病性

ABSTRACT

Pathogenesis related protein (PR) is a kind of defense proteins existing in higher plants, playing an important role in the process of plant pathogens,and responses to external pressure to adapt to adverse environment . The fifth family (PR-5) is a group of very unique proteins, with β-1,3-glucanase activity, protease activity, antifungal, inhibit alpha amylase and inhibition of reverse transcriptase and other biological activities.

Based on the poplar genomic sequence information, we cloned two thaumatin like protein genes PeTLP1 and PeTLP2 in the PR-5 family, and constructed their over expression vector respectively.The poplar Nanlin 895 (Populus×euramericana cv ′Nanlin895′) was selected as receptor materials, by use of Agrobacterium tumefaciens method to carry out the study on genetic transformation in poplar, and the main results are as follows:

(1) Cloning of PeTLP1 and PeTLP2 gene and construction of its plant expression vector, providing a suitable expression vector for genetic transformation of poplar disease resistance related genes.

(2) The study on genetic transformation of PeTLP1 and PeTLP2 gene.With the genetic transformation conditions,We got 59 and 72 transgenic plants respectively.

(3) The molecular detection of transgenic plants.PCR reaction showed that the fragments have been successfully imported into the most plants; real-time quantitative RT-PCR detection results show that the PeTLP1 and PeTLP2 gene has been successfully integrated into the genome of poplar Nanlin 895.We got 14 and 18 strains respectively.

(4) Analysis of the expression of transgenic plants. The results showed the purpose gene has been over-expressed in the genome of poplar ′Nanlin 895′.

(5) The protein extracted from the transgenic plants was used for antibacterial test. The results showed the proteins of transgenic plants possessed a certain inhibitory effect and the effection had a positive correction with the gene expression.

Key words：PeTLP1；PeTLP2；over-expression；poplar；disease resistance

目 录

1 文献综述 1

1.1 植物基因工程在杨树抗病育种中的应用 1

1.2 杨树真菌病害与防治 1

1.3 病程相关蛋白 2

1.3.1 TLPs基因概述 3

1.3.1.1 TLPs的三维结构 3

1.3.1.2 TLPs的B-1,3葡聚糖酶活性 3

1.3.1.3 TLPs抗真菌机制 4

1.3.2 TLPs基因在植物抗病育种中的应用 4

1.4 基因的过量表达研究及载体构建概述 4

1.4.1 TLPs基因的过量表达载体 4

1.4.2 TLPs基因在植物中的分布 4

1.4.3 大肠杆菌真核表达菌株的特性 5

1.5 植物基因的遗传转化方法 5

1.6 本课题研究的目的、意义 6

2.1 材料 7

2.1.1 植物材料 7

2.1.2 菌株和载体 7

2.1.3 酶和试剂 7

2.1.4 培养基 7

2.1.4.1 细菌培养基 7

2.1.4.2 植物培养基 8

2.1.5 目的基因的序列及分析 8

2.1.6 仪器和设备 8

2.2 实验方法 8

2.2.1 PeTLP1、PeTLP2克隆载体的构建 8

2.2.1.1 RNA提取方法 9

2.2.1.2 逆转录合成第一链cDNA 9

2.2.1.3 目的基因PeTLP1和PeTLP2的获得 10

2.2.1.4 琼脂糖凝胶中DNA片段回收 11

2.2.1.5 大肠杆菌感受态细胞的制备 11

2.2.1.6 目的基因连接克隆表达载体pMD18-T并转化大肠杆菌感受态 12

2.2.1.7 连接产物的检测 13

2.2.2 pBI121-PeTLP1、pBI121-PeTLP2表达载体的构建 13

2.2.2.1 质粒的提取 13

2.2.2.2 目的基因PeTLP1、PeTLP2的酶切 14

2.2.2.3 目的基因片段的纯化 14

2.2.2.4 连接反应 15

2.2.3 连接产物转化大肠杆菌感受态细胞 15

2.2.3.1 PCR检测 15

2.2.3.2 酶切检测 15

2.2.4 pBI121-PeTLP1、pBI121-PeTLP2表达载体转化根癌农杆菌 16

2.2.4.1 根癌农杆菌感受态的制备 16

2.2.4.2 转化根癌农杆菌 16

2.3 结果与分析 17

2.3.1 目的基因的PCR扩增 17

2.3.2 克隆载体连接产物的检测 18

2.3.3 植物表达载体的构建 19

2.3.3.1 PeTLP1、PeTLP2基因与表达载体pBI121的酶切结果与分析 19

2.3.3.2 连接产物检测结果与分析 19

2.3.3.3 pBI121-PeTLP1和pBI121-PeTLP2表达载体的酶切检测 20

3 遗传转化研究 21