摘 要

盐胁迫是当前影响植物正常生长发育的重要的非生物困境之一，而HKT 是植物体内与耐盐胁迫有关的一类Na 或K 转运体或Na -K 共转运体，其过量表达对于提高植物耐盐性具有显著效果。杨树是世界中纬度平原地区分布最为广泛的一类速生用材树种，具有十分重要的经济价值和生态效益。作为林木研究的模式树种，杨树具有高效稳定的遗传转化体系，可通过基因工程技术获得抗旱、耐盐等杨树转基因新无性系，有利于林木抗逆机制的研究和应用推广。

本研究以HKT转基因杨树扦插苗为试验材料，RT-PCR和qRT-PCR检测表明，目的基因PeHKT整合到山新杨基因组，且在转录水平仍处于过量表达。HKT过表达转基因杨树苗中的叶绿素含量在面对盐胁迫时，呈现上升趋势，推测转基因杨树中HKT基因的过表达有利于增加叶绿素含量，促进植物叶片细胞中叶绿素与叶绿体蛋白间结合，以提高植物抗性。超氧化物歧化酶（SOD）和过氧化物酶（POD）活性测定结果表明HKT过表达杨树通过增强了活性氧的清除能力和过氧化酶活性，减少盐胁迫对杨树的伤害，使其保持一定的耐盐性。这些结果表明HKT转基因杨树抗盐能力相对于未转基因对照杨树，抗盐能力有明显提高，具有潜在的应用推广价值。

关键词：HKT基因；过表达；转基因杨树；耐盐性；生理生化测定

Determination of Antimicrobial and Biochemical

Indexes of HKT Transgenic Poplar

ABSTRACT

Salt stress is one of the important abiotic dilemmas that affects the normal growth and development of plants. HKT is a kind of Na / K transporter or Na -K co-transporter, related to salt stress in plants. Overexpression of HKT gene has a significant effect on improving plant salt tolerance. Poplar is one of the most widely distributed species of fast-growing timber in the middle latitudes of the world，which has significant economic and ecological value. As a model plant for tree genetic inheritance, poplar has a highly efficient and stable genetic transformation system, which can obtain some new poplar transgenic lines, such as drought resistance and salt tolerance, through genetic engineering technology. Therefore, it is beneficial to the research of forest resistance mechanism and application promotion.

In this study, annual cottage saplings of poplar were used as experimental materials. The results showed that the pH35GS::PeHKT vector was not lost in the biennial transgenic lines and the PeHKT gene was still overexpressed which confirmed by RT-PCR and qRT-PCR. The chlorophyll content in the transgenic poplar seedlings showed an upward trend when it was confronted with salt stress, which means the HKT gene in transgenic poplar protects the combination of chlorophyll and chloroplast protein in the leaf cell to improve plant resistance and increase chlorophyll content. The determination results of superoxide dismutase (SOD) and the activity of peroxidase (POD) showed that the scavenging ability of reactive oxygen species and the activity of peroxidase were strengthened when the poplars were exposed to the salt stress, which could reduce the damage of salt stress to poplar so that maintaining a certain salt tolerance. These results indicated that the salt tolerance of HKT transgenic poplar was significantly higher than that of non-transgenic poplar, and it had an important practical application value.

Key words: HKT gene, Overexpression, Transgenic poplar, Salt tolerance, Physiological and biochemical determination

目 录

1 文献综述 - 1 -

1.1 植物耐盐机制研究 - 1 -

1.1.1 植物盐胁迫影响 - 1 -

1.1.2 植物耐盐机理 - 2 -

1.2 植物HKT基因研究进展 - 2 -

1.2.1 植物HKT的发现与分类 - 2 -

1.2.2 HKT基因与植物耐盐性关系 - 3 -

1.3 杨树概述 - 4 -

1.4 研究目的与意义 - 5 -

2 HKT转基因杨树的检测 - 6 -

2.1 实验材料 - 6 -

2.1.1 植物材料 - 6 -

2.1.2 实验试剂 - 6 -

2.1.3 仪器设备 - 6 -

2.2 实验方法 - 6 -

2.2.1 DNA提取 - 6 -

2.2.2 RT-PCR反应 - 7 -

2.2.3 RNA提取 - 8 -

2.2.4 cDNA合成 - 9 -

2.2.5 实时定量PCR - 9 -

2.3 结果与分析 - 10 -

2.3.1 RT-PCR检测转基因杨树 - 10 -

2.3.2 qRT-PCR检测转基因杨树 - 11 -

3 HKT转基因杨树耐盐性检测 - 12 -

3.1 实验材料 - 12 -

3.1.1 植物材料 - 12 -

3.1.2 实验试剂 - 12 -

3.1.3 仪器设备 - 12 -

3.2 实验方法 - 12 -

3.2.1 植物叶绿素提取 - 12 -

3.2.2 蛋白定量测定（BCA） - 13 -

3.2.3 过氧化物酶（POD）测定 - 14 -

3.2.4 超氧化物歧化酶（SOD）测定 - 15 -

3.3 结果与分析 - 16 -

3.3.1 NaCl胁迫下转HKT基因杨树的叶绿素变化 - 16 -

3.3.2 NaCl胁迫下转HKT基因杨树的POD活性变化 - 17 -

3.3.3 NaCl胁迫下转HKT基因杨树的SOD活性变化 - 17 -

结 论 - 19 -

致 谢 - 20 -

参考文献 - 21 -

1 文献综述

1.1 植物耐盐机制研究

全世界约有130亿hm²的陆地，其中30亿hm²属于盐碱土。而中国，陆地面积约9.3亿hm²，其中约有0.27亿hm²盐碱土。土壤盐渍化一直以来是一个全球性的问题，由于不合理的耕作、灌溉以及施肥不当等原因，土壤次生盐渍化愈演愈烈，成为当前限制植物生长和农业发展的重要因素^[1]。一般来说，植物盐害类型可分为原初盐害和次生盐害两种。原初盐害的影响部位是细胞的质膜，如膜的选择透性和对离子的选择吸收产生变化，从而引起膜的结构功能变化；次生盐害表现为盐浓度过高引起土壤中的水势降低，结果发生渗透胁迫现象。当环境中盐分过高时，植物体的外部形态结构和内部生理生化特征都可能发生改变，甚至导致严重减产^[4]。因此，如何提高植物的抗盐性已成为各国关注的焦点。

1.1.1 植物盐胁迫影响