摘 要

林木作为现今人类最主要的可再生资源，杨树是林木中的模式物种，具有早期速生、适应性强、分布广、种类和品种多、容易杂交、容易改良遗传性、容易无性繁殖等特点，从而常用于集约栽培，也是世界上广泛栽培的重要造林树种之一，因此杨树具有重要的经济价值。杨树染色体组较小，其分子生物学研究较为深入。但杨树染色体数目较多（2n=2X=38），形态较小，在细胞学研究方面较为滞后。特殊探针的FISH技术是鉴定单染色体有效手段。在哺乳动物和鸟类中，组蛋白基因通常位于基因组中的几个位点上成簇排列或者的单独排列。因此以组蛋白基因为探针的FISH技术将是一种有效的鉴定小染色体的手段。目前该技术在蛙类基因组中得到广泛的应用，但在植物基因组中尚未见报道。本研究以美洲黑杨和小叶杨基因组DNA为探针，利用H3、H4组蛋白基因保守位点设计引物，对H3、H4组蛋白基因进行克隆，并对所得序列进行测序与比对，获得杨树H3、H4组蛋白基因。该研究结果为进一步利用特殊探针FISH技术鉴定杨树染色体单条染色体奠定了基础。

关键词：美洲黑杨；小叶杨；H3、H4组蛋白基因；基因克隆； BLAST

H₃、H₄ histone gene cloning of poplars

ABSTRACT

It is generally accepted that forests are one of the most important renewable resources in today’s world. Among all the plants in the forest, poplars are known as the model plant with the features of early fast growth, strong adaptability, wide distribution, more varieties and types, easier hybridization, easier genetic improvement and easier asexual propagation. With all the advantages above, poplars are often used in intensive cultivation and are one of the most widely cultivated and economically important forest plants in the world. Although poplar genome is small and the researches of molecular biology are more deeply,poplar has a large number of chromosome（2n=2X=38）,with small form and in cytology research is relatively lagging behind.Special probe FISH technology is an effective means to identify single chromosome.In mammals and birds, histone genes usually located in several sites in the genome clusters arranged or separate arrangement.Therefore,the FISH technology with histone gene probe will be an effective means for the identification of small chromosome.At present, this technology has been widely used in the frogs genome, but has not yet been reported in the plant genome.This study uses populus deltoides and populus simonii genomic DNA as probes, uses H3, H4 histone gene loci conservative as design primers to cloning H3, H4 histone gene , and comparison the sequence,to obtain the H3, H4 histone genes of poplas.The study laid a foundation to the the further use of special probe FISH technical to authenticate poplar individual chromosomes.

Key words：Populus deltoides；Populus simonii；histone H3/H4；gene cloning； BLAST

目 录

1 文献综述………………………………………………………………………………………1

1.1 杨树资源概述及开发利用………………………………………………………………1

1.1.1杨树资源概述…………………………………………………1

1.1.2 杨树资源的开发利用…………………………………………………1

1.2 杨树分子生物学研究现状………………………………………………………………2

1.3 杨树细胞学研究现状………………………………………………………………2

1.4 组蛋白的研究现状…………………………………………………………………………3

1.4.1 组蛋白概述…………………………………………………3

1.4.2 组蛋白分类…………………………………………………3

1.5 基因克隆技术…………………………………………………………………………4

1.5.1 基因克隆概述…………………………………………………4

1.5.2 常用的基因克隆方法…………………………………………………5

2 实验材料与方法………………………………………………………………………………9

2.1 实验材料……………………………………………………………………9

2.2 实验方法…………………………………………………………………………9

2.2.1 CTAB法提取DNA…………………………………………………9

2.2.2 PCR………………………………………………10

2.2.3 凝胶电泳…………………………………………………10

2.2.4 凝胶回收…………………………………………………10

2.2.5 连接与转化…………………………………………………11

3 实验结果与讨论………………………………………………………………………………12

3.1 实验结果…………………………………………………………………12

3.2 讨论…………………………………………………………………………20

结论与展望…………………………………………………………21

致 谢…………………………………………………………22

参考文献……………………………………………………23

1文献综述

1.1 杨树资源概述及开发利用

1.1.1 杨树资源概述