中国科学院新疆理化技术研究所机构知识库
Advanced  
XJIPC OpenIR  > 资源化学研究室  > 学位论文
题名: 小麦大穗转化株中外源DNA 片段的检测、分离与克隆
作者: 李静
答辩日期: 2009-06
导师: 赵民安
专业: 有机化学
授予单位: 中国科学院研究生院
授予地点: 北京
学位: 硕士
关键词: 小麦大穗转化株 ; 外源目的DNA 片段 ; Southern 杂交 ; 化学发光生物素探针标记法 ; DNA 微克隆文库 ; 阳性克隆
摘要:
优良品种是现代农业生产的基础,近年来世界农业增长的一半得益于品种选育。小麦作为世界上第一大粮食作物,是人类最主要的食物来源。我国每年小麦消费缺口 达2000~3500万吨,这一数字还在逐年上升,提高小麦种植效益和单产已成为我国小麦育种工作的主要课题。本研究中小麦大穗转化株是由大赖草总DNA 通过花粉管通道法导入春麦761所获得的新株系,它所拥有的大穗、大粒、高蛋白、抗黄萎病等优良性状变异均来自外源DNA片段的导入,本文对大穗转化株中 的外源DNA片段进行检测分离及克隆,目的在于:其一从大穗转化株中分离获得外源DNA片段;其二对外源DNA片段核苷酸序列的结构、来源及与近缘物种相 似基因的同源性进行初步分析;其三进一步推测此外源DNA片段可能在小麦大穗转化株产生性状变异过程中发挥的作用,以期为后续分子育种工作奠定基础。 本论文主要研究内容包括以下几部分: 第一部分探讨了通过化学发光生物素探针标记Southern杂交法检测小麦大穗转化株中外源DNA片段。首先采用CTAB法提取小麦大穗转化株、春麦 761基因组总DNA,过柱纯化,紫外分光光度计定量,稀释成浓度为1μg/μl,再将小麦大穗转化株、春麦761总DNA通过BamHⅠ和HindⅢ双 酶切后,对小麦大穗转化株双酶切产物进行Southern杂交检测外源DNA片段。检测过程包括大赖草生物素探针的制备,小麦大穗转化株双酶切产物凝胶电 泳的原位转膜,预杂交,大赖草生物素标记探针与目的DNA的杂交,化学发光法检测等步骤。最后得到大赖草探针与大穗转化株酶切总DNA的一条特异性杂交条 带,片段长度约为750~1000bp。 第二部分构建了转基因大穗小麦经杂交所显示区域的DNA微克隆文库。对杂交得到的外源DNA条带进行凝胶回收纯化,将其与pUC18质粒载体连接,转化到 E.coli DH5α超级感受态细胞中克隆,蓝白斑筛选阳性克隆子构建小麦大穗转化株经杂交所显示区域的DNA文库。克隆过程还包括采用Inoue法制备超级感受态细 胞,探索连接转化过程中外源DNA片段与pUC18的最佳连接反应,以得到高效的转化率。结果显示,目的DNA片段和pUC18比例在2:1时,转化率可 以达到1×108个转化克隆/μg质粒DNA,筛选得到重组子一千余个,随机挑选176个质粒重组子构成DNA微克隆文库进行分析。 第三部分再次用大赖草总DNA为探针对筛选到的176个重组子进行斑点杂交,以最终确定出含有目的DNA片段的阳性克隆,进而测序分析。此杂交过程除了重 组子的质粒提取和DNA斑点印迹的手工制备以外,其余杂交步骤与第一部分相同。杂交结果显示:176个阳性克隆子斑点杂交化学发光自显影后出现6个斑点, 同时作对照的三个蓝斑未出现斑点,且杂交背景干净,斑点清晰。 第四部分将杂交获得的六个外源DNA测序,结果为同一片段,序列长度为945bp,命名为Dps1,经过Genbank数据库初步分析,它与普通小麦、圆 锥小麦、单粒小麦中1B染色体上转位子序列同源性在80%~83%,而且这些转位子大多与高分子麦谷蛋白基因的表达调控有关,推测Dps1可能与大穗转化 株中较受体春麦761蛋白质含量的提高密切相关。
英文摘要: Choice breeding is the agriculture foundation in modern times and a half of the increase of agriculture product profited from breeding in the world recent years. As a hugest foodstuff crop, wheat is the uppermost food source. we have to improve the benefit and yields in one unit ground because there are 20,000,000~35,000,000 tons comsumption gap in our country which is always ascending. Transgenic Wheat (Triticum aestivum L.) in this research is a new kind of species which was inputed total DNA of Leymus racemosus to spring wheat 761’s by Pollen Tube Pathway, attributing to input of total DNA of Leymus racemosus, it has a lot of great properties and aberrances such as big spike, big grain, high protein, resist cyanosis and so on. The purpose of this article is that study the length, structure, source and homology with relative species of extrinsic target DNA segment by cloning this sequence, at the same time, this nucleotide sequences will be put into Ganbank database to embody. And then, we may speculate on the function in the transgenic wheat of this extrinsic DNA segment in order to do more great groundwork for molecule breeding. There were several parts about research content in thesis. We detected extrinsic DNA segment in transgenic Wheat by Chemiluminescent Biotin-labeled Nucleic Acid Method (CBNAM) in the first part. Total DNA in transgenic Wheat and spring wheat 761 were extracted to 1μg/μl concentration using CTAB way after purification and ration by UV. We also took DNA segment with BamHⅠand HindⅢ to take place Enzyme digestion, on the top of examining the DNA segment by CBNAM. The whole process includes preparation of the Leymus racemosus probe, transgenic Wheat DNA transfering to velum after Enzyme digestion, Southern blotting and Chemiluminescence. As a result, only one band has approximately 750~1000bp from hybridization between transgenic Wheat and Leymus racemosus probe. DNA library about hybridization band between transgenic Wheat and Leymus racemosus genome has been established in the second part. We purified DNA segments from 1.2%Agarose gel, and connected them to pUC18 plasmid, then transformed into competent E.coli DH5α to clone, at last, we built extrinsic DNA segments library according to filtration of α- complementarity principle. The cloning course involved preparation of competent E.coli DH5α by Inoue method, in order to gain higher transform rate, we quested for the best reaction which was concerned with extrinsic DNA segment and pUC18 veceor. Finally, the results show that 176 recombinants were screened which made up of a DNA fragment library. In the third phases, 176 positive clones were tested employing Dot Hybridization to search the target DNA segments. The outcomes of the Hybridization had been put in Genbank for detecting the sequence and embodying. we had to extract recombine vectors to electrophoresis, prepare Biotin-probe to Dot Hybridization, check Nucleotides sequence etal. The result of the Dot Hybridization displayed: 6 spots appears after Chemiluminescence, simultaneously, there has no spot from DNA of blue colony as comparison. Finially, 6 DNA fragments were sequenced ,in the result of they are the same one that is 945bp long,named Dps1. It has Identities between 80%~83% with some genes transposon sequences which are related with High-molecular-weight (HMW)glutenin in the Triticum monococcum, Triticum aestivum and Triticum turgidum by analyzing using Genbank data. On the basis of this, we presume that Dps1 may be a transposon sequence because of inserting of it, the result of higher protein has happened in the Transgenic Wheat.
内容类型: 学位论文
URI标识: http://ir.xjipc.cas.cn/handle/365002/3561
Appears in Collections:资源化学研究室_学位论文

Files in This Item:
File Name/ File Size Content Type Version Access License
李静硕士论文.pdf(1130KB)学位论文--暂不开放View 联系获取全文

作者单位: 中国科学院新疆理化技术研究所

Recommended Citation:
李静. 小麦大穗转化株中外源DNA 片段的检测、分离与克隆[D]. 北京. 中国科学院研究生院. 2009.
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[李静]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[李静]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
文件名: 李静硕士论文.pdf
格式: Adobe PDF
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Powered by CSpace