红花CtDof基因的克

发布时间：2018-03-25 16:20

  本文选题：红花　切入点：CtDof　出处：《长春师范大学》2017年硕士论文

【摘要】：红花(Carthamus tinctorius L.),为菊科红花属草本植物。目前,全世界菊科红花属植物约有25种,而在我国,仅有红花一种。红花具有较强的抗旱和抗寒的能力,主要栽种于我国河南、新疆、四川、浙江等地区。转录因子Dof是植物所特有的一类转录因子,能够响应光的应答,参与代谢调节,从而影响种子的萌发和成熟,还能够参与抗逆反应,提高植物的抗性等,在植物的生长发育过程中发挥着重要的作用。本研究以“吉红一号”红花为实验材料,克隆CtDof基因,对其在不同组织及种子不同发育阶段的表达水平进行分析,同时构建超表达载体转化拟南芥,鉴定其功能。主要研究结果如下:1.在红花不同组织部位之间,红花Dof基因家族中的Unigene100791基因(CtDof1)的相对表达量最高,尤其是在成熟种子中,Unigene100791基因的相对表达量分别是Unigene39146基因的12.2倍,Unigene41817基因的2.2倍,Unigene71654基因的1.8倍,Unigene97057基因的3.9倍;在种子发育16天时Unigene100791基因的相对表达量最高,分别是Unigene39146基因的10.7倍,Unigene41817基因的2.1倍,Unigene71654基因的2.1倍,Unigene97057基因的0.2倍;因此,本研究主要以高表达的Unigene100791基因作为主要研究对象,并进行了后续研究。2.利用RT-PCR技术,成功克隆得到了CtDof1基因的中间片段;利用RACE技术,从红花种子中首次分离做出CtDof1基因的全长cDNA序列,对CtDof1全长cDNA序列进行生物信息学分析,CtDof1基因全长1274 bp,开放阅读框为981bp,编码326个氨基酸。3.构建了CtDof1基因的植物超表达载体pBasta-CtDof1,并将其转入野生型拟南芥,获得转基因拟南芥T2代植株14株。分别对转基因拟南芥和野生型拟南芥的种子进行不同盐浓度处理,结果发现,在盐胁迫的条件下,转基因拟南芥对盐表现出敏感性。4.将pBasta-CtDof1植物超表达载体转入野生型红花中,经筛选鉴定获得转基因红花7株。5.构建了CtDof1基因的原核表达载体,并将重组质粒pEASY-Blunt E1-CtDof1成功转入E.coliBL21(DE3)中,在37℃,IPTG浓度为1 mmoL,诱导时间为6 h时,融合蛋白在E.coliBL21(DE3)中得到了高效的表达,为后续纯化该蛋白奠定了实验基础。
[Abstract]:Carthamus tinctorius L., a herbaceous plant of the genus Carthamus, is mainly planted in Henan, Xinjiang, China. At present, there are about 25 species of Carthamus L. in the world, but there is only one species of safflower in China. Sichuan, Zhejiang and other regions. Transcription factor Dof is a kind of transcription factor that is unique to plants. It can respond to light response, participate in metabolic regulation, thus affect seed germination and maturation, also participate in stress resistance, improve plant resistance, etc. In this study, "Jihong 1" safflower was used as experimental material to clone CtDof gene and analyze its expression level in different tissues and seeds at different stages of development. At the same time, the superexpression vector was constructed and transformed into Arabidopsis thaliana to identify its function. The main results are as follows: 1. The relative expression of Unigene100791 gene (CtDof1) in safflower Dof gene family is the highest among different tissues of safflower. In particular, the relative expression of Unigene100791 gene in mature seeds was 12.2 times that of Unigene39146 gene and 2.2 times of that of Unigene41817 gene and 3.9 times of that of Unigene71654 gene, and the relative expression of Unigene100791 gene was the highest at 16 days of seed development. It is 10. 7 times of that of Unigene39146 gene and 2. 1 times of that of Unigene71654 gene. Therefore, this study mainly focused on the highly expressed Unigene100791 gene as the main research object, and carried out further research. 2. Using RT-PCR technique, The intermediate fragment of CtDof1 gene was cloned successfully, and the full-length cDNA sequence of CtDof1 gene was first isolated from safflower seeds by RACE technique. The CtDof1 full-length cDNA sequence was analyzed by bioinformatics. The total length of CtDof1 gene was 1274 BP, and the open reading frame was 981bp, encoding 326amino acids. The plant overexpression vector pBasta-CtDof1 of CtDof1 gene was constructed and transferred into wild type Arabidopsis thaliana (Arabidopsis thaliana). Fourteen transgenic Arabidopsis T2 plants were obtained. The seeds of transgenic Arabidopsis and wild type Arabidopsis were treated with different salt concentrations. Transgenic Arabidopsis thaliana showed salt-sensitivity .4.The pBasta-CtDof1 plant overexpression vector was transferred into wild type safflower, and 7 transgenic safflower strains. 5. The prokaryotic expression vector of CtDof1 gene was constructed, and the recombinant plasmid pEASY-Blunt E1-CtDof1 was successfully transferred into E. coli BL21DE3). When the concentration of IPTG was 1 mmol / L at 37 鈩，

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