本文選題：紅花　切入點：CtDof　出處：《長春師范大學(xué)》2017年碩士論文

【摘要】：紅花(Carthamus tinctorius L.),為菊科紅花屬草本植物。目前,全世界菊科紅花屬植物約有25種,而在我國,僅有紅花一種。紅花具有較強的抗旱和抗寒的能力,主要栽種于我國河南、新疆、四川、浙江等地區(qū)。轉(zhuǎn)錄因子Dof是植物所特有的一類轉(zhuǎn)錄因子,能夠響應(yīng)光的應(yīng)答,參與代謝調(diào)節(jié),從而影響種子的萌發(fā)和成熟,還能夠參與抗逆反應(yīng),提高植物的抗性等,在植物的生長發(fā)育過程中發(fā)揮著重要的作用。本研究以“吉紅一號”紅花為實驗材料,克隆CtDof基因,對其在不同組織及種子不同發(fā)育階段的表達水平進行分析,同時構(gòu)建超表達載體轉(zhuǎn)化擬南芥,鑒定其功能。主要研究結(jié)果如下:1.在紅花不同組織部位之間,紅花Dof基因家族中的Unigene100791基因(CtDof1)的相對表達量最高,尤其是在成熟種子中,Unigene100791基因的相對表達量分別是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;在種子發(fā)育16天時Unigene100791基因的相對表達量最高,分別是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,本研究主要以高表達的Unigene100791基因作為主要研究對象,并進行了后續(xù)研究。2.利用RT-PCR技術(shù),成功克隆得到了CtDof1基因的中間片段;利用RACE技術(shù),從紅花種子中首次分離做出CtDof1基因的全長cDNA序列,對CtDof1全長cDNA序列進行生物信息學(xué)分析,CtDof1基因全長1274 bp,開放閱讀框為981bp,編碼326個氨基酸。3.構(gòu)建了CtDof1基因的植物超表達載體pBasta-CtDof1,并將其轉(zhuǎn)入野生型擬南芥,獲得轉(zhuǎn)基因擬南芥T2代植株14株。分別對轉(zhuǎn)基因擬南芥和野生型擬南芥的種子進行不同鹽濃度處理,結(jié)果發(fā)現(xiàn),在鹽脅迫的條件下,轉(zhuǎn)基因擬南芥對鹽表現(xiàn)出敏感性。4.將pBasta-CtDof1植物超表達載體轉(zhuǎn)入野生型紅花中,經(jīng)篩選鑒定獲得轉(zhuǎn)基因紅花7株。5.構(gòu)建了CtDof1基因的原核表達載體,并將重組質(zhì)粒pEASY-Blunt E1-CtDof1成功轉(zhuǎn)入E.coliBL21(DE3)中,在37℃,IPTG濃度為1 mmoL,誘導(dǎo)時間為6 h時,融合蛋白在E.coliBL21(DE3)中得到了高效的表達,為后續(xù)純化該蛋白奠定了實驗基礎(chǔ)。
[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，

本文編號：1663932