本文選題：水母雪蓮 + SmTTG1　； 參考：《青海大學(xué)》2017年碩士論文

【摘要】：水母雪蓮(Saussurea medusa Maxim.),又稱水母雪兔子,屬菊科風(fēng)毛菊屬,是一種生長于青藏高原地區(qū)的珍稀、瀕危藥用植物。同時,它又是一種典型的高山植物。植株密被表皮毛是水母雪蓮最具有代表性的形態(tài)特征,它是水母雪蓮對極端環(huán)境的一種適應(yīng)結(jié)構(gòu)。研究水母雪蓮表皮毛的形成和發(fā)育過程,對揭示高山植物適應(yīng)環(huán)境的特殊機制將具有重要的理論意義。為此,本研究以水母雪蓮作為實驗材料,通過RT-PCR,結(jié)合RACE技術(shù),克隆了與表皮毛發(fā)育相關(guān)的基因TTG1,并研究了該基因的表達。主要研究結(jié)果如下:1.克隆到的水母雪蓮TTG1基因全長1346 bp,其中包括1014 bp的開放閱讀框,99 bp的5’端非編碼區(qū),211 bp的3’端非編碼區(qū),22 bp的ploy(A)尾巴。該基因共編碼337個氨基酸殘基,分子量為37.75 KD,等電點為4.88。同源比對顯示,水母雪蓮TTG1基因的核苷酸序列與同一科植物大麗花TTG1基因的核苷酸序列的同源性為75%;推導(dǎo)出來的氨基酸序列與大麗花的同源性為83%,與擬南芥、煙草的同源性為74.56%、73.98%。通過SOPMA工具預(yù)測,水母雪蓮TTG1蛋白包含18.69%的α-螺旋,10.09%的β-轉(zhuǎn)角,43.03%的無規(guī)卷曲,28.19%的延伸鏈。無規(guī)卷曲和延伸鏈為該蛋白的主要二級結(jié)構(gòu)。水母雪蓮TTG1基因的氨基酸序列有五個WD40 repeat,說明該基因可能編碼一種WD40蛋白,將該基因命名為SmTTG1(genbank accession number:KX447632)。2.采用實時熒光定量PCR(qRT-PCR)技術(shù),檢測β-actin、18SrRNA、GAPDH、EF和TUA五個基因在水母雪蓮根、莖、葉、花中的表達情況,運用geNorm、NormFinder和Bestkeeper三個軟件分析這5個基因在這4種組織內(nèi)表達的穩(wěn)定性。結(jié)果顯示,在根、莖、葉、花中表達最穩(wěn)定的是18SrRNA。因此,18SrRNA可以作為水母雪蓮qRT-PCR的校正內(nèi)參基因。3.以18SrRNA作為內(nèi)參基因,運用qRT-PCR技術(shù),測定SmTTG1在根、莖、葉、花中的表達情況。結(jié)果顯示,SmTTG1基因在4種組織中都有表達,其中在葉和花中的表達量最高。4.將SmTTG1基因連接到pColdⅠDNA表達載體,并導(dǎo)入BL21(DE3)大腸桿菌中進行原核表達,表達產(chǎn)物用SDS-PAGE電泳分析。結(jié)果顯示,目的蛋白在大腸桿菌中表達,表達蛋白的分子量大約是38KD,與由核苷酸序列推導(dǎo)的目的蛋白分子量大小相符。
[Abstract]:Saussurea medusa Maxim.Here, also called jellyfish, is a rare and endangered medicinal plant in the Qinghai-Xizang Plateau. At the same time, it is a typical alpine plant. The dense coat of the plant is the most representative morphological feature of the jellyfish Saussurea, which is an adaptive structure of the jellyfish to the extreme environment. It is of great theoretical significance to study the formation and development of the epidermal hair of Saussurea jellyfish in order to reveal the special mechanism of adaptation of alpine plants to the environment. Therefore, the gene TTG1 related to epidermal development was cloned by RT-PCR and RACE technique, and the expression of TTG1 gene was studied by using Saussurea jellyfish as experimental material. The main results are as follows: 1. The cloned TTG1 gene of Saussurea jellyfish is 1346 BP in length, including an open reading frame of 1014 BP, an open reading frame of 99 BP, a 5 '-terminal non-coding region of 211bp, a 3'end non-coding region of 22 BP and a tail of ployphus A (22 BP). The gene encodes 337 amino acid residues with molecular weight of 37.75 KD and isoelectric point of 4.88. The homologous alignment showed that the nucleotide sequence of the TTG1 gene of Saussurea jellyflower was 75 with that of TTG1 gene of the same family plant, the deduced amino acid sequence was 83% of the deduced amino acid sequence with Arabidopsis thaliana, and the homology of tobacco was 73.98 with Arabidopsis thaliana. It was predicted by SOPMA that the TTG1 protein of Saussurea jellyfish contained 18.69% 偽 -helix 10.09% 尾 -rotation angle 43.03% random crimp and 28.19% extended chain. Random coils and extended chains are the main secondary structures of the protein. The amino acid sequence of the TTG1 gene of Saussurea jellyfish has five WD40 repeats, indicating that the gene may encode a WD40 protein named number: KX4476322.2. The expression of 尾 -actinine 18s rRNA GAPDHFEF and TUA genes in the roots, stems, leaves and flowers of Saussurea jellyfish was detected by real-time fluorescence quantitative PCRQRT-PCR.The stability of the expression of the five genes in the four tissues was analyzed by using geNorm Norm Finder and Bestkeeper software. The results showed that the most stable expression was 18s rRNA in roots, stems, leaves and flowers. Therefore, the 18s rRNA can be used as the corrected reference gene of qRT-PCR of Saussurea jellyfish. The expression of SmTTG1 in roots, stems, leaves and flowers was determined by qRT-PCR using 18SrRNA as an internal reference gene. The results showed that the SmTTG1 gene was expressed in all four tissues, and the highest expression was found in leaves and flowers. The SmTTG1 gene was ligated into the pCold 鈪，

本文編號：1971818