水母雪莲SmTTG1基因的克隆及表达研究

发布时间：2018-06-03 06:28

  本文选题：水母雪莲 + SmTTG1　； 参考：《青海大学》2017年硕士论文

【摘要】：水母雪莲(Saussurea medusa Maxim.),又称水母雪兔子,属菊科风毛菊属,是一种生长于青藏高原地区的珍稀、濒危药用植物。同时,它又是一种典型的高山植物。植株密被表皮毛是水母雪莲最具有代表性的形态特征,它是水母雪莲对极端环境的一种适应结构。研究水母雪莲表皮毛的形成和发育过程,对揭示高山植物适应环境的特殊机制将具有重要的理论意义。为此,本研究以水母雪莲作为实验材料,通过RT-PCR,结合RACE技术,克隆了与表皮毛发育相关的基因TTG1,并研究了该基因的表达。主要研究结果如下:1.克隆到的水母雪莲TTG1基因全长1346 bp,其中包括1014 bp的开放阅读框,99 bp的5’端非编码区,211 bp的3’端非编码区,22 bp的ploy(A)尾巴。该基因共编码337个氨基酸残基,分子量为37.75 KD,等电点为4.88。同源比对显示,水母雪莲TTG1基因的核苷酸序列与同一科植物大丽花TTG1基因的核苷酸序列的同源性为75%;推导出来的氨基酸序列与大丽花的同源性为83%,与拟南芥、烟草的同源性为74.56%、73.98%。通过SOPMA工具预测,水母雪莲TTG1蛋白包含18.69%的α-螺旋,10.09%的β-转角,43.03%的无规卷曲,28.19%的延伸链。无规卷曲和延伸链为该蛋白的主要二级结构。水母雪莲TTG1基因的氨基酸序列有五个WD40 repeat,说明该基因可能编码一种WD40蛋白,将该基因命名为SmTTG1(genbank accession number:KX447632)。2.采用实时荧光定量PCR(qRT-PCR)技术,检测β-actin、18SrRNA、GAPDH、EF和TUA五个基因在水母雪莲根、茎、叶、花中的表达情况,运用geNorm、NormFinder和Bestkeeper三个软件分析这5个基因在这4种组织内表达的稳定性。结果显示,在根、茎、叶、花中表达最稳定的是18SrRNA。因此,18SrRNA可以作为水母雪莲qRT-PCR的校正内参基因。3.以18SrRNA作为内参基因,运用qRT-PCR技术,测定SmTTG1在根、茎、叶、花中的表达情况。结果显示,SmTTG1基因在4种组织中都有表达,其中在叶和花中的表达量最高。4.将SmTTG1基因连接到pColdⅠDNA表达载体,并导入BL21(DE3)大肠杆菌中进行原核表达,表达产物用SDS-PAGE电泳分析。结果显示,目的蛋白在大肠杆菌中表达,表达蛋白的分子量大约是38KD,与由核苷酸序列推导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% 伪 -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，

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