ChIFN-γ调控烟草腺毛发育基因功能研究

发布时间：2018-12-15 14:22

【摘要】：鸡γ干扰素(Chicken interferon-gamma,ChIFN-γ)作为一种细胞因子,不仅具有广谱的高抗病毒活性,还起着抗细菌与抗寄生虫的作用,ChIFN-γ的抗病毒和免疫调节机制在动物体内已比较清楚。实验室前期研究发现ChIFN-γ能显著提高烟草的抗虫性。转ChIFN-γ烟草的腺毛密度增加了一倍,腺毛分泌物较野生烟草明显增多,推测这些性状提高了烟草抗虫性。通过检测转ChIFN-γ烟草和野生型烟草的全基因组差异表达谱,结合GO与KEGG Pathway分析筛选出7个抗虫候选基因,进行荧光定量PCR验证最终获得3个可能与烟草腺毛发育相关的基因:CyP71、LBM1和IspH。本研究主要以3个基因的功能为重点,通过基因克隆、遗传转化拟南芥进行基因过表达研究,研究结果如下:通过提取烟草根和叶总RNA,一步法RT-PCR克隆了CyP71、LBM1和IspH。其中CyP71序列全长1545 bp,开放阅读框1545 bp,编码514个氨基酸,以亮氨酸含量最高(11.9%),生物信息学推测蛋白质性质:分子量为58.11 kDa,pI为8.49,蛋白质不稳定系数为38.04,属于稳定蛋白质,无信号肽和跨膜结构域存在。已登录到NCBI的其他物种的CyP71基因中,与马铃薯(Solanum tuberosum)的同源性达最高,达88%以上,系聚类分析表明CyP71在进化过程中相对保守。LBM1序列全长846 bp,开放阅读框846 bp,编码281个氨基酸,丝氨酸含量最高(9.6%),推测蛋白质性质:分子量31.98 kDa,pI为5.08,蛋白质不稳定系数48.07,属于不稳定蛋白,未匹配到信号肽和跨膜结构域。蛋白质结构域分析发现LBM属于MYB类转录因子并存在一个蛋白-蛋白互作的SANT DNA结合的结构域。IspH序列长1386 bp,开放阅读框1386 bp,编码461个氨基酸,谷氨酸和赖氨酸含量最高分别为8.7%和8.5%,分子量51.66 kDa,预测pI为5.59,蛋白质稳定系数29.02,属于稳定蛋白,推测存在线粒体和叶绿体的靶向信号肽。氨基酸序列在茄科植物中高度保守,与马铃薯和番茄的同源性都在92%以上,蛋白质结构域分析结果表明IspH属于Lyt B超家族,这类家族蛋白负责催化MEP途径的最后一步反应。构建含植物过表达载体pSH737-IspH、pSH737-CyP71和pSH737-LBM的农杆菌LBA4404,通过蘸花法遗传转化拟南芥,果荚成熟后收集种子进行筛选,得到CyP71抗性植株38株,LBM1 35株,IspH 30株,3个基因的转化率均在5%以上,其中对IspH抗性植株随机抽取10株进行GUS染色和基因组PCR验证,结果均为阳性。转IspH植株长势较同时期的野生型稍好,叶片颜色较深,GUS染色结果显示,转IspH拟南芥叶片所有的表皮毛细胞GUS报告基因都能被启动表达。体视显微镜下观测,表皮毛细胞均呈蓝色,表明IspH基因与表皮毛的发育直接相关。转IspH拟南芥表皮毛与野生型的大小和分布无显著差异,但数量较多。统计分析发现,25 mm2叶片面积内,转IspH拟南芥表皮毛数量平均为19.6,野生型平均为9.6,转IspH表皮毛数量增加了一倍以上,说明IspH基因的表达与拟南芥表皮毛的发育呈正相关。LBM1与CyP71抗性植株还处于幼苗阶段,该时期下其表皮毛较野生型拟南芥无显著差异,具体的调控作用还待进一步观察。研究为阐释ChIFN-γ调控烟草腺毛发育的作用机制奠定了基础。
[Abstract]:Chicken interferon-gamma (ChIFN-1), as a cytokine, not only has a broad spectrum of high antiviral activity, but also plays an anti-bacterial and antiparasitic role, and the anti-viral and immunomodulating mechanisms of the ChIFN-1 are more clear in the animal body. In the early stage of the laboratory, it was found that the chIFN-1 could significantly improve the insect-resistance of the tobacco. The density of the glandular hairs transferred to the ChIFN-1 tobacco was doubled, and the secretion of the glandular hairs increased significantly compared with that of the wild tobacco, and it was speculated that these traits increased the resistance of the tobacco to the resistance of the tobacco. Seven anti-insect candidate genes were screened by detection of the full-genome differential expression profile of the transgenic ChIFN-1 tobacco and the wild-type tobacco, and seven anti-insect candidate genes were screened by the combination of GO and KEGG Pathway analysis, and the genes related to the development of the tobacco glandular hairs were finally obtained by fluorescence quantitative PCR verification: CyP71, LBM1, and IspH. This study focused on the function of three genes, and through gene cloning and genetic transformation of Arabidopsis, the results of the study were as follows: CyP71, LBM1 and IspH were cloned by one-step RT-PCR by extracting total RNA of tobacco and leaf. The total length of CyP71 sequence is 1545 bp, the open reading frame is 1545bp, the coding is 514 amino acids, the leucine content is the highest (11. 9%), the biological information is the protein property: the molecular weight is 58. 11 kDa, the pI is 8.49, the protein instability coefficient is 38. 04, and belongs to the stable protein. There is no signal peptide and transmembrane domain present. In the CyP71 gene of other species that have been registered in NCBI, the homology with Solanum tuberosum is up to 88%, and the cluster analysis shows that CyP71 is relatively conservative in the process of evolution. The total length of the LBM1 sequence is 846 bp, the open reading frame is 846 bp, the coding is 281 amino acids, the serine content is the highest (9. 6%), the protein property is deduced, the molecular weight is 31.98 kDa, the pI is 5.08, and the protein instability coefficient is 48.07, which belongs to the unstable protein and does not match the signal peptide and the transmembrane domain. The analysis of the protein domain found that the LBM is a domain of the MYB transcription factor and that there is a protein-protein cross-linked SANT DNA binding. The sequence of IspH was 1386bp, the open reading frame was 1386bp, the amino acids were encoded, the content of glutamic acid and lysine was 8. 7% and 8.5%, the molecular weight was 51. 66 kDa, the predicted pI was 5.59, and the protein stability coefficient was 2.02, which was a stable protein, and it was presumed that the target signal peptide of the mitochondria and the chloroplast was present. The amino acid sequence is highly conserved in the solanaceae plant, and the homology with the potato and the tomato is more than 92%. The analysis of the protein domain shows that the IspH is the family of the Lyt B-ultrasound, and the family of family proteins is responsible for the final step reaction of the MEP pathway. the agrobacterium LBA4404 containing the plant-overexpressing vector pSH737-IspH, pSH737-CyP71 and pSH737-LBM is constructed, and the seeds are collected through the genetic transformation of the arabidopsis arabidopsis by a dipping method, and the seeds are collected for screening after the fruit is mature, so that 38 strains of the CyP71 resistant plant, 35 strains of LBM1 and 30 strains of IspH are obtained, and the conversion rate of the three genes is more than 5 percent, in which 10 plants were randomly selected for GUS staining and genomic PCR, and the results were positive. The long potential of the transgenic IspH plants was slightly better than that of the wild type in the same period, and the color of the leaves was deep. The GUS staining showed that the GUS reporter gene of all the epidermal hair cells of the transgenic Isophanan leaves can be activated and expressed. It was observed under the stereomicroscope that the epidermal hair cells were blue, indicating that the IspH gene was directly related to the development of the epidermal hair. There was no significant difference in the size and distribution of the wild type and the size and distribution of the epidermal hair of Arabidopsis thaliana. The results showed that in the area of 25 mm2, the average number of the epidermal hairs of the transgenic Isophans was 19. 6, the average of the wild-type was 9. 6, and the number of the TFs increased by more than one time, indicating that the expression of the IspH gene was positively related to the development of the epidermal hair of Arabidopsis. The BM1 and CyP71 resistant plants are also in the stage of the seedling, and its epidermal hair is no significant difference with the wild type of Arabidopsis, and the specific regulatory action is still to be observed. The study has laid a foundation for elucidating the mechanism of the regulation of the development of the gIFN-1 in the regulation of the development of the glandular hairs of the tobacco.
【学位授予单位】：贵州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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