【摘要】：雞γ干擾素(Chicken interferon-gamma,ChIFN-γ)作為一種細(xì)胞因子,不僅具有廣譜的高抗病毒活性,還起著抗細(xì)菌與抗寄生蟲(chóng)的作用,ChIFN-γ的抗病毒和免疫調(diào)節(jié)機(jī)制在動(dòng)物體內(nèi)已比較清楚。實(shí)驗(yàn)室前期研究發(fā)現(xiàn)ChIFN-γ能顯著提高煙草的抗蟲(chóng)性。轉(zhuǎn)ChIFN-γ煙草的腺毛密度增加了一倍,腺毛分泌物較野生煙草明顯增多,推測(cè)這些性狀提高了煙草抗蟲(chóng)性。通過(guò)檢測(cè)轉(zhuǎn)ChIFN-γ煙草和野生型煙草的全基因組差異表達(dá)譜,結(jié)合GO與KEGG Pathway分析篩選出7個(gè)抗蟲(chóng)候選基因,進(jìn)行熒光定量PCR驗(yàn)證最終獲得3個(gè)可能與煙草腺毛發(fā)育相關(guān)的基因:CyP71、LBM1和IspH。本研究主要以3個(gè)基因的功能為重點(diǎn),通過(guò)基因克隆、遺傳轉(zhuǎn)化擬南芥進(jìn)行基因過(guò)表達(dá)研究,研究結(jié)果如下:通過(guò)提取煙草根和葉總RNA,一步法RT-PCR克隆了CyP71、LBM1和IspH。其中CyP71序列全長(zhǎng)1545 bp,開(kāi)放閱讀框1545 bp,編碼514個(gè)氨基酸,以亮氨酸含量最高(11.9%),生物信息學(xué)推測(cè)蛋白質(zhì)性質(zhì):分子量為58.11 kDa,pI為8.49,蛋白質(zhì)不穩(wěn)定系數(shù)為38.04,屬于穩(wěn)定蛋白質(zhì),無(wú)信號(hào)肽和跨膜結(jié)構(gòu)域存在。已登錄到NCBI的其他物種的CyP71基因中,與馬鈴薯(Solanum tuberosum)的同源性達(dá)最高,達(dá)88%以上,系聚類(lèi)分析表明CyP71在進(jìn)化過(guò)程中相對(duì)保守。LBM1序列全長(zhǎng)846 bp,開(kāi)放閱讀框846 bp,編碼281個(gè)氨基酸,絲氨酸含量最高(9.6%),推測(cè)蛋白質(zhì)性質(zhì):分子量31.98 kDa,pI為5.08,蛋白質(zhì)不穩(wěn)定系數(shù)48.07,屬于不穩(wěn)定蛋白,未匹配到信號(hào)肽和跨膜結(jié)構(gòu)域。蛋白質(zhì)結(jié)構(gòu)域分析發(fā)現(xiàn)LBM屬于MYB類(lèi)轉(zhuǎn)錄因子并存在一個(gè)蛋白-蛋白互作的SANT DNA結(jié)合的結(jié)構(gòu)域。IspH序列長(zhǎng)1386 bp,開(kāi)放閱讀框1386 bp,編碼461個(gè)氨基酸,谷氨酸和賴氨酸含量最高分別為8.7%和8.5%,分子量51.66 kDa,預(yù)測(cè)pI為5.59,蛋白質(zhì)穩(wěn)定系數(shù)29.02,屬于穩(wěn)定蛋白,推測(cè)存在線粒體和葉綠體的靶向信號(hào)肽。氨基酸序列在茄科植物中高度保守,與馬鈴薯和番茄的同源性都在92%以上,蛋白質(zhì)結(jié)構(gòu)域分析結(jié)果表明IspH屬于Lyt B超家族,這類(lèi)家族蛋白負(fù)責(zé)催化MEP途徑的最后一步反應(yīng)。構(gòu)建含植物過(guò)表達(dá)載體pSH737-IspH、pSH737-CyP71和pSH737-LBM的農(nóng)桿菌LBA4404,通過(guò)蘸花法遺傳轉(zhuǎn)化擬南芥,果莢成熟后收集種子進(jìn)行篩選,得到CyP71抗性植株38株,LBM1 35株,IspH 30株,3個(gè)基因的轉(zhuǎn)化率均在5%以上,其中對(duì)IspH抗性植株隨機(jī)抽取10株進(jìn)行GUS染色和基因組PCR驗(yàn)證,結(jié)果均為陽(yáng)性。轉(zhuǎn)IspH植株長(zhǎng)勢(shì)較同時(shí)期的野生型稍好,葉片顏色較深,GUS染色結(jié)果顯示,轉(zhuǎn)IspH擬南芥葉片所有的表皮毛細(xì)胞GUS報(bào)告基因都能被啟動(dòng)表達(dá)。體視顯微鏡下觀測(cè),表皮毛細(xì)胞均呈藍(lán)色,表明IspH基因與表皮毛的發(fā)育直接相關(guān)。轉(zhuǎn)IspH擬南芥表皮毛與野生型的大小和分布無(wú)顯著差異,但數(shù)量較多。統(tǒng)計(jì)分析發(fā)現(xiàn),25 mm2葉片面積內(nèi),轉(zhuǎn)IspH擬南芥表皮毛數(shù)量平均為19.6,野生型平均為9.6,轉(zhuǎn)IspH表皮毛數(shù)量增加了一倍以上,說(shuō)明IspH基因的表達(dá)與擬南芥表皮毛的發(fā)育呈正相關(guān)。LBM1與CyP71抗性植株還處于幼苗階段,該時(shí)期下其表皮毛較野生型擬南芥無(wú)顯著差異,具體的調(diào)控作用還待進(jìn)一步觀察。研究為闡釋ChIFN-γ調(diào)控?zé)煵菹倜l(fā)育的作用機(jī)制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：貴州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2

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