發(fā)布時(shí)間：2018-04-12 06:59

  本文選題：蘇云金芽胞桿菌 + Cry1Ah蛋白　； 參考：《東北農(nóng)業(yè)大學(xué)》2014年碩士論文

【摘要】：蘇云金芽孢桿菌(Bacillus thuringiensis,簡(jiǎn)稱(chēng)Bt)由于其對(duì)靶標(biāo)害蟲(chóng)具有殺蟲(chóng)特異性,而對(duì)人畜無(wú)害,不污染環(huán)境,因而被作為微生物殺蟲(chóng)劑應(yīng)用已有60年的歷史,并且cry基因也已經(jīng)應(yīng)用于轉(zhuǎn)基因作物,同時(shí)蘇云金芽胞桿菌也成為目前全球應(yīng)用最廣泛的微生物殺蟲(chóng)劑。Cry毒素對(duì)靶標(biāo)害蟲(chóng)所具有的殺蟲(chóng)特異性取決于毒素與昆蟲(chóng)中腸上皮細(xì)胞特異性受體之間的相互作用,因此對(duì)于昆蟲(chóng)中腸受體結(jié)構(gòu)與功能的研究不僅可以為毒素與受體間的相互作用提供材料,同時(shí)也為研究Cry毒素的作用機(jī)制奠定基礎(chǔ)。氨肽酶N(animopeptidase N,簡(jiǎn)稱(chēng)APN)是公認(rèn)的Cry蛋白的受體之一,也是研究得最成熟的受體,屬于肽鏈端解酶,在鱗翅目昆蟲(chóng)中可以水解蛋白質(zhì)或多肽N末端的氨基酸。 crylAhl基因是由本實(shí)驗(yàn)室從天然菌株BT8中分離并克隆的,并且是具有自主知識(shí)產(chǎn)權(quán)的新型殺蟲(chóng)晶體蛋白基因,表達(dá)133kDa的Cry1Ah蛋白,并對(duì)棉鈴蟲(chóng)(Helicoverpa armigera)、亞洲玉米螟(Ostrinia furnacalis)和水稻二化螟(Chilo suppressalis)等多種鱗翅目害蟲(chóng)表現(xiàn)出高于商業(yè)化crylAc基因的殺蟲(chóng)活性。同源性比較結(jié)果表明：crylAh全長(zhǎng)基因與cry1Ac全長(zhǎng)基因所編碼的氨基酸序列相似性為86%,并且兩者表現(xiàn)出相似的生物活性,即對(duì)棉鈴蟲(chóng)高毒力,對(duì)家蠶安全低毒。 本實(shí)驗(yàn)室周子珊博士利用配體垂釣平臺(tái),發(fā)現(xiàn)Cry1Ah和CrylAc毒素在棉鈴蟲(chóng)的BBMV上雖然存在著不同的結(jié)合蛋白,但都有一條特異性較強(qiáng)的結(jié)合蛋白條帶,而在家蠶的BBMV上并沒(méi)有檢測(cè)到這種高親和力的蛋白,經(jīng)過(guò)質(zhì)譜鑒定,結(jié)果表明這種蛋白是棉鈴蟲(chóng)中腸蛋白APN1,因此推測(cè)這種結(jié)合蛋白可能是CrylAh和Cry1Ac蛋白對(duì)棉鈴蟲(chóng)產(chǎn)生殺蟲(chóng)特異性與高毒力的原因。 本研究通過(guò)設(shè)計(jì)全長(zhǎng)引物克隆得到棉鈴蟲(chóng)中腸受體apnl基因,并利用原核系統(tǒng)進(jìn)行表達(dá),同時(shí)在體外通過(guò)Western blotting雜交實(shí)驗(yàn),驗(yàn)證了APN1可以與CrylAh和CrylAc毒素結(jié)合。生物信息學(xué)分析表明：本研究克隆并表達(dá)的APN1含有鱗翅目昆蟲(chóng)中腸氨肽酶的結(jié)構(gòu)特點(diǎn),即具有高度保守的GAMEN區(qū)域和HEXXH鋅指結(jié)構(gòu),經(jīng)預(yù)測(cè)其氨基酸序列中存在潛在的N-連接和O-連接的糖基化位點(diǎn)。 利用Western blotting檢測(cè)APN1是否可以與毒素結(jié)合時(shí),我們發(fā)現(xiàn)100kDa和70kDa左右的APN1短片段也可以與Cry1Ah和Cry1Ac蛋白結(jié)合,經(jīng)質(zhì)譜鑒定發(fā)現(xiàn)它們都含有APN1,因此我們認(rèn)為這些短片段是APN1全長(zhǎng)蛋白降解的結(jié)果。 為了進(jìn)一步確定棉鈴蟲(chóng)中腸受體APN1與Cry1Ah蛋白的結(jié)合區(qū),根據(jù)APN1的結(jié)構(gòu)特點(diǎn)將其分成四段進(jìn)行分段克隆、表達(dá)及與CrylAh蛋白的體外結(jié)合實(shí)驗(yàn)。結(jié)果發(fā)現(xiàn)：片段H1、H2和H3在大腸桿菌中成功表達(dá)蛋白。結(jié)合實(shí)驗(yàn)發(fā)現(xiàn)只有片段H3與Cry1Ah蛋白有比較明顯的結(jié)合。 本研究所構(gòu)建的原核表達(dá)體系可以為其它昆蟲(chóng)中腸受體的克隆與表達(dá)奠定基礎(chǔ),同時(shí)也為Cry毒素與受體互作機(jī)制的研究提供思路。體外結(jié)合實(shí)驗(yàn)和Western雜交可以作為體外驗(yàn)證昆蟲(chóng)中腸受體與毒素結(jié)合能力的一種可行性方案,初步確定受體與毒素之間相互作用的關(guān)系。后續(xù)研究是對(duì)片段H3蛋白進(jìn)行純化并與Cry1Ah毒素結(jié)合,最終確定棉鈴蟲(chóng)APN1與CrylAh蛋白的結(jié)合區(qū)。
[Abstract]:Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt) because of its insecticidal specificity to the target pest, and is harmless to human beings and animals, do not pollute the environment, which is used as a microbial insecticide has a history of 60 years, and the cry gene has also been applied to transgenic crops, while Bacillus thuringiensis has become each other effect on specific microbial pesticide insecticidal.Cry toxin is currently the world's most widely used of target pests in between the toxin and the insect midgut epithelial cell specific receptor, therefore research on the structure and function of insect midgut receptors not only can provide materials for interaction between toxin and receptor, but also laid the foundation for the mechanism of action study of Cry toxin. Aminopeptidase N (animopeptidase N, referred to as APN) is one of the most recognized Cry receptor protein, is studied by the most mature peptide, which belongs to Chain end hydrolysate, an amino acid that can hydrolyze the N terminal of protein or polypeptide in Lepidoptera.
The crylAhl gene is from the laboratory from natural strain BT8 isolated and cloned, and novel insecticidal crystal protein gene with independent intellectual property rights, the expression of 133kDa Cry1Ah protein, and the cotton bollworm (Helicoverpa armigera), the Asian corn borer (Ostrinia furnacalis) and Chilo suppressalis (Chilo suppressalis) and other Lepidoptera pests show higher than the commercial insecticidal activity of crylAc gene. The homology comparison showed that the amino acid sequence of the full-length crylAh gene and cry1Ac gene encoding the similarity of 86%, and both showed similar biological activity of cotton bollworm with high toxicity to silkworm, safety and low toxicity.
The laboratory of Dr. Zhou Zishan using ligand fishing platform, found that Cry1Ah and CrylAc toxin in cotton bollworm BBMV binding protein, although there are different, but have a strong specific binding protein bands in the silkworm BBMV did not detect this high affinity protein identification by mass spectrometry. Results show that this protein is the midgut protein APN1, suggesting that this may be the CrylAh binding protein and Cry1Ac protein have insecticidal specificity and high virulence on the cotton bollworm.
This study obtained the midgut receptor apnl gene by cloning full-length primer design, and expressed by prokaryotic system, at the same time, through the experiment of Western blotting hybridization in vitro, proved that APN1 could combine with CrylAh and CrylAc toxin. Bioinformatics analysis showed that: the structure of the research on cloning and expression of APN1 containing the lepidopteran midgut the aminopeptidase, which is the GAMEN and HEXXH regions of highly conserved zinc fingers, the predicted the existence of potential glycosylation sites N- and O- linked its amino acid sequence.
Whether the use of Western blotting APN1 could be detected with toxin binding, we found that APN1 and 100kDa short fragments of about 70kDa can be combined with Cry1Ah and Cry1Ac proteins were identified by mass spectrometry revealed that they all contain APN1, so we think that these short fragments are APN1 full-length protein degradation results.
In order to further determine the binding region of the midgut receptors APN1 and Cry1Ah protein, according to the structure characteristics of APN1 will be divided into four sections section cloning, expression and in vitro binding experiments with CrylAh protein. The results showed that the fragment of H1, H2 and H3 successfully expressed protein in Escherichia coli. Combined with the experiment found that only fragments of H3 and Cry1Ah a combination of protein obviously.
The prokaryotic expression system constructed in this research for cloning and expression of other insect midgut receptors to lay the foundation, but also provide ideas for the research on the interaction mechanism of Cry toxin receptor. A feasible scheme of in vitro binding assay and Western hybridization can be used as in vitro validation of insect midgut receptors and toxin binding ability, preliminary to determine the relationship between each other interactions between the receptor and the toxin. Further research is the fragment of H3 protein was purified and combined with Cry1Ah toxin, and ultimately determine the binding region of cotton bollworm APN1 and CrylAh protein.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：S435.622

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相關(guān)期刊論文 前2條

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2 王桂榮,梁革梅,吳孔明,郭予元;棉鈴蟲(chóng)中腸氨基氨肽酶N基因的克隆及序列分析[J];中國(guó)農(nóng)業(yè)科學(xué);2003年11期

本文編號(hào)：1738659