本文關(guān)鍵詞： 糖蛋白GP-1 抗植物病毒活性 基因組　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：植物病毒病發(fā)生普遍、防治困難,嚴(yán)重危害著農(nóng)業(yè)生產(chǎn),并在世界范圍內(nèi)造成了巨大經(jīng)濟(jì)損失。微生物是抗植物病毒劑研發(fā)的重要資源庫(kù),從微生物代謝物中分離與篩選抗植物病毒活性物質(zhì)一直是國(guó)內(nèi)外學(xué)者們的研究熱點(diǎn)�？{斯鏈霉菌ZX01 (Streptomyces kanasensis ZXO1,以下簡(jiǎn)稱鏈霉菌ZX01)是西北農(nóng)林科技大學(xué)無(wú)公害農(nóng)藥研究服務(wù)中心從新疆喀納斯湖分離得到的一株放線菌,經(jīng)過(guò)前期試驗(yàn)表明鏈霉菌ZX01菌株代謝物具有較好的抗植物病毒活性�；诖�,本論文以鏈霉菌ZX01為供試菌株,主要從抗煙草花葉病毒(Tobacco mosaic virus, TMV)活性成分分離與鑒定、基因組測(cè)序與分析、接合轉(zhuǎn)移體系構(gòu)建、nsdA阻斷突變株構(gòu)建等方面進(jìn)行研究,得出以下主要研究結(jié)果：(1)采用萃取、吸附層析、離子交換層析等分離技術(shù)手段,并結(jié)合活性追蹤,從鏈霉菌ZX01代謝物中分離得到了一種具有抗TMV活性的糖蛋白GP-1。該糖蛋白的分子量為8479 Da,多糖和蛋白含量分別為40.23%和54.36%；蛋白部分由15種氨基酸組成,其中酸性氨基酸Asp和Glu含量較高(15.15%和12.63%),堿性氨基酸Arg和Lys含量較低(0.62%和4.88%)；多糖部分由6種單糖組成,分別是甘露糖,木糖,阿拉伯糖、葡萄糖、氨基葡萄糖和半乳糖(0.38：0.14：0.40：2.62：0.10：1.00)；GP-1中同時(shí)存在N-糖苷鍵和O-糖苷鍵；其二級(jí)結(jié)構(gòu)包含20.10%p-折疊,21.70%p-轉(zhuǎn)角和58.30%無(wú)規(guī)則卷曲,無(wú)α-螺旋存在。100℃處理1h對(duì)GP-1的二級(jí)結(jié)構(gòu)影響不大,但對(duì)GP-1的抗TMV活性有較大影響,60℃以下活性穩(wěn)定,80℃以上活性逐漸喪失。經(jīng)過(guò)質(zhì)譜分析,GP-1是一個(gè)結(jié)構(gòu)較為新穎的糖蛋白。利用超濾、親和層析及HPLC技術(shù)建立了糖蛋白GP-1的快速純化與檢測(cè)方法。(2)采用活體和離體方法測(cè)定了糖蛋白GP-1的抗TMV活性,結(jié)果顯示GP-1對(duì)TMV具有較強(qiáng)的保護(hù)效果,能夠較好地阻止TMV侵染寄主植物。鈍化試驗(yàn)與電鏡試驗(yàn)共同表明GP-1對(duì)TMV粒子具有破壞效果,使得TMV喪失侵染能力。另外,GP-1還能抑制TMV外殼蛋白在寄主植物體內(nèi)的積累,從而減輕發(fā)病癥狀。誘導(dǎo)抗性試驗(yàn)結(jié)果證明GP-1能夠誘導(dǎo)寄主抗病性,并且隨著誘導(dǎo)時(shí)間的延長(zhǎng),誘導(dǎo)抗病性先增強(qiáng)后減弱。GP-1處理后,煙草體內(nèi)的防御酶SOD、PPO和PAL活性均急劇升高,而體內(nèi)的MDA含量整體下降。(3)利用高通量測(cè)序技術(shù)對(duì)鏈霉菌ZX01基因組進(jìn)行測(cè)序,最終得到ZX01基因組草圖序列總長(zhǎng)7,026,279 bp,分布于225 contigs中,G+C含量為73.88%�；蝾A(yù)測(cè)得到6245個(gè)編碼基因,其中4176個(gè)蛋白具有明確的生物學(xué)功能,1997個(gè)蛋白與KEGG庫(kù)中的蛋白同源,3996個(gè)蛋白具有COG分類。ZX01基因組中含有7套核糖體RNA操作元和65個(gè)tRNA基因。(4)利用antiSMASH v3.0對(duì)鏈霉菌ZX01基因組中次級(jí)代謝物生物合成基因簇進(jìn)行預(yù)測(cè),結(jié)果發(fā)現(xiàn)了21條次級(jí)代謝物的生物合成基因簇,分布于19個(gè)contigs中。這些基因簇操縱萜類、聚酮類、磷酸酯類、細(xì)菌素類等物質(zhì)的生物合成。聚酮合酶(PKS)或非核糖體肽酶(NRPS)參與的基因簇有Cluster1、9、16、18和20,這5個(gè)基因簇與已知的抗生素合成基因簇相似性較低。半定量PCR結(jié)果顯示,在一般培養(yǎng)條件下Cluster1、9和20能夠正常表達(dá)、Cluster18不表達(dá)、Cluster16部分表達(dá)；經(jīng)過(guò)γ-丁內(nèi)酯誘導(dǎo)后Cluster 1、9、16、18和20均能表達(dá),其中Cluster9和16超量表達(dá)。另外,還對(duì)糖基化相關(guān)基因和全局調(diào)控基因進(jìn)行了預(yù)測(cè),找到了多種糖基轉(zhuǎn)移酶基因和全局調(diào)控因子。(5)對(duì)接合轉(zhuǎn)移體系中的多種條件進(jìn)行探索與優(yōu)化,得了適合鏈霉菌ZX01遺傳操作的接合轉(zhuǎn)移最佳條件：孢子50℃熱激10 min,然后37℃孵育2-3 h,供體和受體的比例為10：1,在含有10～30 mM MgCl2的高氏一號(hào)培養(yǎng)基平板進(jìn)行接合轉(zhuǎn)移,培養(yǎng)16～18h后覆蓋抗生素,此時(shí)的轉(zhuǎn)化效率最高。(6)利用Overlap PCR技術(shù)構(gòu)建了基因重組載體質(zhì)粒pRV5455 (pKC1139::nsdA UD::KanR),結(jié)合使用大腸桿菌ET12567 (pUZ8002),成功阻斷了鏈霉菌ZX01中的全局調(diào)控基因nsdA,獲得了nsdA缺失的ZX01突變株(ZX01△nsdA)。nsdA缺失不僅會(huì)影響鏈霉菌ZX01菌落形態(tài),提高孢子和菌絲生長(zhǎng)量,還能提高糖蛋白GP-1的產(chǎn)量。綜上所述,鏈霉菌ZX01具有較強(qiáng)的天然產(chǎn)物合成能力,在植物病害防控方面具有廣泛的應(yīng)用前景�；蚪M的測(cè)序和遺傳轉(zhuǎn)化體系的建立,為該菌株的分子遺傳、代謝調(diào)控和工程菌構(gòu)建研究提供了豐富的數(shù)據(jù)資料,更為工程菌的構(gòu)建以其在農(nóng)作物病蟲(chóng)害防控方面的實(shí)際應(yīng)用奠定了較為堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Plant virus diseases were widespread, difficult to control, serious harm to agricultural production, and caused tremendous economic losses in the world. Microorganisms are Antiphytoviral agents developed as important resources, from the metabolites of microorganism isolation and screening of antiviral activity has been a research focus of scholars at home and abroad. Kanas (Streptomyces ZX01 Streptomyces kanasensis ZXO1, hereinafter referred to as ZX01) is the Northwest Agriculture and Forestry University Streptomyces pollution-free pesticide research and service center of Xinjiang Kanas Lake were obtained from an actinomycete, after a preliminary test showed that Streptomyces ZX01 metabolites have better activity against plant virus. Based on this, this thesis with Streptomyces ZX01 as tested strains, mainly from the anti tobacco mosaic virus poison (Tobacco mosaic virus, TMV) isolation and identification of active components, sequencing and analysis of genome, joint transfer system, n SdA blocked mutant construction and other aspects of research, major research findings are as follows: (1) by extraction, adsorption chromatography, ion exchange chromatography and other separation techniques, combined with active tracking, got a molecule with anti TMV activity of glycoprotein GP-1. of the glycoprotein was 8479 Da isolated from Streptomyces ZX01 metabolites. The polysaccharide and protein content were 40.23% and 54.36%; protein consists of 15 amino acids, including amino acid Asp and Glu were higher (15.15% and 12.63%), basic amino acids Arg and Lys were low (0.62% and 4.88%); polysaccharide composed of 6 monosaccharides, respectively, mannose, xylose, Arabia sugar, glucose, galactose and glucosamine (0.38:0.14:0.40:2.62:0.10:1.00); N- glycosidic bond and O- glycosidic bond coexist in GP-1; secondary structure contains 20.10%p- folding, 21.70%p- angle and no 58.30% Coil, no alpha helix.100 treatment effect of two level structure of GP-1 1H is small, but has a great influence on the GP-1 activity against TMV, 60 degrees below 80 DEG C activity and stability, activity lost gradually. By mass spectrometry analysis, GP-1 is a relatively novel glycoprotein structure. Using ultrafiltration and affinity chromatography and HPLC technology to establish the rapid purification of glycoprotein GP-1 and detection method. (2) in vivo and in vitro determination of glycoprotein GP-1 of anti TMV activity, the results show that GP-1 has a protective effect on strong TMV, can effectively prevent TMV infection of host plants. The passivation test and SEM test shows that GP-1 has destructive effect on TMV particles, makes TMV lose infection ability. In addition, GP-1 can inhibit TMV capsid protein in host plants of accumulation, so as to reduce the incidence of symptoms. The induced resistance test results show that GP-1 can induce host resistance, And with the induction time, reduced.GP-1 treatment resistance first increased after induction, defense enzymes in tobacco SOD, PPO and PAL activity were increased dramatically, while the total MDA content of in vivo decreased. (3) the sequence of Streptomyces ZX01 genome using high-throughput sequencing, finally ZX01 genome sequence length was 7026279 BP, distributed in 225 contigs, the content of G+C is 6245 73.88%. gene prediction encoding gene, of which 4176 proteins with biological functions clear, homologous protein 1997 protein and KEGG in the library, 3996 proteins with COG classification of.ZX01 genome contains 7 sets of ribosomal RNA operation and 65 tRNA genes (. 4) prediction of secondary metabolite biosynthesis in Streptomyces genome of ZX01 gene cluster with antiSMASH V3.0, found that the biosynthetic gene cluster of 21 secondary metabolites, distributed in 19 contigs . these gene clusters manipulate terpenoids, polyketide biosynthesis, phosphate ester, bacteriocin and other substances. Polyketide synthase (PKS) or non ribosomal peptide enzyme (NRPS) gene cluster in Cluster1,9,16,18 and 20, antibiotic biosynthesis gene cluster of these 5 clusters with known low similarity. Quantitative PCR results showed that under normal culture conditions of Cluster1,9 and 20 normal expression, the expression of Cluster18, Cluster16 expression; after y-butyrolactone after induction of 1,9,16,18 and Cluster were 20 and 16 which can express Cluster9 over expression. In addition, the glycosylation related genes and global regulatory genes were predicted. Find a variety of glycosyltransferase genes and global regulatory factor. (5) exploration and optimization of various conditions of conjugal transfer system, engaging for genetic manipulation of Streptomyces ZX01 got the best transfer conditions: 50 degrees of heat shock 10 Mi spores N, then 37 C after incubation of 2-3 h, the donor and acceptor ratio was 10:1, containing 10 ~ 30 mM MgCl2 kaoi 1 medium plate conjugation culture for 16 ~ 18h coverage after antibiotics, at this time the highest conversion efficiency (6). The recombinant plasmid pRV5455 was constructed by using Overlap PCR Technology (pKC1139:: nsdA UD:: KanR), using a combination of Escherichia coli ET12567 (pUZ8002), successfully blocked global regulatory gene nsdA of Streptomyces ZX01, the nsdA deletion mutant strain ZX01 (ZX01 nsdA.NsdA) will not only affect the colony morphology of Streptomyces ZX01, improve the growth of spores and hyphae, but also improve the glycoprotein GP-1 the yield of Streptomyces. In summary, ZX01 has a strong ability of synthesis of natural products, and has wide application prospect in the prevention and control of plant diseases. The establishment of genome sequencing and genetic transformation system, the molecular genetic strain, The research of metabolic regulation and engineering bacteria construction provided abundant data and data, and laid a solid foundation for the construction of engineering bacteria and its practical application in the control and prevention of crop diseases and insect pests.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S476
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本文編號(hào)：1446676