喀纳斯链霉菌ZX01抗TMV活性物质分离及全局调控基因nsdA阻断研究

发布时间：2018-01-20 03:34

  本文关键词： 糖蛋白GP-1 抗植物病毒活性 基因组　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

【摘要】：植物病毒病发生普遍、防治困难,严重危害着农业生产,并在世界范围内造成了巨大经济损失。微生物是抗植物病毒剂研发的重要资源库,从微生物代谢物中分离与筛选抗植物病毒活性物质一直是国内外学者们的研究热点。喀纳斯链霉菌ZX01 (Streptomyces kanasensis ZXO1,以下简称链霉菌ZX01)是西北农林科技大学无公害农药研究服务中心从新疆喀纳斯湖分离得到的一株放线菌,经过前期试验表明链霉菌ZX01菌株代谢物具有较好的抗植物病毒活性。基于此,本论文以链霉菌ZX01为供试菌株,主要从抗烟草花叶病毒(Tobacco mosaic virus, TMV)活性成分分离与鉴定、基因组测序与分析、接合转移体系构建、nsdA阻断突变株构建等方面进行研究,得出以下主要研究结果：(1)采用萃取、吸附层析、离子交换层析等分离技术手段,并结合活性追踪,从链霉菌ZX01代谢物中分离得到了一种具有抗TMV活性的糖蛋白GP-1。该糖蛋白的分子量为8479 Da,多糖和蛋白含量分别为40.23%和54.36%；蛋白部分由15种氨基酸组成,其中酸性氨基酸Asp和Glu含量较高(15.15%和12.63%),碱性氨基酸Arg和Lys含量较低(0.62%和4.88%)；多糖部分由6种单糖组成,分别是甘露糖,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中同时存在N-糖苷键和O-糖苷键；其二级结构包含20.10%p-折叠,21.70%p-转角和58.30%无规则卷曲,无α-螺旋存在。100℃处理1h对GP-1的二级结构影响不大,但对GP-1的抗TMV活性有较大影响,60℃以下活性稳定,80℃以上活性逐渐丧失。经过质谱分析,GP-1是一个结构较为新颖的糖蛋白。利用超滤、亲和层析及HPLC技术建立了糖蛋白GP-1的快速纯化与检测方法。(2)采用活体和离体方法测定了糖蛋白GP-1的抗TMV活性,结果显示GP-1对TMV具有较强的保护效果,能够较好地阻止TMV侵染寄主植物。钝化试验与电镜试验共同表明GP-1对TMV粒子具有破坏效果,使得TMV丧失侵染能力。另外,GP-1还能抑制TMV外壳蛋白在寄主植物体内的积累,从而减轻发病症状。诱导抗性试验结果证明GP-1能够诱导寄主抗病性,并且随着诱导时间的延长,诱导抗病性先增强后减弱。GP-1处理后,烟草体内的防御酶SOD、PPO和PAL活性均急剧升高,而体内的MDA含量整体下降。(3)利用高通量测序技术对链霉菌ZX01基因组进行测序,最终得到ZX01基因组草图序列总长7,026,279 bp,分布于225 contigs中,G+C含量为73.88%。基因预测得到6245个编码基因,其中4176个蛋白具有明确的生物学功能,1997个蛋白与KEGG库中的蛋白同源,3996个蛋白具有COG分类。ZX01基因组中含有7套核糖体RNA操作元和65个tRNA基因。(4)利用antiSMASH v3.0对链霉菌ZX01基因组中次级代谢物生物合成基因簇进行预测,结果发现了21条次级代谢物的生物合成基因簇,分布于19个contigs中。这些基因簇操纵萜类、聚酮类、磷酸酯类、细菌素类等物质的生物合成。聚酮合酶(PKS)或非核糖体肽酶(NRPS)参与的基因簇有Cluster1、9、16、18和20,这5个基因簇与已知的抗生素合成基因簇相似性较低。半定量PCR结果显示,在一般培养条件下Cluster1、9和20能够正常表达、Cluster18不表达、Cluster16部分表达；经过γ-丁内酯诱导后Cluster 1、9、16、18和20均能表达,其中Cluster9和16超量表达。另外,还对糖基化相关基因和全局调控基因进行了预测,找到了多种糖基转移酶基因和全局调控因子。(5)对接合转移体系中的多种条件进行探索与优化,得了适合链霉菌ZX01遗传操作的接合转移最佳条件：孢子50℃热激10 min,然后37℃孵育2-3 h,供体和受体的比例为10：1,在含有10～30 mM MgCl2的高氏一号培养基平板进行接合转移,培养16～18h后覆盖抗生素,此时的转化效率最高。(6)利用Overlap PCR技术构建了基因重组载体质粒pRV5455 (pKC1139::nsdA UD::KanR),结合使用大肠杆菌ET12567 (pUZ8002),成功阻断了链霉菌ZX01中的全局调控基因nsdA,获得了nsdA缺失的ZX01突变株(ZX01△nsdA)。nsdA缺失不仅会影响链霉菌ZX01菌落形态,提高孢子和菌丝生长量,还能提高糖蛋白GP-1的产量。综上所述,链霉菌ZX01具有较强的天然产物合成能力,在植物病害防控方面具有广泛的应用前景。基因组的测序和遗传转化体系的建立,为该菌株的分子遗传、代谢调控和工程菌构建研究提供了丰富的数据资料,更为工程菌的构建以其在农作物病虫害防控方面的实际应用奠定了较为坚实的基础。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476
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本文编号：1446676