蝗綠僵菌葡聚糖苷轉(zhuǎn)移酶基因的功能研究
發(fā)布時(shí)間：2018-09-17 16:26
【摘要】：昆蟲容易受病原真菌感染而致病,其攜帶的病原真菌會(huì)進(jìn)一步傳播和擴(kuò)散造成大面積的昆蟲群感染并死亡。因此,利用昆蟲病原真菌防治農(nóng)業(yè)害蟲是一種長(zhǎng)效和環(huán)保的生防措施。真菌細(xì)胞壁是真菌細(xì)胞外周的重要結(jié)構(gòu),為真菌抵御外界不良環(huán)境和寄主免疫反應(yīng)等提供保護(hù),葡聚糖苷轉(zhuǎn)移酶具有水解和轉(zhuǎn)移酶的活性,在葡聚糖分子組裝和維持細(xì)胞壁完整性方面具有重要作用。但是其在真菌生長(zhǎng)發(fā)育過程中的作用機(jī)制還不清楚。闡明葡聚糖苷轉(zhuǎn)移酶在病原真菌中的功能將為提高真菌農(nóng)藥的毒力及抗真菌藥物的篩選提供參考依據(jù)。為此,本課題以蝗綠僵菌為試驗(yàn)材料,采用同源重組技術(shù)構(gòu)建了葡聚糖苷轉(zhuǎn)移酶敲除及回復(fù)菌株,對(duì)其生長(zhǎng)、毒力及抗逆特性進(jìn)行了初步的分析。主要研究?jī)?nèi)容:1.采用生物信息學(xué)分析基因的結(jié)構(gòu)2.構(gòu)建敲除及回復(fù)菌株利用同源重組方法構(gòu)建了ΔMwg2,ΔMwg3,??Mwg1/Mwg2、??Mwg2/Mwg3敲除及回復(fù)菌株。3.基因的功能分析(1)生長(zhǎng)特性分析:對(duì)野生、敲除及回復(fù)菌株的孢子進(jìn)行接種培養(yǎng),測(cè)定其萌發(fā)率和產(chǎn)孢量并觀察其菌絲的結(jié)構(gòu)。(2)抗逆特性分析:對(duì)野生、敲除和回復(fù)菌株的孢子分別進(jìn)行熱激、紫外照射處理,統(tǒng)計(jì)萌發(fā)率,比較其萌發(fā)半抑制時(shí)間的差異。(3)毒力分析:用新鮮成熟的野生、敲除型菌株的孢子配制成孢懸液,采用體內(nèi)注射和體表侵染方法感染東亞飛蝗,統(tǒng)計(jì)半致死時(shí)間,分析毒力差異。主要研究結(jié)果:1.經(jīng)過生物信息學(xué)分析得出,Mwg1:Gen Bank:EFY91318.1,c DNA是1367bp,氨基酸大小是406aa,等電點(diǎn)是5.22p I,分子量是41k Da,其中含有1個(gè)內(nèi)含子;Mwg2:Gen Bank:EFY92943.1,c DNA是998bp,氨基酸大小是277aa,等電點(diǎn)是5.03p I,分子量是26k Da,其中含有1個(gè)內(nèi)含子;Mwg3:Gen Bank:EFY86494.1,c DNA是1669bp,氨基酸大小是511aa,等電點(diǎn)是4.89p I,分子量是52k Da,其中含有2個(gè)內(nèi)含子。2.本實(shí)驗(yàn)構(gòu)建了?Mwg2、?Mwg3和CP1、CP2、CP3菌株,同時(shí)構(gòu)建了??Mwg1/Mwg2、??Mwg2/Mwg3菌株3.葡聚糖苷轉(zhuǎn)移酶基因?qū)染G僵菌生長(zhǎng)特性的影響(1)萌發(fā)與產(chǎn)孢方面:?Mwg1,?Mwg3與WT和CP相比,產(chǎn)孢量增加,萌發(fā)提前;而?Mwg2與WT和CP相比,產(chǎn)孢量減少,萌發(fā)延遲;??Mwg1/Mwg2和??Mwg2/Mwg3與WT相比,產(chǎn)孢量增加,萌發(fā)提前。(2)菌絲結(jié)構(gòu)方面:?Mwg1與WT和CP相比,菌絲末端更細(xì)長(zhǎng),菌絲膈間距離增大;?Mwg2與WT和CP相比,分支數(shù)明顯增加;?Mwg2與WT和CP相比,菌絲末端更細(xì)長(zhǎng),菌絲膈間距離增大;??Mwg1/Mwg2、??Mwg2/Mwg3與WT相比,菌絲末端更細(xì)長(zhǎng),菌絲膈間距離增大。4.聚糖苷轉(zhuǎn)移酶基因?qū)染G僵菌抗逆特性的影響(1)紫外耐受性方面:?Mwg1、?Mwg3、??Mwg1/Mwg2、??Mwg2/Mwg3與WT和CP相比,紫外處理后孢子萌發(fā)率提前,紫外耐受力增強(qiáng);?Mwg2與WT和CP相比,紫外處理后孢子萌發(fā)率延遲,紫外耐受力減弱。(2)濕熱耐受性方面:?Mwg1與WT和CP相比,濕熱處理后孢子萌發(fā)率提前,濕熱耐受力增強(qiáng);?Mwg2與WT和CP相比,濕熱處理后孢子萌發(fā)率延遲,濕熱耐受力減弱;?Mwg2、??Mwg2/Mwg3與WT和CP相比無顯著性差異;??Mwg1/Mwg2與WT相比,濕熱處理后孢子萌發(fā)率提前。(3)細(xì)胞壁敏感劑方面:在加有細(xì)胞壁敏感劑剛果紅的1/4SADAY平板上,?Mwg1與WT和CP相比,菌落形態(tài)明顯減小,生長(zhǎng)緩慢;??Mwg1/Mwg2與WT相比,也表現(xiàn)為生長(zhǎng)緩慢,而其他敲除菌株無顯著性影響;各個(gè)菌株在添加有Na Cl、Sor的1/4SADAY平板上生長(zhǎng)情況與WT相比,無顯著性差異,說明葡聚糖苷轉(zhuǎn)移酶不影響綠僵菌對(duì)高滲透壓的耐受性。5.聚糖苷轉(zhuǎn)移酶基因?qū)染G僵菌毒力的影響主要采用了體表侵染和體內(nèi)注射,分別制備敲除和WT菌株的孢懸液,感染東亞飛蝗,分析葡聚糖苷轉(zhuǎn)移酶基因?qū)染G僵菌毒力的影響。研究結(jié)果表明:體表侵染時(shí),?Mwg1、?Mwg2、?Mwg3與WT相比,毒力無差異;但??Mwg1/Mwg2、??Mwg2/Mwg3與WT相比,毒力明顯減弱。體內(nèi)注射時(shí),?Mwg2與WT相比,毒力無差異;但?Mwg3與WT相比,毒力增強(qiáng);??Mwg1/Mwg2、??Mwg2/Mwg3與WT相比,毒力增強(qiáng)。
[Abstract]:Insects are susceptible to pathogenic fungi. The pathogenic fungi carried by insects can further spread and cause large-scale insect infections and deaths. Therefore, the use of insect pathogenic fungi to control agricultural pests is a long-term and environmentally friendly biocontrol measure. Fungal cell wall is an important structure in the periphery of fungal cells, which protects fungi from the outside world. Dextransferase has the activities of hydrolysis and transferase. It plays an important role in the assembly of dextran molecules and the maintenance of cell wall integrity. However, the mechanism of its role in fungal growth and development is still unclear. Therefore, we constructed a glucan transferase knockout and recovery strain by homologous recombination technology with Metarhizium anisopliae as experimental material, and preliminarily analyzed its growth, virulence and stress resistance characteristics. Construction of knockout and recovery strains using homologous recombination method to construct Mwg2, _Mwg3,?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 knockout and recovery strains. 3. Functional analysis of genes (1) Growth characteristics: wild, knockout and recovery strains were inoculated and cultured, their germination rate and spore production were measured and observed. Structure of mycelium. (2) Stress resistance analysis: Spores of wild, knockout and recovery strains were treated by heat shock and ultraviolet radiation respectively, and the germination rate was counted and the difference of semi-inhibition time was compared. (3) Toxicity analysis: spore suspension was prepared from spores of fresh and mature wild, knockout strains, and was sensitized by in vivo injection and surface infection. The main results were as follows: 1. Bioinformatics analysis showed that Mwg1: Gen Bank: EFY91318.1, C DNA 1367 bp, amino acid size 406aa, isoelectric point 5.22 pI, molecular weight 41kDa, including 1 intron; Mwg2: Gen Bank: EFY92943.1, C DNA 998 bp, amino acid size 2.22 bp. 77aa, isoelectric point is 5.03pI, molecular weight is 26kDa, which contains one intron; Mwg3: Gen Bank: EFY86494.1, C DNA is 1669bp, amino acid size is 511aa, isoelectric point is 4.89pI, molecular weight is 52kDa, which contains two introns. The effects of glucan transferase gene on the growth characteristics of Metarhizium anisopliae were as follows: (1) In terms of sporulation and sporulation, Mwg1 and Mwg3 produced more spores and germinated earlier than WT and CP, while Mwg2 produced less spores and delayed germination compared with WT and CP, and?? Mwg1/Mwg2 and?? Mwg2/Mwg3 produced more spores and germinated earlier than WT. Compared with CP, Mwg2 had longer mycelial end and larger diaphragm spacing; Mwg2 had more branches than WT and CP; Mwg2 had longer mycelial end and larger diaphragm spacing than WT and CP; Mwg1/Mwg2,?? Mwg2/Mwg3 had longer mycelial end and larger diaphragm spacing than WT. The effects of UV treatment on the ultraviolet tolerance (1) compared with WT and CP, Mwg1, Mwg3, Mwg1 / Mwg2, Mwg2 / Mwg3 had earlier spore germination rate and stronger ultraviolet tolerance; (2) Compared with WT and CP, Mwg2 had longer spore germination rate and weaker ultraviolet tolerance. Compared with WT and CP, the spore germination rate of Mwg2 was delayed and the heat and humidity tolerance was weakened after heat treatment;?Mwg2,?? Mwg2/Mwg3 had no significant difference compared with WT and CP;?? Mwg1/Mwg2 had earlier spore germination rate after heat treatment than WT. (3) Cell wall sensitizer: Congo with cell wall sensitizer Compared with WT and CP, the colony morphology of? Mwg1 decreased significantly and grew slowly on the red 1/4 SADAY plate;?? Mwg1/Mwg2 also grew slowly compared with WT, but other knockout strains had no significant effect; the growth of each strain on the 1/4 SADAY plate with Na Cl and Sor had no significant difference compared with WT, indicating that glucan transferase did not. Effects of glycosyltransferase gene on the virulence of Metarhizium anisopliae were studied. The results showed that the virulence of Metarhizium anisopliae was mainly affected by the infection on the body surface and the injection in vivo. The toxicity of? Mwg1,? Mwg2,? Mwg3 was not different from that of WT, but?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 was significantly weaker than that of WT.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476
本文編號(hào)：2246477
[Abstract]:Insects are susceptible to pathogenic fungi. The pathogenic fungi carried by insects can further spread and cause large-scale insect infections and deaths. Therefore, the use of insect pathogenic fungi to control agricultural pests is a long-term and environmentally friendly biocontrol measure. Fungal cell wall is an important structure in the periphery of fungal cells, which protects fungi from the outside world. Dextransferase has the activities of hydrolysis and transferase. It plays an important role in the assembly of dextran molecules and the maintenance of cell wall integrity. However, the mechanism of its role in fungal growth and development is still unclear. Therefore, we constructed a glucan transferase knockout and recovery strain by homologous recombination technology with Metarhizium anisopliae as experimental material, and preliminarily analyzed its growth, virulence and stress resistance characteristics. Construction of knockout and recovery strains using homologous recombination method to construct Mwg2, _Mwg3,?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 knockout and recovery strains. 3. Functional analysis of genes (1) Growth characteristics: wild, knockout and recovery strains were inoculated and cultured, their germination rate and spore production were measured and observed. Structure of mycelium. (2) Stress resistance analysis: Spores of wild, knockout and recovery strains were treated by heat shock and ultraviolet radiation respectively, and the germination rate was counted and the difference of semi-inhibition time was compared. (3) Toxicity analysis: spore suspension was prepared from spores of fresh and mature wild, knockout strains, and was sensitized by in vivo injection and surface infection. The main results were as follows: 1. Bioinformatics analysis showed that Mwg1: Gen Bank: EFY91318.1, C DNA 1367 bp, amino acid size 406aa, isoelectric point 5.22 pI, molecular weight 41kDa, including 1 intron; Mwg2: Gen Bank: EFY92943.1, C DNA 998 bp, amino acid size 2.22 bp. 77aa, isoelectric point is 5.03pI, molecular weight is 26kDa, which contains one intron; Mwg3: Gen Bank: EFY86494.1, C DNA is 1669bp, amino acid size is 511aa, isoelectric point is 4.89pI, molecular weight is 52kDa, which contains two introns. The effects of glucan transferase gene on the growth characteristics of Metarhizium anisopliae were as follows: (1) In terms of sporulation and sporulation, Mwg1 and Mwg3 produced more spores and germinated earlier than WT and CP, while Mwg2 produced less spores and delayed germination compared with WT and CP, and?? Mwg1/Mwg2 and?? Mwg2/Mwg3 produced more spores and germinated earlier than WT. Compared with CP, Mwg2 had longer mycelial end and larger diaphragm spacing; Mwg2 had more branches than WT and CP; Mwg2 had longer mycelial end and larger diaphragm spacing than WT and CP; Mwg1/Mwg2,?? Mwg2/Mwg3 had longer mycelial end and larger diaphragm spacing than WT. The effects of UV treatment on the ultraviolet tolerance (1) compared with WT and CP, Mwg1, Mwg3, Mwg1 / Mwg2, Mwg2 / Mwg3 had earlier spore germination rate and stronger ultraviolet tolerance; (2) Compared with WT and CP, Mwg2 had longer spore germination rate and weaker ultraviolet tolerance. Compared with WT and CP, the spore germination rate of Mwg2 was delayed and the heat and humidity tolerance was weakened after heat treatment;?Mwg2,?? Mwg2/Mwg3 had no significant difference compared with WT and CP;?? Mwg1/Mwg2 had earlier spore germination rate after heat treatment than WT. (3) Cell wall sensitizer: Congo with cell wall sensitizer Compared with WT and CP, the colony morphology of? Mwg1 decreased significantly and grew slowly on the red 1/4 SADAY plate;?? Mwg1/Mwg2 also grew slowly compared with WT, but other knockout strains had no significant effect; the growth of each strain on the 1/4 SADAY plate with Na Cl and Sor had no significant difference compared with WT, indicating that glucan transferase did not. Effects of glycosyltransferase gene on the virulence of Metarhizium anisopliae were studied. The results showed that the virulence of Metarhizium anisopliae was mainly affected by the infection on the body surface and the injection in vivo. The toxicity of? Mwg1,? Mwg2,? Mwg3 was not different from that of WT, but?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 was significantly weaker than that of WT.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 龔炎杰;王子涵;;釀酒酵母葡聚糖的研究進(jìn)展[J];現(xiàn)代食品科技;2006年01期
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