本文選題：象耳豆根結(jié)線(xiàn)蟲(chóng) + 二齡幼蟲(chóng)��； 參考：《海南大學(xué)》2015年碩士論文

【摘要】：近年來(lái),象耳豆根結(jié)線(xiàn)蟲(chóng)在熱帶和亞熱帶地區(qū)番石榴園中大規(guī)模爆發(fā),給番石榴造成毀滅性的危害和經(jīng)濟(jì)損失,己逐漸上升為熱帶和亞熱帶地區(qū)最重要的根結(jié)線(xiàn)蟲(chóng)種類(lèi)之一。隨著現(xiàn)代高效農(nóng)業(yè)的發(fā)展,日益增加的溫室與設(shè)施大棚導(dǎo)致地溫不斷提高,使象耳豆根結(jié)線(xiàn)蟲(chóng)的入侵與擴(kuò)散成為可能,這將給全國(guó)的農(nóng)作物生產(chǎn)帶來(lái)威脅。因而,弄清其寄主范圍和侵染力強(qiáng)弱,并找出高效的防治方法是防止該病害蔓延的關(guān)鍵。 通過(guò)海南島各類(lèi)寄主植物根結(jié)線(xiàn)蟲(chóng)種類(lèi)鑒定,發(fā)現(xiàn)象耳豆根結(jié)線(xiàn)蟲(chóng)種群已進(jìn)一步擴(kuò)散,取代南方根結(jié)線(xiàn)蟲(chóng),而成為我國(guó)熱帶亞熱帶地區(qū)根結(jié)線(xiàn)蟲(chóng)優(yōu)勢(shì)種群。為了避免化學(xué)殺線(xiàn)蟲(chóng)劑施用對(duì)環(huán)境的污染,本研究運(yùn)用宏基因組技術(shù),直接從與該線(xiàn)蟲(chóng)發(fā)生和防控關(guān)系最為密切的根圍微生物中篩選殺線(xiàn)蟲(chóng)蛋白酶基因資源,并對(duì)其酶的活性做了初步研究,旨在為象耳豆根結(jié)線(xiàn)蟲(chóng)的生物防治開(kāi)辟一條新的途徑。具體研究結(jié)果如下： 1)象耳豆根結(jié)線(xiàn)蟲(chóng)侵染測(cè)定。通過(guò)溫室大棚盆栽番茄,探討了象耳豆根結(jié)線(xiàn)蟲(chóng)最低接種量,以及接種量對(duì)7個(gè)番茄品種侵染差異及生長(zhǎng)的影響。研究的結(jié)果表明,象耳豆根結(jié)線(xiàn)蟲(chóng)對(duì)番茄最低侵染起始濃度為40條/500cm3。7個(gè)番茄品種中,“阿姆斯丹F1”中抗品種,“中雜9號(hào)”、“利生一號(hào)”、“西粉一號(hào)”、“大安吉日”抗病品種；“美國(guó)903”、“金圣”為感病品種。 2)宏基因組文庫(kù)構(gòu)建和篩選。從受象耳豆根結(jié)線(xiàn)蟲(chóng)嚴(yán)重侵染的番石榴園中,健康番石榴根圍取樣,經(jīng)總DNA提取、純化、末端修復(fù)、連接、包裝和轉(zhuǎn)染等過(guò)程,構(gòu)建宏基因組Fosmid表達(dá)文庫(kù)。特征分析結(jié)果表明：該文庫(kù)庫(kù)容31104個(gè)克隆,平均插入片段36.5kb,空載率1.9%,包含超過(guò)1Gbp的微生物基因組信息。同時(shí),以脫脂奶為底物,經(jīng)透明圈表型選擇,篩選出含蛋白酶陽(yáng)性克隆子111株,其中9株對(duì)象耳豆根結(jié)線(xiàn)蟲(chóng)具有較強(qiáng)毒殺作用,包括21fa6、5ba8、5fa8、20aa2、60ha9、14ba10、83fa2、83ha8和178fa8,尤以21fa6克隆對(duì)線(xiàn)蟲(chóng)的毒殺效果最好。 3)含蛋白酶基因克隆子21fa6活性探索。以21fa6克隆子為研究對(duì)象,搖床轉(zhuǎn)數(shù)150r/min,培養(yǎng)溫度35℃是最佳活化條件；搖床轉(zhuǎn)速200r/min,培養(yǎng)溫度為35℃時(shí),是克隆菌最適產(chǎn)酶活最佳條件；加入誘導(dǎo)物脫脂奶、誘導(dǎo)劑FAS雙重作用下,48h時(shí),發(fā)酵液蛋白吸收值達(dá)到最大值。21fa6蛋白酶克隆子的初步研究,為蛋白酶基因片段亞克隆、測(cè)序、活性蛋白酶分離純化奠定了基礎(chǔ)。 4)對(duì)21fa6構(gòu)建和篩選出亞克隆(spro114a5)。通過(guò)對(duì)基因結(jié)構(gòu)進(jìn)行了初步分析發(fā)現(xiàn)：spro114a5是一種分泌型胞外蛋白酶,與來(lái)自于Maricaulis maris MCS10(accession no. YP_756822at NCBI)的絲氨酸蛋白酶S15僅有45%的同源性,有其保守的催化三元組：Asp469,His541和Ser348。殺線(xiàn)蟲(chóng)室內(nèi)生物測(cè)定實(shí)驗(yàn)表明：克隆發(fā)酵液在無(wú)需誘導(dǎo)劑,無(wú)需克隆載體上的啟動(dòng)子,就能起到殺線(xiàn)蟲(chóng)的作用。說(shuō)明該蛋白酶基因有獨(dú)立的啟動(dòng)子,同時(shí),也說(shuō)明該蛋白酶是一種分泌型胞外絲氨酸蛋白酶。
[Abstract]:In recent years, the Elephant Ear bean root knot nematode outbreak in the tropical and subtropical guava orchard caused devastating damage and economic loss to guava. It has gradually risen to one of the most important species of root knot nematode in tropical and subtropical areas. With the development of modern high efficiency agriculture, increasing greenhouse and facilities have led to the field. The increase of temperature makes it possible to invade and spread the root knot nematode of the Elephant Ear bean, which will threaten the crop production throughout the country. Therefore, it is crucial to find out the host range and the intensity of the infection and to find the effective control method to prevent the disease.
Through the identification of the species of root knot nematode of various host plants in Hainan Island, it was found that the Elephant Ear bean root knot nematode population has further spread and replaced the southern root knot nematode, and has become the dominant population of root knot nematode in tropical subtropics of our country. In order to avoid the pollution of the chemical nematode, this study uses the macro genome technology to direct the study. The nematode gene resources were screened in the most closely related root circumference microorganism, and the activity of the enzyme was preliminarily studied. The aim of this study was to open up a new way for the biological control of the root knot nematode. The results are as follows:
1) the infection of the root knot nematode of the elephant ear. Through the greenhouse potted tomato, the minimum inoculation amount of the root knot nematode of the Elephant Ear bean root knot nematode, and the effect of the inoculation on the difference of infection and growth of the 7 tomato varieties were discussed. The results showed that the lowest infection of the root knot nematode to tomato was in 40 /500cm3.7 tomato varieties. The resistant variety of F1, "Zhong Zi No. 9", "Li Sheng No. 1", "West powder No. 1", "Da'an auspicious" resistance variety, "American 903", "Jinsheng" as a susceptible variety.
2) construction and screening of the macrogenome library. From the guava orchard, which was heavily infected by the Elephant Ear bean root knot nematode, the healthy guava root circumference sampling, the total DNA extraction, purification, terminal repair, connection, packaging and transfection, the Fosmid expression library of the Gou Jianhong genome. The characteristics analysis showed that the library capacity was 31104 clones and the average inserts were inserted. Segment 36.5kb, with a no-load rate of 1.9%, containing more than 1Gbp microbial genome information. At the same time, 111 strains of protease positive clones were screened by the selection of skimmed milk, and 9 of them had strong toxicity, including 21fa6,5ba8,5fa8,20aa2,60ha9,14ba10,83fa2,83ha8 and 178fa8, especially 21fa6. Cloning has the best killing effect on nematodes.
3) exploration of the activity of 21fa6 containing protease gene clones. Taking 21fa6 kunon as the research object, the number of rocking bed rotation 150r/min and the culture temperature 35 is the best activation condition. The optimum condition of the optimum enzyme production is the rotational speed of 200r/min, and the culture temperature is 35. The fermentation broth is added to the defatted milk of the inducer, the inducer FAS double action, 48h, and the fermentation liquid. The preliminary study of the protein absorption value reached the maximum value of.21fa6 protease clones, which laid the foundation for the subcloning and sequencing of the protease gene fragment, the separation and purification of active protease.
4) the subclone (spro114a5) was constructed and screened by 21fa6. Through a preliminary analysis of the gene structure, it was found that spro114a5 is a secretory extracellular protease, which has only 45% homology with the serine protease S15 from Maricaulis Maris MCS10 (accession No. YP_756822at NCBI), and has its conservative catalytic three tuple: Asp469. The laboratory test of 41 and Ser348. nematicid showed that the cloned fermentation broth could play a role in nematode without the inducer and without the promoter on the cloned carrier. It indicated that the protease gene had an independent promoter and also indicated that the protease was a secretory extracellular serine protease.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S432.45

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