象耳豆根结线虫侵染能力测定及土壤宏基因组文库杀线虫活性克隆筛选

发布时间：2018-05-30 03:28

本文选题：象耳豆根结线虫 + 二龄幼虫　；参考：《海南大学》2015年硕士论文

【摘要】：近年来,象耳豆根结线虫在热带和亚热带地区番石榴园中大规模爆发,给番石榴造成毁灭性的危害和经济损失,己逐渐上升为热带和亚热带地区最重要的根结线虫种类之一。随着现代高效农业的发展,日益增加的温室与设施大棚导致地温不断提高,使象耳豆根结线虫的入侵与扩散成为可能,这将给全国的农作物生产带来威胁。因而,弄清其寄主范围和侵染力强弱,并找出高效的防治方法是防止该病害蔓延的关键。通过海南岛各类寄主植物根结线虫种类鉴定,发现象耳豆根结线虫种群已进一步扩散,取代南方根结线虫,而成为我国热带亚热带地区根结线虫优势种群。为了避免化学杀线虫剂施用对环境的污染,本研究运用宏基因组技术,直接从与该线虫发生和防控关系最为密切的根围微生物中筛选杀线虫蛋白酶基因资源,并对其酶的活性做了初步研究,旨在为象耳豆根结线虫的生物防治开辟一条新的途径。具体研究结果如下： 1)象耳豆根结线虫侵染测定。通过温室大棚盆栽番茄,探讨了象耳豆根结线虫最低接种量,以及接种量对7个番茄品种侵染差异及生长的影响。研究的结果表明,象耳豆根结线虫对番茄最低侵染起始浓度为40条/500cm3。7个番茄品种中,“阿姆斯丹F1”中抗品种,“中杂9号”、“利生一号”、“西粉一号”、“大安吉日”抗病品种；“美国903”、“金圣”为感病品种。 2)宏基因组文库构建和筛选。从受象耳豆根结线虫严重侵染的番石榴园中,健康番石榴根围取样,经总DNA提取、纯化、末端修复、连接、包装和转染等过程,构建宏基因组Fosmid表达文库。特征分析结果表明：该文库库容31104个克隆,平均插入片段36.5kb,空载率1.9%,包含超过1Gbp的微生物基因组信息。同时,以脱脂奶为底物,经透明圈表型选择,筛选出含蛋白酶阳性克隆子111株,其中9株对象耳豆根结线虫具有较强毒杀作用,包括21fa6、5ba8、5fa8、20aa2、60ha9、14ba10、83fa2、83ha8和178fa8,尤以21fa6克隆对线虫的毒杀效果最好。 3)含蛋白酶基因克隆子21fa6活性探索。以21fa6克隆子为研究对象,摇床转数150r/min,培养温度35℃是最佳活化条件；摇床转速200r/min,培养温度为35℃时,是克隆菌最适产酶活最佳条件；加入诱导物脱脂奶、诱导剂FAS双重作用下,48h时,发酵液蛋白吸收值达到最大值。21fa6蛋白酶克隆子的初步研究,为蛋白酶基因片段亚克隆、测序、活性蛋白酶分离纯化奠定了基础。 4)对21fa6构建和筛选出亚克隆(spro114a5)。通过对基因结构进行了初步分析发现：spro114a5是一种分泌型胞外蛋白酶,与来自于Maricaulis maris MCS10(accession no. YP_756822at NCBI)的丝氨酸蛋白酶S15仅有45%的同源性,有其保守的催化三元组：Asp469,His541和Ser348。杀线虫室内生物测定实验表明：克隆发酵液在无需诱导剂,无需克隆载体上的启动子,就能起到杀线虫的作用。说明该蛋白酶基因有独立的启动子,同时,也说明该蛋白酶是一种分泌型胞外丝氨酸蛋白酶。
[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.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.45

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