【作者】 王建梅；

【导师】 李新江；

【作者基本信息】 河北大学 ， 动物学， 2020， 硕士

【摘要】 蝗虫属于一大类农业害虫,而关于蝗虫肠道菌群结构组成和群落多样性及功能菌的研究很少,且蝗虫食物的主要成分是纤维素、半纤维素,蝗虫食量大,具有开发成植物纤维素、半纤维素的消化生物反应器的潜能,可提高秸秆利用效率,变废为宝。本研究以花胫绿纹蝗Aiolopus tamulus(Fabricius,1789),亚洲小车蝗Oedaleus decorus asiaticus Bey-Bienko,1941,长翅素木蝗Shirakiacris shirakii(Bolívar,1914)为研究对象,采用16S rDNA基因文库构建的方法,对其成虫中、后肠内的微生物16S rDNA的V3-V4区进了高通量测序,研究了不同物种之间肠道微生物多样性异同,同时测定了3种蝗虫对纤维素、半纤维素的消化率并分离、鉴定了肠道中纤维素降解菌,主要研究结果如下:1.3种蝗虫的肠道微生物基因测序结果注释到的主要分类阶元隶属于7个门12个纲20个目42个科54个属。2.在门级水平,变形菌门Proteobacteria和厚壁菌门Firmicutes在3种蝗虫的肠道微生物群落中相对丰度都超过1%,均为优势菌门,放线菌门Actinobacteria、蓝细菌门Cyanobacteria、梭杆菌门Fusobacteria都微量存在于3种蝗虫肠道内,而拟杆菌门Bacteroidetes在长翅素木蝗肠道内未被检测到,软壁菌门Tenericutes仅在亚洲小车蝗肠道内测到。3.在属级水平,花胫绿纹蝗肠道微生物的优势属有3个:克雷伯氏菌属Klebsiella、肠球菌属Enterococcus、肠杆菌属Enterobacter,亚洲小车蝗肠道微生物的优势属有6个:克雷伯氏菌属Klebsiella、肠球菌属Enterococcus、泛菌属Pantoea、沃尔巴克氏体属Wolbachia、肠杆菌属Enterobacter、乳球菌属Lactococcus,长翅素木蝗肠道微生物的优势菌属则有3个:克雷伯氏菌属Klebsiella、乳球菌属Lactococcus、葡萄球菌属Staphylococcus。4.α多样性结果显示长翅素木蝗肠道微生物的物种丰富度相对最高,而花胫绿纹蝗肠道内微生物群落多样性相对最高;β多样性结果表明不同物种的蝗虫其肠道菌群结构不同,其中长翅素木蝗与花胫绿纹蝗肠道菌群结构差异较小,同时蝗虫标本采集时间差异也会导致同一物种蝗虫的肠道菌群结构发生改变。5.采用苔黑酚比色法和蒽酮比色法测定3种蝗虫成虫的粪便及麦苗中的纤维素半纤维素的含量并计算出蝗虫对纤维素和半纤维素的消化率,其中花胫绿纹蝗对纤维素的消化率为43.95%,对半纤维素的消化率为17.21%;长翅素木蝗对纤维素的消化率为44.12%,对半纤维素的消化率为47.65%;亚洲小车蝗对纤维素的消化率为38.01%,对半纤维的消化率为24.99%;3种蝗虫之间纤维素消化率差异不显著(P>0.05),长翅素木蝗的半纤维素消化率显著高于花胫绿纹蝗与亚洲小车蝗的半纤维素消化率(P<0.05),此外,通过对消化率与微生物之间相关性的分析发现克雷伯氏菌属Klebsiella、肠球菌属Enterococcus、乳球菌属Lactococcus都与纤维素的消化具有显著相关性。6.经以羧甲基纤维素钠为唯一碳源,从花胫绿纹蝗肠道内筛选出3株有纤维素降解能力的菌株,对其进行分离、纯化,并利用DNS法分别测定纤维素酶活进行复筛,结合形态学观察、生理生化鉴定及细菌16S rDNA分子生物学鉴定,表明株菌H-1,H-2,H-7为蜡样芽孢杆菌。7.从长翅素木蝗肠道内筛选出2株有纤维素降解能力的菌株,并利用DNS法分别测定纤维素酶活进行复筛,根据形态、生理生化特征及细菌16S rDNA分子生物学鉴定表明菌株S-3、S-5为蜡样芽孢杆菌。更多还原

【Abstract】 Grasshoppers belong to a large category of agricultural pests,but there were few research on the composition of grasshopper intestinal microflora,community diversity and functional bacteria.The main components in grasshoppers food are cellulose and hemicellulose.Due to grasshoppers large appetite and high utilization rate,the intestinal contents of grasshoppers have the potential to be developed into bioreactor,at the same time,it can improve straw utilization efficiency and turn waste into treasure.In this study,Aiolopus tamulus,Oedaleus asiaticus and Shirakiacris shirakii were taken as the research objects,using the method of constructing a 16 S rRNA gene library and Illumina Miseq sequencing technology.The V3-V4 region of the midhindgut of the adult was introduced into high-throughput sequencing for the first time,and the intestinal microbial diversity among different species was studied.At the same time,the digestibility of cellulose and hemicellulose of three species grasshoppers was determined,and cellulose degrading bacteria in midgut and hindgut were isolated and identified.The main research results were as follows:1.The main taxonomic elements anoted by the genne sequencing results of intestinal microorganisms of 3 speceises grasshoppers belongs to 7 phyla,12 classes,20 orders,42 families and 54 genera.2.The relative abundance of Proteobacteria and Firmicutes was more than 1% in the intestinal microbial communities of the three grasshoppers,which were dominant bacteria.Actinobacteria,Cyanobacteria and Fusobacteria exist in the intestinal tracts of the three species grasshoppers in trace amounts.However,Bacteroidetes was not detected in the intestinal tract of Shirakiacris shirakii,Tenericutes was only detected in the intestinal tract of Oedaleus asiaticus,and was not detected in the intestinal tracts of the other two species grasshoppers.3.At the level of genus,there were three dominant genera of Aiolopus tamulus,including Klebsiella,Enterococus,Enterobacter,and 6 dominant genera of Oedaleus asiaticus.Specifically,it included Klebsiella,Enterococcus,Pantoea,Wolbachia,Enterobacter,Lactococus,and three dominant genera of Shirakiacrisshirakii,namely Klebsiella,Lactococus,and Staphylococcus.4.Diversity analysis showed that the species richness in the intestinal tract of Shirakiacris shirakii was the highest,while the diversity of the microbial community in the intestinal tract of Aiolopus tamulus was the highest.Different species of grasshoppers have different intestinal microflara structures,among them,there was little difference of intestinal microflora structure between Shirakiacrisshirakii and Aiolopus tamulus,and at the same time,time differences will also lead to changes in the intestinal microflora structure of the same species of grasshoppers.5.The contents of cellulose hemicellulose in feces and wheat seedlings of the three species grasshoppers were determined by adopting moss black phenol colorimetry and anthrone colorimetry.The digestibility of cellulose/hemicellulose among Aiolopus tamulus,Shirakiacris shirakii and Oedaleus asiaticus were 43.95/17.21%,44.12/47.62% and38.01/24.99%,respectively.There was no significant difference in cellulose digestibility among the three species grasshoppers(P>0.05),the hemicellulose digestibility of Shirakiacris shirakii was significantly higher than the other two species grasshoppers(P<0.05).In addition,by analyzing the correlation between digestibility and microorganisms,we found Klebsiella,Enterococcus and Lactococcus were all significantly related to cellulose digestion.6.With sodium carboxymethyl cellulose as the sole carbon source,three strains capable of producing transparent circle were screened from the intestinal tract of Aiolopus tamulus,and they were separated and purified.The cellulase activity was measured by DNS method and re-screened.Combined with morphological observation,physiological and biochemical identification and molecular biology,the strains H-1,H-2 and H-7 were preliminarily identified as Bacillus cereus.7.Two strains capable of producing transparent circles were screened from the intestinal tract of Shirakiacris shirakii.Cellulase activity was measured by DNS method and re-screened.The two strains were identified according to morphology,physiological and biochemical characteristics and molecular biology.The strains S-3 and S-5 were preliminarilyidentified as Bacillus cereus.更多还原