易切削不锈钢
中文摘要
新鲜桑叶是已知的家蚕最佳饵料，目前为止的人工饲料即便使用大量的桑叶粉也无法完全替代桑叶，大部分的家蚕品种甚至不取食人工饲料。另一方面，饲料饲养的家蚕幼虫体质弱、丝蛋白合成效率低、产丝量少等问题也一直没有得到根本解决。为了进一步探讨解决上述问题，本文利用代谢组学和微生物组学方法，分析了家蚕食性和饮食与肠道菌变化、以及血淋巴代谢差异的关系。研究结果如下：（1）基于对家蚕实用品种菁松B进行的双向歧化选择获得的人工饲料摄食性差异性家蚕品系，比较了菁松B高食性品系在桑叶育与饲料育环境，以及菁松B高食性与低食性品系在桑叶育环境中5龄幼虫血淋巴的代谢组学差异。GC/LC-MS分析结果显示，共鉴定出130种代谢物，其中主要的差异代谢物是氨基酸、有机酸和维生素。与桑叶育蚕相比，饲料育蚕血淋巴中硫胺素等4种维生素含量显著下降，它们与59个差异代谢物质构成了众多的edges。甲硫氨酸亚砜等15种氨基酸及其代谢物含量显著升高，同时甲硫氨酸和赖氨酸等6种必需和非必需氨基酸的含量显著下降。组成丝蛋白最主要的甘氨酸、丙氨酸、天冬酰胺和丝氨酸含量出现了不一致的显著下调或上调变化。与此同时，由氨基酸形成尿酸和尿素的代谢通路关键物质含量显著升高，血淋巴中出现尿酸和尿素富集。另一方面，酸和奎尼酸等有机酸的含量显著下降，碳水化合物和脂类代谢水平，以及胆固醇水平都有低于桑叶育蚕的趋势。本文还筛选出10种雄性优势代谢物，29种与饲料有关的性别间差异代谢物，为进一步研究家蚕性别间对饲料育适应性差异机制提供了线索和靶标。
（2）本文的肠道微生物菌分析，除了调查遗传来源高度相似的家蚕菁松B高食性和菁松B低食性品系外，还引入了能很好摄食人工饲料摄的自然突变体广食品系和几乎不摄食人工饲料的鲁七品种，以进一步理解不同蚕品种的影响。结果显示：桑叶育环境中，即使对人工饲料摄食性具有显著差异的不同家蚕品系之间，肠道微生物的多样性也很相似，最主要的微生物都是蓝藻细菌丰度高达65.2±17.9%，其次是厚壁菌门丰度约占29.2±17.6%，第3与第4位的肠道菌变形菌门和拟杆菌门丰度降至4.2±4.5%和0.6±0.8%。在属水上在丰度大于1%的微生物主要是绿藻，其丰度高达65.0±17.9%，其次是可以增强宿主免疫力肠球菌属（19.4±12.7%）和参与水解糖
和蛋白质的梭菌属（7.6±14.9%）。人工饲料育家蚕的肠道菌的种类比桑叶育蚕显著减少，优势菌的丰度也出现了明显变化。桑叶育环境中的肠道优势菌蓝藻细菌的丰度显著降低，菁松B高食和广食品系家蚕分别降低了67.9±20.7%和99.1±0.6%；厚壁菌门的丰度在菁松B高食品系体内的丰度上调了5.5±1.6倍，在广食品系体内降低了41.7±30.7%；变形菌门的丰度只在广食品系家蚕肠道出现了32.9±8.1倍上调，菁松B 高食品系体内则没有发生显著变化。进一步的深度分类分析结果显示，人工饲料育家蚕肠道细菌近99%还未分类到属水平，人工饲料育菁松B高食品系体内丰度最高的是肠球菌科，丰度上调了10.1±2.8倍；其次是绿藻菌（o），但丰度降低了67.8±20.7%。广食品系家蚕体内肠杆菌科丰度上调了64.0 ±15.4倍，其次是绿藻菌（o）和毛螺菌科，丰度分别降低了99.1±0.6%和99.99%±0.01%。
KEGG数据库进行的PICRUSt功能预测结果显示，桑叶育家蚕的肠道菌主要对宿主的排泄系统和异生素生物降解及代谢产生影响。而人工饲料育家蚕的肠道菌为宿主提供上述功能显著减弱的同时，参与宿主环境适应、细胞生长和死亡、细胞运动、遗传信息加工等功能的微生物显著增多。
本文结果表明，家蚕肠道菌组成和饮食关系密切，桑叶育家蚕肠道菌维持在一个比较稳定的状态，几乎不受人工饲料摄食性遗传差异的家蚕品系影响。人工饲料饲养能够显著改变家蚕肠道的微生物多样性组成和对宿主的功能影响，即使是人工饲料摄食性都很好的不同品系家蚕，在人工饲料饲养后肠道微生物的组成也出现了显著差异。显示饮食是改变家蚕肠道菌的主要因素，人工饲料育家蚕的肠道微生物改变还与宿主家蚕的遗传差异有关。
临床营养师关键词：人工饲料；家蚕；食性差异品系；血淋巴；代谢组学；肠道菌
directx 3d作者：董慧玲
死猪江葬
指导教师：徐世清教授
协助指导教师：崔为正教授
司马杨虎教授
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Abstract
Fresh mulberry leaf is the best natural feed of Bombyx mori larvae. However, for almost all silkworm varieties bred so far, only the intake of artificial diets by larvae has been improved, while the metabolic utilization of artificial diets is still not as good as that of mulberry leaves. Issues such as weak silkworm larvae, low silk protein synthesis efficiency, and low silk yields in silkworms reared on artificial diets have not been resolved. In order to further explore the remaining problems of artificial diets, in this paper, the metabonomics and microbiome methods were used to analyze the relationship between feeding habits and dietary of gut microbiota and hemolymph metabolic differences. The results are as follows:
(1) The metabolomics is based on the disruptive selected the ingestive behavior of Jingsong B silkworm varieties obtained to artificial diet feeding habit differences silkworm strains, compared with reared on fresh mulberry leaves and the artificial diet, Jingsong B high food intake strains. Comparison of Jingsong B high food intake strains and Jingsong B low food intake reared on fresh mulberry leaves when the fifth instar larvae hemolymph metabonomics differences. GC / LC-MS analysis showed that, we unambiguously assigned 130 metabolites, amino acid, organic acid and vitamin are the most different metabolites. The contents of four types of vitamins including thiamine i新乡医学院学报
n the haemolymph of silkworms reared on artificial diet were significantly decreased compared with those reared on mulberry leaves, and they constituted a large number of edges with 59 differential metabolites. The contents of 15 kinds of amino acids and their metabolites including methionine sulfoxide were significantly increased, while the contents of six kinds of essential and non-essential amino acids including methionine and lysine were significantly decreased. The contents of glycine, alanine, asparagine and serine, the most dominant amino acids in silk protein, were significantly either downregulated or upregulated. Meanwhile, the contents of key substances in the metabolic pathways of uric acid and urea formation from amino acids were significantly increased, and uric acid and urea were enriched in the haemolymph. On the other hand, the contents of organic acids such as pipecolic acid and quinic acid were significantly decreased, the levels of carbohydrate metabolism, lipid metabolism, and cholesterol were lower than those in silkworms reared
on mulberry leaves. In this paper, 10 male dominant metabolites and 29 feed-related differential metabolites between sexes were also screened out, which will provide clues and targets for further study on the mechanism of difference in adaptability to artificial diet between silkworm sexes.
(2) In order to understand the impact of silkworm varieties, we used two high similar genetic source of Jingsong B high food intake strain (Hi) and Jingsong B low food intake strain (Lo), one natural mut
ation of artificial diets feeding well Guangshi strain (Gs), and another natural strain of Lu 7 (L7) in which almost all its larvae starved to death on the artificial diets. Results of their gut microbiomics shown as follows:
In the mulberry leaves breeding larvae, the diversity of intestinal microbes was similar in the four silkworm strains, even if there are significant differences in the feed intake to artificial diets. The most abundant microorganism was Cyanobacteria which up to 65.2 ± 17.9% of total gut microorganisms, followed by the Firmicutes (29.2 ± 17.6%), the third and the fourth intestinal bacteria was Proteobacteria(4.2 ±4.5%) and Bacteroidetes(0.6 ±0.8%), respectively. On the genus level，the most predominant bacteria of Streptophyta (o) was up to 65.2 ± 17.9% of total gut microorganisms, followed was Enterococcus (19.4 ± 12.7%) and Clostridium (7.6 ± 12.7%), which was involved in host immunity and hydrolyzing sugar and protein.
The diversity of gut microbiota was significantly decreased in silkworm guts reared on artificial diets, and the abundance of dominant bacteria were significantly changed. Compare with silkworms reared on fresh mulberry leaves, the abundance of Cyanobacteria was decreased 67.9 ±20.7% and 99.1 ±0.6% in artificial diets reared Hi and Gs, respectively. The Firmicutes was up-regulated 5.5 ± 1.6 times in Hi and decreased 41.7 ±30.7% in Gs. The abundance of Proteobacteria was up-regulated 3
2.9 ± 8.1 times in the larval intestine of Gs, while there was no significant change in Hi strain. Further in-depth classification analysis showed that nearly 99% of the intestine bacteria reared on artificial diet were not classified into the genus. In gut of Hi reared on artificial diets, the abundance of Enterococcaceae was up-regulated 10.1 ±2.8 times, while the Streptophyta(o) decreased 67.8 ± 20.7%. Whereas, the Enterobacteriaceae was up-regulated 64.0 ± 15.4 times, but the Streptophyta (o) and Lachnospiraceae was decreased 99.1 ± 0.6% and 99.99% ± 0.01% in Gs, respectively.
The results of PICRUSt in the KEGG database showed that the gut microbiota of
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silkworms reared on fresh mulberry leaves was mainly affected by Excretory System and Xenobiotics Biodegradation and Metabolism. In addition, compared with silkworms reared on fresh mulberry leaves, the diversity of gut microbiota involved in host environmental adaptation, Cell Growth and Death, Cell Motility, genetic information processing and so on were significantly increased.
In general, the composition of gut microbiota and diets have a strong relationship. The gut microbiota in silkworm larvae reared on fresh mulberry leaves maintained in a relatively stable state,
most no effect from genetic differences on artificial diet feeding habits (silkworm strains). The gut microbial diversity and the functional for the host might been great changed in silkworm reared on artificial diets. The results showing that diet is the main factor to change the gut microbiota of silkworm, the changes of intestinal microflora under the larvae reared on artificial diets was related to the genetic differences of the host silkworm.
Key words: Artificial diet; silkworm; feed intake; hemolymph; metabonomics; gut microorganism.
Written by Dong Hui-Ling
战争论文
Supervised by Xu Shi-Qing
Assist Supervised by Cui Wei-Zheng
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