阅读说明：本技术 与小体重鸡只厌食行为高度关联的肠道微生物标记物 (Intestinal microbial markers highly correlated with anorexia behavior in small-weight chickens ) 是由 周浩 孟和 杨凌宇 何川 丁金梅 徐珂 郑煜明 韩铖潇 罗怀希 于 2019-09-20 设计创作，主要内容包括：本发明涉及与小体重鸡只厌食行为高度关联的肠道微生物标记物。所述标记物分别为Rhodovulum属Rhodovulum bhavnagarense菌种和Terrisporobacter属Terrisporobacter glycolicus菌种。该标记物在小体重鸡只肠道中富集,鸡只表现为厌食瘦小,该标记物的发现为微生物减肥产品的研发提供了参考,另外,可在家禽生产中,用于产蛋鸡体重的控制。(The present invention relates to gut biomarker highly correlated with anorexia behavior in small-weight chickens. The markers are Rhodovulum species and Terrisporabacterium species, respectively. The marker is enriched in intestinal tracts of chickens with small weight, and the chickens show anorexia and emaciation, and the discovery of the marker provides reference for the research and development of microbial weight-reducing products, and in addition, the marker can be used for controlling the weight of laying hens in poultry production.)

1. An intestinal biomarker highly correlated with anorexia behavior in small-weight chickens, comprising Rhodovulum bhavnagarense species.

2. The intestinal biomarker highly correlated to anorexia nervosa of small-weight chickens according to claim 1, wherein the specific taxonomic characteristics of Rhodovulum bhavnagense strain are as follows: (ii) Bacteria; proteobacteria; alphaproteobacteria; rhodobacterales; rhodobacter acetae; rhodovulum; rhodovulum bhavnagense.

3. The intestinal biomarker highly correlated to anorexia nervosa in small-weight chickens according to claim 1, wherein Rhodovulum bhavnagense strain contains a DNA sequence as shown in SEQ ID No. 1.

4. An intestinal biomarker highly correlated with anorexia nervosa in small-weight chickens, wherein the intestinal biomarker comprises Terrisporobacter glycocolicus strain.

5. The intestinal biomarker highly correlated to anorexia nervosa of small-weight chickens according to claim 4, wherein the specific taxonomic features of the Terrisporabacter glycocolicus strain are: (ii) Bacteria; terrabacteria group; firmicutes; clostridium; clostridiales; peptost-reptocacee; terrisporobacter; terrisporabacter glycocolicus.

6. The intestinal biomarker highly correlated to anorexia nervosa of small-weight chickens according to claim 4, wherein Terrisporabacter glycocolicus strain contains DNA sequence shown in SEQ ID NO. 2.

7. The method for screening intestinal biomarkers highly correlated to anorexia nervosa of small-weight chickens according to claim 1 or 4, comprising the steps of:

1) the white rook chickens are taken as a common ancestor, and the weight of 56 days old chickens are taken as a target to carry out long-term artificial differential selection, so that the condition of feeding the weight and the feeding environment are kept consistent, and the single character difference is obtained, after 56 generations are selected, the obtained small-weight family chickens only have obvious anorexia and emaciation symptoms, and the weight is only one tenth of the weight of the large-weight family chickens on average;

2) collecting two families of chicken intestinal microorganism samples, and extracting microorganism DNA in intestinal tracts by using a kit to obtain intestinal microorganism genome DNA;

3) constructing a DNA library and then performing metagenome sequencing;

4) analyzing and identifying the intestinal microbial species and the abundance of each species of the small-weight family chicken and the large-weight family chicken;

filtering low-quality sequences in the original sequence, and then comparing the sequences with flora sequences in an NCBI NR database to obtain the sequence and abundance of each species of the intestinal microorganisms in each sample;

5) intestinal microbial screening

Measuring the abundance of intestinal microbial species in the large-weight family chicken and the small-weight family chicken according to the method in the steps 2) -4), and comparing to obtain the most remarkably different intestinal bacteria, namely Rhodovulum Rhodovulum bhavnagarenense and Terrisporabacter Glycolicus in Terrisporabacter;

wherein, filtering the low-quality sequence in the original sequence means that the sliding window quality detection of 4 bases is carried out from the 5' end of reads, and the sliding window with the average value of the base quality lower than 2 is cut; excising bases with the mass of the first and the tail end bases less than 2; filtering bases with the number of bases less than 25.

8. The method for screening intestinal biomarkers highly correlated with anorexia nervosa of small-weight chickens according to claim 7,

the P value of Rhodovulum bhavnagense (Rhodovulum sp.) is 0.000045,

the P value of Terrispora glycocolicus in the genus of Terrispora is 0.0002.

9. The method for screening intestinal biomarkers highly correlated with anorexia nervosa of small-weight chickens according to claim 7, wherein the comparison in step 5) is performed by:

the abundance differences of microorganisms with different classification levels among different groups are calculated by STAMP software, if the corrected P value of one microorganism in the test result is less than 0.01, the microorganism is judged to have significant differences in the distribution of the two groups, and finally, two bacteria which are high in abundance and have the most significant differences in the large-weight chickens are screened out to be used as markers.

10. The method for screening intestinal biomarkers highly correlated with small body weight chicken anorexia nervosa according to claim 7, wherein the difference in abundance between two groups is calculated by STAMP software, and the difference is analyzed by converting absolute abundance into relative abundance.

Technical Field

The invention relates to the field of microbial markers, in particular to an intestinal microbial marker highly correlated with anorexia of small-weight chickens.

Background

In the poultry breeding industry, the hens are too fat and fat, so that the normal egg yield of the hens can be influenced to a great extent, and the economic benefit is influenced. At present, obesity is also an important cause of human chronic diseases such as cardiovascular and cerebrovascular diseases, cancer, diabetes and the like, and is listed as one of ten diseases threatening human health by the world health organization. Over the last 20 years, with the increasing standard of living, people in China have "fattened" with an alarming rate, and obesity has become a serious social problem. Thus, the search for healthily viable methods of controlling obesity is becoming more acute.

It is well known that the diversity, composition, and function of the intestinal microflora are strongly related to the body weight and body metabolism of the host. The intestinal microorganisms help the host digest food, and have a direct effect on the body weight phenotype of the host. According to these characteristics, the use of enterobacteria as food preparations has been studied for improving and promoting the healthy development of animal organisms. The anorexia nervosa behavior of animals is also strongly related to intestinal microorganisms, which can cause the anorexia behavior of the host through the gut-brain axis. In addition, the species diversity of intestinal microorganisms of the lean individual is obviously lower than that of the obese individual, the obese and lean individual can be well identified according to the preference of intestinal bacteria, the accuracy can reach 90%, and the feasibility of finding the intestinal microorganisms obviously related to the animal weight and anorexia is further demonstrated.

In fact, finding and providing microbial markers that are significantly correlated with small body weight anorexia behavior in animals is of great significance in both the poultry farming and human weight loss industries. In the future, the coliform bacteria can also provide reference materials for developing weight control microbial products and can also be used for microecologics for poultry weight control.

Disclosure of Invention

The object of the present invention is to overcome the above-mentioned drawbacks of the prior art and to provide a gut biomarker highly correlated with anorexia nervosa in small-weight chickens.

The purpose of the invention can be realized by the following technical scheme:

an intestinal biomarker highly correlated with anorexia performance in small-weight chickens, said intestinal biomarker comprising Rhodovulum bhavnagarense species (Schizozandra radicata species).

The Rhodovulum bhavnagense strain belongs to the genus Rhodovulum.

The specific taxonomic features of Rhodovulum bhavnagense strain are: (ii) Bacteria; proteobacteria; alphaproteobacteria; rhodobacterales; rhodobacter acetae; rhodovulum; rhodovulum bhavnagense (bacteria kingdom; Proteobacteria; class A Proteobacteria; order Rhodobacteriaceae; genus Rhodooomycetes; species of Buffersonia).

The Rhodovulum bhavnagense strain contains a DNA sequence shown in SEQ ID NO. 1.

An intestinal biomarker highly correlated with anorexia nervosa of small-weight chickens, said intestinal biomarker comprising terrispobacter glycolicus species (glycerol utilization tyrocidious species).

The species Terrisporabacter glycocolicus belongs to the genus Terrisporabacter.

The specific taxonomic features of terrisporobacterium glycocolicus strains are as follows: (ii) Bacteria; terrabacteriagroup; firmicutes; clostridium; clostridiales; peptostreptococcacee; terrisporobacter; terrisporabacter glycocolicus (bacterial kingdom; Geobacillus kingdom; Thelephoraceae; Clostridiales; Peptostreptococcus class; Geobacillus species; Glycerol utilizing Tyrosporus species).

The DNA sequence contained in the Terrisporabacter glycocolicus strain is shown in SEQ ID NO. 2.

The method for screening the intestinal microbial markers highly correlated with the anorexia behavior of the small-weight chickens comprises the following steps:

1) taking white rook chickens as a common ancestor, carrying out long-term artificial differential selection by taking the weight of 56 days as a target, ensuring that the feeding condition of the weight and the feeding environment are kept consistent to obtain single character difference, and obtaining small-weight family chickens only have obvious symptoms of anorexia and emaciation after selecting 56 generations, and the weight of the small-weight family chickens is only one tenth of the weight of the large-weight family chickens on average (the model is a world famous weight bidirectional selection family model, Lillie M and the like, and genomics of 60years of bidirectional weight selection for 8-ek body weight information, 2017,97(3): 781-;

2) collecting two families of chicken intestinal microorganism samples, and extracting microorganism DNA in intestinal tracts by using a kit (TIANGEN DNA stock mini kit) to obtain intestinal microorganism genome DNA;

3) constructing a DNA library and then performing metagenome sequencing;

4) analyzing and identifying the intestinal microbial species and the abundance of each species of the small-weight family chicken and the large-weight family chicken;

filtering low-quality sequences in the original sequence, and then comparing the sequences with flora sequences in an NCBI NR database to obtain the sequence and abundance of each species of the intestinal microorganisms in each sample;

5) intestinal microbial screening

Measuring the abundance of intestinal microbial species in the large-weight family chicken and the small-weight family chicken according to the method in the steps 2) -4), and comparing to obtain the most remarkably different intestinal bacteria, namely Rhodovulum Rhodovulum havnagaense bacteria and Terrisporabacter glycocolicus bacteria in Terrisporabacter;

wherein, filtering the low-quality sequence in the original sequence means that the sliding window quality detection of 4 bases is carried out from the 5' end of reads, and the sliding window with the average value of the base quality lower than 2 is cut; excising bases with the mass of the first and the tail end bases less than 2; filtering bases with the number of bases less than 25.

The P value of Rhodovulum bhavnagense is 0.000045, and the P value of Terrisporabacter glycocolicus is 0.0002.

The comparison method in the step 5) comprises the following steps: the differences in abundance of microorganisms at different taxonomic levels between the different groups were calculated using STAMP software (v2.1.3) which identified key markers on the principle: the Welch' Test method was used to screen for key microorganisms. If the corrected P value of a microorganism is less than 0.01 in the test result, the microorganism is judged to have significant difference in distribution in two groups, and finally two bacteria which are high in abundance and have the most significant difference in intestinal tracts of small-weight chickens are screened out to be used as markers.

It should be noted that the obtained flora abundance table generally shows the absolute abundance of the microorganisms, so that when comparing, the data needs to be normalized, and the absolute abundance is converted into the relative abundance to perform the differential analysis.

Compared with the prior art, the method utilizes the characteristic that the small-weight chicken intestinal microorganisms with anorexia are different from the large-weight chicken intestinal microorganisms to screen the intestinal bacteria marker which can be used for the anorexia related to animals. The marker is enriched in intestinal tracts of chickens with small weight, and the chickens show anorexia and emaciation, and the discovery of the marker provides reference for the research and development of microbial weight-reducing products, and in addition, the marker can be used for controlling the weight of laying hens in poultry production.

Drawings

FIG. 1: the abundance of Rhodovulum bhavnagense strain in large and small body weight family intestinal tracts, and H and L respectively refer to large and small body weight family chickens.

FIG. 2: the abundance of Terrisporabacter glycocolicus strain in intestinal tracts of large and small body weight families, H and L respectively refer to large and small body weight families of chickens.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.