阅读说明：本技术 利用宏基因组技术辅助检测食品中是否含有c2z7型转基因玉米成分的方法 (Method for detecting whether C2Z7 type transgenic corn ingredient is contained in food or not by aid of metagenome technology ) 是由 杜杰 郑帅 刘婷婷 朴春梅 谢道昕 于 2019-11-07 设计创作，主要内容包括：本发明公开了一种利用宏基因组技术辅助检测食品中是否含有C2Z7型转基因玉米成分的方法。本发明从宏基因组角度出发,开发和制定适合检测食物中C2Z7型转基因玉米成分、并能辅助判断其生物安全性的技术指标。本发明的意义在于,通过宏基因组的变化,找到转有aroA基因的玉米对摄食者内环境第一道屏障,即胃肠道系统的影响,并可以为评价此类玉米对于摄食者自身的生理状态的影响(好的或者坏的)提供间接判断依据。(The invention discloses a method for detecting whether C2Z7 type transgenic corn ingredients are contained in food or not by using metagenome technology. The invention develops and formulates a technical index which is suitable for detecting C2Z7 type transgenic corn components in food and can assist in judging the biological safety of the food from the perspective of metagenome. The significance of the invention lies in that the influence of the corn with aroA gene on the first barrier of the inner environment of a food taker, namely the gastrointestinal tract system, is found through the change of the metagenome, and an indirect judgment basis can be provided for evaluating the influence (good or bad) of the corn on the physiological state of the food taker.)

1. The application of the substances for detecting the abundance of the genes and strains shown in the table below in the auxiliary detection of whether the food contains C2Z7 type transgenic corn;

2. products for auxiliary detection of the presence of C2Z7 type transgenic corn in food products, including substances for detecting the abundance of genes and strains shown in the following table;

3. a method for assisting in detecting whether C2Z7 type transgenic corn is contained in food comprises the following steps:

(1) respectively feeding the common feed and the feed to be detected to the mice; the feed to be detected is a mixture of food to be detected and common feed;

(2) after 10 weeks, extracting the total DNA of the mouse feces, and performing high-throughput sequencing;

(3) analyzing the sequencing result to obtain the abundance value of the specific gene and the strain of the sample, and judging according to the standard range shown in the following table; if the standard ranges listed in the following table are met, the food to be tested contains or possibly contains C2Z7 type transgenic corn; on the contrary, the food to be detected does not contain C2Z7 type transgenic corn;

the specific genes, strains and standard abundance ranges are the genes, strains and numerical values shown in the following table;

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4. The method of claim 3, wherein: the feed to be detected is a mixture of 1 volume part of food to be detected and 3 volume parts of common feed.

5. The method of claim 3 or 4, wherein:

the sequencing result analysis method comprises the following steps:

(1) performing quality control filtration on sequencing data;

(2) after the step (1) is completed, further assembling the effective data obtained after filtering by using SOAP denovo software to obtain a Scaffolds sequence;

(3) after the step (2) is completed, breaking the Scaftolds sequence from the N junction to obtain a Scaftolds sequence without N, filtering out fragments below 500bp, further performing open reading frame prediction and filtering on the remaining Scaftolds sequence fragments by using MetaGeneMark software, and performing redundancy removal by using CD-HIT software;

(4) after the step (3) is completed, comparing the obtained representative segments by using the redundancy removal as a reference by using an effective Data result to obtain the reads number of each gene segment in each sample, then filtering out the gene segments of which the reads number is not more than 2, and forming a Unigenes list by using the remaining Clean Data segments to calculate information such as gene abundance and the like, and comparing a KEGG database to perform function annotation and abundance analysis of a metabolic pathway;

(5) after the step (4) is completed, comparing Unigenes to a Microbe NR database of NCBI by using DIAMOND software, then selecting a comparison result that evalue is less than or equal to minimum evalue x 10, applying an LCA algorithm of MEGAN software to perform species annotation on each sequence, and obtaining abundance information and gene number information of each sample on each generic layer level by combining the abundance of Unigenes.

6. The method of claim 5, wherein:

the step of quality control filtering the sequencing data comprises the step of removing reads as follows:

(a1) reads smaller than 40bp and reads with N base content higher than 10 bp;

(a2) reads with sequencing joint sequence overlap over 15 bp;

(a3) mouse (host) derived reads.

7. The method of any of claims 3 to 6, wherein: the sequencing platform is an IlluminaNovaseq6000 high-throughput sequencing platform.

8. The method of any of claims 3 to 7, wherein: the mouse is a BALB/C-Tg (NF kappa B-RE-luc) -Xen mouse.

9. The use of a vector carrying the method according to any one of claims 3 to 8 for the auxiliary detection of the presence of transgenic maize of the C2Z7 type in food products or for the preparation of products for the auxiliary detection of the presence of transgenic maize of the C2Z7 type in food products is described.

10. The use of the species and genes as set forth in claim 1 as markers to aid in the detection of the presence of C2Z 7-type transgenic corn in food products.

Technical Field

The invention relates to a method for detecting whether C2Z7 (CC 2) transgenic corn ingredients are contained in food by using metagenome technology in an auxiliary manner.

Background

According to the definition of the convention of the United nations, "Biosafety protocol", transgenic Organisms are called "Modified Living Organisms" Living Modified Organisms, called LMOs for short, or "Genetically Modified Organisms", GMOs. LMOs or GMOs refer to living organisms having a novel combination of genetic materials obtained by modern biotechnology, and in fact, to organisms whose genetic composition has been altered in nature by introducing foreign DNA into their genomes. At present, the most closely related to the public are various transgenic crops. Transgenic crops are produced by adding genetic information of other organisms (usually in reproductive isolation species, and the genetic information cannot be obtained in a common hybridization mode) to genes of original crops by using a gene editing technology, so that the crops with better quality or the quality which the original crops do not have are produced.

Since the commercialization of transgenic crops in 1996, transgenic crops have been grown in 26 countries worldwide, and as far as 2016, the growing area reaches 1.851 hectare, the crop species are the most in terms of transgenic soybeans, and related countries are the top in terms of the growing area in the united states, and the domestic transgenic crop species are the most. Under the guidance of series file specifications such as agricultural transgenic organism safety management regulations (revised version of 10/7/2017), experimental research, development, planting and import work of transgenic crops are carried out in China, transgenic crops approved to be planted at present are Bt-transgenic cotton and transgenic antiviral papaya, transgenic corns and rice are included in an experimental state, and transgenic soybeans, corns, rapes, beet and cotton are approved to be imported as processing raw materials.

At present, the technology for detecting transgenic crops mainly aims at the specific genetic information of crops, carries out the detection of DNA and RNA, and evaluates the safety of the transgenic crops, including the evaluation of the structure and biological effect of nucleic acid, protein and other components which are contained in the transgenic crops and enter organisms after being ingested.

The metagenome is a combination of genomes of various microorganisms symbiotic with the human body, and a major part thereof is a genome of an intestinal microorganism, particularly an intestinal flora. The intestinal flora is a collection of bacteria of various types in the intestinal tract, and the number of the bacteria is equivalent to the number of cells of a human body. In the long evolution process of human beings, a symbiotic/parasitic relationship is formed between human beings and intestinal flora, the immune state and dietary habits of human bodies can regulate the composition of the flora, and the structural change and metabolic function change of the flora can adversely affect the physiological and disease processes of the human bodies. More importantly, changes in the bacterial flora structure caused by changes in dietary composition can be observed within a week. Therefore, starting from the view point of the metagenome of the intestinal flora, the influence of the diet containing the transgenic corn components on the metagenome is evaluated, a sensitive and accurate detection index can be established, and the influence of the diet on the health of a host can be predicted in an auxiliary way.

A C2Z7 type transgenic corn, named CC2, is a gene of foreign protein aroA (glyphosate-resistant herbicide) based on Z580 type corn, and the glyphosate herbicide encoded by the gene is a broad-spectrum and non-selective herbicide, has strong inhibiting effect on annual and perennial weeds, is easy to decompose by microorganisms, and has no residual toxicity in soil. The gramineous crops such as corn and the like are sensitive to glyphosate, so that the application of the gramineous crops is limited, and therefore, the glyphosate-resistant gene is transferred into the corn, so that the application range of the glyphosate can be enlarged, the production cost can be reduced, the corn is protected from phytotoxicity, and the purposes of increasing the yield and the income are finally achieved. Whether the genes and the corn expressing the corresponding proteins can cause harm after being directly ingested into a human body needs to be comprehensively and deeply evaluated at present. The detection means which is commercialized at present generally uses an enzyme-linked immunosorbent kit to detect protein components of corn seeds, but cannot judge the biological effect of the corn seeds as food.

Disclosure of Invention

The invention aims to provide a method for detecting whether C2Z7 (CC 2) transgenic corn ingredients are contained in food or not by using metagenome technology.

The invention firstly protects the application of the substances for detecting the abundance of the genes and strains shown in the following table in the auxiliary detection of whether the food contains C2Z7 type transgenic corn;

the invention also protects a product for auxiliary detection of whether the food contains C2Z7 type transgenic corn, which comprises substances for detecting the abundance of genes and strains shown in the following table;

the invention also provides a method for auxiliary detection of C2Z7 transgenic corn in food, which comprises the following steps:

(1) respectively feeding the common feed and the feed to be detected to the mice; the feed to be detected is a mixture of food to be detected and common feed;

(2) after 10 weeks, extracting the total DNA of the mouse feces, and performing high-throughput sequencing;

(3) analyzing the sequencing result to obtain the abundance value of the specific gene and the strain of the sample, and judging according to the standard range shown in the following table; if the standard ranges listed in the following table are met, the food to be tested contains or possibly contains C2Z7 type transgenic corn; on the contrary, the food to be detected does not contain C2Z7 type transgenic corn;

the specific genes, strains and standard abundance ranges are the genes, strains and numerical values shown in the following table;

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The feed to be detected is a mixture of 1 volume part of food to be detected and 3 volume parts of common feed.

The common feed can be a rat and a rat feed of Beijing Huafukang biotech GmbH, and the product number is as follows: 1032.

the sequencing result analysis method comprises the following steps:

(1) performing quality control filtration on sequencing data;

(2) after the step (1) is completed, further assembling the effective data obtained after filtering by using SOAP denovo software to obtain a Scaffolds sequence;

(3) after the step (2) is completed, breaking the Scaftolds sequence from the N junction to obtain a Scaftolds sequence without N, filtering out fragments below 500bp, further performing open reading frame prediction and filtering on the remaining Scaftolds sequence fragments by using MetaGeneMark software, and performing redundancy removal by using CD-HIT software;

(4) after the step (3) is completed, comparing the obtained representative segments by using the redundancy removal as a reference by using an effective Data result to obtain the reads number of each gene segment in each sample, then filtering out the gene segments of which the reads number is not more than 2, and forming a Unigenes list by using the remaining Clean Data segments to calculate information such as gene abundance and the like, and comparing a KEGG database to perform function annotation and abundance analysis of a metabolic pathway;

(5) after the step (4) is completed, comparing Unigenes to a Microbe NR database of NCBI by using DIAMOND software, then selecting a comparison result that evalue is less than or equal to minimum evalue x 10, applying an LCA algorithm of MEGAN software to perform species annotation on each sequence, and obtaining abundance information and gene number information of each sample on each generic layer level by combining the abundance of Unigenes.

The step of quality control filtering the sequencing data comprises the step of removing reads as follows:

(a1) reads smaller than 40bp and reads with N base content higher than 10 bp;

(a2) reads with sequencing joint sequence overlap over 15 bp;

(a3) mouse (host) derived reads.

The sequencing platform can be specifically an Illumina Novaseq6000 high-throughput sequencing platform.

The mouse can be a BALB/C-Tg (NF kappa B-RE-luc) -Xen mouse.

The invention also protects the application of the vector carrying any one of the methods in auxiliary detection of whether the food contains C2Z7 type transgenic corn.

The invention also protects the application of the vector describing any one of the methods in preparing products for assisting in detecting whether the food contains the C2Z7 type transgenic corn.

The invention also protects the application of any one of the strains and genes as markers in auxiliary detection of whether C2Z7 type transgenic corn is contained in food.

The invention develops and formulates a technical index which is suitable for detecting C2Z7 type transgenic corn components in food and can assist in judging the biological safety of the food from the perspective of metagenome. The significance of the invention lies in that the influence of the corn with aroA gene on the first barrier of the inner environment of a food taker, namely the gastrointestinal tract system, is found through the change of the metagenome, and an indirect judgment basis can be provided for evaluating the influence (good or bad) of the corn on the physiological state of the food taker.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

7610 maize line (type 76-100) is a line obtained by crossing PHBA6 maize as male parent and Chang 7-2 maize as female parent. PHBA6 corn is a variety cultivated by Pioneer corporation of america (Pioneer Hi-Bred International, Inc.) with a male parent of PHZ51 type corn and a female parent of PHG47 type corn, described in the literature (specifically in the supplementary materials list of the literature): schaefer C M, Bernardo R.P. publication structure and SNP diversity of biological Minnesota mail in seeds [ J ]. Crop Sci.53: 1529-. Agronomy org. Maize type Chang 7-2 is described in the literature: zhang Wen Ying, Huafuping, Shen Min, et al, Breeding of fine maize inbred line Chang 7-2 and utilization thereof [ J ]. proceedings of the Henan institute of occupational technology, 2001(4): 17-19.; the above materials are publicly available from the institute of cardiovascular and pulmonary diseases in Beijing.

Corn type Z580 (zheng 58): are described in the literature: zhang Fang forest, cultivation and application of Zheng 58 of corn fine inbred line [ J ]. J.CROPS, 2001(4): 31-31.; publicly available from the institute of cardiovascular and pulmonary vascular diseases, Beijing.

Type 52C7 (Bt506) maize: the number 111-7-2018 of the bulletin board recorded in the rural area of agriculture: qualitative PCR method for detecting insect-resistant corn Bt506 and derived varieties thereof by transgenic plants and product components thereof; publicly available from the institute of cardiovascular and pulmonary vascular diseases, Beijing.

C2Z7 type (CC-2) maize: the literature is described: research on the survival competitive capacity of transgenic herbicide-resistant corn CC-2, journal of crops, 2014, 6: 64-66; publicly available from the institute of cardiovascular and pulmonary vascular diseases, Beijing.

The invention uses a normal clean grade 8 week old female BALB/C background NF-kB response element-luciferase transgenic mouse (BALB/C-Tg (NF kB-RE-luc) -Xen, Caliper Life Sciences company, USA) as an experimental mouse purchased from Nanjing university model animal research institute.