CN111647664A - Method for non-invasive identification of insect genotype - Google Patents
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Abstract
The invention provides a method for non-invasively identifying insect genotypes. The method comprises the following steps: (a) respectively feeding wild insects and gene-edited insect mutants, wherein each insect individual needs to be independently marked or fed; (b) taking insect skin peeling once as age growth once, and respectively collecting molting corresponding to 1-adult insect period; (c) extracting ecdysis genome DNA by using a DNA extraction kit; (d) and (3) taking genome DNA extracted from ecdysis as a template, designing a primer according to a target gene sequence, carrying out PCR amplification, sequencing an amplification product, and further identifying the genotype. The method can realize noninvasive, early and repeated insect genotype identification by molting, and solves the problems of mechanical damage caused by shearing insect body tissues and incapability of identifying young insects.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for noninvasive identification of insect genotypes.
Background
In recent years, with the continuous and deep research on the CRISPR/Cas9 system of gene editing technology, the system is easy to operate, time-saving, efficient and the likeHas the advantages of being widely applied to a plurality of biological research fields, in particular to locusta migratoria (A)Locusta migratoria) Fruit flies (1)Drosophilidae) Silkworm (silkworm)Bombyx mori) Aedes aegypti (a), Aedes aegypti (b)Aedes aegypti) And the like in the study of various insects. After genome editing is carried out on insects by using CRISPR/Cas9 genome directed editing technology, the genotype identification of the edited individuals is very important.
Currently, the sample tissues required for identifying insect mutants are mainly prepared by extracting genomic DNA from insect whole body or local tissues. For example, in the published literature, Aedes aegypti (A) mosquitoAedes aegypti) Culex pipiens perniciflua (b)Culex quinquefasciatus) (ii) Spodoptera lituraSpodoptera litura) (iii) diamondback mothPlutella xylostella) And cotton bollworm: (Helicoverpa armigera) The gene editing experiment of (1) mostly adopts a mode of extracting genome DNA from an embryo; in the orange of butterfly (Papilio xuthus) Kallima inachus (A) and (B)Vanessa cardui) And Kallima inachus (Kallima Kallissima)Junonia coenia) In lepidopteran insects, local tissues such as head, breast, compound eye and wing muscle tissues are often used as samples for extracting DNA to identify the genotype of the mutant. In migratory locust, the genome DNA in the podophyllum antepodium or haemolymph of migratory locust is extracted to complete the genotype identification.
These traditional identification techniques can cause irreversible mechanical damage to the individual insect, even death of the individual, and are not conducive to the observation of mutant phenotypes. In addition, because young insects are fragile and extremely easy to die, the existing methods are not suitable for early identification of insect genotypes and only have small harm to adult insects, so that after gene editing is carried out by using CRISPR/Cas9, a large number of mutant individuals are required to be fed to adults, the timeliness is poor, and waste of time and resources is easily caused. Locusts migratoria is a worldwide agricultural pest, and is also a typical metamorphosis type insect. Recently, a large range of locust disasters have rolled over more than 20 countries worldwide, with serious consequences on food safety. In recent years, with the completion of the sequencing of locusta migratoria genome, the exploration of novel molecular targets for pest control has become a research hotspot. The CRISPR/Cas9 technology is combined to carry out primary verification on the functions of the target genes at the cellular level, basic data are provided for subsequent in vivo level research, and the method is a main way for completing screening and verification of the target genes of pests. How to complete the identification of the individual genotype of the migratory locust under the noninvasive condition is the key for ensuring the preliminary screening of the experimental phenotype.
Disclosure of Invention
The invention aims to provide a method for non-invasively identifying insect genotypes, and solve the problems that the existing identification method can bring mechanical damage to insect bodies, the early identification of young insects can not be carried out due to the death of the insects caused by the mechanical damage, and the accuracy of the genotype identification is not ideal.
The purpose of the invention is realized as follows: a method for non-invasively identifying an insect genotype comprising the steps of:
(a) respectively feeding wild insects and gene-edited insect mutants, wherein each insect individual needs to be independently marked or fed;
(b) taking insect skin peeling once as age growth once, and respectively collecting molting corresponding to 1-adult insect period;
(c) extracting ecdysis genome DNA by using a DNA extraction kit;
(d) and (3) taking genome DNA extracted from ecdysis as a template, designing a primer according to a target gene sequence, carrying out PCR amplification, sequencing an amplification product, and further identifying the genotype.
In step (a), the insect is an insect that has an ecdysis process, can be independently marked or raised.
In the step (a), the gene editing refers to the genome editing of the insect by using CRISPR/Cas9 genome directed editing technology, TALEN or ZFN genome editing technology.
In the step (b), ecdysis corresponding to 1-year-old insects or ecdysis corresponding to 1-and 2-year-old insects is collected for early identification of insect genotype.
In the step (c), the concentration of the extracted genomic DNA may be measured using a micro ultraviolet spectrophotometer.
According to the invention, noninvasive, early and repeated insect genotype identification can be realized by molting, and the problems of mechanical damage caused by shearing insect body tissues, incapability of identifying young insect bodies and accuracy of genotype identification are solved. Experiments prove that the insect molting at the low age can extract genomic DNA with higher total amount, and the genomic DNA extracted from the molting is compared with the genomic DNA extracted from tissues such as hemolymph, forefoot epididymis and the like, so that the content and the integrity of the genomic DNA extracted from the molting and the genomic DNA extracted from the hemolymph and the like are not obviously different. Sequencing shows that the DNA amplification target fragment sequence extracted from molting is completely consistent with the fragment sequence obtained by the original method, namely the genotype identification result is consistent.
Drawings
Fig. 1 is a photograph of molting of locusta migratoria at different ages.
FIG. 2 is a graph comparing the DNA content of the genome extracted from molting of migratory locusts at different ages.
FIG. 3 is a graph of the results of genomic DNA integrity tests for three different tissues. Wherein, Foreleg pretarsus: the forefoot appendage; hemolymph: haemolymph; ecdysis: and (5) molting.
FIG. 4 is a graph showing the comparison of the concentration of genomic DNA extracted from three tissues of 3-year-old individuals of migratory locusts of different genotypes. The DNA concentration of three tissues of a wild type is compared, the DNA concentration of three tissues of a chimera is compared, the DNA concentration of three tissues of a heterozygote is compared, and the DNA concentration of three tissues of a homozygote is compared.
FIG. 5 is a graph showing the sequencing results of the amplified target DNA fragments extracted from three different tissues. OrcoF: the forefoot appendage; OrcoE: molting; OrcoH: haemolymph.
Detailed Description
In order to better explain the technical scheme of the invention, migratory locusts are taken as an example, and the accompanying drawing is used for describing the novel insect genotype identification mode of the invention, namely noninvasive, early and repeated genotype identification by molting in detail. The experimental procedures and reagents not described in the examples were carried out according to the conventional procedures in the art or the instructions of the manufacturers.
The method for non-invasively identifying the locust genotype specifically operates as follows:
(1) gene editing mutant individuals:
migratory locust using CRISPR/Cas9 genome directed editing technologyOrcoThe genes are edited.
First, in vitro synthesisOrcogRNA of the gene: referring to the instruction of a gRNA synthesis kit (invitrogen, A29377), a suitable target sequence (20 nt upstream of a PAM region is a target region) is searched on a target gene, and corresponding upstream and downstream primers are synthesized according to the requirements of the kit. Synthesizing DNA template of gRNA by PCR technology, finally obtaining by in vitro transcriptionOrcogRNA of a gene.
Then, ova of the wild type migratory locusts are collected within four hours of the birth, the ova bags are placed into a culture dish filled with sterile water, the ova are swept out by a brush to remove impurities on the ova, after the ova are soaked in 75% ethanol for disinfection and washed by the sterile water, the surface moisture of the dry ova is absorbed by paper, and the ova are horizontally placed on an agarose gel plate prepared in advance according to the sequence of head-to-tail connection.
The gRNA synthesized in vitro was mixed with Cas9 protein, and a mixed solution of the gRNA having gene editing activity and Cas9 protein was injected at the beginning of embryonic development by a microinjection system at 23 nL in one injection. After the injection is finished, the injected worm eggs are put into a constant-temperature incubator at 30 ℃ for culture until hatching.
(2) Feeding migratory locusts:
migratory locust constructed by CRISPR/Cas9OrcoThe gene mutant and corresponding wild migratory locust are respectively fed, each migratory locust is independently fed in a metal feeding cage with the length of 10cm × 10cm × 25cm, the feeding temperature is 30 ℃, the relative humidity is 30-40%, the illumination period is 14L: 10D, and the migratory locust is fed with fresh wheat seedlings.
(3) Collecting materials for extracting genomic DNA:
collecting molting of locusta migratoria nymphs from 1 st to adult stage. Every moulting of locusta migratoria nymphs is regarded as one-time growth in the age, namely 5 moulting in the period from 1 year to adult. The molting size is related to the size of the locusta migratoria at that age, namely, the molting volume is gradually increased along with the increase of the age (as shown in figure 1 in particular).
Moulting for each individual and each age period needs to be collected separately in enzyme-free sterile EP tubes for marker differentiation. The molt can be extracted immediately after collection, or stored at-80 deg.C for a short period, and taken out.
(4) Extracting genome DNA:
extracting ecdysis genome DNA of different ages by using a DNA extraction kit (TIANGE, DP 304-03) specifically comprises the following steps:
crushing the molt into fine powder by using an electric grinding rod, adding 200 mu L of buffer GA, and shaking until the molt is completely suspended.
② adding 20 μ L protease K solution, mixing uniformly, placing at 56 deg.C until tissue is dissolved, centrifuging briefly to remove water drop on inner wall of tube cover.
And thirdly, adding 200 mu L of buffer solution GB, fully reversing and uniformly mixing, standing at 70 ℃ for 10 min, strain-clearing the solution, and centrifuging briefly to remove water drops on the inner wall of the tube cover.
Adding 200 mu L of absolute ethyl alcohol, fully shaking and mixing for 15s, wherein flocculent precipitates can appear, and centrifuging briefly to remove water drops on the inner wall of the tube cover.
Fifthly, adding the obtained solution and flocculent precipitate into an adsorption column CB3, centrifuging at 12000rpm for 30 seconds, pouring waste liquid, and putting the adsorption column CB3 into a collecting pipe.
Sixthly, adding 500 mu L of buffer GD into the adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing the adsorption column CB3 into a collecting pipe.
Seventhly, 600 mu L of rinsing liquid PW is added into the adsorption column CB3, centrifugation is carried out for 30s at 12000rpm, waste liquid is poured out, and the adsorption column CB3 is placed into a collecting pipe. And repeating the steps once.
Eighthly, placing the adsorption column CB3 into a collecting pipe, centrifuging at 12000rpm for 2min, pouring the waste liquid, placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material.
Ninthly, transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption film, placing the adsorption film at room temperature for 2-5min, centrifuging the adsorption film at 12000rmp for 2min, and collecting the solution into the centrifugal tube, namely the genomic DNA solution.
Taking 1.5 mu L of the genome DNA solution obtained by the method, measuring the concentration by using an ultraviolet spectrophotometer, making a concentration mark on the tube wall, and storing for a long time at the temperature of minus 20 ℃.
FIG. 2 is a graph showing the comparison of the genomic DNA content extracted from molts of migratory locusts at different ages, and it can be seen that the molts of migratory locusts at a low age can extract a higher total amount of genomic DNA, wherein the DNA content in the molts of migratory locusts at 1 year is sufficient for PCR amplification and sequencing identification (FIG. 2). Integrity comparison of ecdysis-extracted genomic DNA with that of hemolymph and forefoot epididymis of conventional method revealed that the integrity of genomic DNA extracted from ecdysis was similar to that extracted from hemolymph (fig. 3). Since the hemolymph and the poda epizoea of 1-2 instar locusta nymphs are extremely difficult to obtain, genomic DNAs in the hemolymph and the poda epizoea of wild type and mutant 3 instar locusta nymphs are respectively extracted and compared with the extracted DNA of 3 instar ecdysis. The results showed no significant difference in DNA content extracted from molting and hemolymph (fig. 4).
(5) And (3) identifying the genotype:
designing a pair of primers at the upstream and downstream of the migratory locust target site by using Primer Premier5 softwareOrco-F、Orco-R, should be greater than 150 bp from the target site, and not greater than 500 bp in total length.
Taking genomic DNA extracted from ecdysis as template, performing PCR amplification on the target fragment by using KOD DNA Polymerase amplification system (TOYOBO, KOD-101), sequencing the amplification product, and identifying genotype.
Sequencing results show that the DNA amplification fragment sequence extracted from ecdysis is completely consistent with the fragment sequences obtained by the two original methods, namely the genotype identification results are consistent (figure 5). In conclusion, the method has good repeatability, and the obtained result is completely consistent with the two existing methods for extracting DNA with damage, so that the method can completely replace the two existing methods.
Claims (5)
1. A method for non-invasively identifying the genotype of an insect is characterized by comprising the following steps:
(a) respectively feeding wild insects and gene-edited insect mutants, wherein each insect individual needs to be independently marked or fed;
(b) taking insect skin peeling once as age growth once, and respectively collecting molting corresponding to 1-adult insect period;
(c) extracting ecdysis genome DNA by using a DNA extraction kit;
(d) and (3) taking genome DNA extracted from ecdysis as a template, designing a primer according to a target gene sequence, carrying out PCR amplification, sequencing an amplification product, and further identifying the genotype.
2. The method for non-invasively identifying the genotype of an insect according to claim 1, wherein in step (a), the insect is an insect that has an ecdysis process, can be independently tagged or fed.
3. The method for noninvasive identification of insect genotype as claimed in claim 1, wherein in step (a), gene editing refers to genome editing of insects by using CRISPR/Cas9 genome directed editing technology, TALEN or ZFN genome editing technology.
4. The method for non-invasively identifying the genotype of an insect according to claim 1, wherein in the step (b), molts corresponding to 1-year-old insects or molts corresponding to 1-and 2-year-old insects are collected for early identification of the genotype of the insect.
5. The method for non-invasively genotyping of claim 1, wherein in step (c), the concentration of the extracted genomic DNA is determined using a micro uv spectrophotometer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114990110A (en) * | 2022-07-19 | 2022-09-02 | 江西农业大学 | Non-destructive sampling method for field butterfly monitoring |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807580A (en) * | 2006-01-23 | 2006-07-26 | 重庆大学 | Method for separating and purifying insect pathogenic fungoid thallus from infected insect haemolymph |
| CN102031307A (en) * | 2010-11-19 | 2011-04-27 | 西北农林科技大学 | Kit and method for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis |
| CN106857412A (en) * | 2017-03-29 | 2017-06-20 | 中国农业科学院植物保护研究所 | Identify a kind of method whether material has an impact on insect lipids and have which kind of influence |
| CN109593762A (en) * | 2018-12-29 | 2019-04-09 | 中国农业科学院蔬菜花卉研究所 | Diamondback moth APN1 gene and its application are knocked out using CRISPR/Cas9 gene editing system |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807580A (en) * | 2006-01-23 | 2006-07-26 | 重庆大学 | Method for separating and purifying insect pathogenic fungoid thallus from infected insect haemolymph |
| CN102031307A (en) * | 2010-11-19 | 2011-04-27 | 西北农林科技大学 | Kit and method for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis |
| CN106857412A (en) * | 2017-03-29 | 2017-06-20 | 中国农业科学院植物保护研究所 | Identify a kind of method whether material has an impact on insect lipids and have which kind of influence |
| CN109593762A (en) * | 2018-12-29 | 2019-04-09 | 中国农业科学院蔬菜花卉研究所 | Diamondback moth APN1 gene and its application are knocked out using CRISPR/Cas9 gene editing system |
Non-Patent Citations (1)
| Title |
|---|
| 郑美艳: "利用末龄幼虫蜕和蛹壳对单头昆虫基因的无损伤检测", 《应用昆虫学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114990110A (en) * | 2022-07-19 | 2022-09-02 | 江西农业大学 | Non-destructive sampling method for field butterfly monitoring |
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Application publication date: 20200911 |