CN111647664A - Method for non-invasive identification of insect genotype - Google Patents

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

A method for non-invasive identification of insect genotypes

technical field

The invention relates to the field of biotechnology, in particular to a method for non-invasive identification of insect genotypes.

Background technique

In recent years, with the deepening of the research on the gene editing technology CRISPR/Cas9 system, the system has been widely used in many biological research fields due to its advantages of simple operation, time saving and high efficiency, especially for locust ( Locusta migratoria ) , Drosophilidae ( Drosophilidae ), silkworm ( Bombyx mori ), Aedes aegypti ( Aedes aegypti ) and other insect research. After genome editing of insects using CRISPR/Cas9 genome-directed editing technology, it is particularly important to identify the genotype of the edited individuals.

Currently, the sample tissues required for the identification of insect mutants are mainly used to extract genomic DNA from the whole body or local tissues of insects. For example, in the published literature, gene editing of Aedes aegypti , Culex quinquefasciatus , Spodoptera litura , Plutella xylostella , and Helicoverpa armigera Experiments mostly use the method of extracting its genomic DNA from embryos; in lepidopteran insects such as Papilio xuthus , Vanessa cardui and Junonia coenia , the head , thorax, compound eye and wing muscle tissue and other local tissues were used as samples for DNA extraction to identify mutant genotypes. In migratory locusts, the identification of genotype is mainly accomplished by extracting the genomic DNA from the anterior appendages of the forefoot or the hemolymph of the migratory locusts.

These traditional identification techniques will cause irreversible mechanical damage to individual insects, and even cause individual death, which is not conducive to the observation of mutant phenotypes. In addition, because young insects are fragile and easy to die, the existing methods are not suitable for the early identification of insect genotypes, and only cause less damage to adult insects. Therefore, after gene editing using CRISPR/Cas9, It is necessary to rear a large number of mutant individuals to adults, which has poor timeliness and is prone to waste time and resources. The migratory locust is a worldwide agricultural pest and a typical morphological insect. Recently, a large-scale locust plague has swept through more than 20 countries around the world, causing serious consequences for food security. In recent years, with the completion of the locust genome sequencing as an opportunity, the exploration of new molecular targets for pest control has become a research hotspot. Combining CRISPR/Cas9 technology to initially verify the function of target genes at the cellular level provides basic data for subsequent studies at the in vivo level, which is the main way to complete the screening and verification of pest target genes. How to complete the identification of individual genotypes of migratory locusts under non-invasive conditions is the key to ensure the preliminary screening of experimental phenotypes.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a non-invasive method for identifying insect genotypes, so as to solve the problem that the existing identification methods will bring mechanical damage to the worms, and the juvenile worms will die due to mechanical damage and cannot perform early identification and genotype identification. The accuracy is not ideal.

The object of the present invention is achieved in this way: a kind of method for non-invasive identification of insect genotype, comprising the following steps:

(a) Wild-type insects and gene-edited insect mutants are reared separately, and each individual insect needs to be independently labeled or reared separately;

(b) Each time the insect molts is regarded as an instar growth, and the molts corresponding to the 1st instar to the adult stage of the insect are collected respectively;

(c) Extracting the genomic DNA of the molted skin with a DNA extraction kit;

(d) Using the genomic DNA extracted from the molting as a template, primers are designed according to the target gene sequence and PCR amplification is performed, and the amplified product is sequenced to identify the genotype.

In step (a), the insect is an insect that has a molting process and can be independently labeled or reared.

In step (a), gene editing refers to genome editing of insects using CRISPR/Cas9 genome-directed editing technology, TALEN or ZFN genome editing technology.

In step (b), the molts corresponding to the 1st instar stage of the insects or the molts corresponding to the 1st and 2nd instar stages of the insects are collected for early identification of insect genotypes.

In step (c), the concentration of the extracted genomic DNA can be determined by using a micro-ultraviolet spectrophotometer.

The invention can realize non-invasive, early and repeated insect genotype identification by molting, and solve the problems of mechanical damage caused by clipping worm tissue, inability to identify juvenile worms and accuracy of genotype identification. It has been verified by experiments that a relatively high amount of genomic DNA can be extracted from the molting of insects at a young age. The genomic DNA extracted from the molting is compared with the genomic DNA extracted from the haemolymph and the anterior appendage of the forefoot, and it is found that the genome extracted from the molting There was no significant difference in the content and integrity of the genome extracted from DNA and hemolymph. Sequencing showed that the DNA amplification target fragment sequence extracted from the molting was completely consistent with the fragment sequence obtained by the original method, that is, the genotype identification results were consistent.

Description of drawings

Figure 1 is a photo of migratory locusts molting at different ages.

Figure 2 is a comparison diagram of genomic DNA content extracted from molting at different ages of migratory locusts.

Figure 3 is a graph showing the results of genomic DNA integrity testing in three different tissues. Among them, Foreleg pretarsus: forefoot anterior appendage; Hemolymph: hemolymph; Ecdysis: molting.

Figure 4 is a graph comparing the concentrations of genomic DNA extracted from three tissues of 3-year-old migratory locust individuals of different genotypes. Among them, a. Comparison of DNA concentration in three tissues of wild type, b. Comparison of DNA concentration of three tissues of chimera, c. Comparison of DNA concentration of three tissues of heterozygote, d. Comparison of DNA concentration of three tissues of homozygote.

Figure 5 is a diagram of the sequencing results of DNA amplification target fragments extracted from three different tissues. OrcoF: anterior segment of the forefoot; OrcoE: molting; OrcoH: hemolymph.

Detailed ways

In order to better illustrate the technical scheme of the present invention, the following takes migratory locusts as an example, and in conjunction with the accompanying drawings, the novel insect genotype identification method of the present invention, that is, non-invasive, early and repeated genotype identification by molting is introduced in detail. The experimental operations, reagents, etc. that are not described in the examples are all carried out according to the conventional operations in the field or the operation instructions of the manufacturers.

The specific operations of the method for the non-invasive identification of the locust genotype of the present embodiment are as follows:

(1) Gene editing mutant individuals:

Using CRISPR/Cas9 genome-directed editing technology to edit the Orco gene of the migratory locust.

First, synthesize the gRNA of the Orco gene in vitro: refer to the instructions of the gRNA synthesis kit (invitrogen, A29377), find a suitable target sequence on the target gene (the upstream 20nt of the PAM region is the target region), and synthesize the corresponding upstream and downstream according to the requirements of the kit primers. The DNA template of gRNA was synthesized by PCR technology, and finally the gRNA of Orco gene was obtained by in vitro transcription.

Subsequently, collect the eggs of wild-type gregarious migratory migratory locusts within four hours, put the egg bag into a petri dish filled with sterile water, and use a brush to sweep out the eggs to remove impurities on the eggs. After soaking in 75% ethanol for disinfection and cleaning with sterile water, the surface moisture of the dried egg particles was absorbed with paper, and they were placed horizontally on a prepared agarose gel plate in an end-to-end order.

The gRNA synthesized in vitro was mixed with Cas9 protein, and the mixture of gRNA with gene editing activity and Cas9 protein was injected at the beginning of embryonic development through a microinjection system, with a single injection of 23 nL. After the injection, the injected eggs were placed in a constant temperature incubator at 30 °C for incubation until hatching.

(2) Individual feeding of locusts:

The locust Orco gene mutant constructed by CRISPR/Cas9 and the corresponding wild-type locust were reared separately: each individual locust was reared individually in a metal cage of 10cm×10cm×25cm, and the rearing temperature was 30°C and the relative humidity was 30°C. It is 30-40%, the light cycle is 14L: 10D, and it is raised with fresh wheat seedlings.

(3) Collect materials for extracting genomic DNA:

The molting from the first instar to the adult stage of the locust locust was collected. Each molting of the locust locust worm is regarded as an increase in the instar stage, that is, a total of 5 molting times from the 1 instar to the adult stage. The size of its molt is related to the size of the individual migratory locusts at that age, that is, with the growth of the age, the size of the molt will gradually increase (as shown in Figure 1).

The molting of each individual and each age needs to be collected separately in an enzyme-free sterile EP tube and marked for distinction. The molt can be extracted immediately after collection, or it can be stored at -80°C for a short period of time, and it can be taken out during extraction.

(4) Extract genomic DNA:

Use DNA extraction kit (TIANGE, DP304-03) to extract molting genomic DNA of different ages. The specific steps are as follows:

① Break the molt into fine powder with an electric grinding rod, add 200 μL of buffer GA, and shake until it is completely suspended.

②Add 20 μL Proteinase K solution, mix well, place at 56°C until the tissue dissolves, and briefly centrifuge to remove water beads on the inner wall of the tube lid.

③Add 200 μL of buffer GB, invert and mix well, place at 70 °C for 10 min, the solution should become clear, and centrifuge briefly to remove the water droplets on the inner wall of the tube cover.

④Add 200 μL of absolute ethanol, shake and mix well for 15s, flocculent precipitation may appear at this time, briefly centrifuge to remove water droplets on the inner wall of the tube cover.

⑤ Add the solution and the flocculent precipitate obtained above into an adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, pour off the waste liquid, and put the adsorption column CB3 into the collection tube.

⑥ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000rpm for 30s, pour off the waste liquid, and put the adsorption column CB3 into the collection tube.

⑦ Add 600 μL of rinsing solution PW to the adsorption column CB3, centrifuge at 12000rpm for 30s, pour off the waste liquid, and put the adsorption column CB3 into the collection tube. repeat.

⑧Put the adsorption column CB3 into the collection tube, centrifuge at 12000 rpm for 2 minutes, pour out the waste liquid, and place the adsorption column CB3 at room temperature for a few minutes to completely dry the residual rinse solution in the adsorption material.

⑨ Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200μL of elution buffer TE to the middle of the adsorption membrane, place it at room temperature for 2-5min, centrifuge at 12000rmp for 2min, and collect the solution into the centrifuge tube. That is, the genomic DNA solution.

Take 1.5 μL of the genomic DNA solution obtained by the above method, measure the concentration with a UV spectrophotometer, and mark the concentration on the wall of the tube, which can be stored at -20 °C for a long time.

Figure 2 is a comparison diagram of the content of genomic DNA extracted from the molting of migratory locusts at different ages. It can be seen from the figure that a higher total amount of genomic DNA can be extracted from the molting of migratory locusts at a younger age. PCR amplification and sequencing identification were performed (Figure 2). Comparing the integrity of the genomic DNA extracted from the molt with the traditional method of the haemolymph and the genomic DNA extracted from the forefoot anterior segment, it was found that the integrity of the genomic DNA extracted from the molt was similar to that of the haemolymph (Figure 3). . Since the haemolymph and the forefoot anterior appendages of the 1-2 instar locust flies are extremely difficult to obtain, the haemolymph and the genomic DNA in the forefoot anterior appendages of the wild-type and mutant 3 instar locust flies were extracted respectively, and compared with the DNA of the third instar molting Extraction amount for comparison. The results showed that there was no significant difference in DNA content and hemolymph extracted from molting (Fig. 4).

(5) Identify the genotype:

Using Primer Premier5 software, a pair of primers Orco -F and Orco -R were designed upstream and downstream of the locust target site, the distance from the target site should be greater than 150 bp, and the total length should not be greater than 500 bp.

The genomic DNA extracted from the molt was used as the template, and the target fragment was amplified by PCR using the KOD DNA Polymerase amplification system (TOYOBO, KOD-101). The amplified product was sequenced to identify the genotype.

The sequencing results showed that the sequences of the amplified DNA fragments extracted from the molting were completely consistent with those obtained by the two original methods, that is, the genotype identification results were consistent (Figure 5). To sum up, it can be seen that the present invention has good repeatability, and the obtained results are completely consistent with the two existing methods for DNA extraction with damage, and can completely replace the two existing methods.

Claims (5)

1. a method for noninvasive identification of insect genotype, is characterized in that, comprises the following steps: (a) Wild-type insects and gene-edited insect mutants are reared separately, and each individual insect needs to be independently labeled or reared separately; (b) Each time the insect molts is regarded as an instar growth, and the molts corresponding to the 1st instar to the adult stage of the insect are collected respectively; (c) Extracting the genomic DNA of the molted skin with a DNA extraction kit; (d) Using the genomic DNA extracted from the molting as a template, primers are designed according to the target gene sequence and PCR amplification is performed, and the amplified product is sequenced to identify the genotype. 2 . The method for noninvasively identifying insect genotypes according to claim 1 , wherein, in step (a), the insect is an insect that has a molting process and can be independently labeled or reared. 3 . 3. The method for non-invasively identifying insect genotypes according to claim 1, wherein in step (a), gene editing refers to genome editing of insects using CRISPR/Cas9 genome-directed editing technology, TALEN or ZFN genome editing technology . 4. The method for non-invasively identifying insect genotypes according to claim 1, wherein in step (b), collecting the molts corresponding to the first instar stage of the insect or collecting the molts corresponding to the first and second instar stages of the insect to carry out the insect molting process. Early identification of genotypes. 5 . The method for non-invasively identifying insect genotypes according to claim 1 , wherein in step (c), the concentration of the extracted genomic DNA can be measured by using a micro-ultraviolet spectrophotometer. 6 .

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