CN107006432B - Method for rapidly and directionally obtaining low-age wingless myzus persicae - Google Patents
Method for rapidly and directionally obtaining low-age wingless myzus persicae Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 241000721621 Myzus persicae Species 0.000 title claims abstract description 13
- 241000180219 Sitobion avenae Species 0.000 claims abstract description 19
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 15
- 241001124076 Aphididae Species 0.000 claims description 53
- 241000721623 Myzus Species 0.000 claims description 33
- 241000272106 Ophiophagus Species 0.000 claims description 26
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 210000002257 embryonic structure Anatomy 0.000 claims description 4
- 230000002068 genetic effect Effects 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 241000254181 Sitophilus Species 0.000 abstract description 38
- 230000014509 gene expression Effects 0.000 abstract description 14
- 238000011160 research Methods 0.000 abstract description 5
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- 241000196324 Embryophyta Species 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
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- 241000819999 Nymphes Species 0.000 description 4
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- 238000012258 culturing Methods 0.000 description 4
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- APAPGJJZPJQKJJ-NTCAYCPXSA-N (6e)-2,6-dimethyl-10-methylidenedodeca-2,6-diene Chemical compound CCC(=C)CC\C=C(/C)CCC=C(C)C APAPGJJZPJQKJJ-NTCAYCPXSA-N 0.000 description 3
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- 230000037361 pathway Effects 0.000 description 2
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- 101150084750 1 gene Proteins 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 206010015146 Erysipeloid Diseases 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for quickly and directionally acquiring low-age wingless aphelenopsis unjus of sitobium avenae, which comprises the following steps of: s1: single-head raising of wingless avenae for expanding propagation for N generations; s2: and continuously feeding the (N + 1) th generation of myzus persicae, and collecting offspring of the myzus persicae to obtain the low-age wingless pseudo embryo and/or the myzus persicae. The method for directionally acquiring the low-age sitophilus avenae can directionally acquire the pseudo embryo which can be developed into the sitophilus avenae, the 1 st-age sitophilus avenae which can be developed into the sitophilus avenae, the 2 nd-age sitophilus avenae which can be developed into the sitophilus avenae, the 3 rd-age sitophilus avenae of the sitophilus avenae, the 4 th-age sitophilus avenae of the sitophilus avenae and the sitophilus avenae of the sitophilus avenae, provides a foundation for the gene expression research of the pseudo embryo period and the 1 and 2 th-age sitophilus avenae, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of insect biology, in particular to a method for quickly and directionally obtaining low-age wingless aphids of sitobion avenae.
Background
The myzus avenae has typical diffusion polymorphism, is regulated by external environmental factors, and can generate two different biological types in the development process of a single genetic type of the myzus avenae: finned and non-finned. Like other wing second-type insects, winged wheat aphids can carry out long-distance migration diffusion, wingless aphids for propagation and expansion can be formed after the winged wheat aphids reach a destination, large-scale propagation offspring is harmful, the development plasticity is an important survival strategy formed by the aphids in the long-term evolution process for adapting to the changing ecological environment, and if the generation of the winged aphids can be effectively prevented, the grain loss caused by the wheat aphids can be obviously reduced. Therefore, the molecular mechanism of aphid pterotype differentiation potential has been the focus of research and attention in recent years.
The wing-type differentiation of Aphis gramineus was found to have 2 sensitive stages: the pseudo-embryo period (pre-natal) and the 1 and 2 instars (post-natal) are easily influenced by external factors, so that the wing ratio in the population is adjusted. It has also been established by histological studies that in many aphid groups, embryos have wing primordia that degenerate during development to wingless aphids, while the wing primordia of winged aphids continue to develop, begin to thicken, and eventually develop into wings. The development of aphid wings may be a default pathway in vivo, while the differentiation of wing type is achieved by changing the default development pathway if aphid is stimulated by external stimuli before or after birth. Therefore, the research on the molecular mechanism of the Myzus erygii wing-shaped differentiation needs to detect the gene expression change in the wing-shaped differentiation sensitive period, and further deduces the key genes influencing the wing-shaped differentiation and the regulation network thereof. However, studies have found that, in 3 stages, the myzus erysipeloides myzus pseudo-embryo and the 1 st and 2 nd-year myzus persicae cannot be distinguished by the appearance, and that, starting from the 3 rd-year myzus, the breast of a winged individual begins to swell and develop winged buds, so that the winged or wingless aphids can be distinguished. Therefore, the molecular mechanisms for researching the wing-shaped differentiation of aphids at present are aimed at the gene expression analysis after the wing-shaped differentiation of the aphids, and the difference expression change of genes at the key stage of the wing-shaped differentiation cannot be compared.
Disclosure of Invention
The invention provides a method for directionally obtaining low-age sitophilus avenae, which comprises the following steps: s1: single-head raising of wingless avenae for expanding propagation for N generations; s2: and continuously feeding the (N + 1) th generation of myzus persicae, and collecting offspring of the myzus persicae to obtain the low-age wingless pseudo embryo and/or the myzus persicae.
In a further embodiment of the present invention, in S1, during the propagation of N generations, each generation is individually bred.
In a further embodiment of the present invention, the generation N in S1 is 3 to 5, preferably 3. The aphid breeding method has the advantages that continuous propagation is carried out for 3-5 generations, each generation is fed independently, and wingless individuals can be guaranteed to breed the aphids on the basis of providing sufficient food and good living environment; if the propagation generation number is less than 3, the genetic background generated by the initial aphid in the environment can not be completely eliminated, and the wingless ratio of the offspring can not be ensured; in addition, aphids are social organisms in the nature, and if the aphids are fed in an unlimited and independent propagation manner, certain influences on other biological parameters of the aphids can be caused.
In a further embodiment of the present invention, the continuous feeding is single-head feeding in S2.
In a further embodiment of the present invention, in S2: and (3) collecting the pseudo embryo in the (N + 1) th generation of the sitobion avenae to obtain the pseudo embryo capable of being developed into the wingless sitobion avenae.
In a further embodiment of the present invention, in S3: and collecting the myzus avenae Log which are bred within 24h in the initial production of the (N + 1) -th generation myzus avenae Log, namely the 1-year-old myzus avenae Log which can be developed into wingless myzus avenae Log.
In a further embodiment of the present invention, in S3: collecting the myzus avenae F.E.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.G.
It should be noted that the invention adopts a single-head culture feeding mode, and aims to eliminate the influence of the external environment of the aphid of the 1 st generation on the aphid, so that the aphid to be tested is in a single genetic background. In S2: collecting the Ophiophagus avenae of the No. N +1 generation Ophiophagus avenae which is bred within 24h of primary production, ecdysis twice, and developing into Ophiophagus avenae of 3-year age to obtain Ophiophagus avenae of 3-year age of wingless Ophiophagus avenae; or in S2: collecting the Ophiophagus avenae of the No. N +1 generation Ophiophagus avenae which is bred within 24h of primary production, ecdysis for three times, and developing into Aphiophagus avenae of 4-year age to obtain Aphiophagus avenae of 4-year age of wingless Ophiophagus avenae; or in S2: collecting the Ophiophagus avenae which is bred within 24h of the primary production of the (N + 1) -th generation Ophiophagus avenae, ecdysis for four times or more, and developing into adult aphids to obtain the wingless Ophiophagus avenae adult aphids.
The invention also protects the pseudo embryo which can be developed into the wingless avenae, the 1 st-year nymph which can be developed into the wingless avenae, the 2 st-year nymph which can be developed into the wingless avenae, the 3 st-year nymph of the wingless avenae, the 4 st-year nymph of the wingless avenae and the wingless avenae which are obtained by the method.
It should be noted that, the feeding method of the present invention is not only single-head feeding, but also refer to patent numbers: the method for feeding the sitobion avenae disclosed in ZL201410117638.3 specifically comprises the following steps: wheat seeds were spread evenly on the bottom of a 160mm glass petri dish. Soaking the seeds in clear water for 24 hours to promote the seeds to take roots and germinate; after 24h, pouring out the water in the culture dish, covering two layers of white gauze soaked by clear water on the seeds, and washing the germinated seeds for 1 time every other gauze every day; removing gauze when the plant grows to a height (from the stem base to the tip) of 30mm, and continuously washing the plant once a day for keeping the plant root moisture and preventing the seeds from mildewing; when the plant grows to about 60mm of plant height, pulling up the whole wheat seedling (carefully not damaging the root), taking 4-6 wheat seedlings, and putting the rootstalk parts of the wheat seedlings into a 2ml centrifuge tube; soaking absorbent cotton in clear water, preferably without dripping, wrapping the stem base with wet absorbent cotton, sealing the centrifugal tube, and keeping the leaves outside the centrifugal tube to obtain wheat seedling with centrifugal tube. Wrapping the stem base part with wet absorbent cotton, and sealing the centrifugal tube opening to moisturize the root and prevent wheat aphids from entering the centrifugal tube; taking a plastic or glass culture dish with the diameter of 120mm, paving qualitative filter paper with the same specification at the bottom, and spraying clear water on the qualitative filter paper for moisturizing; putting the wheat seedlings with the centrifugal tubes into a culture dish; inoculating the adult sitobion avenae on the leaves of the wheat by using a writing brush, wherein the feeding temperature of the sitobion avenae is 20 ℃, the illumination time is 16L to 8D, and the relative humidity is 65 +/-5%.
The technical scheme provided by the invention has the following beneficial effects: (1) the method for directionally obtaining the low-age sitophilus avenae can directionally obtain the pseudo embryo which can be developed into the sitophilus avenae, the 1 st sitophilus which can be developed into the sitophilus avenae, the 2 st sitophilus which can be developed into the sitophilus avenae, the 3 st sitophilus which can be developed into the sitophilus avenae, the 4 th st sitophilus which can be developed into the sitophilus avenae and the sitophilus avenae which can be developed into the sitophilus avenae, and provides a basis for the gene expression research of the low-age situs avenae in the period of pseudo embryo (pre-natal period) and the 1 and 2 th-age (post-natal period); (2) the method provided by the invention is convenient to operate, simple and feasible, and has wide application prospect.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a comparison chart of expression quantity detection of gene miR-a in different wing-shaped different development periods of sitobion avenae in the embodiment of the invention;
FIG. 2 is a comparison chart of expression quantity detection of gene miR-b in different development stages of different wing types of Aphis gramineus in the embodiment of the invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
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. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.
Examples
S1: selecting 10 wingless sitobion avenae to become aphids, culturing in a culture dish independently for three generations, selecting 3 healthy aphids in each generation, and continuously culturing independently;
s2: respectively continuing single-head breeding of fourth-generation aphids, and respectively collecting offspring:
(1) collecting the pseudo embryo in the fourth generation of the sitobion avenae, namely the pseudo embryo which can be developed into the wingless sitobion avenae;
(2) collecting the myzus avenae Log which is primarily produced within 24h by the fourth generation myzus avenae Log, namely the myzus avenae Log of 1 year which can be developed into wingless myzus avenae Log;
(3) collecting the myzus avenae f.avenae which are bred within 24h in the initial production of fourth generation myzus avenae, and ecdysis once to obtain the myzus avenae of 2 years which can be developed into wingless myzus avenae;
(4) collecting the myzus avenae f.avenae which are bred within 24h in the initial production of fourth generation myzus avenae, ecdysis twice, and developing into myzus avenae f.avenae of 3-year age to obtain myzus avenae of 3-year age of wingless myzus avenae;
(5) collecting the myzus avenae f.mairei which is bred within 24h in the initial production of fourth generation myzus avenae, ecdysis for three times, and developing into 4-year myzus avenae f.mairei to obtain 4-year myzus avenae of wingless myzus avenae;
(6) collecting the green aphids of the fourth generation of the intraspecific 24h, ecdysis for four times or more, and developing into adult aphids to obtain wingless adult aphids of the intraspecific 24 h.
And (3) effect measurement:
1. fin and no-fin adult aphid statistics
The experiment of the embodiment is repeated for 5 batches (namely, the experiment is carried out in 5 months, one batch is carried out in each month), 10 groups are selected for each type of each batch for effect measurement, the fourth generation of the tubular aphid is selected to produce the tubular aphid which is bred within 24h, the developed tubular aphid becomes the aphid, the winged rate and the wingless rate of the formed aphid are identified to represent the effect of the invention, the average value is taken as the result, the actual ratio of the obtained wingless aphids is 100 percent, and the requirements of the construction of a genomics library and the molecular biology identification are completely met.
2. The difference of gene expression levels of two wing types at different ages
In order to further verify the effect, the applicant randomly selects a plurality of genes, verifies the expression quantity change of the genes in different development periods of different wing types of the sitophila avenae through the qRT-PCR technology, can obviously see the difference of the expression quantities of the genes in different development periods of the two wing types, and confirms that the wingless gene obtained by the method provided by the invention can meet the requirement of molecular biological identification. To characterize the reproducibility of the present invention, the finned and finless experiments were repeated for 5 batches (i.e., 5 months apart, one batch per month of the experiment), and 10 groups were selected for each batch for each age and the results were averaged as follows.
A wing-free type: respectively collecting pseudo embryos capable of developing into wingless sitobion avenae, 1-year-old nymphs capable of developing into wingless sitobion avenae, 2-year-old nymphs capable of developing into wingless sitobion avenae, 3-year-old nymphs of wingless sitobion avenae, 4-year-old nymphs of wingless sitobion avenae and wingless sitobion avenae, and respectively selecting 10 groups for determination in each age.
The winged aphid is directionally obtained by the method for obtaining the winged avenae, including the steps of S1, selecting 10 wingless avenae to become aphids, independently culturing the aphids in a culture dish for three generations, respectively selecting 3 healthy avenae in each generation, continuously and independently culturing the aphids, S2, respectively feeding the fourth generation of the avenae to 3 instars, respectively placing 10 of the avenae in a sealed culture dish with the diameter of 120mm, S3, respectively adding 2 mu L of E- β -farnesene with the concentration of 500 ng/mu L into the sealed culture dish, using a solvent of an E- β -farnesene solution as n-hexane, then adding the solvent once every 2h, adding 5 times to obtain a cycle, repeating the S301 step for 4 cycles (namely, treating for 5 days altogether), wherein the time point for adding the E- β -farnesene solution for each cycle is the same as the time point for adding the E- β -first-farnesene solution for the first cycle, the first time point for removing the generation of the avenae during which the first generation period, adding the avenae for obtaining the avenae after the avenae, adding the avenae to obtain the first generation period, and collecting the avenae after the first generation period, the avenae are respectively adding more than 2-24-9-3-24-9-5-2-5-3-th-generation period, and collecting the aphids, respectively, and obtaining the gloea-24-9-th-after the aphids in the avenae, and collecting the aphids in the breeding period, wherein the avenae are the avenae, the avenae are the aphids in the first-3-9-3-9-3-9-th-3-9-th-9-th-9-th.
Gene detection: aiming at winged and wingless aphids, 6 aphids of different ages are respectively selected for determination. Randomly selecting 2 genes miR-a and miR-b, respectively measuring relative expression amounts of the genes through a qRT-PCR technology, setting three biological parallel samples and three technical repeated samples for each gene of each group, respectively averaging results obtained from 10 groups of 5 batches, and specifically obtaining the results shown in table 1, figure 1 and figure 2.
TABLE 1 Gene expression amount measurement
The technical scheme provided by the invention has the following beneficial effects: (1) the method for directionally obtaining the low-age sitophilus avenae can directionally obtain the pseudo embryo which can be developed into the sitophilus avenae, the 1 st sitophilus which can be developed into the sitophilus avenae, the 2 st sitophilus which can be developed into the sitophilus avenae, the 3 st sitophilus which can be developed into the sitophilus avenae, the 4 th st sitophilus which can be developed into the sitophilus avenae and the sitophilus avenae which can be developed into the sitophilus avenae, and provides a basis for the gene expression research of the low-age situs avenae in the period of pseudo embryo (pre-natal period) and the 1 and 2 th-age (post-natal period); (2) the method provided by the invention is convenient to operate, simple and feasible, and has wide application prospect.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains. Unless specifically stated otherwise, the relative steps, numerical expressions, and values of the components and steps set forth in these embodiments do not limit the scope of the present invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention, and all of the technical solutions are covered in the protective scope of the present invention.
Claims (1)
1. A method for directionally obtaining low-age wingless pseudo embryos and/or myzus persicae is characterized by comprising the following steps:
s1: single-head raising of wingless avenae for expanding propagation for N generations; the number of the N generations is 3-5, in the process of expanding propagation of the N generations, each generation is fed in a single-head mode, the aphid is guaranteed to reproduce wingless individuals, and a feeding mode of single-head culture is adopted, so that the influence of the external environment where the aphid of the 1 st generation is located on the aphid to be tested is eliminated, and the aphid to be tested is under a single genetic background;
s2: continuously feeding the (N + 1) th generation of myzus persicae, and collecting offspring of the myzus persicae to obtain low-age wingless pseudo embryos and/or myzus persicae, wherein the continuous feeding is single-head feeding;
in said S2: collecting the pseudo embryo in the (N + 1) th generation of the sitobion avenae, namely the pseudo embryo which can be developed into the wingless sitobion avenae;
in said S2: collecting the Ophiophagus avenae reproduced within 24h of the initial production of the (N + 1) -th generation of the Ophiophagus avenae, namely the Ophiophagus avenae of 1 year which can be developed into the wingless Ophiophagus avenae; or in said S2: collecting the myzus avenae f.avenae which are bred within 24h of the initial production of the (N + 1) -th generation myzus avenae, and ecdysis once to obtain the myzus avenae of 2 years which can be developed into wingless myzus avenae; or
In S2: collecting the Ophiophagus avenae of the No. N +1 generation Ophiophagus avenae which is bred within 24h of primary production, ecdysis twice, and developing into Ophiophagus avenae of 3-year age to obtain Ophiophagus avenae of 3-year age of wingless Ophiophagus avenae; or in S2: collecting the Ophiophagus avenae of the No. N +1 generation Ophiophagus avenae which is bred within 24h of primary production, ecdysis for three times, and developing into Aphiophagus avenae of 4-year age to obtain Aphiophagus avenae of 4-year age of wingless Ophiophagus avenae; or in S2: collecting the Ophiophagus avenae which is bred within 24h of the primary production of the (N + 1) -th generation Ophiophagus avenae, ecdysis for four times or more, and developing into adult aphids to obtain the wingless Ophiophagus avenae adult aphids.
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