CN106857415B - Method for rapidly and directionally obtaining low-age winged nymphs of sitobion avenae - Google Patents
Method for rapidly and directionally obtaining low-age winged nymphs of sitobion avenae Download PDFInfo
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Abstract
The invention relates to a method for quickly and directionally obtaining low-age winged nymphs of sitobis avenae, which comprises the following steps of S1, feeding single-head wingless sitobis avenae for three generations, S2, feeding single-head fourth-generation nymobis avenae to 3 instars, then placing a plurality of the third-generation nymobis into the same culture device, S3, adding E- β -farnesene solution into the culture device for multiple times, collecting low-age winged false embryos and/or nymobis of the sitobis avenae after the E- β -farnesene solution is added, and directionally obtaining the low-age winged nymobis avenae by the method for directionally obtaining the low-age winged nymobis of the sitobis avenae, wherein the method for directionally obtaining the low-age winged nymobis avenae, the 1-age nymobis avenae, the 3-age nymobis avenae, the 4-age winged nymobis avenae and the 1-aged nymobis avenae can be directionally obtained, and the gene of the 3-aged nymobis avenae can be conveniently researched and has a 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 winged 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 winged nymphs of sitobium avenae, which comprises the following steps of S1, feeding single-head wingless sitobium avenae for three generations, S2, feeding single-head fourth generation nymphs to 3 instars, then placing the multiple numbers of the fourth generation nymphs in the same culture device, S3, collecting pseudowinged embryos and/or nymphs of the low-age sitobium avenae after adding E- β -farnesene solution, wherein all the nymphs produced by the adult myzus need to be removed before adding the E- β -farnesene solution.
In a further embodiment of the present invention, in S1, in the process of expanding propagation for three generations, each generation is individually raised.
In a further embodiment of the present invention, the number of S2 is 10 to 15, preferably 10.
In a further embodiment of the invention, S3 specifically comprises S301, adding E- β -farnesene solution into a culture device for the first time, adding the E- β -farnesene solution once every 2 hours, and adding the E- β -farnesene solution for 5 times to form a period, S302, repeating the step S301 for at least 4 periods, preferably 4 periods, wherein the time point of adding the E- β -farnesene solution for the first time in each period is the same as the time point of adding the E- β -farnesene solution for the first time in the first period, S303, collecting a low-age winged false embryo and/or aphids of Myzus avenae after S302 is completed, wherein the E- β -farnesene solution has strong volatility, the E- β -farnesene solution is added into the closed culture device and can be rapidly volatilized into a gas atmosphere in which the aphids is located, the low-age winged embryos and/or aphids cannot be subjected to a high-dose response alarm, and the aphids cannot be subjected to a high-dose of aphid response, and the aphids cannot be reduced in a long-life-winged offspring, and the aphids cannot be subjected to a high-life-induced by a high-increasing rate.
In a further embodiment of the invention, the concentration of the E- β -farnesene solution is 500-1000 ng/. mu.L, and the volume of each addition of the E- β -farnesene solution is 2-3. mu.L.
In a further embodiment of the present invention, the solvent for the E- β -farnesene solution is n-hexane.
In a further embodiment of the invention, the culture device is a closed petri dish with a diameter of 120 mm.
In a further embodiment of the invention, in S3, after E- β -farnesene is added for 5 cycles, a pseudo embryo in the body of the sitobion avenae is collected, namely the pseudo embryo which can be developed into the winged sitobion avenae.
In a further embodiment of the invention, in S3, after adding E- β -farnesene for 5 weeks, collecting the Ophiophagus avenae reproduced within 24h after the first production of Ophiophagus avenae, namely 1-year-old Ophiophagus avenae which can develop into winged Ophiophagus avenae.
In a further embodiment of the invention, in S3, after E- β -farnesene is added for 5 weeks, the Ophiophagus avenae reproduced within 24h after the first production of the Ophiophagus avenae is collected and ecdysis is carried out once to obtain 2-year-old Ophiophagus avenae capable of developing into winged Ophiophagus avenae.
It is to be noted that, in S3, the tubular aphids bred within 24h are collected after E- β -farnesene is added for 5 weeks, and ecdysis is performed twice to develop the 3-year-old tubular aphids, so that the 3-year-old tubular aphids with the tubular aphids are obtained, or in S3, the tubular aphids bred within 24h are collected after E- β -farnesene is added for 5 weeks, and ecdysis is performed for three times to develop the 4-year-old tubular aphids, so that the 4-year-old tubular aphids with the tubular aphids are obtained, or in S3, the tubular aphids bred within 24h are collected after E- β -farnesene is added for 5 weeks, so that the four-time ecdysis is performed, so that the developed tubular aphids with the wings are obtained.
The invention also protects the pseudo embryo which can be developed into the winged sitobion avenae, the 1 st-year nymphae which can be developed into the winged sitobion avenae, the 2 st-year nymphae which can be developed into the winged sitobion avenae, the 3 st-year nymphae which can be developed into the winged sitobion avenae, the 4 th-year nymphae which can be developed into the winged sitobion avenae and the winged sitobion 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-age sitophilus avenae which can be developed into the sitophilus avenae, the 2 st-age sitophilus avenae which can be developed into the sitophilus avenae, the 3 st-age sitophilus avenae of the sitophilus avenae, the 4 st-age sitophilus avenae and the sitophilus avenae to form the adult aphid, and provides a basis for the gene expression research of the low-age sitophilus avenae in the period of pseudo embryo (pre-natal) and the 1 and 2 th-age sitz (post-natal); (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 feeding fourth generation aphids to 3 instars in a single head manner, and then respectively placing 10 heads of aphids in a sealed culture dish with the same diameter of 120 mm;
s3, respectively adding 2 mu L of E- β -farnesene with the concentration of 500 ng/mu L into a sealed culture dish, wherein the solvent of the E- β -farnesene solution is n-hexane, then adding the n-hexane once every 2h, and adding the n-hexane 5 times to form a cycle, repeating S301 for 4 cycles (namely, 5 days of co-processing), wherein the time point of adding the E- β -farnesene solution for the first time in each cycle is the same as the time point of adding the E- β -farnesene solution for the first time in the first cycle, and removing all the aphids produced by adult aphids in the period;
and (3) after 5 days of adding the E- β -farnesene solution, respectively collecting the low-age winged nymphs of the sitobion avenae:
(1) after adding E- β -farnesene for 5 cycles, collecting a pseudo embryo in the body of the sitobion avenae, namely the pseudo embryo which can be developed into the sitobion avenae with wings;
(2) after adding E- β -farnesene for 5 weeks, collecting the Ophiophagus avenae reproduced within 24h of the primary production of the Ophiophagus avenae, namely the 1-year-old Ophiophagus avenae which can be developed into the winged Ophiophagus avenae;
(3) after adding E- β -farnesene for 5 weeks, collecting the Ophiophagus avenae reproduced within 24h of the primary production of the Ophiophagus avenae, and ecdysis for one time to obtain 2-year-old Ophiophagus avenae capable of developing into Ohiophagus avenae with wings;
(4) after adding E- β -farnesene for 5 weeks, collecting the Ophiophagus avenae reproduced within 24h of the primary production of the Ophiophagus avenae, molting twice, and developing into Ophiophagus avenae of 3-year age to obtain Ophiophagus avenae of 3-year age with Alterophagus avenae;
(5) after adding E- β -farnesene for 5 weeks, collecting the Ophiophagus avenae reproduced within 24h of the primary production of the Ophiophagus avenae, ecdysis for three times, and developing into 4-year Ophiophagus avenae to obtain 4-year Ophiophagus avenae with winged Ophiophagus;
(6) and after adding E- β -farnesene for 5 weeks, collecting Myzus avenae Daphne which is produced within 24h by the first time of Myzus avenae, ecdysis for four times or more, and developing into adult aphids to obtain winged Myzus avenae adult aphids.
The control group was prepared by replacing E- β -farnesene with an equal amount of water, and the same procedure was repeated as in example one.
And (3) effect measurement:
1. fin and no-fin adult aphid statistics
The experiments of the examples and the control group are repeated for 5 batches (namely, the experiments are carried out in 5 months, one batch is carried out in each month), 10 groups are selected for each class of each batch for effect measurement, after E- β -farnesene is added for 5 weeks, the myzus avenae developed by collecting myzus avenae which are originally produced and are bred within 24 hours, the myzus avenae become myzus, the effect of the invention is characterized by identifying the winged rate and the wingless rate of the myzus avenae, the result is averaged, the actual ratio of the winged myzus is 98.17% +/-0.54%, and specific data are shown in the following table 1.
TABLE 1 Final and Afterfin aphids statistics
Although the ratio of the winged aphids obtained by the method for obtaining the winged aphids is less than 100%, the winged ratio of each group is more than 95%, and the requirements of the construction of a genomic library and the identification of molecular biology 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 winged genes 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 finned type: respectively collecting pseudo embryos capable of developing into winged sitobion avenae, 1-year-old nymphs capable of developing into winged sitobion avenae, 2-year-old nymphs capable of developing into winged sitobion avenae, 3-year-old nymphs capable of developing into winged sitobion avenae, 4-year-old nymphs capable of developing into winged sitobion avenae and adult sitobion avenae, and respectively selecting 10 groups for determination in each age.
A wing-free type: the method for directionally obtaining the wingless sitobion avenae comprises the following steps: s1: selecting 5 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: and respectively feeding the fourth generation of Onychidae to adult aphids in a single-head manner, and collecting the Onychidae or adult aphids of the Aureobasidae which are bred within 24 hours of the primary production of the Aureobasidium avenae.
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.
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 2, figure 1 and figure 2.
TABLE 2 determination of Gene expression amount
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-age sitophilus avenae which can be developed into the sitophilus avenae, the 2 st-age sitophilus avenae which can be developed into the sitophilus avenae, the 3 st-age sitophilus avenae of the sitophilus avenae, the 4 st-age sitophilus avenae and the sitophilus avenae to form the adult aphid, and provides a basis for the gene expression research of the low-age sitophilus avenae in the period of pseudo embryo (pre-natal) and the 1 and 2 th-age sitz (post-natal); (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 the present invention should be covered by the protection of the present invention.
Claims (1)
1. A method for directionally obtaining a low-age winged pseudoembryo and/or myzus persicae is characterized by comprising the following steps:
s1: the wingless avenae is bred into single aphid, and the three generations of aphids are propagated; in the process of propagating three generations, feeding each generation with a single head;
s2: feeding the fourth generation of myzus persicae to the 3 rd instar in a single way, and then placing 10 myzus persicae in the same culture device;
s3, adding E- β -farnesene solution into the culture device for multiple times, and collecting low-age winged pseudoembryos and/or myzus persicae after the E- β -farnesene solution is added;
the method specifically comprises the following steps:
s301, adding E- β -farnesene solution into the culture device for the first time, then adding the E- β -farnesene solution once every 2 hours, and adding the E- β -farnesene solution for 5 times to form a period, wherein the culture device is a closed culture device which is a closed culture dish with the diameter of 120 mm;
s302, repeating the step S301 for 4 periods, wherein the time point of adding the E- β -farnesene solution for the first time in each period is the same as the time point of adding the E- β -farnesene solution for the first time in the first period, all aphids produced by adult aphids are removed in the period, the concentration of the E- β -farnesene solution is 500-1000 ng/mu L, the volume of the E- β -farnesene solution added for each time is 2-3 mu L, and the solvent of the E- β -farnesene solution is n-hexane;
s303: after S302 is finished, collecting the low-age winged pseudoembryos and/or myzus persicae;
and after the E- β -farnesene solution is added, collecting the myzus avenae myzus erysipelas which is bred within 24h and is originally produced by the myzus avenae myzus erysipelas, and ecdysis once to obtain the myzus avenae myzus erysipelas of 2 years which can be developed into the myzus erysipelas.
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