Mscht9 gene of sorghum aphid, application of Mscht9 gene and method for preventing and treating sorghum aphid

Abstract

The invention belongs to the technical field of insect prevention and control, and particularly discloses an Mscht9 gene of sorghum aphid, application of the Mscht9 gene and a method for preventing and controlling the sorghum aphid. According to the present invention, the life activity of the sorghum aphid is inhibited through the leaf uptake nucleotide sequence such as the dsRNA sequence represented by SEQ ID NO.1, such that the sorghum aphid prevention and control effect is achieved, and the advantages of efficient gene specific interference, environmental protection and the like are provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of insect control, and particularly relates to an Mscht9 gene of Melanaphis sacchari, its application, and a method for controlling Melanaphis sacchari. Background Art

[0002] Sorghum (Sorghum bicolor) originated in Africa and is an important cash crop used for both forage and grain, and is an important source of nutrients and bioactive compounds in human diet. Sorghum has many excellent traits such as high photosynthetic efficiency, drought and waterlogging tolerance, and salt tolerance, so it is called the "camel among crops". It can also be used as an industrial raw material and can be processed into beverages and beer. In the Americas, sorghum is mainly grown as livestock feed.

[0003] Melanaphis sacchari is the main pest on sorghum plants and also the main virus vector in sorghum. Since its outbreak in 2013, Melanaphis sacchari has changed from a minor pest of sorghum to a major pest, posing a great threat to sorghum cultivation around the world and causing serious economic losses. Melanaphis sacchari sucks the sap of sorghum leaves, causing the leaves to lose water and wilt. Long-term infestation by aphids will turn the sorghum leaves black, reduce photosynthetic efficiency, cause the mature leaves to turn yellow and necrotic, delay the development of the plant or even cause death, reduce the number of spikes, and decrease the 1000-grain weight. At the same time, the honeydew secreted by Melanaphis sacchari can cause sooty mold on sorghum, seriously affecting the yield and quality of sorghum. For a long time, chemical control has been the main method for controlling Melanaphis sacchari. The excessive use of chemical pesticides has led to obvious resistance of aphids and polluted the environment.

[0004] Chitin is an important component of the insect cuticle and peritrophic membrane, and is crucial for the normal growth and development of insects. The rhythmic molting of insects, the periodic metabolism of the trachea, and the degradation of the peritrophic membrane in the intestine are all closely related to chitin. Chitin in the midgut peritrophic membrane can promote food digestion and protect it from mechanical damage, and can also prevent dehydration and infection by pathogenic microorganisms. In order to maintain its normal growth and development, insects need to periodically synthesize and degrade chitin to complete molting, and the chitin content in the insect body varies at different growth and development stages. The chitin metabolism in insects maintains a dynamic balance in the body, and an increase or decrease in the chitin content at a certain period will cause disorders in the chitin metabolism of the insect body.

[0005] RNA interference (RNAi) refers to gene silencing mediated by double-stranded RNA (dsRNA). Compared with many traditional methods, RNAi has characteristics and advantages such as high efficiency, high specificity, co-suppression, and species timeliness, and has developed into a frontier direction in the research of biological gene functions. The RNAi technology is called the fourth-generation new pesticide and has been widely used in the research and development of green pest control technologies. At present, a large number of studies synthesize dsRNA in vitro and make larvae ingest it by feeding or injection to interfere with the expression of corresponding chitinase genes to observe its effects on different developmental stages of insects. Existing experiments have proved that synthesizing dsRNA of chitinase in vitro by RNAi technology and delivering it into insects can cause their growth and development to be retarded and pupation to be difficult, providing new ideas for pest control and having important research value. However, at present, there are few application studies on its control of the greenbug. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above problems existing in the prior art, and provide an Mscht9 gene of the greenbug, its application, and a method for controlling the greenbug, which have the advantages of efficient gene-specific interference and environmental friendliness.

[0007] To achieve the above technical purposes and reach the above technical effects, the present invention is realized through the following technical solutions:

[0008] In the first aspect, the present invention provides an Mscht9 gene of the greenbug, and the nucleotide sequence of the Mscht9 gene is as shown in SEQ ID NO.1.

[0009] In the second aspect, the present invention provides the application of a dsRNA sequence with the nucleotide sequence as shown in SEQ ID NO.1 in controlling the greenbug.

[0010] In the third aspect, the present invention provides a method for controlling the greenbug, which includes the following steps: introducing a dsRNA sequence with the nucleotide sequence as shown in SEQ ID NO.1 into the body of the greenbug.

[0011] Further, during the process of introducing a dsRNA sequence with the nucleotide sequence as shown in SEQ ID NO.1 into the body of the greenbug, the introduction method is feeding or injection.

[0012] Further, the introduction method is feeding.

[0013] Further, the feeding is to feed the greenbug by intaking the dsRNA sequence with the nucleotide sequence as shown in SEQ ID NO.1 through the leaves.

[0014] Further, the injection is to inject the dsRNA sequence shown in SEQ ID NO.1, a nucleotide sequence, into the body of the Chinese sorghum aphid using a syringe.

[0015] Further, the Chinese sorghum aphid is a Chinese sorghum aphid larva.

[0016] Further, during the process of introducing the dsRNA sequence shown in SEQ ID NO.1, a nucleotide sequence, into the body of the Chinese sorghum aphid, the concentration of the dsRNA sequence shown in SEQ ID NO.1, a nucleotide sequence, is 100 - 2000 ng / μL.

[0017] Even further, during the process of introducing the dsRNA sequence shown in SEQ ID NO.1, a nucleotide sequence, into the body of the Chinese sorghum aphid, the concentration of the dsRNA sequence shown in SEQ ID NO.1, a nucleotide sequence, is 1000 ng / μL.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The present invention provides an Mscht9 gene of the Chinese sorghum aphid, its application, and a method for controlling the Chinese sorghum aphid, which have the advantages of high - efficiency gene - specific interference and environmental friendliness.

[0020] 2. The method for controlling the Chinese sorghum aphid provided by the present invention can be combined with biological or chemical control methods to improve the comprehensive control effect.

[0021] 3. In the present invention, by absorbing the dsRNA sequence shown in SEQ ID NO.1, a nucleotide sequence, through the leaves, the life activities of the Chinese sorghum aphid are inhibited, thereby achieving the effect of controlling the Chinese sorghum aphid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation to the present invention. In the drawings:

[0023] Figure 1 is a morphological photograph of the Chinese sorghum aphids in the ds Mscht9 treatment group provided in Example 3 of the present invention;

[0024] Figure 2 is a morphological photograph of the Chinese sorghum aphids in the dsGFP control group provided in Example 3 of the present invention;

[0025] Figure 3 is a molting photograph of the Chinese sorghum aphids in the ds Mscht9 treatment group provided in Example 4 of the present invention;

[0026] Figure 4It is a molting photo of the greenbug in the dsGFP control group provided in Example 4 of the present invention;

[0027] Figure 5 It is the expression level of the chitinase gene in the greenbug after gene interference with ds Mscht9 provided in Example 5 of the present invention. Detailed implementation manners

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0029] Chitin is an important component of the insect cuticle and peritrophic membrane, and is crucial for the normal growth and development of insects. The rhythmic molting of insects, the cyclic metabolism of the trachea, and the degradation of the intestinal peritrophic membrane are all closely related to chitin.

[0030] RNA interference (RNAi) refers to gene silencing mediated by double-stranded RNA (dsRNA). Compared with many traditional methods, RNAi has characteristics and advantages such as high efficiency, high specificity, co-suppression, and species timeliness, and has developed into a frontier direction in the research of biological gene functions. The RNAi technology is called the fourth-generation new pesticide and has been widely used in the research and development of pest green control technologies. At present, a large number of studies have synthesized dsRNA in vitro and fed or injected larvae to ingest it to interfere with the expression of corresponding chitinase genes to observe its effects on different developmental stages of insects. Existing experiments have proved that by synthesizing dsRNA of chitinase in vitro through the RNAi technology and delivering it into the insect body, it causes slow growth and development and difficulty in pupation, providing new ideas for pest control and having important research value.

[0031] Since chitin plays an important role in the growth, development, and molting process of insects, chitinase is a key gene in the insect chitin synthesis pathway, which can affect the chitin structure of insects, digest the chitin in the insect nutrient matrix and exoskeleton, and has obvious effects in pest control. RNAi has become a promising and effective method for pest control. By expressing high levels of double-stranded RNA (dsRNA) targeting key genes of the greenbug, it has been proven to effectively control the growth, development, reproduction, etc. of insects.

[0032] Example 1

[0033] This embodiment provides a method for preparing dsRNA of the Mscht9 gene. Specifically, the preparation method includes the following steps:

[0034] S1. Design and preparation of target gene sequence fragments

[0035] Determine the target gene: First, determine the key target gene in the greenbug, that is, the ds Mscht9 gene, and select a unique region (usually 200 - 500bp) to ensure that the region has strong specificity and no high similarity with non-target genes; in this embodiment, the nucleotide sequence of the Mscht9 gene is shown in SEQ ID NO: 1, specifically as follows:

[0036] Nucleotide sequence of the Mscht9 gene (SEQ ID NO: 1):

[0037]

[0038] Primer design: Design primers for the target gene using the Primer 3.0 software tool, and add the T7 promoter sequence to the 5' ends of the forward and reverse primers;

[0039] The designed primer sequences are shown in Table 1:

[0040] Table 1

[0041]

[0042] S2. PCR amplification of the target sequence

[0043] PCR reaction setup: Use primers with the T7 sequence for PCR amplification to obtain the target gene fragment;

[0044] Fragment verification: Use agarose gel electrophoresis to confirm the size of the amplified product to ensure the correct fragment;

[0045] S3. In vitro transcription to synthesize dsRNA

[0046] Transcription reaction setup: Use a T7 RNA polymerase in vitro transcription kit to transcribe dsRNA from the PCR product. Since the primers contain the T7 sequence, both strands will be transcribed simultaneously to form complementary RNA strands;

[0047] Reaction components: PCR template (target gene fragment) containing the T7 sequence, nucleoside triphosphates (NTPs), T7 RNA polymerase, reaction buffer;

[0048] Incubation: Incubate at 37 °C for 4 - 6 hours to synthesize dsRNA;

[0049] S4. dsRNA purification

[0050] DNase treatment: After the in vitro transcription reaction, add DNase to degrade the DNA template and remove residual DNA;

[0051] dsRNA purification: Use phenol-chloroform extraction or an RNA purification kit to purify dsRNA and ensure the removal of impurities;

[0052] Concentration and quality detection: Use a spectrophotometer to measure the concentration of dsRNA and check its integrity on a denaturing agarose gel.

[0053] Example 2

[0054] This example provides a method for controlling Melanaphis sacchari, which is to introduce the dsRNA sequence prepared in Example 1 into Melanaphis sacchari. In this example, the introduction method is feeding, and more specifically, the introduction method is the leaf absorption method. The steps of the leaf absorption method are as follows:

[0055] S1. Preparation of Melanaphis sacchari

[0056] The aphids were collected from the common sorghum variety "Tiegan Kangsi", and transferred to fresh sorghum plants every two weeks to maintain their population. The Melanaphis sacchari were cultured in an artificial climate chamber with a photoperiod of 16L:8D, a temperature of 28 °C, and a relative humidity of 50 - 60%.

[0057] Keep regular watering and fertilization;

[0058] S2. dsRNA Concentration and Dosage

[0059] The dsRNA synthesized by T7 had a concentration of 1000 ng / μL and was fed to the Melanaphis sacchari by the leaf systemic absorption method;

[0060] S3. Sample Collection

[0061] Take fresh sorghum leaves, wrap the stems with wet absorbent cotton and place them in a petri dish. Inoculate healthy Melanaphis sacchari mothers on the sorghum leaves, and remove the mothers after they give birth to aphids;

[0062] S4. Process of Leaf Systemic Absorption Method

[0063] To clarify the effect of ds Mscht on the Melanaphis sacchari, RNA interference in this example started at the 2nd instar of the Melanaphis sacchari; Insert the small sorghum seedlings into a 1.5 ml centrifuge tube containing 1.0 ml of dsRNA, inoculate 2nd instar nymphs on the leaves, then seal the centrifuge tube with a sealing film and transfer it to a petri dish for the experiment;

[0064] S5. Observe the development and abnormal phenomena of the Melanaphis sacchari after leaf systemic absorption of dsRNA.

[0065] Example 3

[0066] In this example, the effect of leaf systemic absorption of dsRNA on the morphological changes of the Melanaphis sacchari was provided, as follows:

[0067] Observe the development of the Melanaphis sacchari after leaf systemic absorption of dsRNA in Example 2, and label it as the ds Mscht9 treatment group;

[0068] Take the development of the Melanaphis sacchari after leaf systemic absorption of double-stranded dsRNA of the green fluorescent protein gene (GFP) as a control (for the Melanaphis sacchari after leaf systemic absorption of double-stranded dsRNA of the green fluorescent protein gene (GFP), the method was carried out with reference to Example 1 and Example 2), and label it as the dsGFP control group;

[0069] The specific experimental results are as Figures 1 to 2 shown;

[0070] Figure 2 Morphological photos of the greenbug aphids after ingestion of double-stranded dsRNA of the green fluorescent protein gene (GFP) through the leaves, i.e., the greenbug aphids in the dsGFP control group. The body color and body shape of the larvae are normal, indicating that the action of dsGFP has no obvious effect on the normal physiological activities of the greenbug aphids;

[0071] Figure 1 Morphological photos of the greenbug aphids after ingestion of dsRNA through the leaves in Example 2, i.e., the greenbug aphids in the ds Mscht9 treatment group. The body shape and body color of the larvae are relatively abnormal compared to Figure 1 the greenbug aphids in the dsGFP control group;

[0072] Via Figure 1 and Figure 2 It is shown that, compared to the ds Mscht9 treatment group, the larvae in the dsGFP control group appear healthier and have no obvious body abnormalities from the appearance; these morphological differences indicate that ds Mscht9 effectively interferes with the gene expression of the greenbug aphids, thereby leading to morphological abnormalities.

[0073] Example 4

[0074] In this example, the effect of ingestion of dsRNA through the leaves on the molting status of the greenbug aphids is provided, as follows: Observe the molting status of the greenbug aphids after ingestion of dsRNA through the leaves for 24 h in Example 2, and label it as the ds Mscht9 treatment group;

[0075] Take the molting status of the greenbug aphids after ingestion of double-stranded dsRNA of the green fluorescent protein gene (GFP) through the leaves for 24 h as a control (the method for the greenbug aphids after ingestion of double-stranded dsRNA of the green fluorescent protein gene (GFP) through the leaves refers to Examples 1 and 2), and label it as the dsGFP control group;

[0076] The specific experimental results are as Figures 3 to 4 shown:

[0077] Figure 3 Molting photos of the greenbug aphids in the ds Mscht9 treatment group; Figure 4 Molting photos of the greenbug aphids in the dsGFP control group. Through Figure 3 and Figure 4 it can be seen that Figure 4 the larvae of the greenbug aphids in Figure 3 can molt normally, while the larvae of the greenbug aphids in

[0078] Example 5

[0079] In this example, after feeding dsRNA in Example 2, the expression analysis of the chitinase gene of the greenbug was provided, with the expressions of water and dsGFP as controls. The specific experimental results are as Figure 5 shown;

[0080] Figure 5 It shows that gene interference was performed on ds Mscht9, with the expressions of water and dsGFP as controls. It was found that at the 24-hour time point, the expression levels of the target genes were all significantly decreased, lower than those of the control group. This indicates that the treatment with ds Mscht9 successfully inhibited the expression of the target genes.

[0081] The Mscht9 gene of the greenbug provided by the present invention, its application and the method for controlling the greenbug have the advantages of highly efficient gene-specific interference, being environmentally friendly, reducing the use of chemical pesticides, and achieving the purpose of green insect control.

[0082] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0083] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. The Mscht9 gene of the sugarcane aphid, characterized in that, The nucleotide sequence of the Mscht9 gene is shown in SEQ ID NO.

1.

2. Application of the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 in controlling Melanaphis sacchari.

3. A method for controlling the sugarcane aphid, characterized in that, This method includes the following steps: introducing the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 into Melanaphis sacchari.

4. A method for controlling Melanaphis sacchari according to claim 3, characterized in that, During the process of introducing the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 into Melanaphis sacchari, the introduction method is feeding or injection.

5. A method for controlling Melanaphis sacchari according to claim 4, characterized in that, The introduction method is feeding.

6. The method for controlling Melanaphis sacchari according to claim 4, wherein The feeding is to feed Melanaphis sacchari by the phloem absorption of the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 in the leaves.

7. A method for controlling the sugarcane aphid according to claim 4, characterized in that, The injection is to inject the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 into Melanaphis sacchari using a syringe.

8. A method for controlling Melanaphis sacchari according to claim 3, characterized in that, The Melanaphis sacchari is the larva of Melanaphis sacchari.

9. A method for controlling the sugarcane aphid according to claim 3, characterized in that, During the process of introducing the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 into Melanaphis sacchari, the concentration of the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 is 100 - 2000 ng / μL.

10. A method for controlling the sorghum aphid according to claim 9, characterized in that, During the process of introducing the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 into Melanaphis sacchari, the concentration of the dsRNA sequence with the nucleotide sequence shown in SEQ ID NO.1 is 1000 ng / μL.

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