11S globulin seed storage protein, coding gene thereof and application of 11S globulin seed storage protein in prevention and treatment of bemisia tabaci

Abstract

The invention discloses a 11S globulin seed storage protein, a coding gene thereof and application of the 11S globulin seed storage protein in prevention and treatment of bemisia tabaci. The nucleotide sequence of the coding gene of the protein is as shown in SEQ ID NO.1, and the amino acid sequence of the coding gene is as shown in SEQ ID NO.2. The invention further discloses a specific fragment of the 11S gene as shown in SEQ ID NO.5, dsRNA synthesis can be performed by utilizing SEQ ID NO.3 and SEQ ID NO.4, and the expression level of the gene can be specifically inhibited by utilizing dsRNA, so that the content of 11S protein and total amino acid of bemisia tabaci is reduced, and finally the reproductive capacity of bemisia tabaci is remarkably reduced. A new target is provided for a bemisia tabaci prevention and control technology based on RNA interference, and a green and sustainable scheme is provided for solving the contradiction between chemical pesticide and field bemisia tabaci resistance.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and relates to Bemisia tabaci 11S globulin seed storage protein, its encoding gene, and the application in controlling Bemisia tabaci. More specifically, it relates to specific dsRNA and the application of producing specific dsRNA in plants through virus induction to control Bemisia tabaci. At the same time, it also has the potential to control Bemisia tabaci by transgenic plants. Background Art

[0002] Bemisia tabaci ( Bemisia tabaci ) is a cosmopolitan agricultural pest with rapid reproduction and strong adaptability, which harms hundreds of agricultural crops, and the harm of Bemisia tabaci is extremely serious globally. Bemisia tabaci has caused economic losses of billions of dollars to global agriculture every year. Currently, the most commonly used control method for Bemisia tabaci in the field is still chemical pesticides. Due to the long-term application and irregular use of chemical pesticides, the resistance of Bemisia tabaci is getting higher and higher, making the harm of Bemisia tabaci in the field more and more serious. This has gradually become a difficult problem in agricultural production applications. Therefore, there is an urgent need for a green, safe, pollution-free and sustainable control strategy for Bemisia tabaci in agricultural production and life.

[0003] RNA interference (RNAi) technology can precisely regulate various physiological characteristics of insects through sequence-specific double-stranded RNA (dsRNA), including key life activities such as development process, immune response and behavior pattern. Currently, RNAi has been used in various pest and disease management strategies to reduce the resistance of pests and diseases to pesticides or damage the development process of pests and diseases. Due to the characteristics of high efficiency, green and safety, the method of using RNAi to control agricultural pests is a hot spot for the development of new green pest control technologies and also a future trend. The screening of target genes for RNAi technology is particularly important. One is the essential gene that plays a pivotal role in the life activities of target pests, and the other is the specific gene with significant sequence differences in non-target organisms. These two criteria are ideal control targets for agricultural pest control.

[0004] 11S globulin is a class of storage proteins in plant seeds, which can provide nitrogen source and amino acids for seed germination and early seedling growth. Some of these storage proteins have anti-microbial or anti-insect abilities, and certain storage proteins can also participate in the construction or stabilization of seed cell walls. The lack of sufficient storage proteins will have an adverse impact on the growth and development of plant seeds. 11S globulin is an important class of storage proteins in plant seeds and plays a key role in plant seed germination. Bemisia tabaci has obtained the encoding gene of plant 11S globulin through horizontal gene transfer. Bt11S . Therefore, with respect to Bemisia tabaci's Bt11SUsing the gene as a target, the expression of the gene in Bemisia tabaci was inhibited by RNAi means, and the amino acid content of Bemisia tabaci was reduced, so as to achieve sustainable and effective control of Bemisia tabaci, provide a new method for the green control technology of Bemisia tabaci and have broad commercial application prospects. Summary of the Invention

[0005] Based on the previous genome and transcriptome sequencing work of Bemisia tabaci, the inventor found a coding gene of 11S globulin in Bemisia tabaci MED ( Bt11S ). Literature retrieval showed that this gene only exists in plants. At the same time, phylogenetic analysis showed that Bt11S had a high amino acid homology and a close evolutionary relationship with plant 11S, suggesting that it was a plant-derived horizontally transferred gene. To verify the function of this gene in Bemisia tabaci, the full-length sequence of Bt11S gene was first cloned. After detection by qPCR technology, it was found that the expression of this gene in Bemisia tabaci adults was significantly higher than that in other growth stages, indicating that it played an important role in Bemisia tabaci. The present invention is a technology for controlling Bemisia tabaci pests using the key gene of 11S globulin in Bemisia tabaci, which is rich in essential amino acids and provides high-quality nitrogen source, and provides a theoretical basis for the green control of Bemisia tabaci. Bt11S Therefore, the present invention provides a Bemisia tabaci 11S globulin, and its amino acid sequence is shown in SEQ ID NO.2.

[0006] The present invention further provides the coding gene of the Bemisia tabaci 11S globulin. Preferably, it also includes a signal peptide. Specifically, its nucleotide sequence is shown in SEQ ID NO.1. This sequence contains an open reading frame (ORF) of 1209bp, encoding a protein of 402 amino acids (including 21 amino acid signal peptides), and its encoded protein sequence is shown in SEQ ID NO.2.

[0007] The present invention further provides an expression vector or recombinant host cell containing the coding gene.

[0008] The present invention also provides the application of the Bemisia tabaci 11S globulin or the coding gene in controlling Bemisia tabaci or preparing products for controlling Bemisia tabaci; the products are, for example, dsRNA products for producing Bemisia tabaci control, or products used for virus-induced plants or transgenic plants.

[0009] The present invention also provides a dsRNA for silencing the Bemisia tabaci 11S globulin gene, which is designed for the coding gene, specifically, it is complementary to the gene segment shown in SEQ ID NO.1 and designed with the nucleotide sequence shown in SEQ ID NO.5 as the target;

[0010] The present invention also provides a dsRNA for silencing the Bemisia tabaci 11S globulin gene, which is designed for the coding gene, specifically, it is complementary to the gene segment shown in SEQ ID NO.1 and designed with the nucleotide sequence shown in SEQ ID NO.5 as the target; More specifically, the dsRNA is obtained by using the cDNA of Bemisia tabaci adults as a template, performing PCR amplification with primers having the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, recovering the PCR product, and then synthesizing the specific dsRNA.

[0011] The present invention further provides a method for controlling Bemisia tabaci, which is achieved by injecting an expression vector of specific dsRNA targeting the coding gene into a plant.

[0012] Specifically, the dsRNA is designed for the coding gene, specifically, it is complementary to the gene segment shown in SEQ ID NO.1 and is designed with the nucleotide sequence shown in SEQ ID NO.5 as the target. More specifically, the dsRNA is obtained by using the cDNA of Bemisia tabaci adults as a template, performing PCR amplification with primers having the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, recovering the PCR product, and then synthesizing the specific dsRNA. Specifically, the plant is tomato, tobacco or cotton.

[0013] The present invention also provides a method for producing transgenic plants for controlling Bemisia tabaci, which is to introduce an expression vector of specific dsRNA targeting the coding gene into a plant to obtain transgenic plants.

[0014] Specifically, the dsRNA is designed for the coding gene, specifically, it is complementary to the gene segment shown in SEQ ID NO.1 and is designed with the nucleotide sequence shown in SEQ ID NO.5 as the target. More specifically, the dsRNA is obtained by using the cDNA of Bemisia tabaci adults as a template, performing PCR amplification with primers having the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, recovering the PCR product, and then synthesizing the specific dsRNA. Specifically, the plant is tomato, tobacco or cotton.

[0015] In a specific embodiment, the forward fragment primer SEQ ID NO.6 and the reverse fragment primer SEQ ID NO.7 are used to clone the fragment of dsBt11S, and then an expression vector is constructed.

[0016] The present invention discovers the specific dsRNA of the 11S globulin gene of Bemisia tabaci by feeding dsRNA Bt11S which can cause a significant decline in the reproductive ability of Bemisia tabaci, thus proving Bt11SThe specific dsRNA can be used as a molecular target for the control of Bemisia tabaci in the field. We demonstrated in tomato experiments that the VIGS technology can inhibit the expression of this gene in Bemisia tabaci and reduce the egg-laying amount of Bemisia tabaci, thus effectively controlling the population growth of Bemisia tabaci and achieving the feasibility of controlling Bemisia tabaci in the field. At the same time, it also has the potential to control Bemisia tabaci by transgenic methods. Therefore, based on the key physiological function genes of Bemisia tabaci Bt11S The VIGS technology is a new strategy for controlling Bemisia tabaci, which has good commercial value and application prospects. Generally speaking, the present invention has the following beneficial effects: The present invention firstly discovers and confirms that the specific interference of the target gene of pests Bt11S , which is safe for higher animals and humans; its insecticidal effect is specific, safe for non-target organisms, environmentally friendly and pollution-free. Therefore, the present invention has obvious technical advantages compared with traditional pest control methods. Brief Description of the Drawings

[0017] Figure 1A : Detection of the expression level of Bt11S gene mRNA in each instar of Bemisia tabaci (egg stage, 1st and 2nd instars, 3rd instar, 4th instar, adult).

[0018] Figure 1B : Detection of the expression level of Bt11S gene mRNA in each tissue of Bemisia tabaci (head, thorax and abdomen).

[0019] Among them, Figure 1A and Figure 1B use EF1-α gene as the internal reference gene, and the significant differences are represented by letters (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey’s test; n = 3).

[0020] Figure 2 : After the adult population of Bemisia tabaci feeds on Bt11S gene dsRNA, the mRNA expression level of Bt11S gene in adult Bemisia tabaci is detected at 48 h and 72 h. The EF1-α gene is used as the internal reference gene, and the significant differences are represented by letters (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey’s test; n = 3).

[0021] Figure 3 : The adult population of Bemisia tabaci feeds on Bt11SAt 72 h after dsRNA of the gene, the egg-laying amount of Bemisia tabaci mating for 3 days was counted, and the significant differences were represented by letters (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey’s test; n = 10).

[0022] Figure 4 : Bemisia tabaci adult populations fed on dsBt11S tomatoes, and the egg-laying amount of Bemisia tabaci adults was counted on the 10th day. Taking Bemisia tabaci fed on tomatoes expressing dsEGFP as the control, the significant differences were represented by letters (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey’s test; n = 10). Detailed implementation manners

[0023] To further illustrate the present invention, it is specifically described in combination with the following embodiments: Example 1: Identification of Bemisia tabaci 11S globulin Bt11S of Based on the Bemisia tabaci MED genome data, a predicted plant-derived Bemisia tabaci 11S globulin was identified Bt11S . Since the genomic sequence contains intron regions and there are errors generated during splicing. We further combined the plant-derived homologous genes and Bemisia tabaci transcriptome sequencing data to Bt11S correct the coding region sequence of the gene and obtain the complete coding region sequence of the gene. Specific primers were designed based on the corrected Bt11S gene sequence. 60 Bemisia tabaci adult samples were collected, and the RNA of Bemisia tabaci adults was extracted according to the Trizol method, and cDNA was reversely transcribed and synthesized through the PrimeScript II First Strand cDNA Kit. Using the cDNA obtained in the previous step as a template, the Bemisia tabaci Bt11S gene was cloned. The PCR products were recovered, purified and sequenced, and the Bemisia tabaci Bt11S gene sequence is shown in SEQ ID NO.1, and the amino acid sequence translated according to the gene sequence is shown in SEQ ID NO.2.

[0024] Example 2: Expression pattern analysis of Bemisia tabaci 11S globulin Bt11S of According to the cloned Bt11SGene sequence SEQ ID NO.1, and a pair of specific primers qBt11S-F and qBt11S-R designed for qPCR detection. Samples of different instars of Bemisia tabaci (egg stage, 1st and 2nd instars, 3rd instar, 4th instar, adults) were collected, RNA was extracted according to the Trizol method, and cDNA was synthesized by reverse transcription. Samples of different tissues of Bemisia tabaci (head, thorax and abdomen) were collected, RNA was extracted according to the Trizol method, and cDNA was synthesized by reverse transcription. Using the above cDNA as a template, qBt11S-F and qBt11S-R as primers, with EF1-α as the internal reference gene, the real-time fluorescence quantitative PCR (qPCR) technique was used to detect Bt11S gene expression patterns. The results showed that Bt11S was significantly highly expressed in adults ( Figure 1A ), and was highly expressed in the head, thorax and abdomen of Bemisia tabaci adults ( Figure 1B ).

[0025] Example 3: 11S globulin of Bemisia tabaci Bt11S Preparation of dsRNA According to the cloned Bt11S gene sequence SEQ ID NO.1, a pair of specific primers dsBt11S-F and dsBt11S-R containing the T7 promoter were designed as shown in SEQ ID NO.3 and SEQ ID NO.4. Using Bemisia tabaci adult cDNA as a template, the above primers were used for PCR amplification of the gene fragment SEQ ID NO.5, the PCR product was recovered, and Bemisia tabaci dsBt11S-specific dsRNA was synthesized by the T7 RiboMAX Express RNAi System (Promega, Madison, WI, USA) kit.

[0026] Example 4: Effect of feeding dsRNAs on the fecundity of Bemisia tabaci adults Sixty newly emerged female Bemisia tabaci were placed in a biological assay device, and 30 μg of synthesized specific dsRNA was added to 60 μl of artificial diet (30% sucrose + 5% yeast extract). Among them, dsBt11S was added to the treatment group, and dsEGFP was used as the control group. When the feeding time of Bemisia tabaci on the special artificial diet was 48 h and 72 h, 60 Bemisia tabaci adults were collected and total RNA was extracted. After reverse transcription to prepare cDNA, qPCR was used to measure Bt11S gene expression levels. The results showed that the best interference efficiency was achieved at 72 h ( Figure 2). Female whiteflies that had fed on dsRNA for 72 h were selected and transferred into microcages in groups of five, with 10 biological replicates for each group. After 72 h, the total number of eggs laid by female whiteflies was recorded. The results showed that inhibiting the Bt11S gene expression could significantly reduce the egg-laying amount of whiteflies ( Figure 3 ).

[0027] Example 5: Effect of feeding on VIGS plants on the egg-laying amount of adult whiteflies According to the information of the VIGS vector pTRV2, appropriate restriction enzyme sites were selected. At the same time, specific primers were designed in the Bt11S gene ORF region to construct the TRV2-Bt11S vector. Using the designed forward fragment primers such as SEQ ID NO.6 and reverse fragment primers such as SEQ ID NO.7, the dsBt11S fragment was cloned. Restriction digestion was performed using EcoR I and BamH I endonucleases, and then the linearized vector was recovered and purified. An In-fusion recombination experiment was carried out using In-fusion recombinase to ligate the specific dsRNA fragment to the TRV2 vector and transfer it into Escherichia coli. After PCR verification, the TRV1 and TRV2-Bt11S vectors were obtained.

[0028] Finally, using the Agrobacterium transformation system, the mixture of TRV1 and TRV2-Bt11S mixed in a 1:1 ratio was injected into tomatoes. When the tomato seedlings grew 4 true leaves, some tomato leaves were collected and RNA was extracted to synthesize cDNA. PCR was used to detect the expression of the Bt11S interference fragment in tomatoes, which was used for the subsequent research on whitefly control.

[0029] Adult whiteflies hatched within 8 h were collected. Five newly hatched female whiteflies were transferred into microcages, and then the microcages were fixed to the VIGS tomato leaves to allow the whiteflies to fully feed on the back of the tomato leaves. Using tomatoes expressing dsEGFP as a control, the egg-laying amount of whiteflies in each microcage was recorded on the 10th day. The results were as Figure 4 shown. After feeding on plants expressing dsBt11S, the egg-laying amount of adult whiteflies decreased significantly.

[0030] The above implementation cases are the basic implementation cases of the present invention and will not impose any formal limitations on the present invention. Those skilled in the art with knowledge of the technical background can, without departing from the core of the technical solution of the present invention, make effective changes and modifications to the practical application of the present invention by using the methods and technical contents described in the above implementation cases. Therefore, all content that does not depart from the technical solution of the present invention, based on the technical essence of the present invention and modified and equivalently replaced based on the above embodiments, still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A whitefly 11S globulin, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the 11S globulin of Bemisia tabaci according to claim 1, preferably further comprising a signal peptide, and the specific nucleotide sequence thereof is shown in SEQ ID NO.

1.

3. An expression vector or recombinant host cell containing the coding gene as claimed in claim 2.

4. Use of the whitefly 11S globulin according to claim 1, or the encoding gene according to claim 2 in controlling whitefly, or preparing products for controlling whitefly; such products are for example dsRNA preparations for controlling whitefly, or products for virus-induced plants or transgenic plants.

5. A dsRNA for silencing the 11S globulin gene of Bemisia tabaci, which is designed for the coding gene as claimed in claim 2, specifically complementary to the gene segment as described in SEQ ID NO.1, and designed with the nucleotide sequence as shown in SEQ ID NO.5 as the target; More specifically, the dsRNA is obtained by using the adult cDNA of Bemisia tabaci as a template, performing PCR amplification using primers of the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, recovering the PCR products, and then synthesizing the specific dsRNA.

6. A method for controlling Bemisia tabaci, characterized in that: This is achieved by injecting an expression vector for a specific dsRNA encoding the gene as claimed in claim 2 into the plant.

7. The method according to claim 6, characterized in that The dsRNA is designed for the coding gene as described in claim 2, specifically has complementarity with the gene segment described in SEQ ID NO.1, and is designed with the nucleotide sequence shown in SEQ ID NO.5 as the target; More specifically, the dsRNA is obtained by the following method: using the adult cDNA of Bemisia tabaci as a template, performing PCR amplification using primers of the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, recovering the PCR products, and then synthesizing the specific dsRNA; In particular, the plant is tomato, tobacco or cotton.

8. A method for producing a transgenic plant for controlling Bemisia tabaci, characterized in that: An expression vector for specific dsRNA encoding the gene as claimed in claim 2 is introduced into the plant to obtain a transgenic plant.

9. The method according to claim 8, characterized in that The dsRNA is designed for the coding gene as described in claim 2, specifically has complementarity with the gene segment described in SEQ ID NO.1, and is designed with the nucleotide sequence shown in SEQ ID NO.5 as the target; More specifically, the dsRNA is obtained by the following method: using the adult cDNA of Bemisia tabaci as a template, performing PCR amplification using primers of the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, recovering the PCR products, and then synthesizing the specific dsRNA; In particular, the plant is tomato, tobacco or cotton.

10. The method according to claim 8 or 9, characterized in that The forward fragment primer SEQ ID NO.6 and the reverse fragment primer SEQ ID NO.7 were used to clone the fragment of dsBt11S, and then the expression vector was constructed.

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