Δ12 fatty acid desaturase and its encoding gene and its application in controlling Bemisia tabaci

Abstract

The present invention discloses an insect Δ12 fatty acid desaturase, the encoding gene of which is <h2 style=";text-align:left;direction:ltr">BtFAD2-9 , the nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2; the present invention also discloses a specific fragment of the FAD2-9 gene, which is used for the synthesis of dsRNA, and the dsRNA can be used to specifically inhibit the expression level of the gene, inhibit the synthesis of fatty acids and their derivatives and the lipid metabolism process of whiteflies, and finally significantly reduce the reproductive capacity of whiteflies. The present invention provides a new target for whitefly control technology based on RNA interference, and provides a green and sustainable solution for solving the contradiction between chemical pesticides and field whitefly resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a whitefly Δ12 fatty acid desaturase and a coding gene thereof, and applications in preventing and controlling whiteflies, and more specifically to applications of specific dsRNA and transgenic plants expressing specific dsRNA in preventing and controlling whiteflies. Background Art

[0002] Bemisia tabaci, a global agricultural pest, can harm hundreds of crops. Its damage has been reported in more than 90 regions around the world, causing hundreds of millions of yuan in economic losses. Bemisia tabaci has a wide distribution range and strong reproductive capacity. Currently, the most commonly used means of control for Bemisia tabaci in the field is still chemical pesticides. The long-term application and irregular use of chemical pesticides have caused Bemisia tabaci to quickly develop resistance to them, making the damage of Bemisia tabaci in the field more rampant. This has gradually become a difficult problem in agricultural production applications. Therefore, agricultural production and life urgently need a green, safe and sustainable Bemisia tabaci control strategy.

[0003] RNAi can affect many characteristics of insects, such as insect development, immunity and behavioral processes, through specific double-stranded RNA (dsRNA). Currently, RNAi has been used in a variety of pest management strategies to reduce the resistance of pests to pesticides or damage the development of pests. Due to its high efficiency and green characteristics, the use of RNAi to control agricultural pests is currently a hot spot for the development of new green pest control technologies. Due to the characteristics of RNAi targeting key target genes, obtaining biological target genes with strong control effects and high species specificity is a necessary condition for the development of agricultural pest control with RNAi technology as the core.

[0004] Polyunsaturated fatty acids (PUFA) are one of the important nutrients for organisms. PUFA is an important component of the membrane structure of organisms. In addition, studies have shown that it is involved in physiological metabolic processes such as plant stress resistance and insect reproduction. Lack of sufficient PUFA will have an adverse effect on the growth and development of organisms. The FAD2 gene encodes a Δ12 desaturase, which is mainly involved in fatty acid biosynthesis and plays a key role in the biosynthesis of PUFA. Due to the lack of the necessary Δ12 desaturase, higher organisms such as humans cannot independently synthesize complex PUFAs and can only obtain sufficient PUFA nutrition through diet and other means. Bemisia tabaci obtained the plant Δ12 desaturase gene BtFAD2-9 through horizontal gene transfer. Therefore, the BtFAD2-9 gene of Bemisia tabaci was used as the target, and RNAi was used to inhibit the expression of this gene in Bemisia tabaci, destroying the balance of PUFA biosynthesis of Bemisia tabaci, thereby achieving sustainable and effective control of Bemisia tabaci, providing a new method for green control technology of Bemisia tabaci and having broad commercial application prospects. Summary of the invention

[0005] Based on the genome and transcriptome sequencing of whiteflies in the early stage of this experiment, the inventors discovered a Δ12 desaturase gene (BtFAD2-9) in whiteflies. Since the Δ12 desaturase encoded by the BtFAD2-9 gene is a key enzyme in PUFA biosynthesis, it does not exist in other insects and mammals, and it maintains a high amino acid homology and a close evolutionary relationship with plant FAD2, it is speculated that it is a plant-derived horizontal transfer gene. In order to verify the function of this gene in whiteflies, the full-length sequence of the BtFAD2-9 gene was first cloned, and after detection using qPCR technology, it was found that the expression of this gene in whitefly adults was significantly higher than in other growth periods. The present invention is a technology for controlling whitefly pests using BtFAD2-9, a key gene for PUFA nutrient synthesis in whiteflies, which provides a theoretical basis for green control of whiteflies.

[0006] Therefore, the present invention provides a whitefly Δ12 desaturase gene BtFAD2-9, the nucleotide sequence of which is shown in SEQ ID NO.1. The method for obtaining the gene is as follows: (1) using the annotation information of the whitefly genome to analyze the fatty acid synthesis pathway of the whitefly. The predicted plant-derived Δ12 desaturase gene BtFAD2-9 is identified; (2) using the laboratory transcriptome database to calibrate the gene sequence; (3) using primer design software to design specific primers. The coding region sequence of the BtFAD2-9 gene is cloned and sequenced to obtain the sequence. The sequence contains an open reading frame (ORF) of 1146 bp, encoding a protein of 381 amino acids, and the protein coding sequence is shown in SEQ ID NO.2.

[0007] The present invention also provides a dsRNA for silencing the Δ12 desaturase gene BtFAD2-9 of Bemisia tabaci, comprising a fragment of the Δ12 desaturase gene BtFAD2-9 (nucleotide sequence as shown in SEQ ID NO.3). The method for obtaining the dsRNA is as follows: (1) designing a specific primer with a T7 promoter sequence at the 5′ end according to the nucleotide sequence SEQ ID NO.1; (2) using the cDNA of the adult Bemisia tabaci as a template, and performing PCR amplification using specific primers; (3) subjecting the amplified product to 1% agarose gel electrophoresis, and recovering the PCR product; (4) synthesizing the dsRNA using a T7 RibomaxTM Express RNAi System (Promega, Madison, WI, USA) kit.

[0008] Based on the above dsRNA synthesis scheme, the present invention also provides an application of dsRNA (synthesized from SEQ ID NO.3) in the control of whiteflies: feeding whiteflies with the specific dsRNA, the results show that the dsRNA can specifically silence the mRNA expression of the whitefly Δ12 desaturase gene BtFAD2-9, and cause a significant decrease in the egg laying of adult whiteflies. It can be seen that the dsRNA can be used for the control of whiteflies.

[0009] Based on the results of the above-mentioned feeding RNAi experiment, the present invention also provides a transgenic plant for silencing the Δ12 desaturase gene of Bemisia tabaci using dsRNA of the Δ12 desaturase gene of Bemisia tabaci. The selected plants are preferably crops, such as tomatoes, tobacco, cotton, etc. The method for obtaining the transgenic plant is as follows: (1) designing a 5′-end specific primer according to the nucleotide sequence SEQ ID NO.1; (2) using the cDNA of the adult Bemisia tabaci as a template, PCR amplification using specific primers; (3) subjecting the amplified product to 1% agarose gel electrophoresis, recovering the PCR product, and recovering the PCR product; (4) modifying the plant RNAi expression vector using a specific nuclease; (5) connecting the target PCR product to the plant RNAi expression vector using In-fusion ligase; (6) transferring the constructed RNAi expression vector into Agrobacterium; (6) using the above-mentioned Agrobacterium to infect tobacco to construct transgenic tobacco expressing dsBtFAD2-9. The purpose of the present invention is to provide the above-mentioned sequence, various vectors, transgenic plants, etc. for the prevention and control of Bemisia tabaci.

[0010] The present invention discovered that the specific dsRNA of BtFAD2-9, a key nutrient synthesis gene of whiteflies, can cause a significant decrease in the reproductive capacity of whiteflies by feeding dsRNA, thereby proving that the specific dsRNA of BtFAD2-9 can be used as a molecular target for field pest control of whiteflies. We used transgenic tobacco experiments to prove that transgenic technology inhibits the expression of this gene in whiteflies and reduces the egg laying of whiteflies, thereby effectively controlling the growth of whiteflies populations and achieving the feasibility of field whitefly control. Therefore, transgenic RNAi technology based on BtFAD2-9, a key physiological function gene of whiteflies, is a new strategy for whitefly control, which has good commercial value and application prospects. In summary, the present invention has the following beneficial effects as follows:

[0011] The present invention has discovered and confirmed for the first time that the specific interference with the pest target gene BtFAD2-9 is safe for higher animals and humans; its insecticide is specific, safe for non-target organisms, environmentally friendly, and pollution-free. Therefore, the present invention has obvious technical advantages over traditional pest control methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Schematic diagram of the transmembrane domain of the Δ12 desaturase BtFAD2-9 from Bemisia tabaci. BtFAD2-9 has a total of 6 transmembrane regions, and the black circle is the conserved histidine cluster structure of the Δ12 desaturase.

[0013] Figure 2A : Detection of the expression level of BtFAD2-9 gene mRNA in various instars of Bemisia tabaci (egg stage, 1st and 2nd instars, 3rd instar, 4th instar, male adults and female adults).

[0014] Figure 2B : Detection of the expression level of BtFAD2-9 gene mRNA in various tissues (head, thorax and abdomen) of Bemisia tabaci.

[0015] in, Figure 2A and Figure 2B EF1-α gene was used as the internal reference gene, and letters indicate significant differences (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Tukey's test; n=3).

[0016] Figure 3: After the adult population of Bemisia tabaci fed on the dsRNA of the BtFAD2-9 gene, the mRNA expression levels of the BtFAD2-9 gene in male and female adults of Bemisia tabaci were detected at 48h and 96h, and the EF1-α gene was used as the internal reference gene. Letters are used to indicate significant differences (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Tukey's test; n=3).

[0017] Figure 4 : The number of eggs laid by B. tabaci after 96 hours of feeding on dsRNA of BtFAD2-9 gene by adults was counted after 3-day mating. Letters indicate significant differences (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Tukey's test; n=10).

[0018] Figure 5 : Adult populations of Bemisia tabaci fed on transgenic tobacco, and the number of eggs laid by adults was counted on the 7th day. Bemisia tabaci fed on transgenic tobacco expressing dsEGFP was used as the control, and letters indicate significant differences (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Tukey's test; n=10). DETAILED DESCRIPTION

[0019] To further illustrate the present invention, the following examples are provided for specific description:

[0020] Example 1: Identification of the Bemisia tabaci Δ12 desaturase gene BtFAD2-9

[0021] The polyunsaturated fatty acid biosynthesis pathway of B. tabaci was analyzed by using the previous genome sequencing results of our laboratory. A predicted plant-derived Δ12 desaturase gene BtFAD2-9 was identified. Since the genome sequence contains intron regions and there are errors caused by splicing. We further combined the plant-derived homologous genes and the transcriptome sequencing data of B. tabaci to correct the coding region sequence of the BtFAD2-9 gene and obtain the complete coding region sequence of the gene. Based on the corrected BtFAD2-9 gene sequence, specific primers such as SEQ ID NO.4 and SEQ ID NO.5 were designed. 100 adult samples of B. tabaci were collected, and RNA of B. tabaci adults was extracted according to the Trizol method, and reverse transcribed and synthesized into cDNA using the PrimeScript II first-strand cDNA kit. The cDNA obtained in the previous step was used as a template cBtFAD2-9-F and cBtFAD2-9-R as primers to clone the B. tabaci BtFAD2-9 gene. The PCR product was recovered, purified and sequenced to obtain the gene sequence of Bemisia tabaci BtFAD2-9 as shown in SEQ ID NO.1.

[0022] Example 2: Analysis of the expression pattern of the Bemisia tabaci Δ12 desaturase gene BtFAD2-9

[0023] Based on the cloned BtFAD2-9 gene sequence SEQ ID NO.1, the specific primer pairs qBtFAD2-9-F and qBtFAD2-9-R for qPCR detection were designed as shown in SEQ ID NO.6 and SEQ ID NO.7. Samples of various instars of whiteflies (egg stage, 1st and 2nd instars, 3rd instar, 4th instar, male adults and female adults) were collected, RNA was extracted according to the Trizol method, and cDNA was synthesized by reverse transcription. Samples of various tissues of whiteflies (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, qBtFAD2-9-F and qBtFAD2-9-R as primers, and EF1-α as an internal reference gene, the expression pattern of the BtFAD2-9 gene was detected by real-time quantitative PCR (qPCR) technology. The results showed that BtFAD2-9 is highly expressed in adults, especially males ( Figure 2A ), and highly expressed in the abdomen of adult whiteflies ( Figure 2B ).

[0024] Example 3: Preparation of dsRNA for the Bemisia tabaci Δ12 desaturase gene BtFAD2-9

[0025] According to the cloned BtFAD2-9 gene sequence SEQ ID NO.1, the interference fragment (SEQ ID NO.3) was designed and selected by specific primers and software analysis, and then dsRNA was synthesized using the t7 kit according to the interference fragment. Specifically, a specific primer pair dsBtFAD2-9-F and dsBtFAD2-9-R containing a T7 promoter was designed as shown in SEQ ID NO.8 and SEQ ID NO.9, and the adult cDNA of Bemisia tabaci was used as a template, and PCR amplification was performed using the above primers, and the PCR product was recovered, and the Bemisia tabaci dsBtFAD2-9 specific dsRNA was synthesized using the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA) kit.

[0026] Example 4: Effect of feeding dsRNAs on egg production of adult Bemisia tabaci

[0027] 150 newly emerged female whiteflies were placed in a biological assay device, and 150 μg of synthetic specific dsRNA was added to 200 μl of artificial diet (30% sucrose + 5% yeast extract). The treatment group was added with dsBtFAD2-9, and the control group was dsEGFP. When the whiteflies fed on the special artificial diet for 48h and 96h, 50 whiteflies were collected and total RNA was extracted. After reverse transcription to prepare cDNA, the expression level of the BtFAD2-9 gene was determined by qPCR. The results showed that the best interference efficiency was achieved at 96h ( Figure 3 ). Female whiteflies that had been fed with dsRNA for 96 hours were selected and transferred into micro-insect cages in groups of five, with 10 biological replicates each. After 72 hours, the total egg production of female whiteflies was recorded. The results showed that inhibiting the expression of the BtFAD2-9 gene in whiteflies could significantly reduce the egg production of whiteflies ( Figure 4 ).

[0028] Example 5: Effect of feeding on transgenic plants on egg production of adult Bemisia tabaci

[0029] According to the information of plant RNAi vector pCAMBIA-RNAi, suitable restriction sites are selected, and specific primers are designed in the ORF region of BtFAD2-9 gene to construct dsBtFAD2-9 plant expression vector. The forward fragment primers such as SEQ ID NO.10 and SEQ ID NO.11 and the reverse fragment primers such as SEQ ID NO.12 and SEQ ID NO.13 are used to clone the forward fragment and reverse fragment of dsBtFAD2-9 respectively. The pCAMBIA-RNAi vector is modified with XhoI and BglII endonucleases, and the pCAMBIA-RNAi vector is modified with BamHI and SalI endonucleases, and then the linearized vector is recovered and purified. In-fusion recombinase is used to perform homologous recombination experiments, and the specific dsRNA fragment is connected to the pCAMBIA-RNAi vector and transferred into Escherichia coli. After PCR verification, the dsBtFAD2-9 plant expression vector is obtained. Finally, the transgenic plant construction experiment was carried out, and the recombinant pCAMBIA-RNAi-dsBtFAD2-9 plasmid was transformed into tobacco using the Agrobacterium transformation system. When the tobacco seedlings grew 4 true leaves, some tobacco leaves were collected, and the DNA of tobacco leaves was extracted using the plant genomic DNA extraction kit (TIANGEN). The transgenic tobacco was detected and screened by PCR, and used for the subsequent research on whitefly control.

[0030] Adult whiteflies within 8 hours of hatching were collected, and 5 newly hatched female whiteflies were transferred to micro-insect cages. The micro-insect cages were then fixed to transgenic tobacco leaves to allow whiteflies to fully feed on the back of the tobacco leaves. Transgenic tobacco expressing dsEGFP was used as a control, and the number of whiteflies laying eggs in each micro-insect cage was recorded on the 7th day. Figure 5 As shown, the egg production of adult Bemisia tabaci was significantly reduced after feeding on transgenic plants expressing dsBtFAD2-9.

[0031] The above implementation cases are basic implementation cases of the present invention and do not impose any formal restrictions on the present invention. A person skilled in the art who is familiar with the knowledge background in this field can 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 without departing from the core of the technical solution of the present invention. Therefore, all modifications and equivalent replacements based on the above embodiments according to the technical essence of the present invention that do not depart from the content of the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for producing a transgenic plant for controlling Bemisia tabaci, characterized in that: This is achieved by introducing into the plant an expression vector for dsRNA specific to a gene encoding an insect Δ12 fatty acid desaturase; The nucleotide sequence of the gene encoding the insect Δ12 fatty acid desaturase is shown in SEQ ID NO.1; The plant is tomato, tobacco or cotton.

2. The method according to claim 1, characterized in that The nucleotide sequence of the interference fragment targeted by the specific dsRNA is shown in SEQ ID NO.

3.

3. The method according to claim 1, characterized in that The dsRNA is obtained by using adult Bemisia tabaci cDNA as a template, performing PCR amplification using primers of nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9, recovering PCR products, and synthesizing specific dsRNA using a T7 Ribomax Express RNAi System kit.

4. Use of insect Δ12 fatty acid desaturase or its encoding gene or gene fragment of insect Δ12 fatty acid desaturase gene in controlling Bemisia tabaci; The nucleotide sequence of the gene encoding the insect Δ12 fatty acid desaturase is shown in SEQ ID NO.1; the nucleotide sequence of the gene fragment of the insect Δ12 fatty acid desaturase gene is shown in SEQ ID NO.3; The application is to produce transgenic plants for controlling whiteflies, and the plants are tomatoes, tobacco or cotton.

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