Delta7-sterol C5 desaturase, coding gene thereof and application of desaturase in prevention and control of bemisia tabaci

Abstract

The invention discloses an insect delta7-sterol C5 desaturase (delta7-steril C5-class-like, SC5DL), and an amino acid sequence of the insect delta7-sterol C5 desaturase is as shown in SEQ ID NO. 2. The insect delta7-sterol C5 desaturase has an amino acid sequence as shown in SEQ ID NO. The invention also discloses a specific fragment of the SC5DL gene, the specific fragment is used for synthesizing dsRNA, the expression level of the gene can be specifically inhibited by utilizing the dsRNA, the cholesterol synthesis of bemisia tabaci can be inhibited, and finally the reproductive capacity of the bemisia tabaci can be obviously 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 a whitefly Δ7-sterol C5 desaturase and a coding gene thereof, and applications in preventing and controlling whiteflies, and more specifically to specific dsRNA and the application of specific dsRNA produced in plants by virus induction to prevent and control whiteflies, and also has the potential of using transgenic plants to prevent and control whiteflies. Background Art

[0002] Bemisia tabaci is a worldwide agricultural pest with strong host adaptability. It can parasitize more than 600 plant species and harm hundreds of agricultural crops. The harm caused by Bemisia tabaci is extremely serious worldwide and has become a major agricultural pest in many regions. Bemisia tabaci has caused billions of dollars in economic losses to global agriculture every year. At present, the most commonly used control method for Bemisia tabaci in the field is still chemical pesticides. However, due to the wide distribution range of Bemisia tabaci, its strong adaptability, strong reproductive capacity and rapid development of resistance to pesticides, as well as the irregular use of chemical pesticides, Bemisia tabaci has rapidly developed different degrees of resistance to pesticides, making the harm of Bemisia tabaci in the field more and more serious. This has gradually become a difficult problem in agricultural production and application. Therefore, agricultural production and life urgently need a green, safe, pollution-free and sustainable strategy for the control of Bemisia tabaci.

[0003] RNAi induces the degradation of homologous mRNA in the body through small non-coding RNA produced by specific double-stranded RNA (dsRNA) to achieve gene-specific silencing, thereby affecting many characteristics of insects, such as insect growth and development, immunity and behavioral processes. At present, RNAi technology has been applied to a variety of pest management strategies, effectively reducing the harm of pests by reducing the resistance of pests to pesticides or interfering with their development process. Compared with traditional pesticides, RNAi control has the characteristics of high specificity, high efficiency, not easy to produce resistance, harmless to non-target organisms, non-toxic and no residue. The use of RNAi to control agricultural pests is the current trend and hot spot in the development of new green pest control technologies. Because RNAi targets key target genes, has high specificity and is environmentally friendly, it is a new means of green pest control with high species specificity and strong control effect for the control of agricultural pests.

[0004] Sterols are a class of steroid compounds that are widely present in organisms and play a vital role in various biological processes. In organisms, sterols are not only essential components of cell membranes, maintaining the fluidity and stability of cell membranes, but also participate in various physiological processes as signal molecules, including the regulation of hormone synthesis and cholesterol metabolism. The lack of sufficient sterols will have an adverse effect on the growth and development of organisms. The SC5DL gene encodes a Δ7-sterol C5 desaturase, which is mainly involved in cholesterol biosynthesis and plays a key role in its biosynthesis. Bemisia tabaci acquired the plant Δ7-sterol C5 desaturase gene BtSC5DL through horizontal gene transfer. Therefore, the BtSC5DL gene of Bemisia tabaci was used as the target, and RNAi was used to inhibit the expression of this gene in Bemisia tabaci, disrupting the balance of cholesterol biosynthesis in 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] The inventors discovered a Δ7-sterol C5 desaturase gene (BtSC5DL) in the MED of Bemisia tabaci. Since the Δ7-sterol C5 desaturase encoded by the BtSC5DL gene is a key enzyme in cholesterol biosynthesis, the BtSC5DL protein has a sequence similarity of greater than 50% with the SC5DL of plants, and a sequence similarity of less than 25% with the SC5DL of Homo sapiens, and maintains a high amino acid homology and a close evolutionary relationship with the SC5DL of plants, it is speculated that it is a plant-derived horizontal transfer gene. In order to verify the function of the gene in Bemisia tabaci, the present invention cloned the full-length sequence of the BtSC5DL gene for the first time, and after detection using qPCR technology, it was found that the expression of the gene in Bemisia tabaci adults was higher than that in other eggs and third and fourth instars. The present invention is a technology for controlling Bemisia tabaci pests using the key gene BtSC5DL for cholesterol synthesis in Bemisia tabaci, which provides a theoretical basis for green control of Bemisia tabaci.

[0006] Therefore, the present invention provides an insect Δ7-sterol C5 desaturase, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] The present invention further provides an insect Δ7-sterol C5 desaturase encoding gene, which encodes the insect Δ7-sterol C5 desaturase.

[0008] Preferably, the nucleotide sequence is as shown in SEQ ID NO.1.

[0009] The present invention provides an expression vector containing the coding gene and a recombinant host cell.

[0010] The invention provides application of the encoding gene in preventing and controlling Bemisia tabaci.

[0011] The present invention provides a gene fragment of a gene encoding a Δ7-sterol C5 desaturase of Bemisia tabaci, which is complementary to the gene segment described in SEQ ID NO.1 and is designed with the nucleotide sequence shown in SEQ ID NO.3 as a target.

[0012] The present invention also provides a dsRNA for silencing the Bemisia tabaci Δ7-sterol C5 desaturase gene, which is designed for the gene fragment.

[0013] Specifically, 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 T7RiboMAXExpress RNAi System kit.

[0014] The present invention provides a method for silencing the gene of Bemisia tabaci by inducing a plant through a virus, which is achieved by introducing into the plant an expression vector of a dsRNA specific to a gene encoding a Bemisia tabaci Δ7-sterol C5 desaturase;

[0015] Specifically, the forward fragment primer SEQ ID NO. 10 and the reverse fragment primer SEQ ID NO. 11 are used to introduce the expression vector of the specific dsRNA for the gene encoding the Δ7-sterol C5 desaturase of Bemisia tabaci.

[0016] The present invention also provides the insect Δ7-sterol C5 desaturase or its encoding gene, the gene fragment of the insect Δ7-sterol C5 desaturase gene, or the use of the dsRNA in the preparation of products for controlling whiteflies, such as dsRNA preparations for producing whiteflies, or products used for virus-induced plants or transgenic plants.

[0017] The present invention discovered that the specific dsRNA of BtSC5DL, a key cholesterol 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 BtSC5DL can be used as a molecular target for field pest control of whiteflies. We used tomato experiments to prove that VIGS 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. It also has the potential for transgenic control of whiteflies. Therefore, VIGS technology based on BtSC5DL, 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:

[0018] The present invention has discovered and confirmed for the first time that the specific interference with the pest target gene BtSC5DL 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

[0019] Figure 1 : Schematic diagram of the transmembrane domain of the whitefly Δ7-sterol C5 desaturase BtSC5DL. BtSC5DL has three transmembrane regions, and the black circle is the conserved histidine cluster structure of 7-sterol C5 desaturase.

[0020] Figure 2A : Detection of the expression level of BtSC5DL gene mRNA in various instars of Bemisia tabaci (egg stage, 1st and 2nd instars, 3rd instar, 4th instar, and adult).

[0021] Figure 2B : Detection of the expression level of BtSC5DL gene mRNA in various tissues (head, thorax and abdomen) of Bemisia tabaci.

[0022] 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).

[0023] Figure 3 : After the adult population of Bemisia tabaci fed on the dsRNA of the BtSC5DL gene, the mRNA expression level of the BtSC5DL gene in the adult Bemisia tabaci was detected at 48h and 72h, and the EF1-α gene was used as the internal reference gene. Letters represent significant differences (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Tukey's test; n=3).

[0024] Figure 4 : The number of eggs laid by B. tabaci after 72 hours of feeding on dsRNA of BtSC5DL 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).

[0025] Figure 5: Adult populations of Bemisia tabaci fed on dsBtSC5DL tomatoes, and the number of eggs laid by adults was counted on day 10. Whiteflies fed on tomatoes expressing dsEGFP were used as controls, 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

[0026] To further illustrate the present invention, the following examples are provided for specific description:

[0027] Example 1: Identification of the Bemisia tabaci Δ7-sterol C5 desaturase BtSC5DL

[0028] Based on the MED genome data of Bemisia tabaci, a predicted plant-derived Δ7-sterol C5 desaturase BtSC5DL 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 Bemisia tabaci to correct the coding region sequence of the BtSC5DL gene and obtain the complete coding region sequence of the gene. Based on the corrected BtSC5DL gene sequence, specific primers such as SEQ ID NO.4 and SEQ ID NO.5 were designed. 60 adult samples of Bemisia tabaci were collected, and the RNA of Bemisia 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 above step was used as a template cBtSC5DL-F and cBtSC5DL-R as primers to clone the Bemisia tabaci BtSC5DL gene. The PCR product was recovered, purified and sequenced, and the sequence of the Bemisia tabaci BtSC5DL gene was obtained as shown in SEQ ID NO.1.

[0029] Example 2: Analysis of the expression pattern of Bemisia tabaci Δ7-sterol C5 desaturase BtSC5DL

[0030] Based on the cloned BtSC5DL gene sequence SEQ ID NO.1, the specific primer pairs qBtSC5DL-F and qBtSC5DL-R for qPCR detection were designed as shown in SEQ ID NO.6 and SEQ ID NO.7. Samples of various instars of whitefly (egg stage, 1st and 2nd instars, 3rd instar, 4th instar, and adults) were collected, RNA was extracted according to the Trizol method, and cDNA was synthesized by reverse transcription. Samples of various tissues of whitefly (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, qBtSC5DL-F and qBtSC5DLR as primers, and EF1-α as an internal reference gene, the expression pattern of the BtSC5DL gene was detected by real-time quantitative PCR (qPCR) technology. The results showed that BtSC5DL was highly expressed in adults ( Figure 2A ), and highly expressed in the head of adult Bemisia tabaci ( Figure 2B ).

[0031] Example 3: Preparation of dsRNA of Bemisia tabaci Δ7-sterol C5 desaturase BtSC5DL

[0032] According to the cloned BtSC5DL gene sequence SEQ ID NO.1, a pair of specific primers containing T7 promoter, dsBtSC5DL-F and dsBtSC5DL-R, were designed as shown in SEQ ID NO.8 and SEQ ID NO.9. Using the adult cDNA of Bemisia tabaci as a template, PCR amplification was performed using the above primers, and the PCR product was recovered, and the Bemisia tabaci dsBtSC5DL-specific dsRNA was synthesized using the T7 RiboMAX Express RNAiSystem (Promega, Madison, WI, USA) kit.

[0033] Example 4: Effect of feeding dsRNAs on egg production of adult Bemisia tabaci

[0034] 60 newly emerged female whiteflies were placed in a biological assay device, and 30 μg of synthetic specific dsRNA was added to 60 μl of artificial diet (30% sucrose + 5% yeast extract). The treatment group was added with dsBtSC5DL, and the control group was added with dsEGFP. When the whiteflies fed on the special artificial diet for 48h and 72h, 60 whiteflies were collected and total RNA was extracted. After reverse transcription to prepare cDNA, the expression level of the BtSC5DL gene was determined by qPCR. The results showed that the best interference efficiency was achieved at 72h ( Figure 3). Female whiteflies that had fed on dsRNA for 72 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 BtSC5DL gene in whiteflies could significantly reduce the egg production of whiteflies ( Figure 4 ).

[0035] Example 5: Effect of feeding on VIGS plants on egg production of adult Bemisia tabaci

[0036] According to the information of VIGS vector pTRV2, appropriate restriction sites were selected, and specific primers were designed in the ORF region of BtSC5DL gene to construct TRV2-BtSC5DL vector. The fragment of dsBtSC5DL was cloned using the designed forward fragment primer such as SEQ ID NO.10 and the reverse fragment primer such as SEQ ID NO.11. EcoR I and BamH I endonucleases were used for restriction digestion, and then the linearized vector was recovered and purified. In-fusion recombinase was used for homologous recombination experiment, and the specific dsRNA fragment was connected to the TRV2 vector and transferred into Escherichia coli. After PCR verification, TRV1 and TRV2-BtSC5DL vectors were obtained. Finally, the TRV1 and TRV2-BtSC5DL mixed in a ratio of 1:1 were injected into tomatoes using Agrobacterium transformation system. 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 BtSC5DL interference fragments in tomatoes, and it was used for the subsequent research on whitefly control.

[0037] Collect adult whiteflies within 8 hours of hatching, transfer 5 newly hatched female whiteflies to micro-insect cages, and then fix the micro-insect cages to VIGS tomato leaves to allow whiteflies to fully feed on the back of tomato leaves. Using tomatoes expressing dsEGFP as a control, the number of whiteflies laying eggs in each micro-insect cage was recorded on the 10th day. Figure 5 As shown, the egg production of adult Bemisia tabaci was significantly reduced after feeding on plants expressing dsBtSC5DL.

[0038] 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. An insect Δ7-sterol C5 desaturase, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A gene encoding an insect Δ7-sterol C5 desaturase, characterized in that: It encodes the insect Δ7-sterol C5 desaturase as described in claim 1.

3. The coding gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

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

5. Use of the coding gene as claimed in claim 2 or 3 in controlling Bemisia tabaci.

6. A gene fragment of a gene encoding a Δ7-sterol C5 desaturase from Bemisia tabaci, characterized in that: It is complementary to the gene segment described in SEQ ID NO.1 and is designed with the nucleotide sequence shown in SEQ ID NO.3 as the target.

7. A dsRNA for silencing the Δ7-sterol C5 desaturase gene of Bemisia tabaci, characterized in that: It is designed for the gene fragment as described in claim 6.

8. The dsRNA according to claim 7, characterized in that The dsRNA is obtained by using adult cDNA of Bemisia tabaci 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.

9. A method for silencing the gene of whitefly by inducing plant silencing through virus, characterized in that: This is achieved by introducing into the plant an expression vector for dsRNA specific to the gene encoding the Δ7-sterol C5 desaturase of Bemisia tabaci; Specifically, the forward fragment primer SEQ ID NO. 10 and the reverse fragment primer SEQ ID NO. 11 are used to introduce the expression vector of the specific dsRNA for the gene encoding the Δ7-sterol C5 desaturase of Bemisia tabaci.

10. Use of the insect Δ7-sterol C5 desaturase or its encoding gene according to claim 1, the gene fragment of the insect Δ7-sterol C5 desaturase gene according to claim 6, or the dsRNA according to claim 7 or 8 in the preparation of a product for controlling Bemisia tabaci, such as a dsRNA preparation for controlling Bemisia tabaci, or a product for virus-induced plants or transgenic plants.

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