Gene related to cadmium sensitivity of bemisia tabaci and application thereof

Abstract

The invention discloses a gene related to cadmium sensitivity of bemisia tabaci and application of the gene, and relates to genetic engineering and biological control technologies, the gene is BtHSP1 and / or BtCTSB2, the nucleotide sequence of the BtHSP1 gene is as shown in SEQ ID No.1, and the nucleotide sequence of the BtCTSB2 gene is as shown in SEQ ID No.2. The invention also provides dsRNA (double-stranded ribonucleic acid) for interfering the expression of the BtHSP1 gene and the BtCTSB2 gene. The death rate and the cell damage degree of the bemisia tabaci under cadmium stress are improved by inhibiting the expression of the bemisia tabaci BtHSP1 gene or promoting the expression of the bemisia tabaci BtCTSB2 gene. The BtHSP1 and BtCTSB2 genes have the effect of regulating and controlling the cadmium stress resistance of the bemisia tabaci and can be used as targets for preventing and controlling the bemisia tabaci under the cadmium stress, and a new idea is provided for preventing and controlling the bemisia tabaci in a cadmium-polluted area.

Description

Technical Field

[0001] This invention relates to a genetic engineering technique and a biological control technique for whiteflies, and more specifically, to a gene related to cadmium sensitivity in whiteflies and its application. Background Art

[0002] Cadmium pollution has become a global environmental problem, and its biotoxicity, persistence, and accumulation pose a serious threat to ecosystems and human health. Herbivorous insects, as important links in the food chain and food web of ecosystems, are important mediators for the migration and accumulation of cadmium in the environment.

[0003] The whitefly (Bemisia tabaci) is a serious global agricultural pest that feeds on the phloem sap of plants, directly sucking the sap. It transmits over 320 plant viruses, damaging more than 1,000 plant species and causing crop yield reductions of 30%-50%, sometimes even resulting in total crop failure (Brown et al., 1995; Perring, 2001; Wan et al., 2016; Xia et al., 2021). Whiteflies can accumulate cadmium from the soil within themselves through plants and transfer it to the next trophic level via the food chain (Khan et al., 2024), becoming an "invisible carrier" of cadmium pollution. This characteristic not only exacerbates the pollution risk of agricultural ecosystems but also highlights the unique role of whiteflies in cadmium pollution remediation.

[0004] Cadmium stress significantly altered the symbiotic microbiota of whiteflies, with a decrease in the abundance of beneficial bacteria *Rickettsia*. Cadmium stress also significantly reduced the hatching rate of whiteflies, impacting population development. Currently, cadmium pollution is widespread in vegetable-growing areas, and whiteflies are a prevalent and significant pest of vegetables, particularly in greenhouse vegetables. Control of whiteflies is difficult due to factors such as resistance development and generational overlap, and the mechanisms of whitefly resistance to cadmium stress remain unstudied.

[0005] In the existing technology, there are no reports of effective biological control of whiteflies under cadmium stress. Summary of the Invention

[0006] The first technical problem to be solved by this invention is to provide the application of genes related to cadmium sensitivity in the preparation of drugs for the control of whiteflies under cadmium stress. Genes related to cadmium sensitivity in whiteflies are screened out and used as targets. By interfering with their expression, the mortality rate of normal whiteflies under cadmium stress is increased, thereby achieving the purpose of prevention and control.

[0007] The second technical problem to be solved by the present invention is to provide a dsRNA that can interfere with the expression of genes related to cadmium sensitivity in whiteflies.

[0008] The third technical problem to be solved by this invention is to provide an application of dsRNA in the preparation of a drug for controlling whiteflies under cadmium stress. By inhibiting or promoting the expression of cadmium-sensitive genes in whiteflies, the degree of cellular abnormality is increased, thereby achieving biological control of whiteflies under cadmium stress.

[0009] The technical solution adopted by the present invention to solve the first technical problem is the application of genes related to cadmium sensitivity of whiteflies in the biological control of whiteflies under cadmium stress or in the preparation of drugs for the control of whiteflies under cadmium stress. The genes are BtHSP1 and / or BtCTSB2. The nucleotide sequence of the BtHSP1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the BtCTSB2 gene is shown in SEQ ID No. 2.

[0010] Furthermore, the whitefly is a Q-type whitefly.

[0011] Furthermore, this can be achieved through RNAi technology.

[0012] Furthermore, by targeting BtHSP1 and / or BtCTSB2, the mortality rate of whiteflies under cadmium stress was increased by inhibiting the expression of the BtHSP1 gene or promoting the expression of the BtCTSB2 gene.

[0013] The technical solution adopted by the present invention to solve the second technical problem is a dsRNA used to interfere with the expression of BtHSP1 and / or BtCTSB2 genes in whiteflies, wherein the genes are BtHSP1 and / or BtCTSB2, the nucleotide sequence of the BtHSP1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the BtCTSB2 gene is shown in SEQ ID No. 2.

[0014] Furthermore, the method for preparing the dsRNA includes:

[0015] Using the cloned full-length sequences of BtHSP1 and / or BtCTSB2 genes as templates, PCR amplification was performed using primers, and the amplification products were recovered from the gel and then used for in vitro synthesis of dsRNA.

[0016] The primers include:

[0017] The nucleotide sequence of the upstream primer of the BtHSP1 gene is shown in SEQ ID No. 19, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 20.

[0018] The nucleotide sequence of the upstream primer of the BtCTSB2 gene is shown in SEQ ID No. 25, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 26.

[0019] The technical solution adopted by the present invention to solve the third technical problem is the application of the aforementioned dsRNA in the preparation of a drug for controlling whiteflies under cadmium stress.

[0020] Furthermore, by inhibiting the expression of the BtHSP1 gene or promoting the expression of the BtCTSB2 gene in whiteflies, the mortality rate and degree of cell damage in whiteflies under cadmium stress were increased.

[0021] Furthermore, the whitefly is a Q-type whitefly.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention provides information on the correlation between the BtHSP1 and / or BtCTSB2 genes of the whitefly and its cadmium susceptibility. Decreased BtHSP1 gene abundance increases cadmium susceptibility, while decreased BtCTSB2 gene abundance decreases cadmium susceptibility and alleviates cadmium damage. Inhibiting BtHSP1 gene expression or promoting BtCTSB2 gene expression significantly increases the mortality rate of normal whiteflies under cadmium stress, as well as the mortality rate and degree of cell damage. Using BtHSP1 and / or BtCTSB2 genes as targets for biocontrol provides a new approach to whitefly control in cadmium-polluted areas and is also of significant importance for assessing environmental pollution, protecting agriculture, and protecting wild ecosystems. Attached Figure Description

[0024] Figure 1 The effect of cadmium on egg production and hatching rate of whiteflies;

[0025] Among them, A. the number of eggs laid by whiteflies within 7 days after 48 hours of treatment with different concentrations of cadmium; B. the hatching rate of eggs after 48 hours of treatment with different concentrations of cadmium;

[0026] CK: Control group, 30% sucrose and 5% yeast extract; LD: Low-dose group, 30% sucrose, 5% yeast extract and 4.12 μmol / L cadmium chloride; HD: High-dose group, 30% sucrose, 5% yeast extract and 37.93 μmol / L cadmium chloride.

[0027] Figure 2 The microbial composition of whiteflies treated with different cadmium concentrations.

[0028] Figure 3 The expression levels of the BtHSP1, BtHSP2, BtCTSB1, and BtCTSB2 genes in the whitefly were determined by interfering with their expression.

[0029] Figure 4Biological markers of whiteflies after interference with the BtHSP1, BtHSP2, BtCTSB1, and BtCTSB2 genes;

[0030] Among them, A. Adult mortality rate after 48 hours of disturbance; B. Egg production of surviving female adults after 48 hours of disturbance within 7 days; C. Sensitivity test of whiteflies surviving after 48 hours of disturbance to cadmium.

[0031] Figure 5 HE-stained sections of whiteflies surviving after cadmium (LC50) treatment with BtHSP1 and BtCTSB2 gene interference.

[0032] Among them, A. Whitefly tissue without cadmium treatment; B. Whitefly tissue that survived cadmium treatment for 48 hours and was fed with a feeding solution containing dsEGFP for 48 hours; C. Whitefly tissue that survived cadmium treatment for 48 hours and was fed with a feeding solution containing dsBtHSP1 for 48 hours; D. Whitefly tissue that survived cadmium treatment for 48 hours and was fed with a feeding solution containing dsBtCTSB2 for 48 hours. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to embodiments, but the embodiments of this invention are not limited thereto. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. It should be noted that, unless otherwise specified, the reagents and other materials used in this embodiment are all common commercially available products.

[0034] In this embodiment of the invention, the whiteflies were adult Mediterranean whiteflies (B. tabaciMED) collected in a chili pepper field in Changsha, Hunan Province, China in 2020. These whiteflies were reared in a greenhouse (temperature 26±2℃, 14 hours of light: 10 hours of darkness, relative humidity 80%) using chili pepper plants (the chili pepper variety "Zhongjiao 4") as hosts. The experimental B. tabaciMED population was confirmed by mitochondrial COI barcoding.

[0035] In this embodiment of the invention, statistical analyses of the reproductive capacity, egg hatching rate, gene expression, and adult mortality of whiteflies were performed using one-way ANOVA, and the Fisher's minimum significance test (LSD) was conducted in SPSS 23.0 (SPSS Inc., Chicago, Illinois, USA) (P < 0.05). All data are expressed as mean minus standard error (SE). Transcriptome data were analyzed on the Majorbio cloud platform (www.majorbio.com).

[0036] In this embodiment of the invention, the technical term "interference" refers to "blocking, inhibiting or reducing the expression level of a target gene".

[0037] Example 1: Biological manifestations of whiteflies at different cadmium concentrations

[0038] Three cadmium concentrations were selected: 0 (cadmium-free negative control, labeled CK), LC10 (concentration killing 10% of adult whiteflies, 4.12 μmol / L, labeled low dose or LD), and LC50 (concentration killing 50% of adult whiteflies, 37.93 μmol / L, labeled high dose or HD). Newly emerged adult whiteflies were exposed to these concentrations for 48 hours. Afterward, a portion of the surviving adult whiteflies were used to observe their developmental characteristics, while another portion was used for transcriptome sequencing. In the experiment used to observe developmental characteristics, 10 female whiteflies were randomly selected from each treatment and placed on healthy pepper plants in a growth chamber for 7 days. The total number of eggs laid and the number of eggs hatched were recorded. Each treatment was repeated 4 times. The results are as follows: Figure 1 As shown, cadmium treatment stimulated oviposition in whiteflies, with both the high-dose (HD) and low-dose (LD) groups showing a trend of increased oviposition compared to the control group (CK). Cadmium treatment also reduced the hatching rate of whitefly eggs, with the hatching rate of whitefly eggs treated with high-dose cadmium (HD) being significantly lower than that of the control group (CK). The abundance of the beneficial dominant endosymbiotic bacterium *Rickettsia* was as follows... Figure 2 As shown.

[0039] Example 2: Application of dsRNA in the cadmium susceptibility of whiteflies

[0040] Total RNA samples were extracted from the control group (CK), low-dose (LD), and high-dose (HD) treatments using the MJZol Total RNA Extraction Kit (Shanghai Meiji Biotechnology Co., Ltd.). Sequencing libraries composed of high-quality RNA samples (OD260 / 280 ≥ 1.8, OD260 / 230 ≥ 1.0) were constructed. Transcriptome sequencing and analysis were performed by Shanghai Meiji Biotechnology Co., Ltd.

[0041] The sequencing platform was Illumina NovaSeq 6000.

[0042] Transcriptome analysis was performed using the following software:

[0043] fastp(https: / / github.com / OpenGene / fastp)、

[0044] HiSat2(http: / / ccb.jhu.edu / software / hisat2 / index.shtml)、

[0045] RSEM(http: / / deweylab.github.io / RSEM / ),

[0046] DESeq2(http: / / bioconductor.org / packages / stats / bioc / DESeq2 / ),

[0047] Goatools(https: / / github.com / tanghaibao / GOatools)、

[0048] KOBAS (http: / / kobas.cbi.pku.edu.cn / home.do).

[0049] The reference genome is the whitefly (GCF 001854935.1).

[0050] Based on differential gene expression analysis among cadmium treatments, as well as GO and KEGG functional annotation and enrichment analysis, the genes BtHSP1, BtHSP2, BtCTSB1, and BtCTSB2 were identified as potentially associated with the whitefly's response to cadmium stress. These four genes were selected for RNA interference. Primers were designed using Primer Premier 5.0 and are listed in Table 1. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0051] Real-time quantitative PCR analysis was performed using HiScript Q RT SuperMix for qPCR (+gDNAwiper) to synthesize cDNA from total RNA samples. Real-time quantitative PCR was conducted using an ABI 7300 system (Applied Biosystems), with a reaction volume of 20 μL. Each reaction contained 0.8 μL of each primer (primer sequences are shown in Table 1), 0.4 μL of 50×ROX reference dye 1 (Nanjing Novizan Biotechnology Co., Ltd., Nanjing, China), 2 μL of cDNA, 6 μL of ddH2O, and 10 μL of 2×ChamQ SYBR Color qPCRMasterMix (Nanjing Novizan Biotechnology Co., Ltd., Nanjing, China).

[0052] The real-time quantitative PCR amplification program was as follows: 95℃ for 5 minutes, followed by 40 cycles, each cycle consisting of 95℃ for 5 seconds, 55℃ for 30 seconds, and 72℃ for 40 seconds. The amplification efficiency of all gene-specific primers was 90%-110%. The reference gene was 60S ribosomal protein L29 (RPL29) (GenBank accession number: EE596314).

[0053] Table 1

[0054]

[0055]

[0056] dsRNA synthesis: Total RNA was extracted from newly emerged adult whiteflies, and cDNA was synthesized by reverse transcription. PCR was performed using BtHSP1 and / or BtCTSB2 gene interference primers. The PCR products were recovered by gel extraction and sequenced to determine the nucleic acid sequence. The gel-extracted products were then used as templates for dsRNA synthesis. dsRNA was synthesized according to the operating procedures of the T7 RiboMAX Express RNAi kit. The synthesized dsRNA was detected and stored at -20℃ for later use.

[0057] RNA interference experiment: The initially synthesized dsRNA was diluted to 500 ng / μL with a solvent containing 5% yeast extract and 30% sucrose. 200 μL of the above feeding solution containing 500 ng / μL of target gene dsRNA was placed in the feeding device. 120 newly emerged adult whiteflies (male-to-female ratio 1:1) were randomly selected and placed in a feeding device. After 48 hours of feeding, surviving whiteflies were randomly selected for the following experiments: (i) 10 female whiteflies were randomly selected and transferred to the leaves of healthy pepper plants. The total number of eggs laid by the 10 female whiteflies was recorded after 7 days. Each treatment was repeated four times. (ii) Total RNA was extracted from surviving adult whiteflies after interference. cDNA was synthesized by reverse transcription and used as a template for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect the expression of the target gene. Each treatment was repeated four times. (iii) Adult whiteflies that survived interference were randomly collected and treated with a high dose (HD) of cadmium. The cadmium-free treatment was used as a blank control (CK) to monitor their sensitivity to cadmium. The mortality rate of the whiteflies was recorded after 48 hours.

[0058] The results are as follows Figure 3 and Figure 4As shown, after feeding whiteflies with dsRNA, the expression levels of BtHSP1, BtHSP2, BtCTSB1, and BtCTSB2 genes were significantly lower than those in the control group. Successful interference with BtHSP1 and / or BtCTSB2 genes stimulated oviposition in female whiteflies. After feeding whiteflies with dsBtHSP1 and / or dsBtCTSB2 for 48 hours, the mortality rate of adults after BtHSP1 gene interference was significantly higher than that in the control group, and the mortality rate of adult whiteflies under cadmium stress was also significantly increased subsequently. The mortality rate of adults after BtCTSB2 gene interference did not change significantly compared to the control group, but the mortality rate of adult whiteflies under cadmium stress was significantly reduced subsequently. This indicates that interfering with (or inhibiting) the expression of the BtHSP1 gene or promoting the expression of the BtCTSB2 gene can increase the mortality rate of normal whiteflies under cadmium stress.

[0059] Example 3: Application of dsRNA in Cadmium Damage Caused by Whiteflies

[0060] The synthesis of dsRNA is the same as in Example 2.

[0061] Preparation of cadmium-stressed whiteflies: Newly emerged adult whiteflies were randomly collected and treated with a feeding solution containing a high dose (HD) of cadmium, with a cadmium-free feeding solution as a control. After 48 hours of treatment, surviving whiteflies were collected for later use. Each treatment was repeated four times.

[0062] HE staining experiment: The initially synthesized dsRNA was diluted to 500 ng / μL with a solvent containing 5% yeast extract and 30% sucrose. 200 μL of the above feeding solution containing 500 ng / μL of the target gene dsRNA was placed in a feeding device. Adult whiteflies subjected to cadmium stress were placed in the feeding device, with whiteflies fed dsEGFP as a control. After 48 hours of feeding, surviving whiteflies were randomly selected for hematoxylin-eosin (HE) staining, and tissue sections were observed. Results are as follows... Figure 5 Compared with the control group, the study found that feeding on cadmium-containing feed caused abnormal tissue cells in whiteflies. Interference with the BtHSP1 gene resulted in more severe abnormalities in the whitefly tissue cells; however, interference with the BtCTSB2 gene improved these abnormalities. This indicates that interfering with (or inhibiting) the expression of the BtHSP1 gene or promoting the expression of the BtCTSB2 gene can increase the degree of cell damage in whiteflies under cadmium stress, thereby increasing mortality.

[0063] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of genes related to cadmium sensitivity in whiteflies in the biocontrol of whiteflies under cadmium stress or in the preparation of drugs for the control of whiteflies under cadmium stress, characterized in that, The genes are BtHSP1 and / or BtCTSB2, the nucleotide sequence of the BtHSP1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the BtCTSB2 gene is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The whitefly in question is the Q-type whitefly.

3. The application according to claim 1 or 2, characterized in that, This is achieved through RNAi technology.

4. A dsRNA for interfering with the expression of BtHSP1 and / or BtCTSB2 genes in whiteflies, characterized in that, The genes are BtHSP1 and / or BtCTSB2, the nucleotide sequence of the BtHSP1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the BtCTSB2 gene is shown in SEQ ID No.

2.

5. The dsRNA according to claim 4, characterized in that, The method for preparing the dsRNA includes: Using the cloned full-length sequences of BtHSP1 and / or BtCTSB2 genes as templates, PCR amplification was performed using primers, and the amplification products were recovered from the gel and then used for in vitro synthesis of dsRNA. The primers include: The nucleotide sequence of the upstream primer of the BtHSP1 gene is shown in SEQ ID No. 19, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.

20. The nucleotide sequence of the upstream primer of the BtCTSB2 gene is shown in SEQ ID No. 25, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.

26.

6. The use of the dsRNA according to any one of claims 3-5 in the preparation of a drug for controlling whiteflies under cadmium stress.

7. The application according to claim 6, characterized in that, By inhibiting the expression of the BtHSP1 gene or promoting the expression of the BtCTSB2 gene in whiteflies, the mortality rate and degree of cell damage in whiteflies under cadmium stress were increased.

8. The application according to claim 6 or 7, characterized in that, The whitefly in question is the Q-type whitefly.

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