Application of brown planthopper target sequence Nl14 in improving brown planthopper resistance of rice and method

Abstract

The invention provides an application of a brown planthopper target sequence Nl14 in improving resistance of rice to brown planthopper and a method thereof, the rice can silence the function of the brown planthopper target sequence Nl14 shown in a nucleotide sequence such as SEQ ID NO.2 by utilizing a plant-mediated RNA interference technology, and the survival rate, growth and development speed and reproductive capacity of brown planthopper on the rice are remarkably reduced; therefore, the resistance of the rice to the brown planthopper is improved. The method is environment-friendly and economical, has a lasting insect-resistant effect, and is expected to provide powerful guarantee for sustainable production of rice.

Description

Technical Field

[0001] The invention belongs to the application field of agricultural science and technology, and specifically relates to the application and method of a brown planthopper target sequence Nl14 in improving the resistance of rice to the brown planthopper. Background Art

[0002] RNA interference (RNAi) is a form of gene silencing triggered by double-stranded RNA (dsRNA) in all eukaryotic cells. It has been widely studied in insects and has important practical significance for the development of green pest control strategies. The current strategy of controlling insects by synthesizing dsRNA in vitro is affected by many unfavorable factors, such as rapid degradation of dsRNA, low efficiency of dsRNA uptake / processing, and ineffective RNAi, which has limited its application in many pests. Brown planthopper (Nilaparvatalugens, BPH) is a major pest in rice production, posing a serious threat to global rice production. Traditional chemical pesticide control methods are not only costly, but also prone to the development of pest resistance. Therefore, the development of rice varieties resistant to brown planthoppers has become an urgent need. Plant-mediated RNAi is an emerging crop protection technology in which transgenic plants are used to express dsRNA sequences designed for pest target genes, thereby triggering RNAi in target pests and inducing adverse reactions in pests to achieve the purpose of controlling pests. At present, there are very few reports on the use of RNAi technology for rice insect resistance. Summary of the invention

[0003] In order to solve the above defects and shortcomings in the prior art, the present invention provides an application and method of a brown planthopper target sequence Nl14 in improving the resistance of rice to brown planthoppers, thereby improving the resistance of rice to brown planthoppers.

[0004] To solve the above technical problems:

[0005] The first object of the present invention is to provide a use of a brown planthopper target sequence N114 having a nucleotide sequence as shown in SEQ ID NO.2 in improving the resistance of rice to brown planthoppers.

[0006] In the present invention, by screening the RNAi effects of 15 candidate brown planthopper genes, 14-3-3e (Nl14), a member of the 14-3-3 protein family, was identified as a potential plant RNAi target, and this finding was applied to transgenic rice to enhance its resistance to brown planthoppers.

[0007] The second object of the present invention is to provide an expression vector for expressing a nucleotide sequence such as the target sequence Nl14 of SEQ ID NO.2.

[0008] The third object of the present invention is to provide a cell for expressing a nucleotide sequence such as the brown planthopper target sequence Nl14 of SEQ ID NO.2 or an expression vector for expressing a nucleotide sequence such as the brown planthopper target sequence Nl14 of SEQ ID NO.2.

[0009] Furthermore, the method for improving rice resistance to brown planthoppers is plant-mediated RNA interference technology, which specifically includes the following steps:

[0010] Constructing an RNAi expression frame: using the full-length nucleotide sequence of brown planthopper Nl14 as a template, amplifying and constructing an RNAi expression frame of the target sequence, wherein the RNAi expression frame includes a sense fragment, an antisense fragment and a partial fragment of an RNAi vector;

[0011] Construction of RNAi expression vector: insert the RNAi expression frame into the rice RNAi vector pBWA(V)HS-RNAI by enzyme digestion and ligation; then transform into Escherichia coli Top10 competent cells, select positive clones, and extract plasmids for rice genetic transformation;

[0012] Agrobacterium-mediated genetic transformation of rice: The above plasmid is transformed into Agrobacterium EHA105 by electroporation, and then the bacterial solution is used to infect rice callus tissue, and the positive plants are screened as brown planthopper-resistant plants.

[0013] The fourth object of the present invention is to provide a method for improving the resistance of rice to brown planthoppers, using plant-mediated RNA interference technology to enable rice to silence the function of the brown planthopper target sequence Nl14 shown in SEQ ID NO.2, thereby reducing the survival ability of brown planthoppers. The specific process is as follows:

[0014] Constructing an RNAi expression frame: using the full-length nucleotide sequence of brown planthopper Nl14 as a template, amplifying and constructing an RNAi expression frame of the target sequence, wherein the RNAi expression frame includes a sense fragment, an antisense fragment and a partial fragment of an RNAi vector;

[0015] Construction of RNAi expression vector: insert the RNAi expression frame into the rice RNAi vector pBWA(V)HS-RNAI by enzyme digestion and ligation; then transform into Escherichia coli Top10 competent cells, select positive clones, and extract plasmids for rice genetic transformation;

[0016] Agrobacterium-mediated genetic transformation of rice: The above plasmid is transformed into Agrobacterium EHA105 by electroporation, and then the bacterial solution is used to infect rice callus tissue, and the positive plants are screened as brown planthopper-resistant plants.

[0017] Brown planthopper resistance verification: Nl14RNAi transgenic rice was compared with wild-type rice (WT) in an experiment, inoculated with the same number of brown planthoppers, and the growth, development and reproduction of brown planthoppers on these rice plants were observed and recorded. By comparing the degree of damage of the two groups of rice, the ability of transgenic rice to resist brown planthoppers was evaluated.

[0018] The fifth object of the present invention is to provide a use of a brown planthopper target sequence Nl14 having a nucleotide sequence as shown in SEQ ID NO.2 in the preparation of a brown planthopper control drug.

[0019] The beneficial technical effects achieved by the present invention are as follows: the application and method of a brown planthopper target sequence Nl14 provided by the present invention in improving the resistance of rice to brown planthoppers, using plant-mediated RNA interference technology to silence the function of the brown planthopper target sequence Nl14 of the rice nucleotide sequence as shown in SEQID NO.2, significantly reducing the survival rate, growth and development speed and reproductive capacity of brown planthoppers on rice, thereby improving the resistance of rice to brown planthoppers. This method is not only environmentally friendly and economical, but also has a lasting insect-resistant effect, and is expected to provide a strong guarantee for the sustainable production of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The fitness performance of brown planthopper on rice after in vitro injection of Nl14 dsRNA (dsNl14) of the present invention; wherein 1A is the effect on the survival rate of brown planthopper nymphs, 1B is the effect on the egg laying amount of female adults, 1C is the effect on the hatching rate of nymphs, and different lowercase letters indicate that there is a significant difference between the treatment group and the control group;

[0021] Figure 2 This is the effect of brown planthopper feeding on Nl14 RNAi transgenic rice on the transcription level of target gene Nl14 mRNA in brown planthopper. Different lowercase letters indicate significant differences between the treatment group and the control group.

[0022] Figure 3 The statistics of the anti-brown planthopper property of Nl14 RNAi transgenic rice of the present invention are shown in FIG. 3A, wherein 3B is the effect on the weight of female adults of brown planthoppers, 3C is the effect on the survival rate of nymphs of brown planthoppers, and different lowercase letters indicate significant differences between the treatment group and the control group.

[0023] Figure 4 The growth and development indexes of the Nl14 RNAi transgenic rice of the present invention are detected and statistically analyzed, wherein 4A is the effect on plant height, 4B is the effect on tillering number, and 4C is the effect on 1000-grain weight. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments and drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0025] The primer sequences used in the examples of this application are shown in Table 1.

[0026] Table 1

[0027]

[0028] Example 1 Effect of in vitro injection of Nl14 dsRNA on insect resistance of brown planthopper on rice

[0029] In vitro Nl14 dsRNA synthesis: Nl14 dsRNA and dsGFP were synthesized using dsNl14 and dsGFP primers with T7 promoter (sequences are shown in SEQ ID NO.9-SEQ ID NO.12 in Table 1) according to the instructions of the MEGA Script T7 Kit (Ambion, Austin, TX, USA), and about 20 nL Nl14 dsRNA (10 ng / nL) was injected into the body of brown planthoppers using a TransferMan NK2 microinjector (Eppendorf, Germany), and the same amount of dsGFP was injected as a control.

[0030] (1) Determination of the survival rate of brown planthoppers: Select the third-instar nymphs of the same batch and inject them with Nl14 dsRNA and dsGFP. One day later, select the more vigorous brown planthoppers for the test at a rate of 20 per group. The specific operation is as follows: Put a cylindrical glass tube (8 cm high, 4 cm in diameter, with 48 small holes with a diameter of 0.88 mm evenly distributed on the wall) at the base of the stem of Xiushui 11 rice (from Jiaxing Academy of Agricultural Sciences), introduce 20 third-instar nymphs injected with Nl14 dsRNA, and seal the top with a sponge. Each group of experiments was observed for 10 consecutive days. The number of survival and death of brown planthoppers was counted at a fixed time every day (dead individuals were removed after the statistics), and the survival rate of the nymphs was calculated. The results are as follows: Figure 1 As shown in Figure 1A: The survival rate of brown planthoppers showed a downward trend after injection of Nl14 dsRNA, and was significantly lower than that of the dsGFP-injected group and the non-injected group at 4 to 10 days after injection.

[0031] (2) Determination of the egg laying amount and egg hatching rate of brown planthoppers: The fifth-instar nymphs of the same batch were selected and injected with Nl14 dsRNA and dsGFP. One day later, a female adult of brown planthopper with strong vitality was selected to pair with two male insects that were not injected with Nl14 dsRNA to feed on rice. Seven days later, the number of eggs laid in the rice sheaths was counted under a microscope, and the average daily egg laying of a single female adult was calculated. For the remaining treatment groups and control groups of the same batch, the number of newly hatched nymphs was counted every day 7 days after egg laying until no new nymphs were found. The number of unhatched eggs in the sheaths was counted under a microscope, and the nymph hatching rate was calculated. A total of 9 to 15 biological replicates were performed on the same batch. Each experiment was repeated three times. The results are as follows. Figure 1 As shown in Figures 1B and 1C: The egg laying amount and egg hatching rate of brown planthoppers injected with Nl14 dsRNA were significantly lower than those of the dsGFP injection group and the non-injection group, indicating that the target sequence Nl14 plays an important role in the egg laying and egg hatching process of brown planthoppers, and silencing this gene greatly reduces its reproductive capacity.

[0032] The above results indicate that silencing the target sequence Nl14 gene in the insect body can affect the growth, development and reproductive ability of brown planthoppers.

[0033] Example 2 Expression of brown planthopper target sequence N114 dsRNA in rice

[0034] Based on the results of Example 1, we speculate that the expression of Nl14 dsRNA sequence by host rice can trigger RNAi of brown planthoppers, thereby achieving the purpose of controlling pests. Therefore, we cultivated transgenic rice that can stably express Nl14 dsRNA, and obtained T2 generation single copy homozygous positive transgenic rice plant lines (irNl14-3 and irNl14-11) through multiple generations of cultivation, and measured the effect of brown planthoppers feeding on transgenic rice irNl14 on the transcription level of target gene Nl14 mRNA in their own bodies. The detailed process is as follows:

[0035] (1) Gene cloning and RNAi expression vector construction:

[0036] A specific region of Nl14 (200 bp in length) was selected as the target gene and constructed into the RNAi vector pBWA(V)HS-RNAI: the full-length nucleotide sequence of Nl14 cloned in our laboratory (nucleotide sequence as shown in SEQ ID NO.1) was used as a template, and primers (primer sequences are shown in SEQ ID NO.3-SEQ ID NO.8 in Table 1) were designed to clone the sense and antisense fragments of the target gene (nucleotide sequence as shown in SEQ ID NO.2) and the partial sequence fragments of the RNAi vector as the RNAi expression frame, and the RNAi expression frame was inserted into the rice RNAi vector pBWA(V)HS-RNAI by restriction ligation. After that, the competent cells of Escherichia coli Top10 were transformed, and the positive clones with the inserted vector were selected by colony PCR identification, and the plasmids were extracted for rice genetic transformation.

[0037] (2) Agrobacterium-mediated genetic transformation of rice

[0038] First, the plasmid was transformed into Agrobacterium EHA105 by electroporation, and then the callus induced by Nipponbare seeds was infected with the bacterial solution. Then, resistance screening of the callus and induction of differentiation and rooting of the resistant callus were carried out, and finally, hardening and transplanting of the transgenic rice seedlings were carried out.

[0039] (3) Identification of positive plants of Nl14 RNAi transgenic rice in the T0 generation

[0040] DNA from rice leaves was extracted according to the instructions of RNApure Fastplant Kit (CW0598S), and then PCR amplification was performed using universal primers on the RNAi vector (primer sequences are shown in SEQ ID NO.13 and SEQ ID NO.14 in Table 1) to detect whether both the forward and reverse fragments have been transferred into the plant. Through screening and identification, a stable genetic Nl14 RNAi transgenic rice line was obtained.

[0041] (4) Transcription level of target sequence Nl14 mRNA in Nl14 RNAi transgenic rice. T2 generation single-copy homozygous positive transgenic rice plant lines (irNl14-3 and irNl14-11) were obtained through multiple generations of cultivation. The effect of brown planthopper feeding on transgenic rice irNl14 on the transcription level of target gene Nl14 mRNA in itself was determined: 40 third-instar nymphs of brown planthoppers were introduced into five-leaf seedlings of irNl14 transgenic rice of T3 generation, and wild-type WT five-leaf seedlings were used as controls. After brown planthoppers fed for 3 and 6 days, RNApure Fast Tissue&Cell Kit was used to extract RNA from the above two rice species. After reverse transcription, fluorescence quantitative PCR (primer sequences are shown in SEQ ID NO.15-SEQ ID NO.18 in Table 1) was used to detect the relative expression level of Nl14 mRNA in brown planthoppers (2 -ΔCt The internal reference gene was ef2. The biological replicates were performed three times, and each biological replicate consisted of 10 brown planthoppers. Figure 2 As shown in the figure: After feeding on irNl14 transgenic rice for 3 and 6 days, the relative expression level of Nl14 mRNA in brown planthoppers was significantly lower than that in the WT control group, indicating that brown planthoppers feeding on irNl14 transgenic rice can significantly reduce the mRNA transcription level of the target gene Nl14 in their own bodies, achieving the effect of triggering RNAi in brown planthoppers.

[0042] Example 3 Detection of resistance of N114 RNAi transgenic rice to brown planthopper

[0043] (1) Weight measurement of female brown planthoppers: 40-day-old irNl14 transgenic rice T3 seedlings with consistent growth were selected and introduced into 20 newly hatched brown planthopper nymphs, while wild-type WT seedlings were used as controls. One day after their eclosion, 5 female adults were randomly selected from each treatment and weighed (accurate to 0.1 mg), with a total of 10 biological replicates.

[0044] (2) Determination of the survival rate of brown planthoppers: 40-day-old irNl14 transgenic rice T3 seedlings with consistent growth were selected and introduced with 30 2-year-old brown planthopper nymphs. The number of brown planthoppers on the two varieties was counted for 12 consecutive days.

[0045] (3) Determination of the egg-laying capacity of female adults of brown planthopper: 3rd instar nymphs fed on irNl14 transgenic rice until the 4th day after eclosion, and 10 female adults with eggs were selected and placed on new irNl14 transgenic rice seedlings to continue feeding. After 24 hours, the number of eggs in the leaf sheaths at the feeding site was counted under a microscope, and the average daily egg-laying capacity of a single female adult was calculated. A total of 9-15 biological replicates were performed in the same batch, and each experiment was repeated three times.

[0046] The results are as follows Figure 3 As shown: After brown planthoppers feed on irNl14 transgenic rice, their survival rate, female adult weight and egg laying amount can be significantly reduced, indicating that by constructing Nl14 dsRNA transgenic rice to silence the Nl14 gene in brown planthoppers, it can induce adverse reactions in pests and achieve the purpose of pest control.

[0047] Example 4 Evaluation of the Phenotype of N114 RNAi Transgenic Rice

[0048] The transgenic rice cultivated in Example 2 was selected, and the seeds of the rice strain with full grains were shaded and moisturized for germination. Then, the seedlings with uniform germination were selected and transferred to the cement pool of the glass greenhouse for cultivation until the heading stage, and the plant height was counted. When the seeds reached the maturity stage, the number of tillers and the thousand-grain weight were measured. The results are as follows Figure 4 As shown: These phenotypes between transgenic rice expressing Nl14dsRNA and WT did not show significant differences, indicating that expressing Nl14dsRNA in rice has no adverse effects on the phenotype of rice.

[0049] The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. The nucleotide sequence is shown as the brown planthopper target sequence in SEQ ID NO.2 Nl14 Application in improving rice resistance to brown planthopper.

2. An expression vector, characterized in that: The expression vector is used to express a nucleotide sequence such as the target sequence of SEQ ID NO.2 Nl14 .

3. A cell, characterized in that: The cell is used to express the nucleotide sequence of the brown planthopper target sequence of SEQ ID NO.2 Nl14 Or an expression vector, the expression vector is used to express the nucleotide sequence as shown in SEQ ID NO.2 brown planthopper target sequence Nl14 expression vector.

4. The use according to claim 1, characterized in that: The method to improve rice resistance to brown planthoppers is plant-mediated RNA interference technology.

5. The use according to claim 4, characterized in that: Plant-mediated RNA interference technology specifically includes the following steps: Construction of RNAi expression cassette: Brown planthopper Nl14 The full-length nucleotide sequence is used as a template to amplify and construct an RNAi expression frame of the target sequence, wherein the RNAi expression frame includes a sense fragment, an antisense fragment and a partial fragment of an RNAi vector; Construction of RNAi expression vector: Insert the RNAi expression frame into the rice RNAi vector pBWA(V)HS-RNAI by enzyme cutting and ligation; then transform into Escherichia coli Top10 competent cells, select positive clones, and extract plasmids for rice genetic transformation; Agrobacterium-mediated genetic transformation of rice: The above plasmid is transformed into Agrobacterium EHA105 by electroporation, and then the bacterial solution is used to infect rice callus tissue, and the positive plants are screened as brown planthopper-resistant plants.

6. A method for improving rice resistance to brown planthoppers, characterized in that: Using RNA interference technology, the rice nucleotide sequence is silenced as shown in SEQ ID NO.2 as the target sequence of brown planthopper Nl14 function, thereby reducing the survival rate of brown planthoppers.

7. The method for improving rice resistance to brown planthopper according to claim 6, characterized in that: The specific process is as follows: Construction of RNAi expression cassette: Brown planthopper Nl14 The full-length nucleotide sequence is used as a template to amplify and construct an RNAi expression frame of the target sequence, wherein the RNAi expression frame includes a sense fragment, an antisense fragment and a partial fragment of an RNAi vector; Construction of RNAi expression vector: Insert the RNAi expression frame into the rice RNAi vector pBWA(V)HS-RNAI by enzyme cutting and ligation; then transform into Escherichia coli Top10 competent cells, select positive clones, and extract plasmids for rice genetic transformation; Agrobacterium-mediated genetic transformation of rice: The above plasmid is transformed into Agrobacterium EHA105 by electroporation, and then the bacterial solution is used to infect rice callus tissue, and the positive plants are screened as brown planthopper-resistant plants.

8. The nucleotide sequence of the brown planthopper target sequence shown in SEQ ID NO. 2 Nl14 Application in the preparation of drugs for controlling brown planthoppers.

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