Application and method of rice gene OsPIE in improving rice insect resistance

Abstract

The application discloses application and method of rice gene OsPIE in improving rice insect resistance. The gene is a DNA sequence of SEQ ID No. 1, and encodes a protein with 576 amino acid residues. Research finds that the gene is closely related to rice insect resistance, and overexpression of the OsPIE gene in rice can effectively improve direct resistance of the rice to brown planthopper. The application will be widely applied in crop pest control and insect-resistant crop breeding, especially in rice pest control and insect-resistant rice breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant insect resistance, and in particular relates to the application and method of the rice gene OsPIE in improving the insect resistance of rice. Background Technology

[0002] Brown planthoppers (Nilaparvata lugens) are a major pest of rice, primarily harming the crop through sap sucking, oviposition damage, and virus transmission. Due to the lack of highly effective resistant varieties and green control products, current control of brown planthoppers mainly relies on chemical pesticides. This not only threatens rice quality and safety but also leads to a series of problems, including resurgences of brown planthoppers, imbalances in the paddy field ecosystem, weakened natural pest control capabilities, and agricultural environmental pollution, seriously impacting rice production safety in my country. Therefore, developing green control technologies for brown planthoppers is not only an urgent requirement for ensuring my country's food security but also crucial for maintaining agricultural ecological security and achieving sustainable agricultural development. Discovering insect-resistant gene resources and cultivating insect-resistant varieties are important components of achieving green control of rice pests.

[0003] When plants are attacked by herbivorous insects, they rapidly initiate their own induced defense response. This induced defense begins with the recognition of molecular patterns related to herbivorous insects and damage by pattern recognition receptors on the cell membrane surface, subsequently triggering changes in cell membrane surface potential and intracellular calcium levels. 2+ Early signaling events include changes in plant concentration, reactive oxygen species bursts, activation of mitogen-mediated protein kinases, and alterations in hormone signal transduction networks. These signals further trigger a rearrangement of the transcriptome, proteome, and metabolome within the plant, producing direct or indirect defense compounds that enable the plant to exhibit insect resistance. In recent years, with in-depth research, the molecular and chemical mechanisms of plant-induced defense have been continuously revealed, and new insect-resistant genes have been discovered, providing a theoretical basis and genetic resources for the breeding of resistant varieties. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing an application and method for improving the insect resistance of rice using the rice gene OsPIE. Overexpression of the OsPIE gene in rice can enhance its insect resistance. This improvement in insect resistance can be achieved through transgenic technology to overexpress the OsPIE gene in rice.

[0005] The objective of this invention is achieved through the following technical solution: the application of the rice gene OsPIE in improving the insect resistance of rice, wherein the application is to increase the expression level of the rice gene OsPIE, and the insect resistance is to improve the direct resistance of rice to brown planthopper.

[0006] Furthermore, the rice gene OsPIE has the DNA sequence of SEQ ID NO.1, and the protein it encodes has the amino acid sequence of SEQ ID NO.2.

[0007] Furthermore, the rice gene OsPIE enhances rice insect resistance by increasing the expression level of the rice gene OsPIE to increase the synthesis of jasmonic acid, jasmonic acid-isoleucine and abscisic acid, and reduce the oviposition, hatching rate, feeding / oviposition selectivity and feeding amount of brown planthoppers, thereby improving rice insect resistance.

[0008] The present invention also provides a method for improving the insect resistance of rice by increasing the expression level of the rice gene OsPIE, thereby improving the resistance of rice to brown planthopper.

[0009] Furthermore, the specific steps to increase the expression level of the rice gene OsPIE are as follows:

[0010] (1) The full-length ORF of the OsPIE gene was amplified using specific primers OsPIE-F and OsPIE-R. The product was digested with enzymes, purified, and then ligated into a plant expression vector. The vector was then transformed into Agrobacterium.

[0011] (2) Rice callus was infected with Agrobacterium with a vector. After the infected callus was screened for resistance, differentiated, rooted and hardened, T0 generation transgenic rice was obtained. The expression level of OsPIE gene in the T0 generation transgenic rice was increased.

[0012] Furthermore, after two generations of self-pollination of T0 generation transgenic rice, T2 generation seeds were screened by GUS staining, analyzed by PCR, and identified by Southern blot, resulting in two homozygous transgenic lines with single-copy insertion; the expression level of the OsPIE gene was increased in the homozygous transgenic lines.

[0013] This invention also provides a method for preparing transgenic rice overexpressing OsPIE, comprising:

[0014] (1) The full-length ORF of the OsPIE gene was amplified using specific primers OsPIE-F and OsPIE-R. The product was digested with enzymes, purified, and then ligated into a plant expression vector. The vector was then transformed into Agrobacterium.

[0015] (2) Rice callus was infected with Agrobacterium with a vector. After the infected callus was screened for resistance, differentiated, rooted and hardened, T0 generation transgenic rice was obtained, which is OsPIE overexpressing transgenic rice.

[0016] Furthermore, after two generations of self-pollination of T0 generation transgenic rice, the T2 generation seeds were screened by GUS staining, analyzed by PCR, and identified by Southern blot, resulting in two homozygous transgenic lines with single-copy insertion.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] Overexpression of the OsPIE gene in rice significantly enhances its direct resistance to brown planthoppers, validating the application of the OsPIE gene in improving rice insect resistance. This study provides crucial guidance and promotes insect-resistant rice breeding, particularly the development of brown planthopper-resistant rice varieties. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the OsPIE overexpression genetic transformation vector.

[0021] Figure 2 This represents the expression level of the OsPIE gene in rice 8 hours after brown planthopper infestation (mean + standard error, n = 5). WT: parental rice; V7 and V13: OsPIE overexpressing rice lines. An asterisk indicates a significant difference between the overexpressing lines and the parental rice (**: P < 0.01; Student's t-test).

[0022] Figure 3 The values ​​represent the contents of jasmonic acid (A), jasmonic acid-isoleucine (B), and abscisic acid (C) in rice leaf sheaths after brown planthopper infestation (mean + standard error, n = 5). WT: parental rice; V7 and V13: OsPIE overexpressing rice lines. An asterisk indicates a significant difference between the overexpressing lines and the parental rice (*: P < 0.05; **: P < 0.01; Student's t-test).

[0023] Figure 4 This represents the oviposition rate of a single female adult brown planthopper on different rice varieties (mean + standard error, n = 30). WT: parental rice; V7 and V13: OsPIE overexpressing rice lines. An asterisk indicates a significant difference between the overexpressing lines and the parental rice (*: P < 0.05; Student's t-test).

[0024] Figure 5The hatching rate of brown planthopper eggs on different rice varieties is (mean + standard error, n = 30). WT: parental rice; V7 and V13: OsPIE overexpressing rice lines. An asterisk indicates a significant difference between the overexpressing lines and the parental rice (**: P < 0.01; Student's t-test).

[0025] Figure 6 The ratio of female brown planthoppers on different rice varieties to oviposition rate (mean + standard error, n = 10). WT: parental rice; V7 and V13: OsPIE overexpressing rice lines. An asterisk indicates a significant difference between the overexpressing lines and the parental rice (*: P < 0.05; **: P < 0.01; Student's t-test).

[0026] Figure 7 The honeydew secretion of female brown planthoppers after feeding on different types of rice (mean + standard error, n = 20). WT: parental rice; V7 and V13: OsPIE overexpressing rice lines. An asterisk indicates a significant difference between the overexpressing lines and the parental rice (*: P < 0.05; Student's t-test). Detailed Implementation

[0027] This invention utilizes experimental techniques such as PCR and Agrobacterium-mediated transformation, combined with insect biological assays, to study the role of target genes in improving the insect resistance of rice. The results demonstrate that overexpression of OsPIE in rice can enhance resistance to brown planthoppers.

[0028] The present invention will be further illustrated below with reference to specific embodiments. However, it should be noted that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0029] Example 1: Creation of OsPIE-overexpressing transgenic rice

[0030] 1) The full-length ORF of the OsPIE gene was amplified using specific primers OsPIE-F and OsPIE-R. The product was digested with enzymes, purified, and then ligated into the plant expression vector pCAMBIA-1301 (e.g., ...). Figure 1 As shown in the figure, the above fusion vector was transferred into Agrobacterium strain EHA105 by electroporation.

[0031] The primer sequences are as follows (underlined are restriction enzyme sites):

[0032] OsPIE-F: 5'-TGC TCTAGA ATGGCCGCCCGCTTCGG-3'

[0033] OsPIE-R: 5'-AAA GGATCC CTACCCCGGAAGCGAA-3'

[0034] 2) Rice callus tissue was infected with Agrobacterium containing the above-mentioned vector. After resistance screening, differentiation, rooting and hardening, T0 generation transgenic rice overexpressing OsPIE was obtained from the infected callus tissue.

[0035] 3) The T0 generation of transgenic strains overexpressing OsPIE was analyzed, and the two lines with the highest OsPIE expression levels were selected (named V7 and V13). The OsPIE gene expression levels in V7 and V13 are shown in [reference needed]. Figure 2 The seeds were self-pollinated, and the resulting T2 generation seeds were screened by GUS staining, analyzed by PCR, and identified by Southern blot, yielding two homozygous transgenic lines with single-copy insertion.

[0036] Example 2: Overexpression of OsPIE in rice significantly increased the synthesis of jasmonic acid, jasmonic acid-isoleucine and abscisic acid.

[0037] The experiment was conducted in an artificial climate chamber (28±2℃, relative humidity 70-80%, 14h light). Four-week-old rice plants (WT, V7, and V13) were individually transplanted into small plastic cups (diameter × height: 8cm × 10cm) containing nutrient solution for three days. Then, a transparent glass cover (diameter × height: 4cm × 10cm, with 48 evenly distributed 0.88mm diameter ventilation holes) was placed over the leaf sheath of each plant. Ten fertile adult female brown planthoppers were introduced into the glass cover, and the top was sealed with a sponge. At 4, 12, 24, and 48 hours after inoculation, the infested leaf sheaths were harvested, quickly immersed in liquid nitrogen, and stored at -80℃ for later use. Leaf sheaths from rice plants with empty glass covers but without brown planthoppers served as a control. The experiment was repeated five times.

[0038] The extraction and detection methods for jasmonic acid, jasmonic acid-isoleucine, and abscisic acid were based on those of Lu J, Robert CAM, Riemann M, et al. Induced jasmonate signaling leads to contrasting effects on root damage and herbivore performance[J]. Plant Physiology, 2015, 167(3):1100-1116.

[0039] from Figure 3 It is evident that overexpression of OsPIE in rice can significantly increase the content of jasmonic acid, jasmonic acid-isoleucine, and abscisic acid induced by brown planthopper infestation.

[0040] Example 3: Overexpression of OsPIE in rice significantly reduced oviposition in brown planthoppers.

[0041] In this embodiment, the rice variety, experimental conditions, and experimental apparatus were the same as in Example 2. A pair of newly emerged female and male brown planthoppers were placed inside a glass jar, and the top was sealed with a sponge. Fourteen days after inoculation, rice leaf sheaths were cut from the glass jar, and the number of brown planthopper eggs was counted under a microscope. The experiment was performed in 30 biological replicates.

[0042] from Figure 4 It is evident that overexpression of OsPIE in rice can significantly reduce the oviposition rate of female brown planthoppers and enhance the direct resistance of rice to brown planthoppers.

[0043] Example 4: Overexpression of OsPIE in rice significantly reduced the hatching rate of brown planthopper eggs.

[0044] In this embodiment, the rice strain, experimental conditions, and experimental apparatus were the same as in Example 2. Ten oviparous adult brown planthoppers were placed inside a glass jar, and the top was sealed with a sponge. The adult females were removed 24 hours after inoculation. The number of newly hatched nymphs was observed and recorded daily until no more nymphs hatched for three consecutive days. Rice leaf sheaths were cut from the glass jar, and the number of unhatched brown planthopper eggs was counted under a microscope. The experiment was performed in 30 biological replicates.

[0045] from Figure 5 It is evident that overexpression of OsPIE in rice can significantly reduce the egg hatching rate of brown planthopper and improve the direct resistance of rice to brown planthopper.

[0046] Example 5: Overexpression of OsPIE in rice significantly reduced the feeding / oviposition selectivity of brown planthopper.

[0047] In this embodiment, the rice strain and experimental conditions were the same as in Example 2. One parental rice plant and one OsPIE-overexpressing rice plant were planted parallel to each other in small plastic cups containing nutrient solution, with a spacing of approximately 1 cm between the two plants. A glass cover (same specifications as in Example 2) was placed over the leaf sheath of each rice plant, and 15 BPH-carrying adult females were introduced into the glass cover. The top was sealed with a sponge. The number of brown planthoppers on each rice plant was recorded at 1, 2, 4, 8, 12, 24, 36, and 48 hours after inoculation. After 48 hours, all brown planthoppers were removed, and the number of brown planthopper eggs on each rice plant was counted under a microscope. The experiment was performed in 10 biological replicates.

[0048] from Figure 6 It is evident that overexpression of OsPIE in rice can significantly reduce the feeding / oviposition selectivity of female brown planthopper adults and improve the direct resistance of rice to brown planthopper.

[0049] Example 6: Overexpression of OsPIE in rice significantly reduced the feeding amount of brown planthoppers.

[0050] In this embodiment, the rice strain and experimental conditions were the same as in Example 2. The amount of honeydew excreted by the brown planthopper was used to represent its feeding amount. A transparent plastic cover (diameter × height: 5.5cm × 9.5cm) was placed over the leaf sheath of each rice plant. A circular filter paper (diameter 9cm) was placed at the bottom of the plastic cover, and a newly emerged female brown planthopper was placed inside the plastic cover. The top was sealed with a sponge. 24 hours after inoculation, the filter paper was removed, dried, and then 0.1% ninhydrin solution was added (the amino acids in the honeydew react with ninhydrin to produce a blue-purple substance). The reaction was carried out at 60°C for 30 minutes, and finally, the area of ​​the stained region was counted using Image-Pro Plus Image Analysis software. The experiment was repeated 20 times.

[0051] from Figure 7 It is evident that overexpression of OsPIE in rice can significantly reduce the feeding amount of brown planthoppers and improve the direct resistance of rice to brown planthoppers.

[0052] The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.

Claims

1. A rice gene OsPIE Its application in improving the insect resistance of rice is characterized by, The application is to improve rice genes. OsPIE The expression level of the rice gene, wherein the insect resistance is defined as improved resistance of rice to brown planthopper; OsPIE The DNA sequence is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, Rice genes OsPIE The amino acid sequence encoding the protein is shown in SEQ ID NO.

2.

3. The application as described in claim 1, characterized in that, The rice gene OsPIE Improving the insect resistance of rice specifically involves improving the rice genes. OsPIE The expression levels of these substances were increased to enhance the synthesis of jasmonic acid, jasmonic acid-isoleucine, and abscisic acid, and to reduce the oviposition, hatching rate, feeding / oviposition selectivity, and feeding amount of brown planthoppers, thereby improving the insect resistance of rice.

4. A method for improving the insect resistance of rice, characterized in that, Improve rice genes OsPIE The expression level of the rice gene can be increased to improve the resistance of rice to brown planthoppers; OsPIE The DNA sequence is shown in SEQ ID NO.

1.

5. The method as described in claim 4, characterized in that, The improvement of rice genes OsPIE The specific levels of expression are: (1) Amplification using specific primers OsPIE-F and OsPIE-R OsPIE The full-length ORF of the gene was digested with enzymes, purified, and then ligated into a plant expression vector, which was then transformed into Agrobacterium. (2) Rice callus was infected with Agrobacterium tumefaciens containing a vector. After resistance selection, differentiation, rooting, and hardening, the infected callus was used to obtain T0 generation transgenic rice. OsPIE Gene expression levels are increased.

6. The method as described in claim 5, characterized in that, After two generations of self-pollination of T0 generation transgenic rice, the T2 generation seeds were screened by GUS staining, analyzed by PCR, and identified by Southern blot, resulting in two homozygous transgenic lines with single-copy insertion; among the homozygous transgenic lines OsPIE Gene expression levels are increased.

7. An overexpression OsPIE A method for preparing transgenic rice, characterized in that, The rice gene OsPIE The DNA sequence is shown in SEQ ID NO.1, and the preparation method includes: (1) Amplification using specific primers OsPIE-F and OsPIE-R OsPIE The full-length ORF of the gene was digested with enzymes, purified, and then ligated into a plant expression vector, which was then transformed into Agrobacterium. (2) Rice callus tissue was infected with Agrobacterium tumefaciens containing a vector. After resistance selection, differentiation, rooting, and hardening, the infected callus tissue was used to obtain T0 generation transgenic rice, which is the overexpression of Agrobacterium tumefaciens. OsPIE Genetically modified rice.

8. The preparation method according to claim 7, characterized in that, Two homozygous transgenic lines with single-copy insertion were obtained by screening T2 generation seeds produced from T0 generation transgenic rice after two generations of self-pollination, followed by GUS staining, PCR analysis, and Southern blot identification.

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