Rice yield-increasing and insect-resistant gene OsWIH2 and its encoded protein and applications

Abstract

The present invention discloses a rice yield-increasing and insect-resistant gene OsWIH2, its encoded protein, and its application, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the gene OsWIH2 is shown in SEQ ID NO.1, and the amino acid sequence corresponding to the protein encoded by the gene OsWIH2 is shown in SEQ ID NO.2. The present invention can improve the resistance of rice to brown planthoppers through overexpression of the rice OsWIH2 gene, while significantly increasing the rice panicle length, the number of stalks, the grain length, the grain width, and the grain weight, thereby increasing rice yield. The rice OsWIH2 gene of the present invention can provide a breeding strategy for insect-resistant and yield-increasing rice, further ensuring food security.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and breeding technology, and particularly relates to a rice yield-increasing and insect-resistant gene OsWIH2, its encoded protein and application. Background Art

[0002] Rice (Oryza sativa), a vital food crop, ranks among the top three in the world in terms of total production, providing dietary energy for over half of the world's population. With the continued growth of the global population and the acceleration of urbanization, the area of ​​arable land is gradually decreasing. At the same time, the frequent occurrence of extreme weather events caused by global warming further exacerbates the risk of food production decline. The contradiction between the growing population and the shrinking arable land area and the reduction in food production caused by global warming needs to be urgently addressed. The brown planthopper (Nilaparvata lugens), a member of the Hemiptera order and the family Delphacidae, is a monophagous pest that harms rice plants by directly sucking phloem sap, laying eggs, and transmitting rice viruses, threatening global food security. Chemical pesticides are widely used in rice cultivation as an effective method of pest control. However, long-term use of chemical pesticides can lead to increased pest resistance, leading to a resurgence of the pest. Breeding insect-resistant varieties, which confer natural resistance to specific pests through genetic modification of rice, can reduce reliance on agrochemicals, mitigate environmental pollution risks, and promote biodiversity conservation and environmentally friendly agricultural development within agricultural ecosystems. Therefore, breeding for both yield-enhancing and insect-resistant varieties is an effective way to increase grain production and ensure food security, and holds significant strategic significance in rice cultivation. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention aims to provide a rice yield-increasing and insect-resistant gene OsWIH2, its encoded protein and its application.

[0004] To achieve the above object, the present invention provides the following technical solution: a rice yield-increasing and insect-resistant gene OsWIH2, the nucleotide sequence of the gene OsWIH2 is shown in SEQ ID NO.1.

[0005] The present invention also provides a protein encoded by the gene OsWIH2, and the amino acid sequence corresponding to the protein is shown in SEQ ID NO.2.

[0006] The present invention also provides an application of the gene OsWIH2 in improving rice yield and brown planthopper resistance.

[0007] The present invention also provides an application of the gene OsWIH2 in preparing transgenic rice.

[0008] Preferably, the application method includes positive regulation, but is not limited to the overexpression method described in positive regulation.

[0009] The beneficial effects of the present invention are as follows: the present invention provides a rice yield-increasing insect-resistant gene OsWIH2, its encoded protein, and its application. OsWIH2-overexpressing rice lines significantly increased rice panicle length, stalk number, grain length, grain width, and grain weight; greenhouse and field experiments showed that OsWIH2-overexpressing rice lines can reduce the survival rate of brown planthopper nymphs, delay the development of brown planthoppers, and improve rice resistance to brown planthoppers. The present invention opens up a channel for genetic breeding of yield-increasing insect-resistant rice, provides a new production idea for increasing yield and enhancing insect resistance in rice genetic breeding, and has important field application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a gene pattern diagram of the overexpression vector of OsWIH2 overexpressing rice lines;

[0011] Figure 2 Schematic diagram of greenhouse experiments evaluating the effects of OsWIH2-overexpressing rice lines on rice resistance to brown planthoppers; (a) compares the survival rates of brown planthoppers after feeding on ZH11 and three OsWIH2-overexpressing rice lines, and (b) compares the developmental stages of brown planthoppers after feeding on ZH11 and three OsWIH2-overexpressing rice lines;

[0012] Figure 3 Comparative results of greenhouse experiments evaluating the tolerance of OsWIH2-overexpressing rice lines to brown planthopper infestation; (a) shows the comparison results on day 0 and day 3, and (b) shows the comparison results on day 9 and day 12.

[0013] Figure 4 Schematic diagram of rice planting pattern in field experiment;

[0014] Figure 5 The results of a field experiment evaluating the effects of OsWIH2 overexpression on rice resistance to brown planthoppers are shown in Figure 1. (a) shows the results of brown planthopper resistance on the 70th day of rice growth, and (b) shows the results of brown planthopper resistance on the 80th day of rice growth.

[0015] Figure 6 Schematic diagram of the field experiment evaluating the effect of OsWIH2 overexpression on rice agronomic morphology; (a) is a comparison of the statistical results of panicle length among different varieties, (b) is a comparison of the statistical results of primary stalk length among different varieties, (c) is a comparison of the statistical results of secondary stalk length among different varieties, (d) is a comparison of the statistical results of grain number per panicle among different varieties, (e) is a comparison of the statistical results of seed setting rate among different varieties; (f) is a comparison of individual rice panicles among different varieties;

[0016] Figure 7Schematic diagram of the field experiment evaluating the effect of OsWIH2 overexpression on rice yield; among them, (a) is a comparison of the statistical results of grain length of different varieties, (b) is a comparison of the statistical results of grain width of different varieties, (c) is a comparison of the statistical results of 1000-grain weight of different varieties, (d) is a comparison of the grain width of different varieties, and (e) is a comparison of the grain length of different varieties. DETAILED DESCRIPTION

[0017] The above scheme will be further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not intended to limit the scope of the invention. The implementation conditions adopted in the examples can be further adjusted according to specific application conditions. Unspecified implementation conditions are generally those in routine experiments. The experimental data were statistically analyzed using SPSS software. The comparison between the two samples was performed using Student's t-test, and the survival rate was tested using Log-rank test. Asterisks in the figures indicate significant differences between the groups (*, p < 0.05; **, p < 0.01).

[0018] Example 1: Obtaining OsWIH2-overexpressing rice lines by transgenic methods

[0019] (1) Cloning of the OsWIH2 gene

[0020] Leaf samples were collected from japonica rice variety Zhonghua 11 (ZH11), RNA was extracted, and cDNA was obtained by reverse transcription. Specific PCR amplification primers were designed based on the sequence of the OsWIH2 gene. The OsWIH2 gene sequence was PCR amplified using a high-fidelity enzyme and the PCR product of the OsWIH2 gene was recovered and purified. The primer sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4. The obtained fragment and the pCAMBIA1300 vector were digested with restriction endonucleases. The digested fragments containing sticky ends and the vector were recovered by DNA purification. The DNA fragments were then ligated into the pCAMBIA1300 vector by homologous recombination to obtain homologous recombinant plasmids, such as Figure 1 After transformation, single clone detection, and plasmid extraction, the constructed recombinant plasmid was transformed into Agrobacterium EHA105 strain by electroporation and preserved.

[0021] PCR system:

[0022] Ingredients Addition amount Rice cDNA 50ng OsWIH2-CE-F 1μl (10μM) OsWIH2-CE-R 1μl (10μM) Premix Ex Taq 12.5 μl <![CDATA[ddH2O]]> to 25μl

[0023] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; then entering the cyclic amplification stage, 95°C for 20 s; 58°C for 30 s; 72°C for 20 s, for a total of 30 cycles, and finally amplification at 72°C for 10 min, followed by gel recovery and purification.

[0024] Homologous recombination system:

[0025] Ingredients Addition amount pCAMBIA1300 (BamHI linearized) 200ng OsWIH2 PCR product 200ng Clonexpress Mix II 5μl <![CDATA[ddH2O]]> to 10μl

[0026] After reacting at 50°C for 30 min, the cells were transformed into TG1 competent cells, amplified and the plasmid was extracted, and then transformed into Agrobacterium tumefaciens EHA105.

[0027] (2) OsWIH2 gene-transformed rice plants and screening of homozygous lines

[0028] Rice callus tissue was infected with Agrobacterium tumefaciens EHA105 strain containing the OsWIH2 gene, and the resistant callus tissue obtained by screening was cultivated into seedlings and planted in hydroponic solution to obtain OsWIH2 overexpressing rice lines (T0 generation).

[0029] The leaves of the seedlings obtained by callus culture were selected to extract total plant DNA, and the hygromycin resistance gene was identified by PCR using conventional operating methods familiar to those skilled in the art to obtain three positive seedlings with successful transformation. Each strain was planted to obtain seeds. According to Mendel's law of inheritance, the T0 generation seeds were screened with hygromycin, and seedlings (T1 generation) that met the 3:1 ratio were screened. The seeds (T2 generation) produced by self-pollination of the T1 generation were verified using the same method, and three homozygous strains (T3 generation) whose offspring could grow in hygromycin-resistant culture medium were screened, namely OsWIH2-OE1, OsWIH2-OE2, and OsWIH2-OE3. Subsequent experiments were performed using the ZH11 wild type and the OsWIH2 overexpression homozygous strains.

[0030] Example 2: Greenhouse experiment to evaluate the brown planthopper resistance of OsWIH2-overexpressing rice lines

[0031] (1) Evaluation of brown planthopper resistance

[0032] The researchers selected ZH11 wild-type rice plants of appropriate age (about 35 days old) and healthy growth, and used glass covers and round sponges to fix the base of the rice stems. The experiment was conducted after the seedlings had been grown for 3 days. Each glass cover was inoculated with 20 first-instar nymphs that hatched within 1 day, and 6 groups were repeated. The survival status and developmental duration of the nymphs were recorded daily. The experimental results showed that the developmental duration of brown planthoppers fed on the OsWIH2-overexpressing rice line was significantly extended by 3.91%, as shown in Figure 2. Figure 2 As shown in Figure A, the survival rate decreased by 14.72%, which is a very significant difference. Figure 2 As shown in Figure B, it shows that overexpression of OsWIH2 can improve the resistance of rice to brown planthoppers in the greenhouse.

[0033] (2) Evaluation of rice brown planthopper resistance

[0034] Appropriate-age (about 35 days) healthy and vigorous ZH11 wild-type rice plants and OsWIH2-overexpressing rice lines were selected and planted individually in plastic cups, secured with sponges and glass covers. Ten female adults of brown planthoppers carrying eggs were inoculated into each rice plant, and photographs were taken to record the wilting and shrivelling of rice plants after being infested by brown planthoppers. Figure 3 The experimental results showed that after 15 days of brown planthopper feeding, the OsWIH2-overexpressing rice line grew well, while the ZH11 wild-type rice plants had obviously withered and fallen over, indicating that overexpressing OsWIH2 can improve rice's tolerance to brown planthopper damage in the greenhouse.

[0035] Example 3: Field trials evaluating brown planthopper resistance and yield of OsWIH2-overexpressing rice lines

[0036] The field experiment was carried out in the experimental field of Linglong Street, Lin'an District, Hangzhou City, Zhejiang Province (119°61'96"E, 30°19'37"N). The planting pattern was as follows: Figure 4 shown.

[0037] (1) Evaluation of brown planthopper resistance

[0038] Twenty-eight plots were set up in the field plot, of which 10 plots were planted with ZH11 wild-type rice plants and the other 18 plots were planted with rice plants of the OsWIH2 overexpressing rice line. An average of 16 rice seedlings were planted in each plot. To evaluate the brown planthopper resistance of different rice species, the number of brown planthoppers on each plant was counted from random plots. The experimental results showed that the number of brown planthoppers feeding on ZH11 wild-type rice plants was greater than that on the OsWIH2 overexpressing rice line at two different time points, especially on the 70th day of rice growth, the difference between the two reached about 90%, as shown in Figure 2. Figure 5 As shown in Figures (a) and (b), overexpression of OsWIH2 can significantly improve rice resistance to brown planthoppers in the field.

[0039] (2) Yield evaluation

[0040] ZH11 wild-type rice plants and OsWIH2 overexpressing rice lines were planted in the field and harvested after the rice was fully mature. The agronomic traits related to rice yield were statistically analyzed. Figure 6 Figures (a)-(f) and Figure 7 As shown in Figures (a) to (e), the main panicle length of the OsWIH2-overexpressing rice line increased by 8.12% compared with the ZH11 wild-type rice plant, the number of primary and secondary stalks increased by 29.04% and 32.3%, respectively, the grain width increased by 15.92%, and the grain length increased by 9.66%. The 1000-grain weight increased significantly by 10.72%, but there was no significant change in the number of main panicle grains and the fruit set rate between the two lines. The above results indicate that overexpressing OsWIH2 can increase rice yield in the field.

[0041] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The description and examples are to be considered as exemplary only.

[0042] It will be understood that the present application is not limited to the exact construction that has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof.

Claims

1. An application of the gene OsWIH2 in improving rice yield and brown planthopper resistance, characterized in that: The nucleotide sequence of the gene OsWIH2 is shown in SEQ ID NO.1, and the amino acid sequence corresponding to the protein encoded by the gene OsWIH2 is shown in SEQ ID NO.

2.

2. Use of the gene OsWIH2 in preparing high-yield and high-brown planthopper-resistant transgenic rice, characterized in that: The nucleotide sequence of the gene OsWIH2 is shown in SEQ ID NO.1, and the amino acid sequence corresponding to the protein encoded by the gene OsWIH2 is shown in SEQ ID NO.2.

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