Rice yield-increasing insect-resistant gene OsWIH2 as well as encoding protein and application thereof

Abstract

The invention discloses a rice yield-increasing insect-resistant gene OsWIH2 as well as an encoded protein and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the gene OsWIH2 is as shown in SEQ ID NO. 1, and the amino acid sequence corresponding to the protein coded by the gene OsWIH2 is as shown in SEQ ID NO. 2. Through overexpression of the rice OsWIH2 gene, the resistance of rice to brown planthoppers can be improved, meanwhile, the rice spike length, the number of branches, the grain length, the grain width and the grain weight are remarkably increased, and then the rice yield is increased. The rice OsWIH2 gene provided by the invention can provide a breeding thought for insect resistance and yield increase of rice, and further guarantees food safety.

Description

Technical Field

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

[0002] Rice (Oryza sativa), an important food crop for mankind, ranks among the top three food crops in the world in terms of total output, and ensures the dietary energy of more than half of the world's population. With the continuous growth of the global population and the acceleration of urbanization, the area of ​​arable land is gradually decreasing; at the same time, the frequent extreme climate events caused by global warming have further aggravated the risk of food production reduction. The contradiction between the increasing population and the reduction of arable land and food production reduction caused by global warming needs to be resolved urgently. Brown planthopper (Nilaparvata lugens), Hemiptera, Delphacidae, is a monophagous pest that harms rice by directly sucking the phloem juice of rice, laying eggs, and spreading rice viruses, threatening global food security. Chemical pesticides are widely used in rice cultivation as an effective method to control pests, but the long-term use of chemical pesticides can increase pest resistance, leading to the resurgence of pests. The breeding of insect-resistant varieties can give crops natural resistance to specific pests through the improvement of rice's own genes, which can reduce dependence on agrochemicals, reduce the risk of environmental pollution, and promote the maintenance of biodiversity in agricultural ecosystems and the development of environmentally friendly agriculture. Therefore, breeding for increased yield and insect resistance is an effective way to increase food production and ensure food security, and has significant strategic significance in rice cultivation. Summary of the invention

[0003] In view of the deficiencies of the prior art, the object of the present invention is to provide a rice yield-increasing and insect-resistant gene OsWIH2 and its encoded protein and 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 a 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 and its encoded protein and application. OsWIH2 overexpressing rice lines significantly increase the rice panicle length, the number of peduncles, the grain length, the grain width and the grain weight; through greenhouse and field tests, it was found that OsWIH2 overexpressing rice lines can reduce the survival rate of brown planthopper nymphs, delay the development of brown planthoppers, and improve the resistance of rice to brown planthoppers. The present invention opens up a channel for the genetic breeding of rice with increased yield and insect resistance, 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 the gene pattern diagram of the overexpression vector of OsWIH2 overexpressing rice lines;

[0011] Figure 2 Schematic diagram of the greenhouse experiment to evaluate the effect of OsWIH2 overexpression rice lines on rice brown planthopper resistance; (a) is a comparison of the survival rate of brown planthoppers after feeding on ZH11 and three rice lines overexpressing OsWIH2, and (b) is a comparison of the developmental stages of brown planthoppers after feeding on ZH11 and three rice lines overexpressing OsWIH2;

[0012] Figure 3 The results of the greenhouse experiment to evaluate the tolerance of OsWIH2-overexpressing rice lines to brown planthopper feeding are shown in Figure 1. (a) is a comparison of the results on the 0th day and the 3rd day, and (b) is a comparison of the results on the 9th day and the 12th day.

[0013] Figure 4 This is a schematic diagram of the rice planting pattern in the field experiment;

[0014] Figure 5 The figure shows the comparison results of the field experiment to evaluate the effect of OsWIH2 overexpression rice lines on rice brown planthopper resistance; (a) shows the comparison results of rice brown planthopper resistance on the 70th day of rice growth, and (b) shows the comparison results of rice brown planthopper resistance on the 80th day of rice growth;

[0015] Figure 6 Schematic diagram of the field experiment to evaluate the effect of OsWIH2 overexpression on the agronomic shape of rice; (a) is a comparison of the statistical results of ear length of different strains, (b) is a comparison of the statistical results of primary stalk length of different strains, (c) is a comparison of the statistical results of secondary stalk length of different strains, (d) is a comparison of the statistical results of grain number per ear of different strains, (e) is a comparison of the statistical results of fruit setting rate of different strains; (f) is a comparison of individual rice ears of different strains;

[0016] Figure 7Schematic diagram of the field experiment to evaluate 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 thousand-grain weight of different varieties, (d) is a comparison of grain width of different varieties, and (e) is a comparison of grain length of different varieties. DETAILED DESCRIPTION

[0017] The above scheme is 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 present invention. The implementation conditions used in the examples can be further adjusted according to specific application conditions, and the unspecified implementation conditions are usually the conditions in routine experiments. The experimental data were statistically analyzed using SPSS software, and the comparison between the two samples was tested using Student's t-test, and the survival rate was tested using Log-rank test. The asterisks in the figure indicate significant differences between the groups (*, p<0.05; **, p<0.01).

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

[0019] (1) Cloning of OsWIH2 gene

[0020] The leaves of Japonica rice Zhonghua 11 (ZH11) were sampled, RNA was extracted and reverse transcribed to obtain cDNA. Specific PCR amplification primers were designed according to the sequence of the OsWIH2 gene, and the OsWIH2 gene sequence was PCR amplified by 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 fragments and the pCAMBIA1300 vector were single-digested with restriction endonucleases, and the digested fragments containing sticky ends and the vector were recovered by DNA purification. The DNA fragments were connected to the pCAMBIA1300 vector by homologous recombination to obtain homologous recombination plasmids, such as Figure 1 After transformation, single clone detection and plasmid extraction, the constructed recombinant plasmid was transferred into Agrobacterium EHA105 strain by electroporation for preservation.

[0021] PCR system:

[0022] Composition Addition amount Rice cDNA 50ng OsWIH2-CE-F 1μl(10μM) OsWIH2-CE-R 1μl(10μM) Premix Ex Taq 12.5μl <![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] Composition Addition amount pCAMBIA1300 (BamHI linearized) 200ng OsWIH2 PCR products 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, and the plasmids were expanded and extracted, and then transformed into Agrobacterium tumefaciens EHA105.

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

[0028] The rice callus was infected with the Agrobacterium tumefaciens EHA105 strain containing the OsWIH2 gene, and the resistant callus obtained by screening was cultivated into seedlings, which were then planted in a hydroponic solution to obtain an OsWIH2 overexpressing rice line (T0 generation).

[0029] The leaves of the seedlings obtained by callus culture were selected to extract the total plant DNA, and the hygromycin resistance gene was identified by PCR using conventional operation methods well known 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 by hygromycin, and the 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 by the same method, and three homozygous strains (T3 generation) whose offspring could grow in hygromycin resistance medium were screened, namely OsWIH2-OE1, OsWIH2-OE2, and OsWIH2-OE3. After that, the experiments were all conducted using the ZH11 wild type and the OsWIH2 overexpression pure 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 ZH11 wild-type rice plants and OsWIH2-overexpressing rice lines of appropriate age (about 35 days) and strong growth were selected, and the base of the rice stems was fixed with glass covers and round sponges. The experiment was conducted after the seedlings were slowed down 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 development period of the nymphs were recorded every day. The experimental results showed that the development period of brown planthoppers feeding on OsWIH2-overexpressing rice lines was significantly extended by 3.91%, such as 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. This indicates that overexpression of OsWIH2 can improve rice resistance to brown planthoppers in the greenhouse.

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

[0034] Wild-type ZH11 rice plants and OsWIH2-overexpressing rice lines of appropriate age (about 35 days old) with robust growth were selected and planted individually in plastic cups and fixed with sponges and glass covers. Ten female adults of brown planthoppers carrying eggs were inoculated into each rice plant, and photos were taken to record the phenomenon of rice plants withering and dying after being infested by brown planthoppers ( Figure 3 The experimental results showed that after 15 days of feeding by brown planthoppers, the OsWIH2 overexpressing rice lines grew well, while the ZH11 wild-type rice plants had obviously withered and lodged, indicating that overexpressing OsWIH2 can improve the resistance of rice to brown planthoppers in the greenhouse.

[0035] Example 3: Field trials to evaluate the 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 OsWIH2 overexpressing rice lines. An average of 16 rice seedlings were planted in each plot. In order to evaluate the brown planthopper resistance of different rice plants, 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 OsWIH2 overexpressing rice lines at two different time points, especially on the 70th day of rice growth, the difference between the two reached about 90%, such as 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 Assessment

[0040] The ZH11 wild-type rice plants and the OsWIH2 overexpressing rice lines were planted in the field and harvested when the rice was fully mature. The agronomic traits related to rice yield were statistically analyzed. Figure 6 Figure (a)-Figure (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 peduncles increased by 29.04% and 32.3%, respectively, the grain width increased by 15.92%, the grain length increased by 9.66%, and the 1000-grain weight increased significantly by 10.72%. However, there were no significant changes in the number of main panicle grains and the fruit setting 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 description and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application. The description and examples are intended to be exemplary only.

[0042] It should 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. A rice yield-increasing insect-resistant gene OsWIH2, characterized in that: The nucleotide sequence of the gene OsWIH2 is shown in SEQ ID NO.

1.

2. A protein encoded by the gene OsWIH2 according to claim 1, characterized in that: The amino acid sequence corresponding to the protein is shown in SEQ ID NO.

2.

3. Use of the gene OsWIH2 according to claim 1 in improving rice yield and brown planthopper resistance.

4. Use of the gene OsWIH2 according to claim 1 in preparing transgenic rice.

5. The use according to claim 4, characterized in that: The application method includes positive regulation.

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