Application of rice OsPDRA3 gene in improving insect resistance of rice

Abstract

The invention provides application of a rice OsPDRA3 gene in improving insect resistance of rice. According to the invention, an OsPDRA3 gene (LOCOs01g42380) is cloned in a rice genome, and an SMART program is used for analyzing and predicting that the protein has multiple transmembrane domains and ATP enzyme structural domains (AAA < + >) related to various cell activities. The influence of OsPDRA3 on the tolerance of rice brown planthopper is further studied, and by comparing the tolerance of wild type and overexpressed OsPDRA3 transgenic plants to brown planthopper, the result shows that the survival rate of a rice line with overexpressed OsPDRA3 after being treated by brown planthopper is obviously increased compared with that of a wild type rice line (the survival rate is 0%), and reaches 75%-100%. Namely, it is shown that the OsPDRA3 gene can significantly improve the insect resistance of rice, can be widely applied to the fields of plant genetic engineering and plant disease resistance, and has huge economic value and application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and plant disease resistance, specifically relating to rice. OsPDRA3 Application of genes in improving insect resistance in rice. Background Technology

[0002] Rice is a crucial food crop in my country, and more than half of the world's population relies on it as their staple food. Pests and diseases severely impact plant growth and crop economic benefits, making agricultural biological resistance mechanisms a priority development area and major research direction within the Life Sciences Department. The rice-brown planthopper interaction mechanism is a vital topic related to agricultural food security. Rice is my country's most important food crop, accounting for 40% of total grain production. The brown planthopper (BPH, Nilaparvata lugens) is migratory, sucks rice sap, and has strong reproductive and adaptability capabilities. Primarily feeding on the phloem, it is one of the most destructive pests of rice, feeding on cultivated rice (Oryza sativa) and several wild rice species. It is one of the most serious and devastating pests in rice-growing areas of southern my country, causing yield reductions and even large-scale rice mortality. Since the late 1960s, the brown planthopper has risen to become a major rice pest in my country, severely impacting yields and posing a serious threat to food security. On September 15, 2020, the brown planthopper was listed as a Class A crop pest, which seriously threatens food production security.

[0003] Due to factors such as the decline in rice variety resistance caused by the pathogenic variation of brown planthoppers and the scarcity of resistant varieties, the control of brown planthoppers in agricultural production mainly relies on chemical pesticides. However, excessive application of chemical pesticides not only threatens food safety and the ecological environment but also easily leads to high pesticide resistance in brown planthoppers. Currently, developing rice insect-resistant genes is an effective way to improve rice resistance to brown planthoppers and cultivate new germplasm with high insect resistance. Brown planthopper resistance genes and resistance loci have been identified in different rice varieties; currently cloned genes include... Bph14 , Bph26 , Bph3 , Bph29 , Bph9 , Bph32 , Bph6 , Bph30 For example, the gene for resistance to brown planthoppers. Bph6 Encoding a novel, atypical LRR-like protein, this protein regulates the exocytosis of rice cell proteins by interacting with the cytosecretion complex subunit protein OsEXO70E1. Simultaneously, it integrates multiple plant hormone signals to maintain the stability of the phloem cell wall composition and structure, thereby hindering brown planthopper feeding and ultimately achieving broad-spectrum resistance to brown planthoppers. Another recently cloned brown planthopper resistance gene...Bph30 This gene encodes a novel protein containing two LRR domains. It is highly expressed in the sclerenchyma cells of rice leaf sheaths and strengthens the sclerenchyma by promoting the accumulation of cellulose and hemicellulose, thus forming a robust physical barrier that prevents brown planthoppers from penetrating the phloem to feed on sap. However, the resources of insect-resistant genes in rice are currently insufficient. Strengthening research on the molecular mechanisms of the interaction between rice and brown planthoppers can, on the one hand, elucidate the mechanisms by which brown planthoppers adapt to crop insect resistance, and on the other hand, reveal the molecular regulatory mechanisms by which crops respond to insect stress.

[0004] 《 Ospdr9 , which encodes a PDR-type ABC transporter, is induced by heavy metals, hypoxic stress and redox perturbations in rice roots" pointed out in the article, Ospdr9 Encoding a rice ABC protein with an inverted (ABC-TMS6)2 structure. Treatment with polyethylene glycol and heavy metals cadmium (20 μmol) and zinc (30 μmol) rapidly and significantly induced root oxidative damage in rice seedlings. Ospdr9 Expression. Hypoxia stress can also induce root rot in rice. Ospdr9 The expression indicates that salt stress only induced [a certain effect] at low levels. Ospdr9 Cold and heat shocks had no effect. Plant growth regulators jasmonic acid, auxin α-naphthaleneacetic acid, and cytokinin 6-benzylaminopurine can also trigger [the reaction]. Ospdr9 The expression of the antioxidants dithiothreitol and ascorbic acid rapidly and significantly induced the growth of rice roots. Ospdr9 The expression of; strong oxidizing agent hydrogen peroxide can also induce Ospdr9 The expression of polyethylene glycol was increased, but the induction level was only one-third of that of the former. These results suggest that polyethylene glycol, along with heavy metal stresses such as cadmium or zinc, hypoxia stress, salt stress, plant growth regulators, and redox changes, may be involved in the response of rice roots to abiotic stresses. Ospdr9 Expression regulation.

[0005] However, rice is not disclosed in existing technologies. OsPDRA3 Application of genes in improving insect resistance in rice. Summary of the Invention

[0006] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and to provide a rice OsPDRA3 Genes may contain rice OsPDRA3 Application of genetically modified biomaterials in improving the insect resistance of rice.

[0007] The second object of the present invention is to provide rice OsPDRA3 Genes may contain rice OsPDRA3 Application of genetic biomaterials in the creation of insect-resistant rice.

[0008] The third objective of this invention is to provide a method for creating insect-resistant rice.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention queries rice genome websites. OsPDRA3 The gene (gene number: LOC_Os01g42380), with a full length of 7117 bp (as shown in SEQ ID No. 1) and a CDS length of 4374 bp (as shown in SEQ ID No. 2), encodes a 1457aa protein (as shown in SEQ ID No. 3). Its domains were predicted using CDD, PFAM, SMART, and Interproscan programs. The results indicate that the rice... OsPDRA3 The protein possesses multiple transmembrane domains and ATPase domains (AAA+) associated with various cellular activities. Further investigation is needed. OsPDRA3 The influence of genes on tolerance to rice brown planthopper will OsPDRA3 Transformed into rice, resulting in overexpression OsPDRA3 Rice lines, by comparing wild-type and overexpression OsPDRA3 The tolerance of transgenic plants to brown planthoppers was assessed, and the results showed that overexpression of [the planthopper species]... OsPDRA3 Compared to wild-type rice lines (0% survival rate), the survival rate of rice lines treated with brown planthoppers was significantly improved, reaching 75%–100%. This indicates that… OsPDRA3 Genes can significantly improve the insect resistance of rice, and research on rice self-resistance genes is of great significance for obtaining insect-resistant rice varieties.

[0011] Therefore, the present invention provides rice OsPDRA3 Genes may contain rice OsPDRA3 The application of gene-based biomaterials in improving insect resistance in rice, the OsPDRA3 The nucleotide sequence is shown in SEQ ID No. 2.

[0012] This invention also provides rice OsPDRA3 Genes may contain rice OsPDRA3 The application of gene-based biomaterials in the creation of insect-resistant rice, the aforementioned OsPDRA3 The nucleotide sequence is shown in SEQ ID No. 2.

[0013] Furthermore, the aforementioned OsPDRA3 The amino acid sequence encoded by the gene is shown in SEQ ID No. 3.

[0014] Furthermore, the rice-containing OsPDRA3 The biological material containing the gene is rice. OsPDRA3 Gene expression cassettes, vectors, or strains.

[0015] Furthermore, the vector is an overexpression vector.

[0016] Preferably, the overexpression vector is the pFGC-pUC121-GFP-HA vector.

[0017] Furthermore, the strain is an overexpression strain.

[0018] This invention also provides a method for creating insect-resistant rice, the method comprising the following steps: [The method involves] rice... OsPDRA3 Genes may contain rice OsPDRA3 Genes are obtained by transforming biological materials into rice explants, culturing, and screening.

[0019] Furthermore, the transformation method is Agrobacterium-mediated transformation.

[0020] Furthermore, the screening method is resistance culture medium screening and / or real-time quantitative PCR identification.

[0021] Furthermore, the insect resistance is specifically against brown planthoppers.

[0022] Furthermore, the rice variety is Nipponbare.

[0023] Preferably, the present invention provides a method for creating insect-resistant rice, the method comprising the following steps: S1. Will OsPDRA The gene was cloned into the pFGC-pUC121-GFP-HA vector; S2. Rice was infected using Agrobacterium-mediated transformation to obtain transgenic plants with stable expression.

[0024] Specifically, the present invention provides a method for creating insect-resistant rice, the method comprising the following steps: S1. Cloned from the rice genome ​ After the gene was correctly sequenced, it was cloned into the pFGC-pUC121-GFP-HA vector digested with BamI, transformed, and positive strains were screened. S2. Extract plasmids from the positive strain obtained in step S1 and transfect Agrobacterium to obtain a positive Agrobacterium strain. S3. Rice explants were infected using Agrobacterium-mediated transformation, cultured on selection and differentiation media for a period of time, and then the expression levels in overexpressing plants were detected by RT-qPCR. ​The gene expression level was determined to obtain transgenic plants with stable expression.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention provides rice ​ Application of genes in enhancing insect resistance in rice. This invention clones genes into the rice genome. ​ The gene (LOC_Os01g42380), with a full length of 7117 bp and a CDS length of 4374 bp, encodes a 1457aa protein. SMART analysis predicts that this protein contains multiple transmembrane domains and ATPase domains (AAA+) associated with various cellular activities. Further investigation is needed. ​ The impact on tolerance to rice brown planthopper will ​ Transformed into rice, resulting in overexpression ​ Rice lines, by comparing wild-type and overexpression ​ The tolerance of transgenic plants to brown planthoppers was assessed, and the results showed that overexpression of [the planthopper species]... ​ Compared to wild-type rice (0% survival rate), the survival rate of rice treated with brown planthoppers was significantly improved, reaching 75%–100%. This indicates that… ​ Genes can significantly improve the insect resistance of rice and can be widely used in the fields of plant genetic engineering and plant disease resistance, with huge economic value and application prospects. Attached Figure Description

[0026] ​ The SMART program was used to predict the domains of the OsPDRA3 protein, which includes multiple transmembrane domains and ATPase domains (AAA+) associated with various cellular activities.

[0027] ​ To detect overexpressing plants using the RT-qPCR method ( ​ -OE) ​ Gene expression levels. Note: *p<0.05, obtained through a t-test (Student's test).

[0028] ​ BPH treatment for wild-type (NIP) and ​ Overexpression plants ( ​ Phenotypic and plant survival statistics of (-OE). Note: *p<0.05, obtained by t-test (Student's test). Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] Example 1 Rice ​ Cloning of genes According to information from the Rice Genome Annotation Project, synthetic rice... ​ The gene (gene number: LOC_Os01g42380) has a full length of 7117 bp (as shown in SEQ ID No. 1) and a CDS length of 4374 bp (as shown in SEQ ID No. 2), encoding a 1457 aa protein (as shown in SEQ ID No. 3). Its domains were predicted using CDD, PFAM, SMART, and Interproscan programs.

[0032] The results are as follows ​ As shown, rice ​ The protein has multiple transmembrane domains and ATPase domains (AAA+) associated with various cellular activities. ​ The genetic characteristics are shown in Table 1.

[0033] Table 1 ​ Genetic characteristics

[0034] Example 2 Rice ​ Construction of gene overexpression rice materials I. Experimental Methods Cloned from the rice genome ​ After the genes are correctly sequenced, ​ The gene was ligated into the pFGC-pUC121-GFP-HA vector. The specific steps are as follows: The cloned gene... ​ The gene sequence was obtained and ligated into the pFGC-pUC121-GFP-HA vector digested with BamI using homologous recombination. After transformation, the vector was screened and cultured on kanamycin-containing plates. Positive strains were selected by PCR and sent for sequencing. Plasmids were extracted from the correctly sequenced strains and transformed into Agrobacterium, and positive strains were identified. The Agrobacterium-mediated transformation method was used to... ​ Transformed into Nipponbare rice explants, and cultured on selection and differentiation media for a period of time, respectively, to obtain stably expressing transgenic plants. ​ -OE.

[0035] For the obtained transgenic plants ​ -OE sampling was used to extract total RNA from rice leaves. The concentration and purity of the obtained RNA were determined, reverse transcribed, and then detected in overexpressing plants using RT-qPCR.​ Gene expression levels.

[0036] II. Experimental Results The results are as follows ​ As shown, compared to the wild type, ​ In the two strains of -OE ​ The gene expression level was significantly higher than that of the wild type, indicating that the constructed overexpression plants effectively expressed the gene. ​ .

[0037] Example 3 Rice ​ Exploring the function of genes I. Experimental Methods Rice planthopper nymphs at the third instar were used to treat rice plants and their tolerance was observed.

[0038] After rice seedlings germinated in pure water for 10 days, uniformly grown seedlings were harvested and planted in a rice house for 2 weeks (30℃, 16 hours light / 8 hours darkness). Rice seedlings at the 4-leaf stage (wild-type NIP, ...) were selected. ​ The plants were subjected to brown planthopper phenotypic treatment. Different genotypes were treated in pots of the same size, with 12 plants per pot and 8-10 brown planthoppers per plant. The tolerance of different genotypes of rice to brown planthopper treatment was observed and recorded daily.

[0039] II. Experimental Results The results are as follows ​ As shown, after 7 days of treatment with brown planthoppers, wild-type rice NIP showed yellowing leaves and lodging stems, indicating it was more sensitive to brown planthoppers; while compared to wild-type rice, ​ Both overexpression lines exhibited upright stems and green leaves. Plant survival statistics revealed that all NIP plants died, resulting in a survival rate of 0%. ​ The survival rate was 100%. ​ The survival rate was 75%. ​ The survival rate of the overexpression material was significantly higher than that of the wild-type plant.

[0040] The above results indicate that ​ Overexpression resulted in enhanced resistance to brown planthopper treatment. That is, it indicates... ​ Genes can significantly improve the insect resistance of rice and can be widely used in the fields of plant genetic engineering and plant disease resistance, with huge economic value and application prospects.

[0041] The above-described embodiments are preferred embodiments for ease of understanding of the present invention. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. Rice OsPDRA3 Genes may contain rice OsPDRA3 The application of gene-based biomaterials in improving insect resistance in rice is characterized by, The OsPDRA3 The nucleotide sequence is shown in SEQ ID No.

2.

2. Rice OsPDRA3 Genes may contain rice OsPDRA3 The application of gene-based biomaterials in the creation of insect-resistant rice is characterized by, The OsPDRA3 The nucleotide sequence is shown in SEQ ID No.

2.

3. The application according to claim 1 or 2, characterized in that, The rice OsPDRA3 The biological material containing the gene is rice. OsPDRA3 Gene expression cassettes, vectors, or strains.

4. The application according to claim 3, characterized in that, The vector is an overexpression vector.

5. The application according to claim 3, characterized in that, The strain in question is an overexpressing strain.

6. A method for creating insect-resistant rice, characterized in that, The method includes the following steps: [The rice...] OsPDRA3 Genes may contain rice OsPDRA3 Genes are obtained by transforming biological materials into rice explants, culturing, and screening.

7. The method according to claim 6, characterized in that, The transformation method is Agrobacterium-mediated transformation.

8. The method according to claim 7, characterized in that, The screening method is resistance culture medium screening and / or real-time quantitative PCR identification.

9. The application according to claim 1 or 2, or the method according to claim 6, characterized in that, The insect resistance is against brown planthoppers.

10. The application according to claim 1 or 2 or the method according to claim 6, characterized in that, The rice variety mentioned is Nipponbare.

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