OsAGO16 gene and application of protein coded by OsAGO16 gene in rice disease resistance

Abstract

The invention relates to the field of gene engineering, and particularly provides an Argonaute 16 (ADO16) gene and application of an encoding protein of the Argonaute 16 in rice disease resistance. Through CRISPR-Cas9 technology-mediated gene mutation, a mutant with the AGO16 gene knocked out is generated under the background of a rice Nipponbare wild type, and comparison analysis with the Nipponbare wild type shows that expression down-regulation caused by mutation of AGO16 in the rice can significantly increase the susceptibility rate of the rice and accumulation of viruses. Experiments show that AGO16 can effectively regulate accumulation of pathogens, and a new solution is provided for rice disease resistance.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to an AGO16 gene derived from rice and application of the protein encoded by the gene in rice disease resistance. Background Art

[0002] Rice is the most important cash crop and food crop in the world. It is widely planted in Asia, Africa, Latin America and other regions, and plays a key role in global food security and economic development. However, the yield and quality of rice are infected by many diseases, which not only seriously affect the growth and development of rice, but also cause irreversible damage to the ecological environment on which it depends. Among them, Rice stripe virus (RSV) is an important plant virus and a representative species of Tenuivirus. RSV is mainly transmitted by Laodelphax striatellus. After infecting rice, it will cause symptoms such as yellow-green stripes on leaves, dwarfing of plants, and poor panicle development, which seriously affect the growth and yield of rice and cause significant economic losses. So far, there is no effective chemical agent to prevent and control RSV. Once infected, the prevention and control measures mainly rely on controlling vector insects or planting disease-resistant varieties. Therefore, it is of great agricultural and economic significance to deeply study the disease resistance mechanism of rice to pathogens, screen and cultivate disease-resistant varieties, and develop new prevention and control strategies.

[0003] In order to cope with the invasion of pathogens, plants have evolved a series of complex defense mechanisms, among which RNA silencing is one of the key disease resistance mechanisms. RNA silencing includes two main forms: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). These two mechanisms play an important role in inhibiting the expression of pathogenic genes and enhancing plant disease resistance. Argonaute (AGO) protein is the main component of the gene silencing regulatory mechanism. AGO protein binds to small RNA (sRNA) to form RNA-Induced Silencing Complexes (RISCs). RISC can bind to mRNA complementary to the sRNA sequence, thereby promoting its degradation or inhibiting its translation, achieving the effect of gene silencing. Therefore, studying and utilizing the role of AGO protein in RNA silencing provides important ideas and technical means for the development of new disease-resistant plant varieties, and is of great significance for humans to further understand the prevention and control of rice diseases and the rapid development of agricultural health. Summary of the invention

[0004] Based on this, the present invention provides an application of rice AGO16 gene in rice disease resistance. Specifically, the present invention provides the following technical solutions:

[0005] The first aspect of the present invention provides a protein encoded by a rice AGO16 gene, the amino acid sequence of which is shown as SEQ ID NO.2.

[0006] The second aspect of the present invention provides a gene encoding the above protein, whose transcript sequence is shown as SEQ ID NO.1, and whose CDS sequence is shown as SEQ ID NO.3.

[0007] The third aspect of the present invention provides the use of the rice AGO16 gene and the protein encoded by the gene in rice disease resistance.

[0008] In a preferred embodiment, the virus is RSV.

[0009] A fourth aspect of the present invention provides a method for regulating the disease resistance of rice using the AGO16 gene, the method comprising regulating the expression level of the AGO16 protein in rice.

[0010] In one embodiment, the virus is RSV; the transcript sequence of the AGO16 gene is shown in SEQ ID NO.1.

[0011] In a preferred embodiment, CRISPR-CAS9 technology is used to obtain mutant rice of the AGO16 gene, and the mutant rice can significantly increase RSV infection.

[0012] Compared with the prior art, the present invention has achieved the following beneficial technical effects:

[0013] The experiment of the present invention proves that the rice AGO16 gene has an important regulatory effect on rice disease resistance, and mutating the gene to reduce its expression can weaken the rice disease resistance. Therefore, the gene can be used to regulate or study the application of rice disease resistance, and has important implications for screening rice disease resistance drugs. The present invention provides a method for breeding disease-resistant rice varieties, and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0015] Figure 1 Identification of the AGO16 gene structure and the mutant ago16-1 constructed by CRISPR-Cas9

[0016] Figure 2 The rice phenotypes of the NPB wild type and ago16-1 mutant.

[0017] Figure 3 This is the infection status of ago16-1 mutant rice and NPB rice under RSV virus infection. DETAILED DESCRIPTION

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] In the following examples, m / v is g / ml unless otherwise specified.

[0021] The mutants and wild-type sources appearing in the following examples are described as follows:

[0022] The AGO16 gene mutant seed ago16-1 was obtained by CRISPR-Cas9 technology.

[0023] The wild rice variety selected is Nipponbare type.

[0024] In the genomes of the AGO16 gene mutant ago16-1 and the wild-type rice NPB, only the AGO16 gene had a CRISPR-Cas9 mutation, and the other genes were the same.

[0025] Example 1 Construction and identification of rice AGO16 gene mutant plants

[0026] Identification of ago16-1 mutant

[0027] The genomic DNA of NPB wild-type rice (NPB WT) and ago16-1 mutant seedlings were extracted, and PCR amplification was performed using primers on the AGO16 gene.

[0028] The main reagents used were: KOD OneTM PCR Master Mix purchased from Toyobo, DNA marker and gelred nucleic acid dye purchased from Polymer Corporation. Primers were synthesized by BGI Biotechnology Co., Ltd.

[0029] Primer sequences used:

[0030] AGO16-F 5'-ATGGCTGCCAAAATTGGTGAAA-3'(SEQ ID NO.4)

[0031] AGO16-R 5'-ACAAAAGAACATCGAACTGCAG-3'(SEQ ID NO.5)

[0032] AGO16-SF 5'-GGTCCTCCTGAATTTTTGCTG-3'(SEQ ID NO.6)

[0033] After PCR amplification, sequencing was performed using primer AGO16-SF near the CRISPR insertion site, which showed that 4 bases were missing in the 14th exon region of the AGO16 gene in ago16-1 plants. The above mutations will cause the loss of the AGO16 gene.

[0034] like Figure 2 As shown, compared with NPB WT, the ago16-1 mutant had a significantly later heading time and higher plant height, showing obvious developmental abnormalities, indicating that the AGO16 gene affects the growth and development of rice.

[0035] Example 2 Application of Rice AGO16 Gene in Regulating Rice Disease Resistance

[0036] 1. Differences in the ability of ago16-1 mutant rice and wild-type NPB rice to resist RSV

[0037] Wild-type rice NPB and AGO16 mutant rice (ago16-1, a mutant in which 4 bases are missing in the 14th exon region of the AGO16 gene) were planted in custom glass jars. After growing for one week in a greenhouse at 28-30°C and a relative humidity of 60±5%, an appropriate amount of gray leafhoppers (Laodelphaxstriatellus) carrying RSV virus were placed in the glass jars according to the calculation of 3 adults per rice seedling, and the jars were sealed with white gauze to ensure that the gray leafhoppers could not escape after being moved in. After 48 hours, the gray leafhoppers were removed, and after about two weeks, obvious RSV symptoms were visible on the rice. The incidence and severity of the disease were counted, and samples were taken to detect the virus accumulation of wild-type rice NPB and mutant ago16-1 at the RNA and protein levels. The experimental steps are as follows:

[0038] 1) RSV virus was used to infect NPB and ago16-1 rice, and the disease incidence of rice was observed and recorded 2, 3, and 4 weeks after infection;

[0039] 2) Take rice leaves from the diseased part 4 weeks after infection, weigh them, and grind them with liquid nitrogen. 1 / 3 of the ground powder is used for protein extraction, and 2 / 3 is used for RNA extraction (Trizol method);

[0040] 3) RSV CP RNA detection

[0041] RNA of NPB WT and ago16-1 was extracted by Trizol method and reverse transcribed into cDNA.

[0042] qRT-PCR reaction was performed using RSV CP-qR, EF1a-qF, and EF1a-qR primers.

[0043] The reagents used were purchased from Novezan Biotechnology Co., Ltd. The primers were synthesized by BGI Biotechnology Co., Ltd.

[0044] Primer sequences used:

[0045] RSV CP-qF 5'-AAGCCACTGGACGATGGAAA-3'(SEQ ID NO.7)

[0046] RSV CP-qR 5'-TGAGACTCGCTCACACCATC-3'(SEQ ID NO.8)

[0047] EF1a-qF 5'-ACATTGCCGTCAAGTTTGCTG-3'(SEQ ID NO.9)

[0048] EF1a-qR 5'-AACAGCCACCGTTTGCCTC-3'(SEQ ID NO.10)

[0049] The results were analyzed by Graphpad Prism 9. Figure 3 As shown in d, the accumulation of RSV in ago16-1 was significantly higher than that in NPB.

[0050] 3) RSV CP protein detection

[0051] Western blot was used to detect the accumulation of RSV CP protein. The primary antibody was anti-RSV, and the secondary antibody was anti Rabbit; α-Tubulin was used as the internal reference for western blot. The monoclonal antibody against Tubulin was purchased from EASYBIO, with the catalog number BE4006-100, and α-RSV CP was a homemade monoclonal antibody against RSV CP.

[0052] The results are as follows Figure 3 As shown: Compared with wild-type NPB, the incidence of rice in the AGO16 mutant line is significantly higher and the severity of the disease is more severe ( Figure 3 ac); the RSV virus content in the mutant ago16-1 was significantly increased compared with NPB WT ( Figure 3 d); The viral coat protein CP protein increased significantly in the mutant ago16-1 ( Figure 3 e). The above results show that the accumulation of RSV RNA and CP protein in ago16-1 mutant increased significantly, indicating that AGO16 gene has the ability to resist RSV virus.

[0053] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A rice AGO16 gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

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

2.

3. Use of the gene according to claim 1 and / or the protein according to claim 2 in rice disease resistance.

4. The use according to claim 4, characterized in that The virus is Rice stripe virus (RSV).

5. A method for regulating rice disease resistance by using AGO16 gene, characterized in that: The method comprises regulating the disease resistance of the plant by controlling the expression level of the AGO16 gene as claimed in claim 2 in rice.

6. The method according to claim 5, characterized in that The pathogen is RSV; the transcript sequence of the AGO16 gene is shown in SEQ ID NO.

1.

7. An application of the AGO16 gene in screening rice disease resistance drugs, characterized in that: The pathogen is RSV.

8. Use of the gene according to claim 1 and / or the protein according to claim 2 in preparing a rice disease-resistant product.

9. A transgenic rice, characterized in that: The mutant rice of AGO16 gene was obtained by using CRISPR-CAS9 technology, and the mutant rice can significantly increase the infection of RSV. The transcript sequence of the AGO16 gene is shown in SEQ ID NO.1.

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