Use of plant TIM protein, TIM gene encoding said protein or its homologous gene in antiviral of plants

Abstract

The present invention discloses the application of plant TIM protein or the TIM gene or its homologous gene in plant antiviral. The present invention also discloses a method for obtaining antiviral plants, which is to down-regulate the expression or activity of plant TIM protein. The present invention discloses a method for identifying whether the plants obtained by the described method have antiviral ability, by identifying whether the plants contain TIM protein or TIM gene. By means of transgenic method, the gene editing vector and gene silencing vector carrying the target gene are respectively introduced into the target plants, and homozygous plants with GmTIM gene knockout of soybean and plants with NbTIM down-regulated expression of Nicotiana benthamiana are obtained, and these plants can significantly inhibit virus infection.

Description

Technical Field

[0001] The present invention belongs to the application field of agricultural science and technology, and specifically relates to the application of plant TIM protein or TIM gene encoding the protein or its homologous gene in plant antiviral. Background Art

[0002] Soybean mosaic virus (SMV) is one of the most common viral diseases in the world's soybean production areas. Under natural conditions, the economic losses caused by SMV are 35%-50%, and can reach as high as 50%-100% in severe cases. In addition to directly causing serious soybean yield reduction and germplasm decline, the infection of SMV also greatly reduces the immunity of soybeans to other pathogens (Arif and Hassan, 2002; Liao et al., 2002). Therefore, SMV has become the main factor restricting soybean production. At present, there is no safe and effective chemical agent to control soybean mosaic virus disease, and breeding disease-resistant varieties is the most economical and effective method to control soybean mosaic virus disease.

[0003] Soybean mosaic virus belongs to the RNA virus (Potyviruses) of the genus Potyvirus, and the strain variation is relatively fast, making it easy to escape the host disease-resistant genes. Therefore, when a single resistant variety is planted over a large area, the resistance dominated by dominant resistance genes is easily broken by the evolution of pathogens, leading to the outbreak of large-scale diseases. This vicious cycle forces breeders to seek new resistance strategies. Recessive resistance genes are essential genes for the survival (replication, movement, and assembly, etc.) of plant viruses. It is known that about 200 antiviral genes, and about half of them are recessively inherited, indicating that in the process of plant-virus interaction, compared with other pathogens, the recessive resistance strategy is more common and the recessive resistance is more persistent (Hashimoto et al., 2016; Truniger and Aranda, 2009). Obtaining genetic resistance by genetically modifying the host genes (recessive resistance genes) necessary for virus infection has become a new strategy for antiviral breeding. For example, the translation initiation factor eIF4E and its homologous genes, which have been studied in detail at present, are essential host factors for Potyviruses that infect many important crops, and have been used as important recessive resistance genes for gene editing breeding (Robaglia and Caranta, 2006; Bastet et al., 2017).

[0004] The TIM family of plant mitochondrial proteins forms the main protein translocation complex on the inner mitochondrial membrane. Such as the translocase of the inner mitochondrial membrane carrier protein (TIM22 complex) and the translocase of the inner mitochondrial membrane leader sequence (TIM23 complex). The TIM23 complex regulates the insertion of proteins with leader sequences into the inner membrane, where the Tim23 subunit is responsible for transporting proteins into the matrix and can also insert certain proteins into the inner membrane (Bauer et al., 1996; Donzeau et al., 2000); the TIM22 complex is responsible for the transport and insertion of hydrophobic membrane proteins, where the Tim22 subunit is responsible for inserting mitochondrial metabolite transporters such as ADP / ATP and phosphate transporters into the inner membrane (Bauer et al., 2000; Duncan et al., 2013).

[0005] There is no report on the application of TIM proteins and TIM genes in plant antiviral defense so far. Summary of the Invention

[0006] Object of the Invention: The technical problem to be solved by the present invention is to provide the application of a TIM gene or its homologous gene in plant antiviral defense.

[0007] The technical problem to be solved by the present invention is to provide a method for obtaining antiviral plants.

[0008] The last technical problem to be solved by the present invention is a method for identifying whether the plants obtained by the described method have antiviral ability.

[0009] The present invention can make plants produce resistance and have broad-spectrum disease resistance through the silencing of tobacco TIM genes or the knockout of soybean TIM genes.

[0010] Technical Solution: To achieve the above object, the present invention provides the application of plant TIM proteins or TIM genes encoding the proteins or their homologous genes in plant antiviral defense.

[0011] Among them, the amino acid sequence of the plant TIM protein is shown in SEQ ID NO.1 or SEQ ID NO.26, and the nucleotide sequence of the TIM gene is shown in SEQ ID NO.2 or SEQ ID NO.5.

[0012] The present invention provides a method for obtaining antiviral plants, the method comprising: down-regulating the expression or activity of a plant TIM protein, the amino acid sequence of the plant TIM protein being shown in SEQ ID NO.1 or SEQ ID NO.26; preferably, the method for down-regulating the expression or activity of the plant TIM protein is to knockout or silence the TIM gene in the genome of the plant, the nucleotide sequence of the TIM gene being shown in SEQ ID NO.2 or SEQ ID NO.5.

[0013] Among them, the down-regulation method is to specifically knockout the GmTIM gene to interfere with the expression of the GmTIM gene. Preferably, the nucleotide sequence of the knocked-out GmTIM gene is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0014] Among them, the virus is soybean mosaic virus (SMV) of the genus Potyvirus, turnip mosaic virus (TuMV), tobacco mosaic virus (TMV) of the genus Tobamovirus or potato virus X (PVX) of the genus Potexvirus.

[0015] Among them, the plants include but are not limited to soybean or Nicotiana benthamiana.

[0016] Among them, the down-regulation method further includes silencing the homologous gene NbTIM by constructing dsRNA of its transcript. The nucleotide sequence of NbTIM is shown in SEQ ID NO.5, and the sequence of its transcript dsRNA is shown in SEQ ID NO.6.

[0017] The present invention also includes a method for identifying whether the plants obtained by the described method have antiviral ability, by identifying whether the plants contain TIM protein or TIM gene.

[0018] Among them, the identification method is detected by RT-qPCR or Western blotting.

[0019] Among them, the primer sequences for target detection of turnip mosaic virus (TuMV) are shown in SEQ ID NO.7 and SEQ ID NO.8, the primer sequences for target detection of tobacco mosaic virus (TMV) of the genus Tobamovirus are shown in SEQ ID NO.9 and SEQ ID NO.10, the primer sequences for target detection of potato virus X (PVX) of the genus Potexvirus are shown in SEQ ID NO.11 and SEQ ID NO.12, and the primer sequences for target detection of soybean mosaic virus (SMV) of the genus Potyvirus are shown in SEQ ID NO.13 and SEQ ID NO.14.

[0020] Beneficial effects: Compared with the prior art, the outstanding effect of the present invention lies in that the present invention discovers for the first time the functions of plant TIM genes including soybean TIM gene and tobacco TIM gene. The knockout of soybean TIM gene and the silencing of tobacco TIM gene can endow plants with resistance and broad-spectrum disease resistance.

[0021] The following further describes the application of silencing / knocking out plant TIM in regulating plant antiviral and the method for cultivating transgenic plants in the present invention in combination with the accompanying drawings and specific implementation methods. Description of the Drawings

[0022] Figure 1 To knockout GmTIM to endow soybean with resistance to soybean mosaic virus (SMV). (A) Sequencing confirms the knockout of GmTIM in the knockout plants; (B) Phenotypes of WT and GmTIM knockout plants 10 days after SMV inoculation. The WT plants also show mosaic symptoms, while the GmTIM knockout plants have not shown symptoms yet; (C) Phenotypes of WT and GmTIM knockout plants 30 days after SMV inoculation. The GmTIM knockout plants are still not diseased; (D) RT-qPCR detects the SMV RNA level in the plants; (E) Western blotting detects the accumulation amount of SMV protein in the above plants.

[0023] Figure 2 To silence NbTIM to endow Nicotiana benthamiana with resistance to turnip mosaic virus (TuMV). (A) RT-qPCR detects the expression level of NbTIM in NbTIM-RNAi transgenic plants; (B) Phenotypes of WT and NbTIM-RNAi transgenic plants 14 days after TuMV-GFP inoculation; (C) RT-qPCR detects the TuMV RNA level in the plants; (D) Western blotting detects the accumulation amount of TuMV protein in the above plants.

[0024] Figure 3 To silence NbTIM and confer Nicotiana benthamiana resistance to Tobacco mosaic virus (TMV). (A) RT-qPCR confirmed the silencing of NbTIM in NbTIM-RNAi transgenic plants; (B) phenotypes of WT and NbTIM-RNAi transgenic plants 10 days after inoculation with TMV-GFP; (C) RT-qPCR confirmed that the TMV RNA level in NbTIM-silenced plants was significantly lower than that in the wild type; (D) Western blotting confirmed that the accumulation of TMV protein in NbTIM-silenced plants was significantly lower than that in wild-type plants.

[0025] Figure 4 To silence NbTIM and confer Nicotiana benthamiana resistance to Potato virus X (PVX). (A) RT-qPCR detected the expression level of NbTIM in wild-type plants and NbTIM-RNAi transgenic plants; (B) phenotypes of WT and NbTIM-RNAi transgenic plants 14 days after inoculation with PVX-GFP; (C) RT-qPCR confirmed that the PVX RNA level in NbTIM-silenced plants was significantly lower than that in the wild type; (D) Western blotting confirmed that the accumulation of PVX protein in NbTIM-silenced plants was significantly lower than that in wild-type plants. Detailed implementation manners

[0026] The implementation manners of the present invention will be described in detail below in conjunction with examples. The following examples are only used to illustrate the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] Example 1 Knockout of GmTIM to confer plant resistance to viruses

[0028] Based on the sequence of soybean GmTIM (shown in SEQ ID NO.2), the target primers were designed as follows: 5’-CGCAGCAGAACGTAGATACTGTTTTAGAGCTAGAAATAGCAAG-3’ (SEQ ID No.15) and 5’-AGTATCTACGTTCTGCTGCGAATCCATATGTTTTCCTGGGAC-3’ (SEQ ID No.16). Referring to the specific method and the same conditions in the literature (Zhang P, Du H, Wang J, Pu Y, Yang C, Yan R, Yang H, Cheng H, Yu D. Multiplex CRISPR / Cas9-mediated metabolic engineering increases soya bean isoflavone content and resistance to soybean mosaic virus. Plant Biotechnol J. 2020 Jun;18(6):1384-1395. doi:10.1111 / pbi.13302.), the target fragment was recombined into the CRISPR-Cas9 vector pGmUbi-Cas9 to obtain the pGmUbi-Cas9-GmTIM vector. 1 μg of the pGmUbi-Cas9-GmTIM vector was mixed with 100 μl of Agrobacterium competent cells (EHA105 strain) and transferred into a clean electroporation cuvette. Electroporation was carried out at 2500 V using an electroporation device. Immediately, pre-cooled LB liquid medium was added. After recovery at 28 °C for 2 hours, it was spread on a resistant medium (LB medium with kanamycin resistance) and grown for 48 hours to screen for positive Agrobacterium. Positive single colonies were picked and transferred into a liquid resistant medium (LB medium with rifampicin and kanamycin resistance), cultured overnight at 28 °C and 200 rpm. The OD600 of Agrobacterium was adjusted to 1.0, and soybeans were inoculated to obtain infected soybean cotyledon nodes. Callus was obtained through differentiation culture, and then small seedlings were obtained through rooting culture. They were transferred and continuously cultured to obtain T0 generation regenerated soybean plants. Part of the leaf samples were taken, and the DNA of the regenerated soybean plants was extracted by the CTAB method. Using this as a template, a 700 bp fragment was amplified using the primer pair 5’-TATCACCGATAAGCCCACTGAGGC-3’ (SEQ ID No.17) and 5’-CACCATCATCAACAACAAATCAGTAGCT-3’ (SEQ ID No.18), and sequencing and alignment were performed to detect whether the regenerated plants were edited. By sequencing, there were deletions of 4 bases (shown in SEQ ID NO.3) and 14 bases (shown in SEQ ID NO.4) near the target sequences of two gene-edited lines (gmtim-1 and gmtim-9), asFigure 1 A. The wild type (Williams 82) and GmTIM-edited plants were respectively inoculated with SMV by rubbing. After 10 days, typical mosaic symptoms appeared in the wild type plants, but the knockout plants showed no symptoms ( Figure 1 B), and after 30 days, typical mosaic and shrinkage appeared in the wild type plants, but the knockout plants still showed no symptoms ( Figure 1 C). The results of RT-qPCR and western blotting showed that both viral RNA and coat protein were negative in GmTIM-knockout soybeans ( Figure 1 D-E), indicating that knockout of GmTIM confers resistance to soybeans. Collectively, the above results indicate that knockout of GmTIM confers resistance to the virus in plants.

[0029] Example 2 Silencing of NbTIM confers resistance to the virus in plants

[0030] Using RNA interference (RNAi) technology, with the NbTIM gene as the target gene (SEQ ID No. 5), an RNA interference vector was constructed.

[0031] Total RNA was extracted from Nicotiana benthamiana and reverse transcribed to obtain cDNA. Using the cDNA as a template, the nucleotide sequence from the 4th to 437th positions of the NbTIM self-transcript (SEQ ID No. 6) was amplified by PCR using two pairs of primers as the complementary DNA double strands of the hairpin structure. The primer pair 1 used was: 5’-CGGGATCCGATGATGCTGCAGAGCTAAG-3’

[0032] (SEQ ID No. 19) and 5’-GGGCCCCTCCTTGCCATTGTCAGTTC-3’ (SEQ ID No.

[0033] 20). The reaction system was 25 μL, including 12.5 μL of PCR Mix, 10 ng of cDNA template, 1 μL of each primer, and water to make up 25 μL. The reaction conditions were: pre-denaturation at 94 °C for 3 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, for 30 cycles; and finally extension at 72 °C for 7 min. The obtained PCR product was fNbTIM (forward). For convenient vector construction, BamHI and ApaI restriction enzyme sites were added to the 5’ and 3’ ends of fNbTIM by PCR respectively. The primer pair 2 was: 5’-ACGCGTCGACCTACTACGACGTCTCGATTCC-3’ (SEQ ID No.

[0034] (21) and 5'-CGACGCGTGAGGAACGGTAACAGTCAAGA-3' (SEQ ID No. 22). The resulting PCR product was rNbTIM (reversed). fNbTIM was double digested with BamHI and ApaI (reacted at 37°C for 30 minutes), rNbTIM was double digested with SalI and MluI, and ligated (at 22°C, for 15 minutes) into the pCambia2301 vector double digested with BamHI and SalI to obtain the RNA interference vector pCambia2301-NbTIM.

[0035] Mix 1 μg of the above RNA interference vector pCambia2301-NbTIM with 100 μl of Agrobacterium competent cells (EHA105 strain), transfer them into a clean electroporation cuvette, use an electroporation device to perform electroporation transformation at 2500 V, immediately add pre-cooled LB liquid medium, recover at 28°C for 2 hours, then spread them on a resistant medium and grow for 48 hours to screen for positive Agrobacterium. Pick a positive single colony and transfer it into a liquid resistant medium, culture it overnight at 28°C and 200 rpm, adjust the OD600 of the Agrobacterium to 1.0, and inoculate Nicotiana benthamiana. Obtain the infected Nicotiana benthamiana leaves, differentiate and culture to obtain callus, then obtain small seedlings through rooting culture, transfer and continue the culture to obtain the T0 generation regenerated plants of Nicotiana benthamiana, and use the primer pair 5'-CGGGATCCGATGATGCTGCAGAGCTAAG-3' (SEQ ID No. 19) and 5'-ACGCGTCTGTAATCAATCCAAATGTAAGATCAATG-3' (SEQ ID No. 23) to detect whether the marker gene has been integrated into the genome; in addition, use the primer pair 5'-CAGTTGGTGGATTCGTTGC-3'

[0036] (SEQ ID No. 24) and 5'-TTGTCAGTTCTCGTTGTCGC-3' (SEQ ID No. 25) to perform fluorescence quantitative PCR (RT-qPCR) detection of the target gene in the regenerated plants of Nicotiana benthamiana transformed with the RNAi vector to determine whether the transcription level of the target gene is down-regulated due to silencing.

[0037] To confirm the role of TIM in virus infection, we selected two lines (Line 3 and Line 9) with effective silencing of NbTIM for further experiments. The results of RT-qPCR detection of the expression level of NbTIM in NbTIM-RNAi transgenic plants are shown in Figure 2A; The onset of disease in NbTIM-silenced plants was delayed compared to wild-type plants, with milder symptoms, less green fluorescence in systemic leaves. When the wild-type plants had already started to die, the virus in NbTIM-silenced plants had just spread to the systemic leaves and the plants were in good condition. Samples from the same part of the systemic leaves were collected for further RNA and total protein extraction. RT-qPCR results showed that the viral RNA level in plants with silenced NbTIM was significantly lower than that of the control ( Figure 2 C); Western blotting was used to detect the accumulation level of TuMV protein in the above plants. 14 days after inoculating WT (wild-type Nicotiana benthamiana leaves) and NbTIM-RNAi transgenic plants with TuMV-GFP, Western blotting results also confirmed that the accumulation level of coat protein in NbTIM-silenced plants was significantly lower than that of the control plants ( Figure 2 D).

[0038] After inoculating WT (wild-type Nicotiana benthamiana leaves) and NbTIM-RNAi plants with TMV-GFP (provided by the Plant-Virus Interaction Group, Institute of Plant Protection, Chinese Academy of Agricultural Sciences) for 10 days, the disease symptoms in NbTIM-silenced plants were milder; the results are shown in Figure 3 .

[0039] After inoculating WT (wild-type Nicotiana benthamiana leaves) and NbTIM-RNAi plants with PVX-GFP (provided by the Plant-Virus Interaction Group, Institute of Plant Protection, Chinese Academy of Agricultural Sciences) for 14 days, the disease symptoms in NbTIM-silenced plants were milder; the results are shown in Figure 4 .

[0040] The above results indicate that silencing of TIM in tobacco confers resistance to viruses of different genera, showing broad-spectrum characteristics.

Claims

1. Plant TIM protein or a protein encoding the same TIM The application of the gene in plant antiviral, the amino acid sequence of the plant TIM protein is shown in SEQ ID NO. 1 or SEQ ID NO. 26, the TIM The nucleotide sequence of the gene is shown in SEQ ID NO. 2 or SEQ ID NO. 5, the plant is soybean or Nicotiana benthamiana, and the virus is soybean mosaic virus (SMV), turnip mosaic virus (TuMV) of the genus Potyvirus, tobacco mosaic virus (TMV) of the genus Tobacco mosaic virus, or potato virus X (PVX) of the genus Potyvirus.

2. A method for obtaining virus-resistant plants, characterized in that: The method comprises: down-regulating the expression or activity of a plant TIM protein, wherein the amino acid sequence of the plant TIM protein is as shown in SEQ ID NO. 1 or SEQ ID NO. 26; the method for down-regulating the expression or activity of a plant TIM protein is to knock out a TIM Gene or silence TIM Gene, the TIM The nucleotide sequence of the gene is shown in SEQ ID NO. 2 or SEQ ID NO. 5, the virus is soybean mosaic virus (SMV), turnip mosaic virus (TuMV), tobacco mosaic virus (TMV) of the genus Potyvirus, or potato virus X (PVX) of the genus Potyvirus, and the plant is soybean or Nicotiana benthamiana.

3. The method according to claim 2, characterized in that Downregulation method is specific knockout TIM Gene interference TIM Gene expression.

4. The method according to claim 3, characterized in that After knockout TIM The nucleotide sequence of the gene is shown in SEQ ID NO. 3 or SEQ ID NO.

4.

5. The method according to claim 2, characterized in that: Down-regulation method is to construct dsRNA of its transcript to homologous gene Nb The silence, Nb The nucleotide sequence is shown in SEQ ID NO. 5, and the sequence of its transcript dsRNA is shown in SEQ ID NO.

6.

6. A method for identifying whether a plant obtained by the method according to any one of claims 2 to 5 has antiviral ability, characterized in that: By identifying whether the plant contains TIM protein or TIM The plant TIM protein has an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 26, TIM The nucleotide sequence of the gene is shown in SEQ ID NO. 2 or SEQ ID NO. 5, the plant is soybean or Nicotiana benthamiana, and the virus is soybean mosaic virus (SMV), turnip mosaic virus (TuMV) of the genus Potyvirus, tobacco mosaic virus (TMV) of the genus Tobacco mosaic virus, or potato virus X (PVX) of the genus Potyvirus.

7. The method according to claim 6, characterized in that The identification method is detected by RT-qPCR or Western blotting.

8. The method according to claim 6, characterized in that The target detection primer sequences of the turnip mosaic virus (TuMV) are shown in SEQ ID NOs. 7 and 8, the target detection primer sequences of the tobacco mosaic virus (TMV) of the genus Tobacco Mosaic Virus are shown in SEQ ID NOs. 9 and 10, the target detection primer sequences of the potato virus X (potato virus X, PVX) of the genus Potato X virus are shown in SEQ ID NOs. 11 and 12, and the target detection primer sequences of the soybean mosaic virus (Soybean mosaic virus, SMV) of the genus Potyvirus are shown in SEQ ID NOs. 13 and 14.

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