CN111849928B - A functional domain related to CWMV replicase region pathogenesis - Google Patents

Abstract

The invention relates to a molecular detection technology of crop virus diseases, belongs to the technical field of agricultural science, and particularly relates to a CWMV replicase region pathogenic related functional domain, wherein a CWMV replicase region pathogenic related sequence (VRpep) is expressed as a formula I: x ₁ ‑MFHNGTCELPKDSAFLDYTADNSGTWMYGKPSRFGHSYGVGFSLDT‑X ₂ I, the pathogenicity of CW1-7 on CWMV RNA1 is determined by the TRV vector-mediated gene silencing-based method. On the basis, through ATG deletion mutation and early termination expression experiments, the effective pathogenic factor is finally determined to be polypeptide, and is positioned at 939-984 amino acids (VRpep) of replicase P153 protein of CWMV RNA1 chain, and the definition of the pathogenic related functional domain from CWMV provides a new idea for developing an anti-disease strategy of soil-borne virus.

Description

A CWMV replicase region pathogenicity-related functional domain

Technical Field

The invention relates to a molecular detection technology for crop virus diseases, belongs to the field of agricultural science and technology, and specifically relates to a pathogenicity-related functional domain of a CWMV replicase region.

Background Art

In China's winter wheat regions, the pathogens of wheat virus diseases spread by Polymyxa graminearum are wheat yellow mosaic virus (WYMV) of the genus Bymovirus and Chinese wheat mosaic virus (CWMV) of the genus Furovirus. WYMV outbreaks have a serious impact on wheat production safety in the affected areas. WYMV is widely distributed in my country, and although CWMV is limited to a few areas such as Shandong and Jiangsu, field surveys in recent years have shown that CWMV has a clear expansion trend.

Viruses absolutely parasitize in plant cells and need host cells to provide energy, materials, and places. This extreme dependence on the host causes interference and physiological changes caused by the host's defense response, which can cause acute or chronic plant diseases. When the virus infection causes physiological abnormalities in the host and disease symptoms appear, it is considered to have a pathogenic effect. All proteins encoded by plant viruses, such as replicase, movement protein, and coat protein, may be the pathogenic factors of the virus. Host pathogenicity caused by replicase has been reported in many plant viruses, such as Plum pox virus (PPV), Oilseed rape mosaic virus (ORMV), Grapevine fanleaf virus (GFLV) and Pepper mild mottle virus (PMMoV). At the same time, the polypeptide encoded by the virus may also be the pathogenicity determining factor of the host plant. For example, the pathogenicity factor of Citrus tristezavirus (CTV), which causes seedling yellowing syndrome, is located at the 3' end of the viral genome. CELIA R.A.DUFF-FARRIER et al. constructed a chimeric viral infectious clone combining the genomic components of two strains (EU and CH) of Pepino mosaic virus (PepMV) and demonstrated that the pathogenicity determining factor of PepMV is the 11-26 amino acids at the N-terminus of CP.

The CWMV genome contains two positive single-stranded RNAs, and the gene encoding the replicase is located on RNA1, encoding two replication-related proteins with molecular weights of 153KDa and 212KDa respectively. P212 consists of two parts, P153 and P55. P153 has three functional domains, including a region with methyltransferase activity, a variable region, and a region with helicase activity; P55 is an RNA-dependent RNA polymerase (RdRp), which is related to viral replication; during in vitro translation of the virus, there is a certain probability that P153 and P55 fuse into a large read-through protein P212, which functions as an RNA replication complex.

Soil-borne wheat virus disease is one of the important diseases in winter wheat areas in my country. Given that the dormant spores of the transmission vector Polymyxomyces graminearum have thick walls and strong stress resistance, and chemical agents are less effective, the most important way to prevent and control soil-borne wheat virus disease is to identify and promote resistant varieties. However, there are currently few wheat varieties resistant to CWMV. Therefore, identifying and clarifying the pathogenic factors and mechanisms of CWMV will provide a theoretical basis for the development and promotion of wheat varieties with high resistance to soil-borne virus diseases.

Summary of the invention

The present invention provides a CWMV replicase region pathogenic function region, which is represented by Formula I:

X ₁ -MFHNGTCELPKDSAFLDYTADNSGTWMYGKPSRFGHSYGVGFSLDT-X ₂ I

wherein _X1 is HEAMWYLQCKIVSDRTTLRSIIDDHLRG (SEQ ID No. 10) or is missing;

_X2 is:

RQRVSKCELVKLMWNHDSRGQVNQKPVNTRAFQYLLLSELSFMMNEMIIYRNLQQVMRKRERTKQARITLRDGVPGCGKSTWILNNANPT (SEQ ID No. 11) or missing.

Preferably, Formula I is SEQ ID No.2, and its corresponding nucleotide sequence is SEQ ID No.1.

The primer set used for cloning SEQ ID No.1 provided by the present invention is:

153-F (SEQ ID No. 5): 5’-CCTGCAGCACGAGGCGATGTGGTAT-3’

153-R (SEQ ID No. 6): 5’-CCTGCAGTGTGGGATTTGCATTGTTC-3’

The present invention further provides a method for identifying the pathogenic functional domain of the CWMV replicase region, wherein the method utilizes a gene silencing system mediated by tobacco rattle virus (TRV) to identify the pathogenic factor sequence of the CWMV replicase region, further experiments confirm that the pathogenic factor is a polypeptide, and the shortest effective pathogenic polypeptide is screened.

Further preferably, the method for identifying the pathogenicity-related functional domain of the CWMV replicase region is:

Step 1: The replicase P153 of CWMV RNA1 (GenBank: AJ012005.1) in NCBI was divided into five polypeptide sequences (P153-1 to P153-5), and the positive chain nucleotide sequences encoding these five polypeptides and RdRp (P55) were constructed into TRV vector (TRV-RNA2), and co-infiltrated Nicotiana benthamiana with TRV-RNA1 Agrobacterium. TRV:00 infiltrated tobacco was set as the control, and the symptom phenotype of Nicotiana benthamiana was continuously observed. The results showed that after Agrobacterium infiltration, only TRV:P153-4 could significantly aggravate the symptoms of Nicotiana benthamiana compared with the control.

Step 2: The reverse complementary sequence of P153-4 was constructed into the TRV vector by the same method, and it was found that it had no ability to enhance the pathogenicity of TRV; the 8 effective translation start codons corresponding to P153 in the P153-4 sequence and the extended sequence at the 5' end were deleted and mutated (TRV: CW1-7M1 to TRV: CW1-7M7), and Nicotiana benthamiana was infected by Agrobacterium co-infiltration method; after infection, the symptoms of Nicotiana benthamiana were observed, confirming that the TRV enhanced symptoms induced by CW1-7M1 and CR1-7 (CW1-7), CW1-7M2 to CW1-7M7 were not significantly different from the control symptoms; indicating that the effective pathogenicity-related factor of P153-4 is a polypeptide.

Preferably, Formula I is SEQ ID No. 4, the shortest effective sequence of the pathogenic factor is amino acids 939-984 of P153 (VRpep), and its corresponding nucleotide sequence is SEQ ID No. 3. The pathogenic factor can also effectively enhance the infection and replication of Potato virus X (PVX) on Nicotiana benthamiana.

The primer set used for cloning SEQ ID No.3 provided by the present invention is:

VRpep-F (SEQ ID No. 7):

5’-CCTGCAGATGTTTCACAATGGAACTTGT-3’

VRpep-R (SEQ ID No. 8):

5’-CCTGCAGTTACGTGTCCAGTGAAAAACC-3’.

In order to further clarify the shortest effective pathogenicity-related polypeptide of CWMV P153-4, the method adopted by the present invention is as follows: based on CW1-7P2, the 5' end sequence thereof was prematurely translated and terminated, and four recombinant vectors TRV:CW-pep1, TRV:CW-pep2, TRV:CW-pep3, and TRV:CW-pep4 were constructed respectively (Figure 3), and Nicotiana benthamiana was infiltrated by Agrobacterium; tobacco symptoms were observed, indicating that the expression of the three polypeptides CW-pep1, CW-pep2 and CW-pep3 could cause tobacco dwarfing, stem base necrosis and other symptoms, while CW-pep4 could not cause the occurrence of the above symptoms; therefore, the shortest effective pathogenicity polypeptide of P153-4 is CW-pep3 (VRpep), and the amino acid sequence of VRpep is SEQ ID No.4.

We used the same method to detect the effect of VRpep on the pathogenicity of another viral vector, PVX, and the results showed that VRpep could also enhance the symptoms of PVX in Nicotiana benthamiana (Figure 5). Molecular methods were used to detect the accumulation of viruses in Nicotiana benthamiana expressing VRpep, and RT-qPCR and Western Blot tests showed that the expression of TRV and PVX at the transcriptional and protein levels were significantly higher than the control (Figures 4 and 5).

More preferably, the method for screening the pathogenicity factor of the CWMV replicase region comprises the steps of:

1. The replicase P153 of CWMV RNA1 (GenBank: AJ012005.1) in NCBI was divided into five polypeptide sequences (P153-1 to P153-5), and the corresponding positive chain nucleotide sequences encoding these five polypeptides and RdRp (P55) were constructed into TRV vector (TRV-RNA2) respectively, and co-infiltrated Nicotiana benthamiana with TRV-RNA1 Agrobacterium, and TRV:00 infiltrated tobacco was set as the control, and the symptom phenotype of Nicotiana benthamiana was continuously observed. The results showed that after 7 days of Agrobacterium infiltration, compared with the control, only TRV:P153-4 could significantly aggravate the symptoms of Nicotiana benthamiana (Figure 1). The nucleotide sequence of this effective pathogenicity-related factor is SEQ ID No.1, and the amino acid sequence is SEQ ID No.2.

The primer set used for cloning the effective pathogenicity-related factor provided by the present invention is:

153-F(SEQ ID No.5):5’-CCTGCAGCACGAGGCGATGTGGTAT-3’

153-R(SEQ ID No.6):5’-CCTGCAGTGTGGGATTTGCATTGTTC-3’

2. To clarify whether the effective pathogenicity-related factor of the CWMV P153-4 sequence is a polypeptide or a viral small interfering RNA (vsiRNA), the reverse complementary sequence of P153-4 was constructed into the TRV vector by the same method, and it was found that it had no ability to enhance the pathogenicity of TRV. In addition, we deleted and mutated the 8 effective translation start codons corresponding to P153 in the P153-4 sequence and the extended sequence at the 5' end (TRV: CW1-7M1 to TRV: CW1-7M7, TRV: CW1-7P2, Figure 2), and infected Nicotiana benthamiana by Agrobacterium co-infiltration. After 7 days of infection, the symptoms of Nicotiana benthamiana were observed. The results showed that except for the TRV enhanced symptoms caused by CW1-7M1 and CR1-7 (CW1-7), there was no significant difference between CW1-7M2 to CW1-7M7 and the control symptoms. At the same time, if the second ATG (CW1-7P2) was retained during the mutation process, its ability to enhance TRV symptoms on tobacco was restored, indicating that the effective pathogenicity factor of P153-4 is a polypeptide rather than vsiRNA, and that the second start codon plays an important role in its pathogenicity.

3. To further clarify the shortest effective pathogenic polypeptide of CWMV P153-4, this experiment terminated the translation of the 5' end sequence of CW1-7P2 in advance, and constructed four recombinant vectors TRV:CW-pep1, TRV:CW-pep2, TRV:CW-pep3, and TRV:CW-pep4 (Figure 3), and infiltrated Nicotiana benthamiana with Agrobacterium. The tobacco symptoms were observed 7 days after infection. The results showed that the expression of CW-pep1, CW-pep2 and CW-pep3 polypeptides could cause tobacco dwarfing, stem base necrosis and other symptoms, while CW-pep4 could not cause the above symptoms. Therefore, the shortest effective pathogenic polypeptide of P153-4 is CW-pep3, with a total of 46 amino acids. Since CW-pep3 is located in the variable region (VR) of the P153 protein, it is named VRpep. The nucleotide sequence of VRpep is SEQ ID No.3, and the amino acid sequence is SEQ ID No.4, corresponding to the 939-984 amino acids of the CWMV replicase protein P153. The primer set used for VRpep is:

VRpep-F(SEQ ID No.7):5’-CCTGCAGATGTTTCACAATGGAACTTGT-3’

VRpep-R (SEQ ID No. 8): 5’-CCTGCAGTTACGTGTCCAGTGAAAAACC-3’.

4. We used the same method to detect the effect of VRpep on the pathogenicity of another viral vector, PVX. The results showed that VRpep can also enhance the symptoms of PVX in Nicotiana benthamiana (Figure 5). Molecular methods were used to detect the accumulation of viruses in Nicotiana benthamiana expressing VRpep. RT-qPCR and Western Blot tests showed that the expression of TRV and PVX at the transcriptional and protein levels were significantly higher than the control (Figure 4, Figure 5).

The beneficial effects of the present invention are as follows: a sequence CR1-7 (CW1-7) with pathogenic function was identified in the P153 region of the CWMV replicase, and further experiments confirmed that its effective pathogenic factor is a 46-amino acid polypeptide VRpep. In addition to TRV, VRpep can also promote the infection and replication of PVX on Nicotiana benthamiana, indicating that the ability of VRpep to enhance viral pathogenicity may be non-specific. The results obtained in this study will help provide ideas for the development of new anti-disease strategies for soil-borne viral diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing symptoms caused by the pathogenic sequence on CWMV replicase on Nicotiana benthamiana; wherein TRV:P153-4 indicates TRV expressing P153-4; TRV:00 is the control.

Fig. 2 Schematic diagram of the P153-4 deletion mutation.

Fig. 3 Schematic diagram of premature translation termination of CW1-7P ₂ , where the expression of CW1-7pep4 in Nicotiana benthamiana cannot aggravate TRV symptoms.

Figure 4 VRpep promotes the systemic infection of TRV to Nicotiana benthamiana; A: Symptoms of Nicotiana benthamiana 7 days after Agrobacterium infiltration with TRV:VRpep; B, C: RT-qPCR and Western blot detection of the accumulation of TRV CP in TRV:00 and TRV:VRpep.

Figure 5 VRpep promotes the systemic infection of PVX to Nicotiana benthamiana; A: Symptoms of Nicotiana benthamiana 5 days after Agrobacterium infiltration with PVX:VRpep; B, C: RT-qPCR and Western blot detection of the accumulation of PVX CP in PVX:GUS and PVX:VRpep.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific implementation schemes. These embodiments should be understood as only the best examples of the present invention, and are not intended to limit the present invention in any way. Any improvements, modifications and equivalent substitutions made within the principles of the present invention should be included in the scope of the present invention.

Example 1: Extraction of Total RNA by Trizol and Establishment of cDNA Library

II Q RT Supermix for qPCR(Vazyme)试剂盒说明书将符合要求的RNA逆转录为高质量的cDNA。Wheat samples infected with CWMV were collected from the diseased experimental field in Rongcheng, Shandong. 2 g of wheat leaf tissue was ground in liquid nitrogen, and the total RNA of the wheat leaves was extracted using the trizol (Invitrogen) method. After the quality was tested by formaldehyde denaturing gel electrophoresis and met the requirements, the total RNA was extracted according to the method of

II Q RT Supermix for qPCR (Vazyme) Kit Instructions Reverse transcribe the RNA that meets the requirements into high-quality cDNA.

Example 2: Primer design

DNAMAN software was used to analyze the multiple cloning site of the viral vector TRV and the CWMV RNA1 replicase gene, and the sequence of pst I and its protective base, namely CCTGCAG, were added before the primer sequence used to screen the pathogenic factor. The specific primer list is shown in Table 1.

Table 1. Primer sequences for TRV vector construction

Example 3: Amplification and recovery of target fragments

SVGel and PCRClean-UpSystem试剂盒说明书进行回收，回收产物在与TsingKe公司T载体pClone007进行连接。连接产物转化大肠杆菌后在氨苄青霉素抗性的LB平板上筛选阳性克隆。PCR对随机挑取平板上的菌落进行鉴定，含有大小正确片段的质粒去测序，验证序列的正确性。The primers designed above were used to carry out amplification reaction with the susceptible wheat cDNA as template; the amplification system was 1μl of susceptible wheat cDNA, 2×PCR Buffer for KOD FX Neo 25μl, 2mM dNTPs 10μl, primmer-F 1μl, primmer-R 1μl, KOD FX Neo (1U/μl) 1μl, dH ₂ O 16μl. The PCR reaction conditions were 94℃ 3min, 94℃ 30s, 55℃ 30s, 72℃ 30min, 35 cycles, 72℃ 10min. The amplified product was electrophoresed at 150 volts for 40 minutes on a 2% agarose gel, stained with nucleic acid dye, and the amplification results were observed and analyzed using a gel imager. If the amplified band was obvious and specific, the specific amplified band was cut under a long-wave ultraviolet lamp and analyzed according to

The SVGel and PCRClean-UpSystem kit instructions were followed for recovery, and the recovered product was connected to the T vector pClone007 of TsingKe. The ligated product was transformed into E. coli and positive clones were screened on an ampicillin-resistant LB plate. PCR was used to identify the colonies on the randomly selected plates, and the plasmids containing the correct size fragments were sequenced to verify the correctness of the sequence.

Example 4: Recombinant vector construction

The silencing vector PASH18 plasmid was digested with pst I and then phosphorylated. The reaction system was 5 μg of vector plasmid, 5 μl of 10×FastDigest Buffer, 5 μl of FastDigest pst I, and 50 μl of nuclease-free water. After treatment at 37°C for 30 min, the dephosphorylation enzyme Thermosensitive Alkaline Phosphatase was added and treated at 37°C for another 30 min.

The target fragments obtained by digesting the T vector containing the target gene (sense strand and antisense strand) described in Example 3 with pst I were ligated with the silencing vector PASH18 treated with the above enzyme digestion under the action of T4 ligase, and the ligation products were transformed into Escherichia coli and positive clones were screened on a plate containing tetracycline resistance. The transformation was successful into Escherichia coli.

Example 5: Agrobacterium co-infiltration of tobacco

Take 500ng of the expression vector plasmid successfully connected above, add it to the competent EHA105 of Agrobacterium, and screen the positive clones with LB plates containing Kan and Rif antibiotics after electroporation. The Agrobacterium containing the target gene recombinant vector plasmid and the TRV-RNA1 Agrobacterium bacterial solution are eluted and resuspended with sterile water and transient transfection solution, and the OD value is adjusted to 1.0, and then mixed at a volume ratio of 1:1. Select a Nicotiana benthamiana plant with a seedling age of about 3 weeks, pierce a small hole on the back of the third and fourth leaves with a syringe needle, and slowly infiltrate the tobacco leaves with the bacterial solution from the small hole with a syringe without a needle. Make a mark, culture the Nicotiana benthamiana in a normal growth environment (24℃, 16h light, 8h dark), and continue to observe the symptoms of the plants.

Example 6: Fluorescence quantitative PCR (RT-qPCR)

According to the sequences of TRV RdRp and PVX CP genes, specific quantitative primers were designed, and the internal reference gene was tobacco UBC. The primer sequences are shown in Table 2.

Table 2. Primers and sequences involved in RT-qPCR

Table2.Primers Sequences involved in RT-qPCR

qPCR Master Mix说明书进行定量PCR检测，10μl检测体系如下：Reference ChamQ ^TM

qPCR Master Mix instructions for quantitative PCR detection, 10μl detection system is as follows:

The above reagents were added to a 384-well quantitative plate (Applied Biosystems) in sequence, mixed, and reacted in a real-time fluorescence quantitative PCR instrument according to the following procedure:

Three independent experiments were performed, each with three biological replicates, and two technical replicates were performed for each biological replicate. The results were analyzed using the 2 ^-ΔΔCt relative quantitative method, and the expression level of each gene mRNA was normalized to the expression level of the housekeeping gene UBC.

Example 7: Western blot

Western blot was performed on leaves of Nicotiana benthamiana infected with the TRV system at 7 dpi and leaves of Nicotiana benthamiana infected with the PVX system at 5 dpi, respectively. The experimental method was the same as 2.2.10. The primary antibody used for Western blot detection of TRV accumulation was TRVCP, diluted 1:5000; the secondary antibody was Anti-Rabbit IgG (Transgen Biotech). The primary antibody used for Western blot detection of PVX accumulation was PVX CP, diluted 1:5000; the secondary antibody was Anti-Rabbit IgG (Transgen Biotech).

Sequence Listing

<110> Ningbo University

<120> A pathogenicity-related functional domain of a CWMV replicase region and its screening method

<160> 35

<170> SIPOSequenceListing 1.0

<210> 1

<211> 492

<212> DNA

<213> artificial

<220>

Claims (1)

1. A CWMV replicase domain pathogenesis-related domain represented by formula I:

X ₁ -MFHNGTCELPKDSAFLDYTADNSGTWMYGKPSRFGHSYGVGFSLDT-X ₂ I

wherein X ₁ Is HEAMWYLQCKIVSDRTTLRSIIDDHLRG or absent;

X ₂ comprises the following steps:

RQRVSKCELVKLMWNHDSRGQVNQKPVNTRAFQYLLLSELSFMMNEMIIYRNLQQVMRK RERTKQARITLRDGVPGCGKSTWILNNANPT or absent.

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