Use of StpCaP1 gene to improve resistance of potato to PVY and PVS viruses

Abstract

The application provides application of StpCaP1 gene in improving resistance of potato to PVY and PVS viruses. The nucleotide length of the CDS sequence of the StpCaP1 gene is 609 bp. By constructing an RNAi vector, an e potato No. 3 is genetically transformed to verify the transgenic function, and the results prove that the expression of the StpCaP1 gene is not affected by the growth and development of the plant, and the accumulation of two different virus genus viruses PVY and PVS in the plant can be significantly inhibited after virus inoculation, which shows that the potato plant silencing the StpCaP1 gene has a significant effect on the resistance to PVY and PVS viruses, and is a functional gene that can be applied to potato virus resistance breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular disease resistance breeding, specifically relating to... StpCaP Application of gene 1 in improving potato resistance to PVY and PVS viruses. Background Technology

[0002] potato( Solanum tuberosum L.) is characterized by its strong adaptability, rich nutrition, and dual use as both a grain and a vegetable (Xie Conghua and Liu Jun 2021). Potatoes are produced through asexual reproduction, and viruses can accumulate in tubers over generations, causing seed tuber degeneration and severely impacting tuber yield and quality, becoming one of the limiting factors for the rapid development of my country's potato industry (Wang and Zhang 2010). The main viruses infecting potatoes and causing serious losses in my country include six positive RNA viruses (PVY, PLRV, PVX, PVA, PVS, and PVM) and one type of virus (spindle tuber viroid, PSTVd) (Kreuze et al 2020). For example, PVY is one of the most economically destructive viruses affecting potatoes globally (Scholthof et al 2011), belonging to... Potyviridae division Potyvirus The genus *PVS* consists of a single-stranded positive RNA of approximately 10 kb in size and a capsid protein, forming a curved, filamentous virus particle about 750 × 11 nm in length. It has a wide host range and when infecting potatoes, it typically causes symptoms such as mosaic patterns, necrosis of lateral leaves, and wrinkled terminal leaves, leading to a 20%-50% reduction in potato yield. When co-infected with other potato viruses, the yield reduction can even reach over 80% (Li Mengmeng et al., 2015). Betaflexiviridae division Carlavirus The virus, a genus of viruses, consists of single-stranded positive-sense RNA (approximately 8.5 kb) and a coat protein, forming curved rod-shaped particles measuring 610-710 × 12-15 nm. Most potato varieties do not show visible symptoms after PVS infection, but it can cause a 20%-30% yield reduction (Martelli et al., 2007). Statistics show that large-scale yield reductions caused by seed potato degeneration due to virus accumulation account for an estimated 50% or more of the total potential potato yield (Dieudonné et al., 2018). The virus infects potato plants at the cellular level, and chemical agents are difficult to control effectively in the field. Traditional control methods (such as producing virus-free seed potatoes and controlling aphids and other virus-transmitting vectors) also require strict management and high investment. Utilizing the host's own disease resistance mechanisms and breeding resistant varieties is a more economical and effective virus control strategy.

[0003] Because viruses have a simple structure, possessing only a finite genome size and a limited number of encoded proteins, they cannot independently complete processes such as translation, replication, and transport. Therefore, they must recruit certain proteins from host plants to assist them in completing the entire infection cycle. These host proteins that support viral replication are collectively called host factors or susceptibility factors. Natural or induced mutations in host factors that result in recessive alleles will confer corresponding viral resistance on the plant. Eukaryotic translation initiation factors (ECIF) eukaryotic translation initiation factor, eIF ) 4E Its isomers are the earliest discovered and most widely used viral susceptibility genes in crops, but due to eIF4E The widespread existence of isomers leads to a certain degree of functional redundancy within their family, with individual... eIF4E Recessive mutations in genes do not always confer complete viral resistance in plants (Mayberry et al. 2011). Furthermore, because... eIF4Es In potatoes, which play a crucial role in plant growth and development, the loss of their function can lead to embryonic necrosis phenotypes. In recent years, researchers have begun to explore and identify other potential viral susceptibility genes (Patrick et al. 2014; Hashimoto et al. 2016). Currently, there are almost no reports on the discovery and utilization of susceptibility genes in potatoes to enhance plant resistance to potato viruses. Therefore, identifying potato virus susceptibility genes and clarifying their impact on resistance to major potato viruses has significant biological and economic implications.

[0004] This application screened genes that were significantly induced to be expressed by PVY virus in potatoes after infection, based on transcriptome sequencing and quantitative fluorescence analysis. StPCaP1 Currently, regarding StPCaP1 There are no reports in the literature regarding the function of genes in potato resistance to viral diseases.

[0005] RNA interference (RNAi) refers to the suppression of target gene expression by small double-stranded RNAs that stimulate the specific degradation of complementary target mRNAs, leading to phenotypic changes. These phenotypic variations then allow for the study of target gene function. Compared to gene knockout methods, RNAi can suppress gene expression in contemporary plants, enabling the silencing and functional analysis of target genes. It has been widely applied in research on potatoes, tobacco, and tomatoes, among others.

[0006] Therefore, this application utilizes RNAi technology to silence [something] in potatoes. StPCaP1 , for research StPCaP1 Does gene silencing affect potato resistance to several major potato viruses? The results showed... StPCaP1 Gene silencing has little impact on basic plant processes such as growth and development, making it suitable for breeding. Further research has found that... StPCaP1Silencing plants significantly reduced the accumulation of both PVY and PVS viruses, demonstrating for the first time that silencing... StPCaP1 It can enhance the resistance of potatoes to two important potato viruses from two different genera.

[0007] Problems with existing technologies: This application screened genes that were significantly induced to be expressed by PVY virus in potatoes after infection, based on transcriptome sequencing and quantitative fluorescence analysis. StPCaP1 Currently, regarding StPCaP1 The function of genes in potato resistance to viral diseases has not been reported in the literature. Therefore, this application utilizes RNAi technology to silence genes in potatoes. StPCaP1 , for research StPCaP1 Does gene silencing affect potato resistance to several major potato viruses? The results showed... StPCaP1 Gene silencing has little impact on basic plant processes such as growth and development, making it suitable for breeding. Further research has found that... StPCaP1 Silencing plants significantly reduced the accumulation of both PVY and PVS viruses, proving that silencing... StPCaP1 It can enhance the resistance of potatoes to these two important potato viruses. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention provides a potato StPCaP1 Application of genes in enhancing potato resistance to PVY and PVS viruses. Using cDNA from leaves of the cultivated potato variety Emabu 3 as a template, sequences published in the potato genome database were cloned. StPCaP1 Genes were extracted and an RNAi interference vector was constructed. These genes were then genetically transformed into the Emasu 3 potato variety for transgenic function verification, revealing that silent genes were present. StABL1 The gene can enhance the plant's resistance to PVY and PVS, mainly manifested in reduced virus accumulation and milder plant symptoms.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: 1. Potatoes StPCaP1 Gene, Omaloupe No. 3 StPCaP1 The gene CDS sequence is shown in SEQ ID NO.1 of the sequence listing, with a nucleotide length of 609 bp. The sequence ID published in the Potato Genome Database (http: / / spuddb.uga.edu / ) is Soltu.DM.10G000120.1. The two sequences are completely identical.

[0010] 2. StPCaP1 The method for constructing a gene interference vector in plants includes: obtaining the interference sequence by PCR from the cDNA of potato cultivar E3, and the interference... StPCaP1The sequence of the fragment used is shown in SEQ ID No. 2 of the sequence listing. The PCR product was recovered. Using this as a template, a second round of PCR amplification was performed using universal primers: attB1 and attB2. The gel-recovered product was recombined into the pDNOR221 vector via a BP reaction. The positive clone pDNOR221- StPCaP1 Recombinantly into the pHellsgate8 vector via LR reaction, heat-transformed into E. coli DH5α, and sequenced for verification. The correctly sequenced positive clone pHellsgate8-StPCaP1 was electroporated into Agrobacterium GV3101, and clones that tested positive by PCR were preserved for further genetic transformation.

[0011] 3. StPCaP1 The verification methods for the application of genes in enhancing potato resistance to major potato virus diseases include: (1) potato genetic transformation, (2) detection of transgenic positive lines, (3) identification of developmental phenotype of transgenic potato plants, and (4) identification of virus resistance of transgenic potato plants.

[0012] 4. Application of the StpCaP1 gene in enhancing potato resistance to PVY virus, wherein the application is achieved through RNAi silencing. TST1 Gene expression, or gene knockout through gene editing.

[0013] 5. Application of the StpCaP1 gene in improving potato resistance to PVS virus, wherein the application is achieved through RNAi silencing. TST1 Gene expression, or gene knockout through gene editing.

[0014] 6. A vector containing the sequence shown in SEQ ID NO. 1.

[0015] 7. Potato StpCaP1 protein, wherein the potato StpCaP1 protein is encoded by the sequence shown in SEQ ID NO. 1 of the sequence listing.

[0016] Compared with the prior art, the beneficial effects of the present invention are: silence. StPCaP1 The subsequent plant growth and tuber yield were unaffected, indicating that this gene is a susceptible gene that can be used for breeding production and is silenced. StPCaP1 The accumulation of PVY and PVS viruses in the plants was significantly reduced, and the symptoms of disease were significantly alleviated, indicating that the inhibition of PVY and PVS viruses was effective. StPCaP1 The potato plants exhibit remarkable resistance to PVY and PVS viruses, making it a functional gene that can be used in potato antiviral breeding applications. Attached Figure Description

[0017] Figure 1: Schematic diagram of the construction process of the interference vector pHellsgate8-StPCaP1.

[0018] Figure 2 : StPCaP1 Expression levels in the transgenic lines were detected. Data are presented as mean ± standard deviation (n=3). ef1α As an internal reference (*P<0.05, **P<0.01, ***P<0.001, student t test).

[0019] Figure 3 : StPCaP1 Developmental phenotypic identification of interfering transgenic lines. Among them, Figure 3 A: Growth phenotypes of E3 and interference lines after 3 months, scale bar is 10cm. Figure 3 B: Single-plant tuber harvesting phenotypes of E3 and interference lines; Figure 3 Number of potatoes harvested per plant in C, E3 and interference lines; Figure 3 Fresh potato weights per plant in lines D, E3, and interference lines. Data are presented as mean ± standard deviation (n≥12). Duncan's new multiple range analysis was performed, and p<0.05 was observed.

[0020] Figure 4 : StPCaP1 Identification of resistance to PVY and PVS viruses in interfering transgenic lines. Among them, Figure 4 A: StPCaP1 Symptoms of the interfering transgenic lines and the control at 4 weeks after PVY inoculation, scale bar 2.5cm; Figure 4 B: ELISA (top) and qRT-PCR (bottom) were used to determine the viral accumulation of PVY; Figure 4 C:S tPCaP1 Symptoms of the interfering transgenic lines and the control at 4 weeks after PVS inoculation, scale bar 2.5 cm; Figure 4 D: ELISA (top) and qRT-PCR (bottom) were used to determine the viral accumulation in PVS. Data are presented as mean ± standard deviation (n=3). qRT-PCR data are presented as follows: ef1α As an internal reference (*P<0.05, **P<0.01, Student's test).

[0021] Figure 5 : StPCaP1 Identification of resistance to PVX and PVM viruses in interfering transgenic lines. Among them, Figure 5 A: StPCaP1 Symptoms of the interfering transgenic lines and the control at 4 weeks after PVX inoculation, scale bar 2.5 cm; Figure 5 B: ELISA (top) and qRT-PCR (bottom) were used to determine the viral accumulation of PVX; Figure 5 C:S tPCaP1 Symptoms of the interfering transgenic lines and the control at 4 weeks after PVM inoculation, scale bar 2.5 cm; Figure 5 D: ELISA (top) and qRT-PCR (bottom) were used to determine the viral accumulation of PVM. Data are presented as mean ± standard deviation (n=3). qRT-PCR data are presented as follows: ef1α As an internal reference (*P<0.05, **P<0.01, Student's test).

[0022] Figure 6 for StPCaP1 Gene CDS region, where bold text represents interfering fragments. Detailed Implementation

[0023] Unless otherwise specified, the methods and apparatus used in the following embodiments of this invention are conventional methods and apparatus; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. To make the objectives, technical solutions, and advantages of the invention clearer, the specific embodiments of the invention are described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the accompanying drawings and described according to the accompanying drawings are merely exemplary, and the invention is not limited to these embodiments. It should also be noted that, in order to avoid obscuring the technical solution of the invention due to unnecessary details, only processing steps closely related to the solution according to the invention are shown in the accompanying drawings, while other details that are not closely related are omitted.

[0024] Example 1 This embodiment provides potatoes. StPCaP1 Genes, including: potato StPCaP1 Primers were designed based on the sequence (Soltu.DM.10G000120.1) published in the Potato Genome Database (http: / / spuddb.uga.edu / ) to amplify the full-length CDS sequence of this gene from the cultivated potato variety E3. The primer sequences are as follows: StPCaP1 F (5'-ATGGGATACTGGAAAGCAAAGGT-3') StPCaP1 R (5'-TCACGCCTTGGGTGCTTC-3') The obtained CDS sequence is shown in SEQ ID No. 1 of the sequence listing, with a nucleotide length of 609 bp. Figure 6 ).

[0025] Example 2 This embodiment provides potatoes. StPCaP1 Methods for constructing gene-plant interference vectors include: This application selects StPCaP1 A 250 bp fragment from the 5' end of the CDS sequence was used as the interference target fragment, which was obtained from the E3 cDNA by PCR. The PCR primers used were: Ri-StPCaP1 F (5'-ATGGGATACTGGAAAGCAAAGGT-3') Ri-StPCaP1 R(5'-CATCTGAGTTTTTCTTCAGCCCTG-3') put one's oar in StPCaP1 The sequence used is shown in SEQ ID No. 2 of the sequence listing. The PCR product was recovered. Using this as a template, a second round of PCR amplification was performed using universal primers: attBl (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3') and attB2 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'). The gel-recovered product was recombined into the pDNOR221 vector via a BP reaction (1.4 μL target fragment, 1 μL pDNOR221 plasmid, 0.6 μL BP enzyme, recombination at 25°C for 16 h, followed by 0.3 μL proteinase K, reaction at 37°C for 10 min). The positive clone pDNOR221- StPCaP1 The recombinant DNA was introduced into the pHellsgate8 vector via an LR reaction (1.2 μL of the initiation vector, 1.2 μL of phellsgate8, 0.6 μL of LR enzyme, recombination at 25°C for 16 h, followed by the addition of 0.3 μL of proteinase K and reaction at 37°C for 10 min). The resulting DNA was then heat-transformed into *E. coli* DH5α and sequenced for verification. The correctly sequenced positive clone, pHellsgate8-StPCaP1, was electroporated into *Agrobacterium* GV3101. Clones that tested positive by PCR were preserved for further genetic transformation.

[0026] Example 3 This embodiment provides interference with potatoes. StPCaP1 The application of genes in enhancing potato virus resistance includes: 1. Potato genetic transformation Genetic transformation involved inoculating Agrobacterium GV3101 of pHellsgate8-StPCaP1 onto YEB medium supplemented with 50 mg / L Spe and 50 mg / L Rif, and culturing at 28°C on a shaker at 200 rpm until the OD600 reached approximately 0.6. The culture was then centrifuged at 4000 rpm for 6 min, and the precipitate was resuspended in 3% MS liquid medium. E3 tubers grown for 12-16 weeks and approximately 0.5 cm in diameter were cut into 1-2 mm thick slices and immersed in the Agrobacterium bacterial suspension for 10 min, shaking every 5 min. After immersion, the potato slices were removed and the surface bacterial suspension was blotted dry with sterile filter paper. The resistant shoots were transferred to co-culture medium S1: 3% MS + 1 mg / L IAA + 0.2 mg / L GA3 + 0.5 mg / L 6-BA + 2 mg / L ZT in petri dishes and cultured in the dark at 23°C for 2 days. Then, they were transferred to medium S2: S1 + 75 mg / L Kam + 400 mg / L Cef + 200 mg / L Tim, and cultured at 2000 lux light intensity, 16 h / d photoperiod, and 23°C until resistant shoots regenerated. When the resistant shoots reached 0.5–1 cm in length, they were cut off and transferred to rooting medium S3: 3% MS + 100 mg / L Kam + 400 mg / L Cef + 200 mg / L Tim for selection. DNA was extracted from the rooted shoots, and transgenic positive plants were detected by PCR using 35S universal primers. The transgenic culture media are shown in Table 1.

[0027] Table 1. Potato transgenic culture medium

[0028] 2. Detection of transgenic positive lines In a clean bench, cut 1-2 leaflets from the seedlings to be tested and placed them in 2 ml centrifuge tubes. DNA was extracted using the CTAB method. After DNA extraction, it was used as a template for PCR detection of transgenic lines. Vector primer 35S: 5'-GACGCACAATCCCACTATCC-3' and gene primer: StPCaP1-R: 5'-TCACGCCTTGGGTGCTTC-3' were used for PCR amplification. Electrophoresis was used to obtain 3 transgenic plants. Total RNA was extracted from transgenic and control plants using a plant RNA rapid extraction kit (Zhuangmeng). Then, cDNA was generated by reverse transcription using the RT MasterMix with AccuRT (ABM) kit. Using cDNA as a template, quantitative primers were used to target the gene. StPCaP1Expression levels were detected using the following primers: StPCaP1-QF: 5'-CCGGCCCAATTCTGTTTGTGR-3' / StPCaP1-QR: 5'-CTCCTTCTCCTTCACCGACG-3'. Results showed that 4 out of 5 positive transgenic lines exhibited expression levels... StPCaP1 The expression level was significantly suppressed ( Figure 2 ).

[0029] 3. Developmental phenotype and virus resistance identification of transgenic potato plants This application will include 3 StPCaP1 Interference transgenic lines and controls (using the E3 recipient material for transgenics) were simultaneously grown in the same glass greenhouse under identical management conditions. Each line was planted in 18 pots. The plants grew well, and a portion of the plants were evaluated at 3 months of growth and tuber formation. StPCaP1 The effects of genes on potato growth, development, and tuber formation. Results showed that the growth status of RiStPCaP1-2 / -3 plants differed from the control E3. Figure 3 A) and tuber shape ( Figure 3 There was no difference in B), and further statistical analysis also showed that the number of tubers per plant in both the RiStPCaP1 line and the control was 2-5. Figure 3 C), and the yield of a single plant is mainly distributed between 80-160g ( Figure 3 D) indicates that there was no significant difference in the number of tubers per plant and yield of transgenic plants compared to the control, suggesting that interference... StPCaP1 Genes do not affect the growth and development of potato plants.

[0030] Another group of plants were rub-inoculated with PVY, PVS, PVX, and PVM at approximately 4 weeks of growth to identify the resistance of the interfering lines to these viruses. The results showed that at 4 weeks post-PVY inoculation, the upper systematic leaves of wild-type (E3) plants exhibited obvious mosaic symptoms, with curled leaf margins and elongated leaves, while no obvious symptoms were observed in the three transgenic lines. Figure 4 A). ELISA and qRT-PCR were used to further detect the expression levels of viral proteins and RNA in the upper systemic leaves of the plants. The results showed that on day 10 after PVY inoculation, a significant PVY virus accumulation signal was detected in E3, while PVY was not detected in any of the three transgenic lines. On days 15 and 20 after PVY inoculation, the PVY virus content shown by ELISA and qRT-PCR in wild-type plants further increased, indicating a large accumulation of the virus. Although the three transgenic lines also began to gradually accumulate the virus, the expression levels of PVY virus proteins and RNA were consistently significantly lower than those in the wild-type control, indicating that silencing the virus was a key factor. StPCaP1 The gene can significantly inhibit the accumulation of PVY in potatoes. Figure 4B); E3 and the interference strain did not show obvious symptoms after inoculation with PVS ( Figure 4 (C) However, ELISA and qRT-PCR results showed that PVS accumulation in the interference line was almost undetectable on days 10 and 15 after PVS inoculation, while PVS had accumulated significantly in E3. Only on day 20 did the interference line show slight PVS accumulation, but it was still significantly lower than that in E3, indicating that silencing... StPCaP1 The gene can significantly inhibit the accumulation of PVS in potatoes. Figure 4 D); Neither E3 nor the interference strain showed obvious symptoms after inoculation with PVX. Figure 5 A) Furthermore, ELISA and qRT-PCR results showed that the virus accumulated significantly in both E3 and the interfering strain on day 10 after PVX inoculation, gradually increasing over time. There was no significant difference in virus accumulation between the transgenic and E3 strains, indicating that silencing... StPCaP1 Genes do not affect the accumulation of PVX in potatoes. Figure 5 B); Neither E3 nor the interventional strain showed obvious symptoms after inoculation with PVX. Figure 5 (C) Furthermore, ELISA and qRT-PCR results showed that the virus had not yet begun to accumulate in E3 and interference plants on day 10 after PVM inoculation. By day 15, the virus began to accumulate in both E3 and interference plants, gradually increasing over time. There was no significant difference in virus accumulation between the E3 and interference lines, indicating that silencing... StPCaP1 The gene does not affect the accumulation of PVM in potatoes. Figure 5 D). Therefore, silence... StPCaP1 The gene-containing potato plants showed significant resistance to PVY and PVS viruses, making it a functional gene that can be applied to potato antiviral breeding.

[0031] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0032] Main References [1] Xie Conghua, Liu Jun. A review of the development and innovation of potato science and technology in China [J]. Journal of Huazhong Agricultural University, 2021, 40(4): 11. [2] Li Mengmeng, Chen Shihua, Wu Xingquan. Research progress on potato tuber necrosis and ring spot disease. Jiangsu Agricultural Sciences, 2015, 43: 143-146 [3] Dieudonné Harahagazwe, Condori B, Barreda C, et al. How big isthe potato (Solanum tuberosum L.) yield gap in Sub-Saharan Africa and why? Aparticipatory approach[J]. Open Agriculture, 2018, 3. [4] Hashimoto M, Neriya Y, Yamaji Y, Namba S. Recessive Resistance toPlant Viruses: Potential Resistance Genes Beyond Translation InitiationFactors. Front Microbiol , 2016, 7:1695 [5] Kreuze J F, Souza-Dias J A C, Jeevalatha A, et al. Viral diseasesin potato[J]. The potato crop: its agricultural, nutritional and socialcontribution to humankind, 2020: 389-430. [6] Mayberry LK, Allen ML, Nitka KR, Campbell L, Murphy PA, BrowningKS. Plant Cap-binding Complexes Eukaryotic Initiation Factors eIF4F andeIFISO4F. Journal of Biological Chemistry , 2011, 286:42566-42574 [7] Martelli G P, Adams M J, Kreuze J F, et al. Family Flexiviridae:A Case Study in Virion and Genome Plasticity[J]. Annual Review ofPhytopathology, 2007, 45(45):73. [8] Patrick RM, Mayberry LK, Choy G, Woodard LE, Liu JS, White A,Mullen RA, Tanavin TM, Latz CA, Browning KS. Two Arabidopsis loci encode novel eukaryotic initiation factor 4E isoforms that are functionally distinct from the conserved plant eukaryotic initiation factor 4E. Plant Physiol , 2014,164:1820–1830 [9] Scholthof HB, Alvarado VY, Vega-Arreguin JC, Ciomperlik J,Odokonyero D, Brosseau C, Jaubert M, Zamora A, Moffett P: Identification ofan Argonaute for antiviral RNA silencing in Nicotiana benthamiana. Plant Physiol , 2011 .

[10] Wang Q and Zhang W. An Economic Analysis of Potato Demand inChina. Amer J of Potato Res, 2010, 87:245-252.

Claims

1. The application of silencing the StpCaP1 gene in improving potato resistance to PVS virus, characterized by... The CDS sequence of the StpCaP1 gene is shown in SEQ ID NO.

1.

2. The application of silencing the StpCaP1 gene in simultaneously enhancing potato resistance to both PVS and PVY viruses, characterized by... The CDS sequence of the StpCaP1 gene is shown in SEQ ID NO.

1.

3. The application according to any one of claims 1 and 2, characterized in that... The application achieves resistance to the virus by interfering with the expression of the StpCaP1 gene, and the fragment used for interference expression is shown in SEQ ID No. 2.

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