CN117603998A - Application of StpCaP1 gene in improving resistance of potatoes to PVY and PVS viruses - Google Patents

Abstract

The invention provides application of StpCaP1 gene in improving resistance of potato to PVY and PVS viruses. The CDS sequence of StpCaP1 gene has a nucleotide length of 609bp. The RNAi vector is constructed, the genetic transformation of the Hubei potato No. 3 is used for carrying out transgenic function verification, and the result proves that the expression of the StpCaP1 gene is interfered without influencing the growth and development of plants, and the accumulation of PVY and PVS viruses in the plants can be obviously inhibited after virus inoculation, so that the effect of the silencing StpCaP1 gene on resisting PVY and PVS viruses is obvious, and the gene is a functional gene applicable to potato antiviral breeding.

Description

Application of StpCaP1 gene in improving resistance of potatoes to PVY and PVS viruses

Technical Field

The invention belongs to the fields of plant genetic engineering and molecular disease resistance breeding, and in particular relates to application of StpCaP1 gene in improving resistance of potatoes to PVY and PVS viruses.

Background

The potato (Solanum tuberosum L.) has the advantages of high adaptability, rich nutrition, and dual functions of food and vegetable. Potatoes are produced by asexual propagation, viruses can be accumulated in tubers along with generation, so that seed potatoes are degenerated, the output and quality of the tubers are seriously influenced, and the potato seeds become one of the restriction factors for the rapid development of potato industry in China (Wang and Zhang 2010). Viruses that infect potato and cause serious losses in China mainly include 6 kinds of sense RNA viruses such as Potato Virus Y (PVY), potato leaf curl virus (PLRV), potato Virus X (PVX), potato Virus A (PVA), potato Virus S (PVS) and Potato Virus M (PVM) and 1 kind of viruses (spindle tuber virus, PSTVD) (Kreuze et al 2020). For example, PVY is one of the most economically damaging viruses of potato worldwide (Scholthof et al 2011), belongs to the genus Potyvirus of the family Potyviridae, consists of a sense single-stranded RNA of about 10kb in size and a coat protein into curvy linear virus particles of about 750X 11nm in length, has a wide host range, usually causes symptoms such as flower leaf, side leaf necrosis, top leaf wrinkling and the like in plants when the potato is infected, and can cause 20% -50% yield reduction of potato, and even 80% or more yield reduction when the potato is infected together with other potato viruses (Li Mengmeng and the like 2015); PVS belongs to the genus Carlavirus of the family Betaflexviidae, and consists of a single strand of sense RNA of about 8.5kb in size and a curved rod-like virus particle of 610-710X 12-15nm in coat protein, most potato varieties show no visible symptoms after infection with PVS, resulting in a 20% -30% yield reduction (Marteli et al, 2007). It has been counted that the large-scale reduction in yield due to seed potato degradation caused by virus accumulation is estimated to be 50% or more of the potential total yield of potatoes (Dieudonee et al 2018). The virus infects potato plants on the cellular level, chemical agents are difficult to effectively control under field conditions, the traditional control method (such as producing detoxified seed potatoes, controlling virus transmission media such as aphids and the like) also needs strict management and high investment, and the method utilizes the disease-resistant mechanism of a host to cultivate resistant varieties and is a more economic and effective virus control strategy.

Because the virus has a simple structure, only has a genome and a coding protein number with limited size, and cannot independently complete the processes of translation, replication, transportation and the like, certain proteins of a host plant are required to be recruited to assist the host plant to complete the whole infection cycle, the host proteins supporting virus proliferation are collectively called host factors (host factors) or susceptibility factors (susceptibility factors), and recessive alleles caused by natural mutation or induced mutation of the host factors can endow plants with corresponding virus resistance. Eukaryotic translation initiation factor (eukaryotic translation initiation factor, eIF) 4E and its isoforms were the earliest gene found to be most widely used in crops for viral disease, but due to the wide presence of eIF4E isoforms, there is some functional redundancy within the family, and recessive mutations in individual eIF4E genes do not always confer complete viral resistance to plants (Mayberry et al 2011). Moreover, due to the important role of eIF4Es in plant growth and development, loss of function may lead to an embryonic necrotic phenotype, researchers have begun to explore and identify other potential viral susceptibility genes in recent years (Patrick et al 2014; hashimoto et al 2016). There are few reports of digging and utilizing susceptibility genes in potatoes to increase plant resistance to potato viruses so that identification of potato virus susceptibility genes and definition of their resistance effects on primary potato viruses are of great biological and economic significance.

The application screens the gene StPCaP1 which is obviously induced to express by viruses after the potatoes are infected by PVY viruses based on transcriptome sequencing and fluorescent quantitative analysis. At present, no relevant literature report exists on the function of the StPCaP1 gene in potato antiviral diseases.

RNA interference (RNAi) refers to the inhibition of target gene expression by small double-stranded RNA by stimulating specific degradation of complementary target mRNA, causing a phenotypic change, and further studying the function of the target gene based on the phenotypic variation. Compared with the gene knockout method, RNAi can inhibit the gene expression of the current generation plants, thereby realizing silencing and functional analysis of the target genes, and has been widely applied to researches on potatoes, tobacco, tomatoes and the like.

Thus, the present application utilizes RNAi technology to silence StPCaP1 in potatoes to investigate whether StPCaP1 gene silencing affects potato resistance to the major several potato viruses. The result shows that StPCaP1 gene has no great influence on basic processes such as plant growth and development after silencing, and is suitable for breeding. Further, it was found that both PVY and PVS virus accumulation was significantly reduced after StPCaP1 silencing plants, demonstrating for the first time that silencing StPCaP1 enhances potato resistance to two different viral species of important potato viruses.

Problems of the prior art: the application screens the gene StPCaP1 which is obviously induced to express by viruses after the potatoes are infected by PVY viruses based on transcriptome sequencing and fluorescent quantitative analysis. At present, no relevant literature report exists on the function of the StPCaP1 gene in potato antiviral diseases. Thus, the present application utilizes RNAi technology to silence StPCaP1 in potatoes to investigate whether StPCaP1 gene silencing affects potato resistance to the major several potato viruses. The result shows that StPCaP1 gene has no great influence on basic processes such as plant growth and development after silencing, and is suitable for breeding. Further, it was found that the accumulation of PVY and PVS viruses was significantly reduced after StPCaP1 silencing plants, demonstrating that silencing StPCaP1 enhances potato resistance to both important potato viruses.

Disclosure of Invention

In view of the deficiencies of the prior art, the present invention provides the use of the potato StPCaP1 gene in increasing resistance of potatoes to PVY and PVS viruses. Through a sequence published in a potato genome database, a cDNA of a plant No. 3 leaf of a cultivated species of Hubei potato is used as a template, a StPCaP1 gene is cloned, an RNAi interference vector is constructed, genetic transformation of Hubei potato No. 3 is used for carrying out transgenic function verification, and the silencing StABL1 gene is found to improve the resistance of plants to PVY and PVS and is mainly characterized by reduced virus accumulation, reduced plant morbidity symptoms and the like.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

1. the CDS sequence of the potato StPCaP1 gene, the jaw potato No. 3 StPCaP1 gene is shown in a sequence table SEQ ID NO.1, and the nucleotide length is 609bp. The published sequence ID of the potato genome database (http:// spuddb. Uga. Edu /) is Soltu. DM.10G000120.1. The sequences are identical.

The construction method of StPCaP1 gene plant interference vector includes: the interference sequence is obtained by PCR in the cDNA of potato cultivar E3, and the fragment sequence used by the interference StPCaP1 is shown in a sequence table SEQ ID No. 2. The PCR product was recovered. Using this as a template, using universal primers: attBl, attB2, a second round of PCR amplification was performed, the gel recovery product was recombined to pDNOR221 vector by BP reaction, positive clone pDNOR221-StPCaP1 was recombined to pHellsgate8 vector by LR reaction, e.coli DH5a was transformed by heat shock, and sequencing verified. The correctly positive clone pHellgate 8-StPCaP1 was sequenced to shock transform Agrobacterium GV3101 and the clones detected as positive by PCR were saved for further genetic transformation.

Application of StPCaP1 Gene in enhancing resistance of Potato to Main Potato Virus disease A method of verifying the application of StPCaP1 Gene to enhancing resistance of Potato to Main Potato Virus disease comprising: (1) genetic transformation of potatoes, (2) detection of transgenic positive lines, (3) identification of developmental phenotypes of transgenic potato plants, and (4) identification of virus resistance of transgenic potato plants.

Use of a stppap 1 gene for increasing resistance of potato to PVY virus by silencing TST1 gene expression by RNAi, or by gene editing to knock out the gene.

Use of a stppap 1 gene for increasing resistance of potato to PVS virus by silencing TST1 gene expression by RNAi, or by gene editing knockout.

6. A vector contains a sequence shown in a sequence table SEQ ID NO. 1.

7. The potato StpCaP1 protein is encoded by a sequence shown in a sequence table SEQ ID NO. 1.

Compared with the prior art, the invention has the beneficial effects that: the growth and development of plants and potato yield of the plants after the StPCaP1 is silenced are not affected, so that the gene is a disease-sensing gene which can be used for breeding production, the accumulation of PVY and PVS viruses in the plants after the StPCaP1 is silenced is obviously reduced, the disease symptoms of the plants are obviously relieved, and the effect of inhibiting the PVY and PVS viruses of potato plants of the StPCaP1 is extremely obvious, so that the gene is a functional gene which can be used for potato antiviral breeding application.

Drawings

FIG. 1 shows a schematic diagram of the construction process of the interference vector pHellgate 8-StPCaP 1.

Fig. 2: and (3) detecting the expression quantity of the StPCaP1 interference transgenic line. The data are shown as mean ± standard deviation (n=3), with ef1α as internal reference (P <0.05, P < 0.01, P < 0.001, student t test).

Fig. 3: stPCaP1 interferes with developmental phenotype identification of the transgenic lines. Wherein, fig. 3A: e3 and interference line plants 3 months of growth phenotype, scale 10cm, fig. 3B: e3, a single potato harvesting phenotype of the interference strain; FIG. 3C, E3 and number of individual potato harvests of the interference line; FIG. 3D, E3 and interference lines individual potato fresh weights. The data are shown as mean.+ -. Standard deviation (n.gtoreq.12), analyzed by Duncan's new complex polar method, p <0.05.

Fig. 4: stPCaP1 interferes with the identification of resistance to PVY and PVS viruses of transgenic lines. Wherein, fig. 4A: stPCaP1 interfered with symptoms of transgenic lines and controls plants 4 weeks after PVY inoculation, scale 2.5cm; fig. 4B: ELISA (up) and qRT-PCR (down) to determine the viral accumulation of PVY; fig. 4C: stPCaP1 interfered with transgenic lines and controls plant symptoms 4 weeks after PVS inoculation, scale 2.5cm; fig. 4D: ELISA (upper) and qRT-PCR (lower) were used to determine the viral accumulation of PVS. Data are shown as mean ± standard deviation (n=3), qRT-PCR data with ef1α as internal reference (< 0.05, < 0.01, < P, student t-test).

Fig. 5: stPCaP1 interferes with the identification of resistance to PVX and PVM viruses of transgenic lines. Wherein, fig. 5A: stPCaP1 interfered with symptoms of transgenic lines and controls plants 4 weeks after PVX inoculation, scale 2.5cm; fig. 5B: ELISA (upper) and qRT-PCR (lower) to determine the viral accumulation of PVX; fig. 5C: stPCaP1 interfered with symptoms of transgenic lines and controls plants 4 weeks after PVM inoculation, scale 2.5cm; fig. 5D: ELISA (upper) and qRT-PCR (lower) were used to determine the viral accumulation of PVM. Data are shown as mean ± standard deviation (n=3), qRT-PCR data with ef1α as internal reference (< 0.05, < 0.01, < P, student t-test).

FIG. 6 shows the CDS region of StPCaP1 gene, wherein the interfering fragment is bold.

Detailed Description

The methods and apparatus used in the following examples of the present invention are conventional methods and apparatus unless otherwise specified; the equipment and the reagent are conventional equipment and reagents purchased by reagent companies. In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention will be given 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 drawings and described in accordance with the drawings are merely exemplary and the invention is not limited to these embodiments. Here, it should be noted that, in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only the processing steps closely related to the solution according to the present invention are shown in the drawings, while other details having little relationship are omitted.

Example 1

This example provides a potato stpcp 1 gene comprising:

the potato StPCaP1 gene was designed based on published sequences (Soltu. DM.10G000120.1) from the potato genome database (http:// spuddb. Uga. Edu /) to amplify the CDS full-length sequence of the gene from potato cultivar Eyew 3 (E3), the primer sequences were as follows:

StPCaP1 F(5’-ATGGGATACTGGAAAGCAAAGGT-3’)

StPCaP1 R(5’-TCACGCCTTGGGTGCTTC-3’)

the CDS sequence obtained by sequencing is shown as a sequence table SEQ ID No.1, and the nucleotide length is 609bp (figure 6).

Example 2

The embodiment provides a construction method of a potato StPCaP1 gene plant interference vector, which comprises the following steps:

the application selects a fragment with the length of 250bp at the 5' -end of the StPCaP1 CDS sequence as an interference target fragment, and obtains the interference target fragment from the cDNA of E3 through PCR, wherein the PCR primer is as follows:

Ri-StPCaP1 F(5’-ATGGGATACTGGAAAGCAAAGGT-3’)

Ri-StPCaP1 R(5’-CATCTGAGTTTTTCTTCAGCCCTG-3’)

the sequence used for interference StPCaP1 is shown in a sequence table SEQ ID No. 2. The PCR product was recovered. Using this as a template, using universal primers: attBl (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3'), attB2 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'), and the gel recovery product was recombined into the pDNOR221 vector by BP reaction (fragment of interest 1.4ul, pDNOR221 plasmid lul, BP enzyme 0.6ul, recombination 16h at 25 ℃, addition of 0.3ul proteinase K, reaction 10min at 37 ℃), positive clone pDNOR221-StPCaP 1) by LR reaction (entry vector 1.2 ul, phellsgate 8.2 ul, LR enzyme 0.6ul, recombination 16h at 25 ℃, addition of 0.3ul proteinase K, reaction 10min at 37 ℃). ) Recombinant into pHellsgate8 vector, heat shock transformed into E.coli DH5a, and sequencing verification. The correctly positive clone pHellgate 8-StPCaP1 was sequenced to shock transform Agrobacterium GV3101 and the clones detected as positive by PCR were saved for further genetic transformation.

Example 3

This example provides the use of the interfering potato stpcp 1 gene to enhance potato virus resistance comprising:

1. genetic transformation of potato

Genetic transformation Agrobacterium GV3101, pHellsgate8-StPCaP1, was inoculated into YEB medium supplemented with 50mg/L Spe and 50mg/L Rif, and cultured on a shaker at 28℃and 200r/min to 0D600 of about 0.6. Centrifuge 4000r/min for 6min, and re-suspend the pellet with 3% MS liquid medium. Cutting E3 test tube potato with diameter of about 0.5cm and growth period of 12-16 weeks into 1-2mm thick slices, soaking in the above Agrobacterium solution for 10min, and shaking every 5 min. And taking out the potato chips after the dip dyeing is finished, and sucking the surface bacterial liquid by using sterile filter paper. Transferring into a 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 culture dish, and transferring into an S2 culture medium after dark culture at 23 ℃ for 2 d: S1+75mg/L Kam+400mg/L Cef+200mg/L Tim, and culturing under 2000lux light intensity, 16h/d photoperiod and 23 ℃ until the resistant bud is regenerated. When the length of the resistant bud reaches 0.5-1cm, cutting the resistant bud and transferring the cut resistant bud into a rooting culture medium S3:3%MS+100mg/L Kam+400mg/L Cef+200mg/L Tim for screening. DNA extraction is carried out on the rooted buds, and the transgenic positive plants are detected by a 35S universal primer PCR. The transgenic medium is shown in Table 1.

TABLE 1 Potato transgenic Medium

2. Transgenic positive line detection

1-2 small leaves of the seedlings to be detected are cut in a 2ml centrifuge tube in an ultra-clean workbench. DNA was extracted by CTAB method. After DNA extraction was completed, PCR transgenic strain detection was performed using it as a template, using vector primer 35s:5'-GACGCACAATCCCACTATCC-3', the reverse primer is a gene primer: stPCaP 1-R5'-TCACGCCTTGGGTGCTTC-3', amplified by PCR, and detected by electrophoresis to obtain 3 transgenic plants. The total RNA of transgenic plants and control plants was extracted from RNA using a plant RNA rapid extraction kit (Zhuang Meng), and cDNA was then produced by reverse transcription using RT MasterMix with AccuRT (ABM) kit. The expression level of StPCaP1 was detected by using cDNA as a template and a quantitative primer of StPCaP1-QF 5'-CCGGCCCAATTCTGTTTGTGR-3'/StPCaP1-QR 5'-CTCCTTCTCCTTCACCGACG-3', which showed that 4 of 5 positive transgenic lines had significantly suppressed StPCaP1 expression level (FIG. 2).

3. Identification of developmental phenotype and viral resistance of transgenic potato plants

According to the method, 3 StPCaP1 interference transgenic lines and a control (receptor material E3 used for transgenesis) are simultaneously planted in the same glass greenhouse, 18 pots are planted in each line under the same management condition, the plant growth vigor is good, and the influence of the interference StPCaP1 genes on the growth and development of potatoes and potato growth is evaluated when a part of plants grow for 3 months and potato growth. The results show that the plant growth states (figure 3A) and the potato block morphologies (figure 3B) of the RistPCaP1-2/-3 and the control E3 are not different, further statistical analysis also shows that the number of single-plant tubers of the RistPCaP1 strain and the control is 2-5 (figure 3C), and the single-plant yield is mainly distributed between 80-160g (figure 3D), which shows that the single-plant tubers of the transgenic plant are not significantly different from the single-plant tubers and the control, and the interference of the StPCaP1 gene does not influence the growth and development of potato plants.

Another part of the plants was rubbed PVY, PVS, PVX and PVM around 4 weeks of growth to identify the resistance of the interfering strain to these viruses. The results indicated that at week 4 post-PVY inoculation, the upper systematic leaves of wild type (E3) plants developed significant floral leaf symptoms, and leaf edges curled, leaves were elongated, and no significant symptoms were observed in any of the 3 transgenic lines (FIG. 4A). The expression levels of viral proteins and RNA in the upper system leaves of the plants were further detected by ELISA and qRT-PCR techniques, respectively. The results indicated that on day 10 post-PVY inoculation, a significant PVY virus accumulation signal was already detectable in E3, whereas the presence of PVY was not detected in all 3 transgenic lines; the PVY virus content in wild type plants, shown by ELISA and qRT-PCR, was further increased at days 15 and 20 after PVY inoculation, indicating that virus accumulated in large amounts; although 3 transgenic lines also began to accumulate virus gradually, the expression levels of PVY viral proteins and RNA were consistently significantly lower than the wild-type control, suggesting that silencing the StPCaP1 gene significantly inhibited PVY accumulation in potato (fig. 4B); the method comprises the steps of carrying out a first treatment on the surface of the Neither E3 nor the interfering strain showed obvious symptoms after PVS inoculation (FIG. 4C), but ELISA and qRT-PCR results showed that little accumulation of PVS in the interfering strain was detected on days 10 and 15 after PVS inoculation, whereas PVS in E3 had accumulated in large amounts, and PVS in the interfering strain had accumulated slightly by day 20, but was still significantly lower than E3, indicating that silencing the StPCaP1 gene significantly inhibited PVS accumulation in potato (FIG. 4D); neither E3 nor the interfering strain showed obvious symptoms after PVX inoculation (FIG. 5A), and ELISA and qRT-PCR results showed that virus accumulated in large amounts in E3 and the interfering strain on day 10 after PVX inoculation, gradually increased over time, and there was no obvious difference in transgene accumulation from that in E3, indicating that silencing the StPCaP1 gene did not affect PVX accumulation in potato (FIG. 5B); neither E3 nor the interfering lines showed obvious symptoms after PVX inoculation (FIG. 5C), and ELISA and qRT-PCR results indicated that virus had not started to accumulate in both E3 and interfering plants at day 10 after PVM inoculation, and by day 15, virus had started to accumulate in both E3 and interfering plants, with progressive increase over time, and there was no obvious difference in the amount of virus accumulated between E3 and interfering lines, indicating that silencing the StPCaP1 gene did not affect PVM accumulation in potato (FIG. 5D). Therefore, the potato plant with the silent StPCaP1 gene has remarkable effect on resisting PVY and PVS viruses, and is a functional gene applicable to potato antiviral breeding.

The foregoing is merely exemplary of the application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the application and are intended to be comprehended within the scope of the application.

Main reference

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Claims (8)

1. The application of the StpCaP1 gene in improving the resistance of potatoes to PVS viruses is characterized in that the CDS sequence of the StpCaP1 gene is shown as a sequence table SEQ ID NO. 1.
2. The application of the StpCaP1 gene in simultaneously improving the resistance of potatoes to PVS and PVY viruses is characterized in that the CDS sequence of the StpCaP1 gene is shown in a sequence table SEQ ID NO. 1.
3. 3. Use of the stpcp 1 gene according to any of claims 1 and 2, characterized in that said use achieves resistance to viruses by interfering with stpcp 1 gene expression, the fragment used for interfering with expression being shown in the sequence listing SEQ ID No. 2.
4. 4. The construction method of the potato StpCaP1 gene plant interference vector is characterized by comprising the following steps: PCR is carried out on cDNA of potato cultivar E3 to obtain an interference sequence; the fragment sequence used by the interference StpCaP1 is shown in a sequence table SEQ ID No. 2; the PCR product was recovered. Using this as a template, using universal primers: performing a second PCR amplification of attBl and attB2, recombining the gel recovery product into a pDOR 221 vector through BP reaction, recombining positive clone pDOR 221-StpCaP1 into a pHellsgate8 vector through LR reaction, performing heat shock transformation on escherichia coli DH5a, and performing sequencing verification; the correctly positive clone pHellgate 8-StpCaP1 was sequenced to shock transform Agrobacterium GV3101 and the clones detected as positive by PCR were saved for further genetic transformation.
5. Application verification method of stppap 1 gene in improving resistance of potato to PVY and PVS viruses, comprising: (1) genetic transformation of potatoes; (2) transgenic positive line detection; (3) developmental phenotype identification of transgenic potato plants; (4) identification of viral resistance of transgenic potato plants.
6. 6. The potato StpCaP1 gene is characterized in that the CDS sequence of the StpCaP1 gene is shown in a sequence table SEQ ID NO. 1.
7. 7. A vector is characterized in that the vector contains a sequence shown in a sequence table SEQ ID NO. 1.
8. 8. The potato StpCaP1 protein is characterized in that the potato StpCaP1 protein is encoded by a sequence shown in a sequence table SEQ ID NO. 1.