TWI432575B - A gene-transfer vector comprising the helper-component protease gene of papaya ringspot virus for broad-spectrum virus resistance in crops and use thereof - Google Patents

Description

Papaya rotifer virus synergistic protease gene transfer vector for providing crop broad antiviral traits and application thereof

The present invention relates to a recombinant vector for providing resistance to a virus in a plant, which comprises a coding sequence of a Papaya ringspot virus helper-component protease gene (PRSV HC-Pro gene). Fragmentation sequence fragment. The invention also relates to a recombinant microorganism. The invention also relates to a method for rendering plants resistant to viruses. The invention also relates to the use of a full length gene of a synergistic protease or a gene fragment thereof for the manufacture of a pair of virus resistant plants.

Papaya ( Carica papaya L.) is a highly economical crop, and its viral diseases are not treated by drugs. In recent years, due to the improvement of plant tissue culture technology, breakthroughs in plant gene transfer technology, plus pathogen induced disease resistance theory (pathogen) The germination of -derived resistance (PDR) (Sanford and Johnson 1985), which transferred part of the viral genome to the host plant chromosome, has achieved anti-viral infection and has become a popular method. Fitch et al. (Fitch et al., 1992) first transferred the CP gene of the PRSV Hawaiian isolate into papaya by microprojectile bombardment, but their gene-transplanted papaya for the PRSV Yongkang isolate of the Taiwanese system ( YK isolate) is not resistant, and its sequence-homology-dependent resistance becomes a limiting factor for the application of this gene to other regions.

The applicant previously obtained the gene of the coat protein (CP) gene of Taiwan PRSV YK isolate by Agrobacterium sp. (Cheng, 1996), after challenge inoculation and analysis. It is proved that these gene-transgenic papaya strains have good resistance to Taiwan PRSV YK, and are also good for other PRSV viruses in different regions such as Hawaii (HA), Mexico (MX) and Thailand (TH) isolates. Resistance (Bau et al., 2003). The sphingin gene-transgenic papaya showing YK provides a high degree of resistance to PRSV (Bau et al. 2004). In the prior art, the application of the sheath protein gene to papaya can be obtained, although a transgenic plant can be obtained, and the principle of resistance is that the transgenic CP gene stimulates the papa defense response and the transcriptional gene silence mechanism (post) -transcriptional gene silencing (PTGS), which gives papaya the ability to break down the invading PRSV RNA genome and has antiviral properties. However, this principle has the problem of sequence homology specificity. It is not resistant to PRSV with distant CP gene. The shortcomings of specific resistance of this virus strain are still common problems (Tennant et al. 2001). Therefore, sheath protein transfectants from different strains of virus have only regional application value.

The applicant's research team used Agrobacterium- mediated to introduce the sheath protein gene of Taiwan PRSV YK system into immature embryos of papaya (Cheng et al. 1996), and obtained strains with different degrees of resistance to PRSV. In addition to the good resistance to the native YK system, several strains have good resistance to viruses originating from different regions such as Hawaii (HA), Thailand (TH) and Mexico (MX). Sex (Bau et al. 2003). The obtained sheath protein gene transgenic papaya strain of the PRSV YK system was confirmed by field experiments to provide a high degree of resistance to PRSV (Bau et al. 2004).

Applicants found during the field trial of the sheath protein gene transgenic papaya of the PRSV YK system, and found that the PRSV YK sheath protein gene transgenic papaya was killed by the super papaya virus strain, and its attackable by the sheath was explored. The cause of resistance mediated by coat protein gene is caused by a helper-component protease (HC-Pro). HC-Pro is a gene silencing suppressor that has the ability to inhibit post-transcriptional gene silencing (PTGS) (Anadalakshmi et al., 1998; Brineti et al., 1998; Shi Et al., 1997), the authors speculate that the ability of HC-Pro of PRSV 5-19 to inhibit gene silencing is super strong, and the gene silencing mechanism of PRSV YK CP gene transgenic papaya is inhibited by 5-19 HC-Pro. It is unable to function, so that the resistance of the genetically transformed papaya is inhibited and invalidated. Based on the fact that providing resistance to the sheath protein gene alone is not sufficient to deal with super virus strains, a new gene transfer strategy against supervirus has been developed in the art.

In view of the fact that the resistance of the sheath protein gene transgenic plants to the virus in the prior art is homology-dependent resistance, the resistance is carried by the super-strong virus of the super gene silence inhibitor HC-Pro. The problem of killing of strains has not been solved, and the object of the present invention is to provide a gene transfer vector and application thereof, using the papaya virus-associated protein (HC-Pro) as a target gene, and using a non-translating construction strategy to HC- The Pro gene is constructed in a two-dimensional vector, and the resulting gene-transforming plant stimulates the post-transcriptional silence (PTGS) against the gene silencing inhibitor HC-Pro gene, and the virus protects itself against the most important genes in the host defense response. The gene-transplanted plant thus produced has the traits of a wide range of anti-viral strains, that is, the ability to resist viruses of different geographical regions and different systems, and can solve the application of the field-strong PRSV strain to kill the scorpion protein transgenic papaya. problem.

Summary of invention

In order to achieve the above object, in one aspect, the present invention provides a recombinant vector for providing resistance to a virus in a plant, comprising a control sequence and an operably linked papaya wheel operably linked to the control sequence A coding sequence fragment of a Papaya ringspot virus (PRSV) helper-component protease gene (HC-Progene).

In another aspect, the invention provides a recombinant microorganism produced by transformation of a microorganism with a recombinant vector as described above.

In still another aspect, the present invention provides a method for making a plant resistant to a virus, comprising the steps of: transferring a recombinant vector as described above into a plant to render the plant non-translated Sexual expression of the transcript of the coding sequence fragment of the synergistic protease gene of the papaya virus of the recombinant vector, but does not exhibit a synergistic protease, which is produced by the RNA vector followed by transcriptional gene silencing (PTGS). Produces resistance to viruses.

In still another aspect, the present invention provides a plant cell having a nucleic acid molecule selected from the group consisting of a nucleic acid sequence having 85% or more homology with SEQ ID NO: 5, and SEQ ID NO: 6 A nucleic acid sequence having more than 85% homology and a nucleic acid sequence having a homology of 85% or more as set forth in SEQ ID NO: 7.

In a further aspect, the present invention provides a full-length gene of a Papaya ringspot virus (PRSV) helper-component protease (HC-Pro) or a gene fragment thereof for producing a pair of viruses. The use of plants.

Based on the above, the present invention is a strategy for attacking a viral HC-Pro gene by using a gene targeting the HC-Pro gene, providing a broad resistance of plants to different strain viruses, and solving the problem of non-dependent homology resistance, thereby enabling The resulting gene transfer plants have cross-regional application value.

Detailed description of the invention

A recombinant vector for providing resistance to a virus in a plant, comprising a control sequence and a coding sequence fragment of a papaya rot virus synergistic protease gene operably linked to the control sequence.

According to the present invention, the papaya virus-associated protease is a gene silencing suppressor having the ability to inhibit post-transcriptional gene silencing (PTGS) (Anadalakshmi et al., 1998; Brigneti et al., 1998; Shi et al., 1997).

According to the present invention, the coding sequence fragment of the papaya virus virus synergistic protease gene can be constructed in the plastid by any method so as not to express a protein, for example, but not limited to: the coding sequence fragment is located in the control Downstream of the sequence, and the 5' end of the coding sequence fragment of the papaya virus synergistic protease gene has at least one stop codon; preferably, having a complex termination code; more preferably, having two consecutive terminations code.

According to the present invention, the coding sequence fragment of the papaya virus virus synergistic protease gene has the sequence shown in SEQ ID NO: 8.

In accordance with the present invention, the term "operably linked" as used herein means that the expression of a gene is controlled by a spatially linked control sequence. The control sequence may be located at the 5' end [upstream] or 3' end [downstream] of the gene such that the gene is under the control sequence described. As is known in the art, the distance can be appropriately adjusted without affecting the function of the control sequence. According to the present invention, the "coding sequence" means a region of a gene which is transcribed into a message RNA (mRNA) and which is translated into a protein, and thus the "coding sequence fragment" means the full length of the aforementioned region or Fragment.

In accordance with the invention, the term "homology" as used herein refers to the degree defined herein as the association between two sequences, which may be determined by the same ratio between the sequences.

The term "control sequence" as used herein, refers to a sequence that enables a gene or fragment thereof to be turned on or off, thereby affecting the expression of the gene or fragment thereof; for example, the control sequence may be a promoter or terminator ( Terminator).

According to the invention, wherein the control sequence may be any promoter suitable for use in the transgenic HC-Pro of the invention, such as, but not limited to, the 35S promoter. The control sequence may be any promoter expressed in plants, such as, but not limited to, from the Cauliflower mosaic virus 35S promoter (CaMV 35S promoter).

According to the present invention, the coding sequence fragment can be modified in any way that causes the coding sequence fragment to not express a protein, preferably, but not limited to, having at least one termination code inserted at its 5' end (stop Condon) or contain reading frame shift.

According to the present invention, the coding sequence fragment preferably has a group selected from the group consisting of: (i) a homology with a sequence of SEQ ID NO: 5 of 85% or more. a nucleotide sequence; (ii) a nucleotide sequence having a homology of 85% or more with the sequence of SEQ ID NO: 6; and (iii) a sequence of 85% or more with the sequence of SEQ ID NO: The homologous nucleotide sequence.

According to the present invention, the aforementioned recombinant vector further comprises a drug resistance gene in addition to the above control sequence, and the drug resistance gene is any antibiotic gene or screening gene, such as, but not limited to, npt II gene kanamycin (kanamycin) ) a drug resistance gene.

According to the present invention, the recombinant vector can be obtained by any binary vector by inserting a coding sequence fragment of a papaya virus virus synergistic protease gene by genetic engineering technology, and the binary vector is included in any plant. Promoters or terminators, such as, but not limited to, pBI121 and vectors derived therefrom. As is known in the art, the binary vector is a selection vector used to produce a transgenic plant having Escherichia coli and Agrobacterium tumefaciens. The ability to replicate in a CaMV 35S promoter and a Nos terminator, preferably between the CaMV 35S promoter and the Nos terminator of the binary vector.

In a preferred embodiment of the recombinant vector of the present invention, the recombinant vector pBI-519HCF for providing plant resistance to viruses is deposited in a host type in the development of the food industry of the Republic of China. Registered number BCRC940581.

In another preferred embodiment of the recombinant vector of the present invention, the recombinant vector pBI-519HCN or pBI-519HCC for providing plant resistance to viruses can be used as a template with pBI-519HCF as a template. Engineering techniques such as the use of primer pair 519HCStopA (SEQ ID NO: 1) and 519HCSacA (SEQ ID NO: 3); or primer pair 519HCStopB (SEQ ID NO: 4) and 519HCSacB (SEQ ID NO: 2), by polymerase chain The lock reaction and the general selection technique are obtained; or the restriction enzyme splicing and colonization are based on pBI-519HCF.

A recombinant microorganism of the present invention is produced by transforming a microorganism into a recombinant vector as described above, wherein the microorganism is preferably a bacterium, and more preferably a disarmed Agrobacterium tumefaciens ).

According to the invention, said means having a disarmed Agrobacterium tumor metastasis genes (vir genes) and the T-DNA, but without Agrobacterium Agrobacterium oncogene (oncogenes) is (Agrobacterium tumefaciens).

A method for making a plant resistant to a virus of the present invention comprises the steps of: transferring a recombinant vector as described above into a plant such that the plant exhibits the recombination in a non-translatable manner The transcript of the coding sequence fragment of the synergistic protease gene of the papaya virus of the vector, but does not exhibit a synergistic protease, whereby the plant produces resistance to the virus.

According to the present invention, the terms "introducing" and "transfer" as used herein are used interchangeably, meaning that a nucleotide fragment, a vector, and the like are converted into any known one. The technology is introduced into tissue cells.

According to the present invention, the step of transferring a recombinant vector as described above into a plant, as known in the art, may be by, for example, but not limited to, Agrobacterium, particle gun, electroporation (electoporation), microinjection (microinjection) and ultrasonic method.

According to the present invention, the "untranslational expression or not translationally expressed" means that the gene expression is performed only before the gene translation stage, that is, the intact protein is not expressed, so the means Including the coding sequence fragment as a reading frame shift and an insertion termination code, which only produces a transgenic transcript but does not produce a transgenic protein, but the transcript is decomposed into small due to "post-transcriptional gene silence" The fragment-interfering RNA (siRNA) is a transcript in which the transgenic gene is not detected by the virus-resistant strain, but a large number of small fragments of siRNA are produced.

According to the present invention, the plant body is derived from a plant selected from the group consisting of Cucurbitaceae and Caricaceae . Preferably, the plant body is derived from Carica papaya L., including its roots, stems, leaves and embryos.

According to the present invention, the step of transferring a recombinant vector as described above into a plant comprises: introducing the recombinant vector into Agrobacterium sp. to obtain recombinant Agrobacterium; and using the recombinant agricultural The bacillus infection is transferred to the plant to obtain a plant body with the coding sequence fragment.

The full length of a papaya virus virus synergistic protease gene of the present invention or a gene fragment thereof for use in the manufacture of a plant resistant to viruses.

Preferably, the full length gene has the sequence set forth in SEQ ID NO: 8. Preferably, the full-length gene or a gene fragment thereof is selected from a nucleotide sequence having a group consisting of: (i) one having more than 85% of the sequence shown in SEQ ID NO: Nucleotide sequence of homology; (ii) a nucleotide sequence having 85% or more homology to the sequence of SEQ ID NO: 6; and (iii) one and SEQ ID NO: The sequence shown in 7 has a nucleotide sequence of 85% or more homology.

Preferably, the use of the present invention comprises introducing a non-translatable construct comprising a full-length gene of the papaya virus-promoting protease or a gene fragment thereof into a plant.

According to the present invention, the non-translating construct system is derived from the full-length gene of the papaya rot virus synergistic protease or a gene fragment thereof, which is selected from the expression vector, but which does not exhibit papaya virus . Non-translating constructs may use the mechanism of RNA silencing by the expression of RNA to achieve the purpose of counteracting a virus with substantially the same synergistic protein as papaya virus.

Preferably, the untranslatable construct is a binary vector into which a termination code and a papaya rot virus synergistic full-length gene or a gene fragment thereof containing a reading frame displacement are inserted.

A recombinant plant cell of the present invention, wherein a nucleic acid molecule selected from the group consisting of a nucleic acid sequence having 85% or more homology with the sequence of SEQ ID NO: 5, SEQ ID NO: a nucleic acid sequence having a nucleic acid sequence having a homology of 85% or more and a nucleic acid sequence having a homology of 85% or more in the sequence of SEQ ID NO: 7; more preferably, the gene is inserted from the gene body selected from a nucleic acid molecule in the group consisting of a nucleic acid sequence having 90% or more homology to the sequence of SEQ ID NO: 5, a nucleic acid sequence having 90% or more homology to the sequence of SEQ ID NO: 6, and A nucleic acid molecule having a nucleic acid sequence having a homology of 90% or more as represented by SEQ ID NO: 7.

Preferably, the recombinant plant cell is transformed from a plant cell to a recombinant vector as described above or a recombinant microorganism as described above. More preferably, the plant cell line is derived from plant callus and somatic embryos.

The invention will be further illustrated by the following examples, which are to be construed as illustrative and not restrictive.

Example

1. Preparation of a nontranslatable construct of the synergistic protein HC-Pro gene

The HC-Pro gene (SEQ ID NO: 8) of PRSV5-19 was constructed by RT-PCR amplification. Vector (Invitrogen), constructed in a non-translatable manner, designed a set of specific primers 519HCStopA (5'-CGCATGAACATGC TCTAGA TGAACGAT TGATGA GAAAAAT T TCTG-3') (SEQ ID NO: 1) and 519HCSacB (5'- GTGGTTGGATCAAA GAGCTC ACCGACAATGTAGTGTTTCATTTC-3') (SEQ ID NO: 2), designing two stop codes (stop codons, TGATGA ), Xba I cleavage ( TCTAGA ) and adding a base ( T ) to the upstream primer (519HCStopA) The entire protein reading frame shift (reading frame-shift), the downstream primer (519HCSacB) designed Sac I cleavage site ( CTCGAG ), and the PCR-amplified TOPO vector on the HC-Pro full-length gene (after 1371 bp) plus a base T of 1372 bp (SEQ ID NO: 5), which was cleaved with Xba I and Sac I restriction enzymes and then introduced into pBI121 (Clontech, CA) cleaved with the same restriction enzyme, and finally obtained the non-translatable construct "pBI-519HCF", which was transferred. Hosted in the form of a host deposited in the Center for Biological Resource Conservation and Research of the Republic of China Food Industry Development Institute, the registration number is BCRC940581, as shown in the first figure, containing two termination codes (such as the ** mark) and a nucleotide T (bold), the resulting structure The sequencing and restriction enzymes were confirmed and returned.

A pair of specific primers were also designed, the upstream primer was 519HCStopA (SEQ ID NO: 1), and the downstream primer was 519HCSacA (5'-TTTGTGGAGAATAT GAGCTC ACCAATGGCAACCTTTCGAATG-3') (SEQ ID NO: 3), and the HC-Pro gene was amplified by PCR. N-terminal 750 bp fragment [also a non-translating construct for reading frame shift], plus one thiol T of 751 bp (SEQ ID NO: 6), cleavage with Xba I and Sac I restriction enzymes, and cleavage with the same restriction enzyme pBI121 vector, and finally obtained the non-translatable construct "pBI-519HCN", as shown in the first figure, (containing two termination codes (such as ** mark) and one nucleotide T (bold), the resulting construction The body is sequenced and the restriction enzyme is confirmed to be correct.

A pair of specific primers were also designed. The upstream primer was 519HCStopB (5'-GACAGAAATGGGC TCTAGA TGTGGGGTGAA TGATGA TATCATCGCCAAAAG) (SEQ ID NO: 4), the downstream primer was 519HCSacB (SEQ ID NO: 2), and the upstream primer (519HCStopB) was designed. Two termination codes ( TGATGA ), Xba I cleavage ( TCTAGA ) and one additional thiol ( T ) to shift the reading frame of the entire protein, PCR amplification of the C-terminal 750 bp fragment of the HC-Pro gene (also non-translated for reading frame shift) A constitutive construct, a thiol T of 751 bp (SEQ ID NO: 7), which was cleaved with Xba I and Sac I restriction enzymes, and then introduced into the pBI121 vector cleaved with the same restriction enzyme to obtain a non-translatable construct "pBI- 519HCC" (containing two termination codes (such as the ** mark) and one nucleotide T (bold), the resulting construct is ready for use after sequencing and restriction enzyme confirmation.

The non-translatable constructs of the full length, N-terminus and C-terminus of the HC-Pro gene comprising PRSV 5-19 were respectively electroporated into Agrobacterium tumefaciens LBA4404 (Invitrogen), and the resulting transgenic plants were obtained. Agrobacterium contains non-translatable chimeras and can be used for papaya transformation.

2. Preparation of transgenic papaya

In order to shorten the time of transfer and screening, a rapid papaya gene transfer method developed by the applicant's laboratory in recent years (Kung et al., 2009) was used, which was obtained by using papaya, which is known to have excellent traits in the field. Its top buds are used to propagate papaya seedlings in a laboratory tissue culture system. Incubate for two weeks in a medium containing 0.02 mg/l α-naphthylacetic acid and 0.2 mg/l of Murashige-Skoog medium containing 0.02 mg/l α-naphthaleneacetic acid and 0.2 mg/l benzylaminopurine (MSNB). Thereafter, it was transferred to an MS medium containing indole-3-butyric acid (IBA), and induced to develop rootogen after one week of dark culture at 28 ° C, at this time in the root induction period. [Auxin dependent], then moved to a pearlite containing 1/2 volume of MS to grow the roots, at this time in the root development period [unrelated to auxin (Auxin independent)], two weeks later, cut the root tip of the MS solid medium (Murashige-Skoog medium containing 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.1 mg/l 6-benzylaminopurine) The callus and somatic embryos were induced in 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.1 mg/l benzylaminopurine (MSDB medium). Papaya somatic embryos were obtained in this way.

In the following examples, the somatic embryos developed from the adventitious roots of papaya are used as a transfer material, and the corundum is added to the aseptic water for one minute to cause the somatic embryo to produce a wound (Cheng et al., 1996). The Agrobacterium tumefaciens was mixed and allowed to stand for 5 minutes, and cultured for 2 days in a medium containing 5 mg/l 2,4-dichlorophenoxyacetic acid (2-4D). The Agrobacterium was rinsed with sterile water, and the somatic embryos were transferred to a medium containing 500 mg/l carbenicillin and 5 mg/l 2-4D. After two weeks, 50 mg/l kanamycin was added. To screen for successful transformation of the strain. After two months of screening with 100 mg/l of kanamycin, the kanamycin was removed for one month, so that the successful transformation of the somatic embryos can restore the original regeneration ability. The cells were transferred to MSNB medium containing 50 mg/l of connamycin and allowed to germinate to obtain a "geneogenic gene transfer plant".

3. Polymerase chain reaction to detect pseudogene transgenic plants

The gene DNA of the pseudo-gene transgenic plant was extracted as a template, and the pBI-HCF, pBI-HCN and pBI-HCC constructs obtained above were used as a positive control, and the HC-Pro sequence according to the target gene was used. The specific primer pair designed, including 519HCStopA/519HCSacB, 519HCStopA/519HCSacA and and 519HCStopB/519HCSacB primer pairs, respectively, polymerase chain reaction (HCase) of HC-Pro full-length gene, N-terminal fragment and C-terminal fragment (polymerase The increase in chain reaction, PCR). On the other hand, in order for the primer npt II 5 'primer 5'-CCCCTCGGTATCCAATTAGAG-3' (SEQ ID NO: 9) and npt II 3 'primer 5'-CTGGAGTTCTTCGCCA-3' (SEQ ID NO: 10) increase npt II gene And the product of PCR was analyzed by gel electrophoresis. According to the results of gel electrophoresis analysis, the plants with the expected fragments were amplified by the aforementioned PCR method. As a result, it was confirmed that 15 strains of the HC-Pro full-length gene, 31 strains of the HC-Pro N fragment gene, and 14 strains of the HC-Pro C fragment gene were transferred.

4. Evaluation of antiviral capacity of genetically transformed papaya in greenhouse

The 60 strains of the successful transformation plants were subjected to large-scale reproduction of the microorganisms, and the greenhouse virus resistance evaluation test was carried out. The same strain was mechanically inoculated with the super-pathogenic PRSV 5-19 isolate and the PRSV YK isolate, and inoculated 7 After a week, the lines with no disease development and good resistance were selected, and the gene-transgenic papaya had three kinds of anti-pathogenic responses to the virus:

(1) Susceptible lines (S): After 14 days of inoculation, there were severe invasive signs as susceptible strains.

(2) Delayed-type resistant line (DR): delayed-resistant strains with 1 to 2 weeks delay in the non-transgenic papaya plants (NT) compared with the control group. .

(3) Highly resistant line (HR): However, no disease occurred after 28 days of inoculation as a highly resistant strain.

The second figure illustrates the development of the symptoms of some of the 60 gene transgenic lines obtained as described above after inoculation of PRSV 5-19 and PRSV YK viruses in the greenhouse for four weeks, including a high resistance. Strains F3-2-2 and a susceptible strain F3-12-1 and 18-2-4 (YK-CP gene transgenic lines) and non-transgenic papaya (NT), wherein A region shows F3-2- 2. F3-12-1, 18-2-4 (YK-CP gene transgenic lines) and non-transgenic papaya (NT) were inoculated with PRSV 5-19 virus strain, wherein the control group NT had severe withering type Symptoms (column A, column a, the following note is Aa, the rest is analogous), and another control group, PRSV YK-CP gene transplant 18-2-4, also produces severe intrinsic symptoms (second Ab) of the figure, while the control group 18-2-4 inoculated with PRSV YK had good resistance (Bb of the second figure).

The results of the evaluation of the 60 strains obtained above are shown in Table 1, among which there are 31 gene transgenic lines (including 4 pBI-519HCF gene transgenic lines and 20 pBI-519HCN gene transgenic lines). The lineage and 7 pBI-519HCC gene transgenic lines) produced intrinsic signs.

Among the 60 lines, 31 gene transfer lines are susceptible strains (S) for PRSV 5-19, such as F3-12-1 (as shown by Ac in the second figure), and 31 Thirty strains of each strain were susceptible to PRSV YK (S), thus producing the same vein-like symptoms as NT (as shown in Ba and -c in the second figure), and the other strain N10-3- One pair of PRSV 5-19 is a susceptible strain (S), but has delayed resistance to PRSV YK and is a delayed resistant strain (DR).

Among the 60 lines, 10 gene transgenic lines were resistant to PRSV 5-19, 7 of which were delayed-resistant strains (DR) to PRSV YK, while the other 3 lines were against PRSV. YK is a highly resistant strain (HR).

Among the 60 strains, the other 19 strains showed no signs after inoculation of PRSV 5-19 and PRSV YK (A-d in the second panel and B-d in the second panel), and were highly resistant strains (HR). The above strains were further inoculated with virus. Seven weeks after inoculation, a total of 23 strains had no signs of PRSV YK, and 19 of them were also free of 5-19.

As shown in Table 1, seven of the 15 pBI-5-19HCF gene-transplanted papaya lines were highly resistant to 5-19 and YK, with an anti-disease ratio of 46.6% and 31 pBI-5-19HCN genes. There are 9 strains of transgenic papaya strain which are highly resistant to the two viruses, the disease resistance ratio is 29%, and 14 pBI-5-19HCN gene transgenic papaya strains have 3 strains. The resistance and disease resistance ratio was 21.4%. Based on the above results, it can be seen that all three constructs can provide resistance to papaya anti-Papaya virus supervirus strain PRSV 5-19 and typical virus strain PRSV YK.

5. Indirect enzyme-linked immunosorbent assay (ELISA)

Indirect enzyme-bound immunosorbent assay was performed by the method of Yeh and Gonsalves (Yeh and Gonsalves, 1984). Leaf tissue was obtained from the leaves of each PRASV-infected papaya strain, and the material 42 days after inoculation was collected and coated with a coating buffer [50 mM sodium carbonate containing 0.01% sodium azide). Buffer with 0.01% sodium azide)] diluted 100 times, and added to an ELISA 96-well plate, reacted at 37 ° C for 1 hour, the antigen was attached to the plate, and diluted with 2000 times anti-PRSV CP rabbit anti-serum ( Yeh et al., 1984), indirect enzyme-linked immunosorbent assay. The alkaline phosphatase-linked goat anti-rabbit IgG (Alkaline Phosphatase-conjugated Goat anti-rabbit immunoglobulin G, AP-Goat anti-rabbit Ig G) (KPL, Inc., Gaithersburg, MD, USA) was diluted 5000 times as The second antibody detects the rabbit antibody. The alkaline phosphatase substrate (Sigma-Aldrich Corporation, St. Louis, MO, USA) was added for coloration, and the absorption spectrum at 405 nm was measured with a microplate reader (SLT Lab Instruments, Salzburg, Austria). Each plant of each strain was analyzed by ELISA using a polyclonal antibody against CP protein of PRSV. The analysis found that the CP protein of the virus could not be detected in the highly resistant plants, and the reading value was the same as that of the healthy plants. The non-transgenic papaya inoculated with PRSV 5-19 or YK had more than twice the reading value.

6. Southern blotting analysis of transgene insertion sets

Sixteen gene-transplanted papaya strains with high resistance to PRSV 5-19 and PRSV YK were selected, including 5 pBI-519HCF strains, 9 pBI-519HCN strains and 2 pBI-519HCC strains. The analysis of the number of transgene insertion sets was performed in the following manner.

First, the DNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) was used to extract the genetic DNA of the virus-resistant gene-transplanted papaya to limit the enzyme Ase I cleavage and then electrophoresis on 0.8% agarose gel (agarose gel) Electrophoresis) The DNA was separated, the gel was demineralized, untwisted, and acid-base neutralized, and then the DNA on the gel was transferred to Hybond-N ⁺ membrane (Amersham Phamacia Biotech, UK) and crosslinked under UV light ( Cross-linking) For 5 minutes, pre-hybridization at 60 °C for 1 hour, the full-length fragment of HC-Pro of pBI-519HCF was amplified by the 519HCStopA/519HCSacB primer pair. 25 ng of HC-Pro full-length fragment was added, and a specific probe of the α- ³² P-calibrated PRSV 5-19 HC-Pro gene prepared by Primer It II random primer labeling kit (Stratagene, Lajolla, CA, USA) was added at 60 ℃ heterozygous for the reaction, after rinsing, a nylon film (nylon membranes) into a separator containing the reinforcing automatically displayed video clip ^{(Hyperscreen TM, Amersham Phamacia Biotech)} , to X-ray film (Hyperfilm MP, Amersham Phamacia Biotech, UK) Exposure to -80 ° C for 48 hours, comparing the difference in the number of transgene insertions between resistant and susceptible strains.

As shown in the third figure, the number of transgene insertions (copy No.) is shown in the figure, in which non-transgenic papaya (NT) is used as a control group, and four strains of the tested plants are F2-1-4, F2- 1-7, F3-2-2 and N11-1 have a single set of transgene insertions, and other strains have more than two sets of transgene insertions.

7 Anti-Papaya Round Virus Ability Analysis

High-resistance gene-transplanted papaya plants were inoculated with PRSV from different strains of Taiwan PRSV (5-19 and YK) or other different regions, including Mexican (MX), Thai (TH) and Hawaiian (HA) isolates, via HC -Pro and CP sequence alignments were screened for a broad variety of papaya lines resistant to these viruses.

Referring to Table 2 and Figures 5 to 5D, the HC-Pro genes of these viruses YK, MX, TH, HA and the 5-19 HC-Pro gene have a sequence identity of 86.1 to 96.8%. 5-19 CP gene has a sequence identity of 89.4 to 95.9%. The plants to be tested were F2-1-4, F2-7-1, N11-1 and F3-2-2 with a single transgene insertion set, and NT and 18-2-4 were used as a control group.

After 14, 28 and 42 days of virus inoculation, the development of the disease was observed. The results are shown in Table 3. The virus inoculated with different strains of NT was infected in 14 days, and 18-2-4 was highly resistant to YK. For example, MX, TH and HA have delayed resistance, but are susceptible to 5-19; F2-1-4, N11-1 have good resistance to YK and 5-19, and other strains of TH and HA have Delayed resistance, but more susceptible to MX; and F3-2-2 is very resistant to all viruses, showing that HC-Pro can be used as a target gene to fight against super-viral strains and can simultaneously fight against other The ability of papaya to check the virus, including only 86.1% of the same degree of virus.

8. Northern blotting to detect transgene transcript (mRNA) and siRNA expression

Twelve gene-transferred lines containing the constructs obtained from the above constructs, wherein each of the constructs of the gene-transplanted papaya contains three highly resistant strains and one susceptible strain, and their transgenic transcripts are detected in the following manner and The amount of siRNA expression.

In ULTRASPEC ^TM RNA system (Biotecx Laboratories, Houston, TX, USA) papaya extract total RNA (Total RNA) transgenic, 15μg of total RNA for each sample to be measured increases, containing 1.2% formaldehyde agarose gel electrophoresis film Separated. RNA was transferred to Hybond-N ⁺ membrane (Amersham Phamacia Biotech, UK), cross-linked with a UV lamp for 5 minutes, then pre-hybridized at 60 ° C for 1 hour, and α- prepared by Primer It II random primer labeling kit (Stratagene) was added. ^{The 32} P-labeled specific probe of the 5-19 HC-Pro gene coding region was reacted at 60 ° C for 16 hours, and the unhybridized probe was removed by rinsing. The nylon film was placed in an automatic developing sheet holder (HyperscreenTM ⁾ containing a reinforcing separator, and exposed to X-ray film (Hyperfilm MP) at -80 ° C for 48 hours. According to the results of the northern blotting method, the transcripts of the resistant and susceptible strains were compared, and whether the disease resistance mechanism was caused by post-transcriptional gene silencing (PTGS).

The same total RNA was used to detect the amount of transgenic siRNA, and each sample was amplified with 30 μg of total RNA, and polyacrylamide gel electrophoresis [15% polyacrylaminde] (29%, 19:1), 1×TBE (8.9 mM Tris, 8.9 mM boric acid, 20 mM EDTA), 8 M urea] was isolated from RNA and transferred to Hybond-N+ membrane (Amersham Phamacia Biotech, UK) Cross-linked with UV light for 5 minutes, then added to ULTRAHyb-Oligo solution (Ambion Inc., Austin, TX) for pre-hybridization at 42 °C for 1 hour, and added α- ³² P-labeled 5-19 HC-Pro gene coding region. The specific probe was reacted at 42 ° C for 16 hours. After rinsing, the nylon film was placed in an automatic developing blade holder containing a reinforcing spacer, and exposed to X-ray film at -80 ° C for 48 hours. Compare the difference in the accumulation of anti-infective line gene transgenic siRNA. This analysis is based on The Prestain Marker (BioDynamics Laboratory Inc., Tokyo, JAPAN) was used as a molecular marker.

The fourth panel, panel A, shows the results of the analysis of the transgenic transcripts. The high-resistance gene transgenic papaya has fewer or no transcripts, but C9-4-1, C9-5- Although 3 and C10-13 have higher transcript accumulation, their transcripts are dragged, which is presumed to be caused by decomposition of mRNA. The B panel of the fourth panel shows the analysis results of siRNA expression, in which the susceptible plants are not detected by siRNA, while F3-2-2, C9-4-1 and C9-5-3 have high accumulation of siRNA (eg Figure 4, Area B). This result shows that HC-Pro using non-translating constructs can achieve antiviral ability by means of plant gene silence.

9. HC-Pro transgene inherits to offspring and provides resistance

Two strains containing single transgene insertion sets (F2-1-4 and F3-2-2) were selected for selfing, and self-crossing progeny R1 was obtained after selfing, and the two lines were selfed for 90 days. The immature seeds of the selfed progeny R1 were cultured in MSNB medium of 100 mg/l kanamycin to identify whether the progeny was inherited to the nptII gene, and if the nptII gene was inherited to the offspring, it was cultured for two weeks. The immature seeds will produce green buds and can successfully grow papaya seedlings. If the seeds have no nptII gene, they will remain white and not germinate. The papaya seedlings are moved to the greenhouse and inoculated with PRSV 5-19 to analyze their antiviral ability.

The analysis found that F2-1-4 a total of 117 immature seeds have 88 anti-connamycin resistance, 3-2-2 a total of 102 immature seeds have 77 anti-connamycin resistance, these Papaya was inoculated into the greenhouse and inoculated with PRSV 5-19. It also showed good resistance. It showed that HC-Pro, which uses non-translatable constructs, can achieve antiviral ability by means of plant gene silence, and this disease resistance will be inherited. In the children.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention.

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The first figure shows the full-length and partial genes of the synergistic protein HC-Pro of PRSV 5-19 constructed into a simple map of pBI-519HCF, pBI-519HCN and pBI-519HCC in a non-translated manner.

The second panel is a comparison of the resistance of the highly resistant 5-19 HC-Pro gene transgenic papaya line F3-2-2 to PRSV 5-19 and PRSV YK.

The third panel shows a gel electrophoresis pattern of the number of transgenic insertions into the transgenic papaya gene by Southern blotting.

The fourth panel shows a gel electrophoresis pattern of the transgenic transcripts (transcripts, mRNA) and siRNA expression of the genetically transformed papaya by the northern blotting method.

Figures 5 to D show the HC-Pro nucleic acid sequence alignment maps of PRSVs in different regions, wherein "..." represents the same region as the HC-Pro sequence of PRSV 5-19.

Claims (8)

A recombinant vector for providing resistance to a virus in a plant, comprising a binary vector comprising a control sequence and a Papaya ringspot virus operably linked to the control sequence ( Papaya ringspot virus , PRSV) a coding sequence fragment of a helper-component protease gene (HC-Progene), wherein the coding sequence fragment of the papaya virus synergistic protease gene is expressed in a non-translatable manner, And the 5' end of the coding sequence fragment of the papaya virus virus synergistic protease gene is inserted into at least one termination code or reading frame shift; and the coding sequence fragment is SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. A recombinant vector deposited in the Center for Biological Resource Conservation and Research of the Republic of China Food Industry Development Institute, the accession number is BCRC940581, wherein the coding sequence fragment is SEQ ID NO: 5. A recombinant microorganism produced by a microorganism transformed with a recombinant vector as described in claim 1 or 2. The recombinant microorganism according to claim 3, wherein the microorganism is a disarmed Agrobacterium tumefaciens. A method for making a plant resistant to a virus, comprising the steps of: transposing a recombinant vector according to claim 1 or 2 into a plant to make the plant non-translatable The transcript of the coding sequence fragment of the synergistic protease gene of the papaya virus of the recombinant vector, but does not exhibit a synergistic protease, thereby causing the plant to produce resistance to the virus. The method of claim 5, wherein the plant body is selected from the group consisting of Cucurbitaceae and Caricaceae plants. The method of claim 5, wherein the step of transferring the recombinant vector as described in claim 1 or 2 to a plant comprises: introducing the recombinant vector into Agrobacterium, To obtain recombinant Agrobacterium; and to propagate the plant body with the recombinant Agrobacterium infection to obtain a plant body carrying the coding sequence fragment. A recombinant plant cell having a nucleic acid molecule selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 5, the nucleic acid sequence of SEQ ID NO: 6, and SEQ ID NO: 7 A nucleic acid molecule of the nucleic acid sequence shown.