KR101140618B1 - Method for breeding Chinese cabbage varieties resistant to Turnip mosaic virus and Chinese cabbage varieties produced by the method - Google Patents

Abstract

The present invention is a turnip mosaic virus mutated specific amino acids of eIF (iso) 4E protein derived from Chinese cabbage ( Turip) mosaic virus) checking the resistance of Chinese cabbage varieties of cabbage-derived eIF (iso) 4E specific amino acid mutations by way sarcoma of the cabbage varieties resistant to turnip mosaic virus protein, turnip mutation for breeding of cabbage varieties resistant to mosaic virus in The present invention relates to a method capable of inhibiting interaction between VPg (Virus genome linked protein) and eIF (iso) 4E protein of turnip mosaic virus by mutating specific amino acids of eIF (iso) 4E protein derived from cabbage and cabbage.

Chinese cabbage, eIF (iso) 4E protein, mutation, turnip mosaic virus, breeding, molecular marker

Description

Method for breeding Chinese cabbage varieties resistant to Turnip mosaic virus and Chinese cabbage varieties produced by the method}

The present invention relates to a cabbage varieties produced by breeding methods and the method of Chinese cabbage varieties resistant to turnip mosaic virus, and more specifically, a certain amino acid of the eIF (iso) 4E protein of the cabbage-derived mutant turnip mosaic virus (Turnip mutated cabbage varieties resistant to mosaic virus ), specific amino acids of eIF (iso) 4E protein derived from Chinese cabbage, for breeding cabbage varieties resistant to turnip mosaic virus, and for breeding cabbage varieties resistant to turnip mosaic virus. Molecular markers that can be tested and methods of inhibiting interaction of the virus genome linked protein (VPg) and eIF (iso) 4E protein of turnip mosaic virus by mutating specific amino acids of eIF (iso) 4E protein from Chinese cabbage .

In 2006, Chinese cabbage production amounted to 634 billion won, which is one of the four most grown vegetables after red pepper. Chinese cabbage is mainly intensively produced, so it is very vulnerable to various pests, especially viruses. Viruses that damage cabbage include the turnip mosaic virus (Turnip mosaic virus), Cucumber mosaic virus (Cucumber mosaic virus), And plantain mosaic virus (Ribgrass mosaic virus), Especially the turnip mosaic virus belonging to the genus PotyvirusTurnip mosaic virus) The damage is serious. In Korea, it is one of the main culprit of causing loss of yield, such as Gangwon highland cabbage farming, and lowering the merchandise due to deterioration of quality.

In most cases, viral diseases have not yet been adequately controlled, and the best way farmers can do is to use clean seeds or plants, and to prevent physical contact or the influx of viruses. But the surest way to control the virus is to use resistant varieties.

Viral resistance of plants has dominant resistance, recessive resistance and non-host resistance, of which there is still little research on thermal resistance. Resistant resistance is a resistance to the host factor of the host, compared to the dominant resistance determined by the R gene more radically block the mechanism of infection of the virus has a long-lasting and has the advantage of developing a variety of varieties resistant to more viruses.

Many papers have reported that TuMV (turnip mosaic virus) infectivity of Chinese cabbage is determined by interaction of plant transcription initiation factors eIF4E and eIF (iso) 4E with VPg (Virus genome linked protein), a characteristic protein of Potyvirus. . Recently, it has also been found that certain amino acid mutations in cabbage and eIF (iso) 4E can disrupt the interaction with TuMV VPg, leading to the thermophilic resistance of the virus. Identifying these specific amino acids that regulate viral recessive resistance mechanisms will enable the selection and breeding of more stable and efficient resistant cabbages in the future.

Korean Patent Laid-Open Publication No. 2007-0055889 discloses a causative virus-associated SCAR marker of Chinese cabbage virus disease (TuMV-C4).

The present invention has been made in accordance with the above-mentioned demands, and while studying to develop a cabbage variety resistant to TuMV, the cabbage having thermal resistance to TuMV by mutating a specific amino acid of eIF (iso) 4E protein derived from cabbage The present invention has been completed by revealing the possibility of developing varieties.

In order to solve the above problems, the present invention is a specific amino acid of the eIF (iso) 4E protein of the cabbage-derived mutant turnip mosaic virus (Turnip mosaic cabbage varieties resistant to virus ).

The present invention also provides a method for breeding cabbage varieties resistant to turnip mosaic virus by mutating certain amino acids of eIF (iso) 4E protein from cabbage.

The present invention also provides molecular markers capable of examining mutations for the breeding of cabbage varieties resistant to the turnip mosaic virus.

In addition, the present invention provides a method of inhibiting the interaction of VPG (Virus genome linked protein) and eIF (iso) 4E protein of turnip mosaic virus by mutating specific amino acids of eIF (iso) 4 E protein derived from Chinese cabbage.

According to the present invention, by identifying specific amino acids of the eIF (iso) 4E protein that provides thermal resistance to the turnip mosaic virus, it is possible to select and breed more stable and efficient TuMV resistant cabbage in the future.

In order to achieve the object of the present invention, the present invention is composed of the amino acid sequence of SEQ ID NO: 1, 49th (Trp), 95th (Trp), 150th (Lys) or 191 of the eIF (iso) 4E protein derived from Chinese cabbage second (Asp) amino acid mutations turnip provides a resistant cabbage varieties mosaic virus (turnip mosaic virus).

In the present invention, first of all, seven amino acids were designated by analyzing the amino acid portion of the main cap binding pocket and the nucleotide sequence of the cabbage found in the baby pole. Site-directed mutagenesis was used to mutate the candidate amino acids in the cap binding pocket to eucine in eIF (iso) 4E of the TuMV susceptible line. As a result of confirming the interaction with the yeast two hybrid, eIF (iso) 4E mutated to leucine of Trp 95, Asp 191, Trp 49, and Lys 150 was broken in VPg interaction. In other words, this indicates that the corresponding amino acid sites are necessary for VPg to bind eIF (iso) 4E.

Therefore, the cabbage varieties mutated with the amino acid sites may be resistant to turnip mosaic virus because VPM of TuMV does not interact with eIF (iso) 4E of cabbage. The mutation may include substitution of the amino acid with another amino acid, deletion of the amino acid, insertion of another amino acid, preferably the mutation is substitution with the other amino acid.

In the present invention, the 49th, 95th, 150th or 191th amino acid may be substituted with leucine, but is not limited thereto. Preferably, the 95th amino acid may be substituted with leucine or arginine, or the 150th amino acid may be substituted with leucine or glutamic acid, more preferably the 95th amino acid is substituted with leucine or arginine, and the 150th amino acid is leucine or It may be substituted with glutamic acid.

SB-18, 19, and 22, which were mutated at Trp 95 and Lys 150 in Chinese cabbage wild cultivars, showed significant inhibition of viral proliferation.

The present invention also mutated the 49th (Trp), 95th (Trp), 150th (Lys) or 191st (Asp) amino acids of the eIF (iso) 4E protein from Chinese cabbage, which consists of the amino acid sequence of SEQ ID NO: 1 step turnip mosaic virus (turnip mosaic virus) provides a breeding methods of resistant Chinese cabbage varieties, containing for.

In the method of breeding the cabbage variety of the present invention, the 49th, 95th, 150th or 191th amino acid may be substituted with leucine, but is not limited thereto. Preferably, the 95th amino acid may be substituted with leucine or arginine, or the 150th amino acid may be substituted with leucine or glutamic acid, and more preferably the 95th amino acid is substituted with leucine or arginine, and the 150th amino acid may be leucine or It can be substituted with glutamic acid.

The present invention also provides the 49th (Trp), 95th (Trp), 150th (Lys) or 191th (Asp) amino acid sequence of eIF (iso) 4E protein derived from Chinese cabbage, which consists of the amino acid sequence of SEQ ID NO: 1. It provides consisting of amino acids, turnip mosaic virus (turnip mosaic virus) molecular markers that can be tested for a mutation breeding resistant Chinese cabbage varieties. Mutations in the 49th (Trp), 95th (Trp), 150th (Lys) or 191th (Asp) amino acids of SEQ ID NO: 1 turnip mosaic virus ( Turnip mosaic Since a cabbage variety resistant to virus ) can be produced, the TuMV resistant cabbage variety can be bred by examining whether the mutations have occurred in the amino acid sites, so that the amino acid sites can be used as molecular markers.

The present invention also mutated the 49th (Trp), 95th (Trp), 150th (Lys) or 191st (Asp) amino acids of the eIF (iso) 4E protein from Chinese cabbage, which consists of the amino acid sequence of SEQ ID NO: 1 Turnip mosaic virus comprising the step of making mosaic It provides a method to inhibit the interaction of virus genome linked protein (Vpg) with eIF (iso) 4E protein.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One: Site directed mutagenesis Chinese cabbage eIF ( iso 4E mutants  making

TuMV VPg interacts with Arabidopsis eIF (iso) 4E to infect and proliferate plants early, and in this process, cap binding pocket of eIF (iso) 4E will play an important role in VPg interaction. In the present invention, first, the amino acid portion of the main cap binding pocket found in Arabidopsis and the gene sequence of the Chinese cabbage were analyzed and the following seven candidate amino acids were designated. Site-directed mutagenesis was used to mutate the candidate amino acids in the cap binding pocket to eucine in eIF (iso) 4E of the TuMV susceptible line.

Table 1.Table of amino acid cap binding pocket candidates in Chinese cabbage

plant Key amino acids in cap binding pocket Baby pole Trp46 Trp92 Lys102 Thr104 Lys149 Asp195 Ser194 cabbage Trp49 Trp95 Lys105 Thro107 Lys150 Asp191 Ser192

Example  2: cabbage eIF ( iso 4E mutants of yeast two hybrid Through TuMV  Confirm VPg Interaction

 Seven mutated eIF (iso) 4E mutants, wild type, and TuMV VPg were cloned into yeast vectors pGADT7 and pGBKT7, and the yeast two hybrid line was used to confirm the interaction with yeast two hybrids. As a result, it was confirmed that eIF (iso) 4E in which the parts of Trp 95, Asp 191, Trp 49, and Lys 150 were mutated to leucine was broken with VPg. This indicates that the corresponding amino acid sites are necessary for VPg to bind eIF (iso) 4E.

Example  3: Comparative Analysis of Chinese Cabbage Amino Acid Sequences in 11 Cabbage Wild Species

As a result of confirming the eIF (iso) 4E sequences of 11 wild cabbage strains obtained by the National Institute of Horticultural Science, it was confirmed that mutations occurred in two candidate amino acids of Lys 150 and Trp 95 in six cabbage varieties. For SB 15, 18, 19, and 20, the offspring of wild cabbage, eIF (iso) 4E Trp 95 was replaced with arginine or leucine, Lys 150 with leucine or glutamic acid, and both SB 16 and 23 were Lys 150 This was changed to Leucine. These amino acids play an important role in TuMV infection, consistent with candidate amino acids predicted to play an important role in the interaction with VPg in previous yeast two hybrid results, and mutations in this region are expected to contribute to TuMV resistant traits.

Example  4: Cabbage wild species from nine varieties TuMV  Resistant / Body Phenotypic Test

Phenotypes of virus resistance / pathogenicity in nine wild cabbage strains were assayed. The virus was TuMV CHN4 stained and inoculated with carborundum at the stage of cabbage 3-4 sheets. Two weeks after the inoculation, a new leaf was used for DAS ELISA. SB-18, 19, and 22, which were mutated at two candidate amino acids, Trp 95 and Lys 150, were found to be resistant to viral proliferation. SB-16 and SB-23, which were mutated only at Lys 150, were resistant to SB-16 and SB-23 was inhibited to some extent. The remaining plants without mutations in both amino acids were found to be pathogenic to the virus.

Table 2. A table of amino acid sequence analysis of wild cabbage strains

 TuMV Resistive System  SB-16, 18, 19, 22  TuMV pathogenic strain  SB-17, 20, 21, 24, 25  Strains with mutations in Trp95 and Lys150  SB-18, 19, 22  Lineage with mutation at Lys150  SB-16, 23

Figure 1 shows the synthesis of eIF (iso) 4E cDNA by RT-PCR from RNA isolated from nine wild cabbage strains, followed by cloning into a TOPO vector to sequence the plasmid, and the amino acid sequence of the two pathogenic cabbage varieties eIF (iso This is a comparison with 4E.

Figure 2 is a view taken three weeks after inoculation of plants with TuMV. Two pathogenic cabbage varieties were striking as controls, and four plants were SB-16, 18, 19, and 22, respectively, resistant to TuMV.

Figure 3 is a state of DAS ELISA using the new leaves two weeks after inoculation of plants with TuMV. A total of 3 repetitions were performed, n is the struck cabbage without inoculation, and p is the struck cabbage. Numbers 16, 17, 18, 19, 20, 22, 23, 24, 25 are wild cabbage strains SB 16, SB 17, SB 18, SB 19, SB 20, SB 22, SB 23, SB 24, SB 25 It means.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION)          SNU R & DB FOUNDATION <120> Method for breeding Chinese cabbage varieties resistant to Turnip          mosaic virus and Chinese cabbage varieties produced by the method <130> PN09150 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 199 <212> PRT <213> Brassica campestris <400> 1 Met Ala Thr Glu Asp Val Asn Glu Ala Leu Ala Ala Ala Glu Val Pro   1 5 10 15 Ala Thr Glu Thr Thr Glu Lys Gln Pro Ala Asp Lys Leu Glu Arg Lys              20 25 30 Trp Ser Phe Trp Phe Asp Asn Gln Ser Lys Pro Lys Gln Gly Ala Ala          35 40 45 Trp Gly Ala Ser Leu Arg Lys Ala Tyr Thr Phe Asp Thr Val Gln Asp      50 55 60 Phe Trp Gly Leu His Glu Thr Ile Phe Ile Pro Ser Lys Leu Thr Pro  65 70 75 80 Asn Ala Glu Ile His Met Phe Lys Ala Gly Val Glu Pro Lys Trp Glu                  85 90 95 Asp Pro Glu Cys Ala Asn Gly Gly Lys Trp Thr Phe Val Val Thr Ser             100 105 110 Asn Arg Lys Pro Ala Leu Asp Lys Ala Trp Leu Glu Thr Leu Met Ala         115 120 125 Leu Val Gly Glu Gln Phe Asp Glu Ala Asp Glu Ile Cys Gly Val Val     130 135 140 Ala Ser Val Arg Pro Lys Gln Asp Lys Leu Ser Leu Trp Thr Arg Thr 145 150 155 160 Lys Ser Asn Glu Ala Val Leu Met Gly Ile Gly Lys Lys Trp Lys Glu                 165 170 175 Ile Leu Asp Val Thr Asp Lys Ile Thr Phe Thr Asn His Asp Asp Ser             180 185 190 Arg Arg Thr Arg Phe Thr Val         195  

Claims (10)

delete delete delete delete Sited mutagenesis of the 49th (Trp), 95th (Trp), 150th (Lys) or 191th (Asp) amino acids of the eIF (iso) 4E protein from Chinese cabbage, consisting of the amino acid sequence of SEQ ID NO: 1 directed mutagenesis) method a method for producing a Chinese cabbage plants resistant to turnip mosaic virus (turnip mosaic virus), comprising the step of mutation through. 6. The method of claim 5, wherein said 49th, 95th, 150th, or 191th amino acid is substituted with leucine. The method of claim 5, wherein the 95th amino acid is substituted with leucine or arginine, or the 150th amino acid is substituted with leucine or glutamic acid. The method of claim 5, wherein the 95th amino acid is substituted with leucine or arginine and the 150th amino acid is substituted with leucine or glutamic acid. The 49th (Trp), 95th (Trp), 150th (Trp) of the eIF (iso) 4E protein from Chinese cabbage, consisting of the amino acid sequence of SEQ ID NO: 1 prepared by the method of any one of claims 5-8 Lys) or 191st (Asp) amino acid mutations turnip mosaic virus (a cabbage plant resistance to turnip mosaic virus). delete

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