JP2011130697A - Method of creating tomato having jointless trait - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel tomato having a jointless trait and a method of creating the same. <P>SOLUTION: There are disclosed: DNA encoding a protein consisting of a specific amino acid sequence derived from tomato that inhibits the expression of tomato DNA (LeMADS-MC gene); DNA comprising an amino acid sequence in which one or several amino acids in the amino acid sequence are substituted, deleted, inserted or added, and encoding a protein having activity of controlling the absciss layer formation of peduncle; and DNA encoding a protein having activity of controlling the absciss layer formation of peduncle. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a tomato having a jointless character and a production method thereof.

Some fruit vegetables and fruit trees form a delamination at the infarct portion. When such a fruit is harvested, the fruit is detached at the delamination of the stalk portion, and is harvested in a state where the so-called “seed” part is combined with the fruit.
For example, in the case of tomatoes, in fresh tomatoes provided to consumers in the state of fruits, the fact that the “seed” part is bound to the fruits leads to an appeal of freshness to consumers.
However, in the case of tomatoes for processing, it is necessary to remove this “seal” part before it is processed so that it does not affect the quality of the processed product. Later, it will be necessary to remove "spots" from the fruit, which will be very hard work.

It is known that tomatoes have mutants ( jointless , jointless2 ) exhibiting a “jointless trait” that does not have a delamination of the flora . When this mutant tomato is harvested, it detaches from the bottom of the “spot”, so that a fruit without the “spot” can be harvested.
Of these mutants, genes involved in the expression of the jointless trait is isolated with respect Jointless, that encodes a MADS box proteins has been elucidated (Non-Patent Document 1). This gene is known to sit on chromosome 11. Jointless is not used for breeding practical varieties because it has a leafy character in which a growth point is generated at the tip of the inflorescence and leaves are generated.
On the other hand, map-based cloning has progressed for jointless2 , and a transcription factor gene has been found as a candidate (Non-patent Document 2). It has been clarified that this gene sits on chromosome 12 (Non-patent Document 3). In addition, it is known that jointless2 has no effect on other traits.
Therefore, recently, in order to eliminate the work to remove “spot” before processing, and to improve the work efficiency significantly, tomatoes introduced with “jointless traits” derived from jointless2 mutants by mating breeding are used as processing varieties. Widely used.

By the way, it has been clarified that two genes encoding a MADS box transcription factor are arranged side by side at the ripening inhibitor ( rin ) locus of tomato ( Solanum lycopersicum ) (Non-patent Document 4). The upstream gene of the two genes has been identified as the LeMADS-RIN gene related to maturation control, and the downstream gene as the LeMADS-MC gene related to the size of the sepal (Non-Patent Document 4). It is known that the LeMADS-MC gene sits on chromosome 5.
The rin mutant, on the other hand, lacks about 1.7 kb, including the entire final exon of LeMADS-RIN gene and the transcription termination signal. When LeMADS-RIN gene is expressed, mRNA transcription has no transcription termination signal. proceeded contains the entire LeMADS-MC gene flanked for, thus resulting mRNA is known to be a LeMADS-RIN gene and LeMADS-MC gene fusion mRNA lacking the first exon lacking a final exon (Non-Patent Document 5).
This rin mutant was found among natural mutants in the United States in the 1960s (Non-Patent Document 6), (1) it is completely the same as normal until the fruit enlargement, (2) lycopene is completely Not produced, (3) not softened, (4) no increase in ethylene production is observed (Non-patent Document 7).

L. Mao et al., Nature 406, 910-913 (2000) T. J. Yang et al., Chromosoma 114, 103-117 (2005) Budiman et al., Theor. Appl. Genet., 108, 910-913 (2004) J. Vrebalov et al., Science 296, 343-346 (2002) J. J. Giovannoni, Plant Cell 16 Suppl., S170 (2004) R. W. Robinson & M. L. Tomes, Rep. Tomato Genet. Coop. 18, 36-37 (1968) E. C. Tigchelaar et al., HortScience 13, 508-513 (1978)

  This invention makes it a subject to provide the novel tomato which has a jointless character, and its production method. Moreover, this invention makes it a subject to provide the method of selecting easily the tomato which has a jointless character.

In the development of tomatoes having improved shelf life, the present inventors have been studying the rin mutants described above. As a result, normal genes ( LeMADS-RIN genes) and mutant genes (mutant rin genes) the rin F ₁ plants both are expressed, by introducing a complementary antisense DNA to sequence portions derived from LeMADS-MC gene of mutant rin gene sequence, the expression of the mutant rin gene Recombinant tomatoes with reduced levels were produced.
And when this recombinant tomato was observed, it was unexpectedly found that a delamination was not formed in the fruit pulp, that is, a jointless character was imparted to the tomato.

Based on this unexpected discovery, the present inventors speculated that the LeMADS-MC gene might be involved in the expression of the jointless trait, and the normal tomato variety, that is, the normal gene ( LeMADS-RIN gene) When the same antisense DNA as described above was introduced into a tomato variety expressing the LeMADS-MC gene, it was confirmed that the tomato variety had a jointless character. Under these circumstances, the present inventors have found that delamination formation of the infarction portion of tomato is inhibited by suppressing the expression of LeMADS-MC gene in tomato, leading to the present invention.

That is, the present invention is as follows.
[1] A method for producing a tomato ( Solanum lycopersicum ) having a jointless trait, comprising a step of suppressing the expression of any of the following DNAs (a) to (d):
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.
[2] including a step of suppressing the expression of the DNA by introducing a DNA encoding an RNA complementary to the transcription product of the DNA of any one of (a) to (d) into the cell, [1 ] The production method of the tomato as described in.
[3] The DNA encoding the RNA has a base sequence complementary to a base sequence consisting of at least 30 or more consecutive bases of the base sequence represented by Nos. 182 to 748 of the base sequence shown in SEQ ID NO: 1. The production method of the tomato as described in [2] containing.
[4] The DNA encoding the RNA includes a base sequence complementary to the base sequence represented by Nos. 211 to 680, 182 to 630, or 296 to 748 of the base sequence shown in SEQ ID NO: 1. The method for producing tomatoes according to [3].

[5] Tomato ( Solanum lycopersicum ) having a jointless trait in which the expression of any of the following DNAs (a) to (d) is suppressed;
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.
[6] The tomato according to [5], wherein DNA encoding RNA complementary to the transcription product of any of the DNAs (a) to (d) is retained in the cell.
[7] The DNA encoding the RNA includes a base sequence complementary to a base sequence consisting of at least 30 consecutive bases of the base sequence represented by Nos. 182 to 748 of the base sequence shown in SEQ ID NO: 1. [6] The tomato according to [6].
[8] The DNA encoding the RNA includes a base sequence complementary to the base sequence represented by Nos. 211 to 680, 182 to 630, or 296 to 748 of the base sequence shown in SEQ ID NO: 1. [7] The tomato according to [7].

[9] A seed obtained from the tomato according to any one of [5] to [8].
[10] A processed product made from the tomato according to any one of [5] to [8].

[11] A method for producing a tomato ( Solanum lycopersicum ) having a jointless character, comprising cultivating the seed according to [9].

[12] DNA encoding RNA complementary to the transcription product of DNA of any of the following (a) to (d);
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.
[13] A vector comprising the DNA of [12].

[14] detecting the expression level of any of the following DNAs (a) to (d) in the test tomato and selecting a test tomato whose expression level of the DNA is smaller than that of a normal type tomato A method for selecting tomato ( Solanum lycopersicum ) having a jointless character, including:
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.
[15] A forward primer comprising a base sequence complementary to a base sequence containing at least 15 consecutive bases among the complementary sequences of the base sequence represented by Nos. 167 to 633 of the base sequence described in SEQ ID NO: 1, and SEQ ID NO: 1. A test tomato using a primer set comprising a reverse primer comprising a base sequence complementary to a base sequence comprising at least 15 consecutive bases of the base sequence represented by Nos. 192 to 748 of the base sequence according to 1 The selection of tomatoes having a jointless trait according to [14], which comprises detecting the expression level of any one of the DNAs (a) to (d) by performing PCR using the cDNA prepared from the template as a template Method.
[16] A tomato having a jointless trait ( Solanum) comprising detecting the presence of any of the following DNAs (a) to (d) in a test tomato and selecting the test tomato without the DNA: lycopersicum ) selection method;
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.
[17] A forward primer comprising a base sequence complementary to a base sequence comprising at least 15 consecutive bases among the complementary sequences of the base sequences represented by Nos. 14 to 64 of the base sequence shown in SEQ ID NO: 1, and SEQ ID NO: Test tomato using a primer set comprising a reverse primer consisting of a base sequence complementary to a base sequence comprising at least 15 consecutive bases of the base sequence indicated by Nos. 698 to 748 of the base sequence described in 1. Selection of tomatoes having jointless traits according to [16], comprising detecting the presence of any of the DNAs (a) to (d) by performing PCR using the genomic DNA prepared from the template as a template Method.
[18] The forward primer is a primer comprising the base sequence shown in SEQ ID NO: 3 or a primer comprising a base sequence having at least 90% identity with the base sequence, and the reverse primer is shown in SEQ ID NO: 4. The method for selecting a tomato having a jointless trait according to [17], which is a primer comprising a base sequence or a primer comprising a base sequence having at least 90% identity with the base sequence.

According to the present invention, it is possible to easily produce or produce various varieties of tomatoes having jointless traits, and provide tomatoes having novel jointless traits.
In addition, according to the present invention, a tomato having a jointless character can be easily selected.

It is a photograph which shows the result of the expression analysis (electrophoresis) of LeMADS-MC gene in a recombinant plant. From left to right, normal tomato leaves / flowers / flowers / fruits / grass / fruits, known jointless mutants ( jointless (j)) leaves / flowers / flowers / collars / grass / fruits, joints of the present invention The result of the expression analysis in the leaf of a recombinant tomato to which a loess trait was imparted is shown. It is the photograph of the form of the fruit of the recombinant tomato (left, center) and normal type tomato (right) to which the jointless character of this invention was provided. It is a photograph which shows the result of the expression analysis (electrophoresis) of the transgene in the progeny of the recombinant tomato provided with the jointless trait of the present invention. From left, size marker (λ Hind III) (M), progeny of recombinant tomato (4-ha 1-11), normal plant (W).

The method for producing a tomato having a jointless trait according to the present invention includes a step of suppressing the expression of a gene encoding a protein having an activity of controlling the delamination formation of fruit pulp.
In the present invention, the jointless trait refers to a trait in which a delamination (joint part) is not formed in the fruit pulp.

The base sequence of cDNA of the above gene is shown in SEQ ID NO: 1. Previously, the above gene was identified as the LeMADS-MC gene involved in the regulation of sepal size (see “Background”), but the LeMADS-MC gene is involved in the delamination of the infarction . That was not known. In the present specification, the above-mentioned “gene encoding a protein having an activity to control the delamination formation of fruit folds ” is sometimes referred to as “ LeMADS-MC gene” for convenience.
The open reading frame of the above gene is a region of No. 14 to 748 (735 bases) in the base sequence shown in SEQ ID NO: 1, and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (244 residues).

As found by the present inventors, the above gene ( LeMADS-MC gene) controls the delamination of the infarction , thereby inhibiting the delamination of the tomato infarction by suppressing its expression, Tomatoes with jointless traits can be produced.

Specifically, the following DNA expression is suppressed.
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.
The protein consisting of the amino acid sequence of SEQ ID NO: 2 has an activity to control the delamination formation of fruit pulp.

(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA.
In the present invention, the target DNA that suppresses expression is not limited to that encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. That is, even if it is a variant of this DNA, as long as it codes the protein which has the activity which controls the delamination formation of an infarction, it can become a target. Here, whether or not the DNA mutant "encodes a protein having an activity to control the delamination of the fruit bud" is determined whether the delamination is formed in the fruit bud of the tomato expressing the mutant It can be confirmed by observing whether or not (whether or not a jointless character is given). That is, when a delamination is formed in the tomato peduncle that expresses a mutant of a certain DNA, it can be determined that the mutant encodes a protein having an activity to control the delamination of the prunus. .
Here, “several” is usually 2 to 10, preferably 2 to 7, more preferably 2 to 5, and more preferably 2 to 3.

(C) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 1.
One example of DNA encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2 is DNA containing the coding region of the base sequence of SEQ ID NO: 1.

(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.
In the present invention, the target DNA that suppresses expression is not limited to that containing the coding region of the nucleotide sequence of SEQ ID NO: 1. That is, even if it is a variant of this DNA, as long as it codes the protein which has the activity which controls the delamination formation of an infarction, it can become a target.
Here, “under stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Examples of the conditions include 0.1 × SSC / 0.1% SDS solution / 65 ° C. and 0.2 × SSC / 0.1% SDS solution / 68 ° C.

  The DNA shown in (d) comprises a base sequence having 80% or more, more preferably 90% or more, particularly preferably 95% or more identity to the base sequence of SEQ ID NO: 1, and Also included is a DNA encoding a protein having an activity of controlling the delamination formation of. Sequence identity can be determined by FASTA search or BLAST search.

In the present invention, “suppressing DNA expression” includes suppressing transcription of DNA and suppressing translation into protein.
As a method for suppressing the expression of DNA, a method of introducing DNA encoding RNA complementary to the transcription product (mRNA) of the DNA into cells (antisense method, RNAi method), the transcription product specifically Examples include a method of introducing a DNA encoding a ribozyme to be cleaved into a cell, a gene disruption method, and a co-suppression method.

The RNA complementary to the transcript does not need to be complementary to the entire length of the transcript, for example, at least 30, preferably at least 100, more preferably at least 100 consecutive nucleotide sequences of SEQ ID NO: 1. What is necessary is just to be complementary to the base sequence which consists of 400 bases.
In particular, since the region numbered 182 to 748 in SEQ ID NO: 1 is specific for DNA encoding a protein having the activity of controlling the delamination formation of fruit pulp, the RNA may be at least 30 consecutive in this region. It is preferable that the base sequence is complementary to a base sequence consisting of at least 100, more preferably at least 400 bases.
Examples of such RNA include those containing a base sequence complementary to the base sequence consisting of the bases shown in Nos. 211 to 680, 182 to 630, or 296 to 748 of SEQ ID NO: 1.
Here, the term “complementary” is not limited to the case where it is completely complementary to the transcript as long as the expression of DNA can be suppressed. For example, it is at least about 90%, preferably at least about 95%. This includes cases where they have complementarity.

  The RNA complementary to the transcript includes antisense RNA and dsRNA.

Moreover, the recombinant vector containing DNA which codes the said RNA can be utilized for transformation of a tomato.
As a vector for introducing DNA encoding an antisense RNA (antisense vector), a vector usually used for transformation of tomatoes containing a suitable promoter that functions in plant cells and preferably a terminator can be used. , “PBI121” having a CaMV35S promoter, NOS terminator, and the like can be used.
When dsRNA-encoding DNA is introduced into a vector, DNA encoding antisense RNA and DNA encoding sense RNA can be introduced into one vector, or antisense RNA can be encoded into another vector, respectively. It is also possible to introduce DNA encoding DNA and sense RNA.

An antisense vector can be prepared, for example, as follows.
A full-length or partial region of the coding region of the nucleotide sequence of SEQ ID NO: 1 is amplified using the PCR method to obtain a DNA fragment. Preferred regions for designing the base sequence of DNA encoding antisense RNA are as described above. The amplified DNA fragment is ligated in the antisense direction downstream of an appropriate promoter in the vector by a ligase reaction using a restriction enzyme site. Further, it is preferable to place a terminator at the 3 ′ end of the DNA.

Various methods known as plant transformation methods, such as the Agrobacterium method, electroporation method, polyethylene glycol method, liposome method, and particle gun method, can be used to transform tomatoes using recombinant vectors. Can be used.
As materials for transformation, tissues such as tomato protoplasts, cultured cells, callus, roots, stems, leaves and seeds can be used.

  When using protoplasts, coculture with Agrobacterium holding the recombinant vector or by fusing with spheroplasted Agrobacterium, when using cultured cells, the recombinant vector If the tissue is used by co-culturing with Agrobacterium holding the tissue, the leaf section is immersed in the Agrobacterium suspension holding the recombinant vector for a certain period of time to infect the tissue with Agrobacterium. It can be transformed by co-culturing.

  Plants can be regenerated from cells, callus, and tissues transformed in this manner by a known tissue culture method.

  For example, when leaf sections (leaf discs) are used as the plant material for the transformation, after the leaf sections are infected with Agrobacterium, they are used as plant hormones such as inorganic salts, vitamins, saccharides, cytokinins, kanamycin, etc. Stem and leaves can be formed by culturing using a medium containing a selective drug, a bactericide and the like. Next, rooting can be induced by culturing the foliage using a medium obtained by removing plant hormones such as cytokinin from the above medium.

  The term “tomato” as used in the present invention includes all of tomato plants, organs, tissues, calli, and cultured cells.

Whether or not the tomato thus obtained has been transformed is determined by detecting the expression level of the transgene by RT-PCR method, Northern hybridization method, Western blotting method or the expression of the target DNA. It can be confirmed by detecting its presence.
For example, the expression level of the target DNA can also be detected by extracting the RNA of the test tomato and performing Northern hybridization using an RNA probe designed based on the nucleotide sequence of SEQ ID NO: 1.

  Further, in the present invention, the “tomato having a jointless trait in which the expression of any of the DNAs of (a) to (d) is suppressed” may be transformed as long as the expression of the DNA is suppressed. The tomatoes obtained by cultivating cells and tissues of the tomatoes and the progeny tomatoes, and the tomatoes and progeny tomatoes that were mated using the same tomatoes as their parents, and the tomatoes of the progeny tomatoes and the progeny tomatoes are included. .

Moreover, this invention provides the manufacturing method of the tomato which has the jointless character including growing the seed obtained from the tomato which has such a jointless character, and cultivating this seed.
Moreover, this invention provides the manufacturing method of the processed product which uses the tomato which has the said jointless character as a raw material, and the processed product which uses the tomato as a raw material including processing the tomato which has the said jointless character. Examples of such processed products include heated products of tomato fruits, juices and extracts, foods and drinks containing these, food additives, feeds, medicines, cosmetics, and the like.

The present invention also detects the expression level of any of the DNAs (a) to (d) in the test tomato and selects a test tomato whose expression level of the DNA is smaller than that of the normal tomato. A method for selecting a tomato having a jointless character is provided.
For detection of the expression level of DNA, RT-PCR, Northern hybridization, and Western blotting can be used.

For example, the expression level of the DNA can be detected by designing a primer specific to the nucleotide sequence of SEQ ID NO: 1 and performing PCR using cDNA prepared from the test tomato as a template.
For example, a primer set capable of amplifying a base sequence consisting of 100 bases or more, preferably at least 400 bases in the region of Nos. 182 to 748 in SEQ ID NO: 1 can be designed, and PCR can be performed using this primer set. Such a primer set is merely an example, but a base comprising 15 or more, preferably 20 or more consecutive bases in a complementary sequence of the base sequence represented by Nos. 167 to 633 of the base sequence shown in SEQ ID NO: 1. Complementary to a base sequence comprising 15 or more, preferably 20 or more consecutive base sequences of the forward primer consisting of a base sequence complementary to the sequence and the base sequence represented by 192 to 748 of the base sequence described in SEQ ID NO: 1. A primer set consisting of a reverse primer consisting of a typical base sequence can be mentioned.

More specific examples include the following primer sets.
A forward primer consisting of a base sequence complementary to the complementary sequence of the base sequence shown by Nos. 211 to 231 of the base sequence shown in SEQ ID No. 1 and the bases shown by Nos. 660 to 680 of the base sequence shown in SEQ ID No. 1 A primer set consisting of a reverse primer consisting of a base sequence complementary to the sequence.

  A forward primer consisting of a base sequence complementary to the complementary sequence of the base sequence shown in SEQ ID NO: 1 at positions 182 to 202 and the base shown in SEQ ID NO: 1 at positions 610 to 630 A primer set consisting of a reverse primer consisting of a base sequence complementary to the sequence.

  A forward primer consisting of a base sequence complementary to the complementary sequence of the base sequence indicated by Nos. 296 to 316 of the base sequence shown in SEQ ID No. 1 and bases indicated by Nos. 728 to 748 of the base sequence shown in SEQ ID No. 1 A primer set consisting of a reverse primer consisting of a base sequence complementary to the sequence.

Here, “complementary sequence” refers to a sequence that is completely complementary.
The term “complementary” includes not only the case of being completely complementary as long as it functions as a primer but also the case of having a complementarity of at least about 90%, preferably at least about 95%.

The present invention also includes detecting a presence of any of the DNAs (a) to (d) in a test tomato and selecting a test tomato that does not contain the DNA. Provide selection methods.
For detection of the presence of DNA, PCR method or Southern hybridization method can be used.

For example, the presence of the DNA can be detected by designing a primer specific to the nucleotide sequence of SEQ ID NO: 1 and performing PCR using a genomic DNA prepared from the test tomato as a template.
Specifically, from a base sequence complementary to a base sequence comprising 15 or more, preferably 20 or more consecutive bases of the base sequence represented by Nos. 14 to 64 of the base sequence shown in SEQ ID NO: 1. And a reverse primer comprising a base sequence complementary to a base sequence comprising 15 or more, preferably 20 or more consecutive base sequences of the base sequence represented by Nos. 698 to 748 of the base sequence described in SEQ ID NO: 1. PCR can be performed using a primer set consisting of
The definitions of “complementary sequence” and “complementary” are as described above.

Examples of the forward primer include a primer comprising the base sequence shown in SEQ ID NO: 3, or a primer comprising a base sequence having at least about 90% identity, preferably at least about 95% identity with the base sequence. Examples of the reverse primer include a primer comprising the base sequence shown in SEQ ID NO: 4, or a primer comprising a base sequence having at least about 90% identity, preferably at least about 95% identity with the base sequence.
In addition, for the test tomato selected in this way, the tomato having a jointless trait can be further selected by detecting the expression level of the DNA by the above-described method.

Hereinafter, the present invention will be described in more detail with reference to examples.
<1> Construction of binary vector
Unless otherwise stated, procedures for general operations such as DNA fragment processing, DNA fragment binding, DNA fragment cloning, DNA sequence confirmation, E. coli treatment (transformation, culture, plasmid extraction, etc.) It was performed according to the method described in Molecular Cloning 3 ^rd edition. The tomatoes used in the following tests were all sold from the Tomato Genetics Resource Center (University of California, Davis) (URL: http://tgrc.ucdavis.edu/).

1) Cloning of LeMADS-MC gene fragment
Using First-Strand cDNA Synthesis Kit (GE Healthcare Bio-Sciences), cDNA was synthesized from 2 μg of total RNA of normal-ripening fruit (variety: LA2838A) (SEQ ID NO: 1). A PCR reaction was performed using GeneAmp® PCR System 9700 (registered trademark, Applied Biosystems ⁾ as a template with a 20-fold diluted solution. After completion of the PCR reaction, 5 μL was electrophoresed on a 1% agarose gel to confirm amplification. The primer sequences and PCR reaction conditions are as follows.
The amplified PCR fragment was cloned into the Eco RV cleavage site of pBluescript SK (+) and confirmed to have no PCR error by DNA sequence analysis (this PCR fragment is called pBT-ASrin4).

(i) LeMADS-MC (AF448521) primer sequence For the convenience of vector preparation, a Sac I recognition site was added to the MC001 primer 5 ′ end, and a Bam HI recognition site was added to the MC002 primer 5 ′ end.
MC001 primer: 5'-GAG CTC TTG AAC GAT ATG AAA G-3 '(SEQ ID NO: 5)
MC002 primer: 5'-GG ATC CTC CTT GCT TCT GCT AC-3 '(SEQ ID NO: 6)

(ii) PCR reaction conditions A reaction solution having the composition shown in Table 1 was used per sample, and the reaction was performed according to the schedule shown in Table 2.

2) Construction of binary vector
The pBT-ASrin4 obtained in 1) was treated with restriction enzymes with Bam HI and Sac I to obtain a LeMADS-MC cDNA fragment. This results, after a pBI121 vector was digested with Bam HI and Sac I by coupling with the ligation reaction to produce a binary vector of interest (pBI-ASrin4), E. coli was transformed with this binary vector.

<2> Introduction of binary vectors into Agrobacterium
PBI-ASrin4 was introduced into Agrobacterium EHA105 strain by electroporation method using MicroPulser electroporator (Bio-Rad Laboratories). Agrobacterium was plated on a YEB plate (containing 40 mg / mL kanamycin) and incubated for 2 to 3 days in the dark to select a strain into which pBI-ASrin4 had been introduced.

<3> Tomato Transformation Tomato transformation was performed according to the following paper.
highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics.Sun HJ, Uchii S, Watanabe S, Ezura H. Plant Cell Physiol. 2006 Mar; 7 (3): 426-31.

1) Aseptic seeding of tomato seeds Dissolve 1.5 g of fine powder Hyponex (registered trademark, Hyponex Japan Co., Ltd.) and 5.0 g of sucrose in water, adjust to pH 5.8 with 1M NaOH, make 1 L, then add 8 g of agar and autoclave Sterilize at 121 ° C for 15 minutes in a 300 mL plant box (Techjam Co., Ltd.) 30 m
L was dispensed to prepare a seeding medium.
Meanwhile, about 100 tomato seeds (variety: LA2838A) were wrapped in 5 cm square gauze, stapled, and immersed in a 70% aqueous ethanol solution for 2 minutes. Further, it was disinfected by immersing it in Kitchen Hiter (registered trademark, Kao Corporation) diluted 10 times for 45 minutes. Next, tomato seeds wrapped in gauze were rinsed three times with sterilized water, and then immersed in sterilized water to absorb water overnight.
Use a sterilized Kim Towel (registered trademark, Nippon Paper Crecia Co., Ltd.) to remove excess water adhering to the tomato seeds, sow 16 to 25 seeds at a time in the above seeding medium, and in a room for 16 hours at 25 ° C. Germinated.

2) Cultivation of Agrobacterium retaining the target DNA Agrobacterium retaining pBI-ASrin4 was inoculated into an LB plate medium containing kanamycin (100 mg / L) and cultured at 28 ° C. until the colonies became sufficiently large.
Among colonies grown on the LB plate, one colony of Agrobacterium was inoculated into a liquid LB medium containing kanamycin (100 mg / L) separately prepared with a platinum loop, and cultured with shaking at 28 ° C. for 24 hours.

3) Preparation of Agrobacterium suspension
The bacterial solution (1 mL) cultured for 24 hours with shaking was centrifuged (5000 rpm, 5 minutes) to collect Agrobacterium. After removing the liquid LB medium from the supernatant, the same amount of MS medium was added to suspend Agrobacterium. Agrobacterium suspension was prepared by diluting it 40 times with MS medium supplemented with 100 μM acetosyringone and 10 mM mercaptoethanol.

4) Infection of cotyledon slices by Agrobacterium The center of the germinated tomato cotyledons was cut perpendicular to the veins, and two cotyledon slices (leaf discs) were prepared from one cotyledon. The cotyledon section was immersed in the Agrobacterium suspension for 10 minutes to infect Agrobacterium.

5) Co-cultivation After infection treatment, place the cotyledon slices on a sterilized Kim towel, suck the suspension, and arrange 15 sections per petri dish so that the surface of the leaves is on the co-culture medium. Wrapped and co-cultured in a 25 ° C culture room for 4 days.
The co-culture medium was mixed with 1 L of MS medium and 30.0 g of sucrose, adjusted to pH 5.8, added with 3 g of Gellite (registered trademark, Wako Pure Chemical Industries, Ltd.) and sterilized at 121 ° C for 15 minutes in an autoclave. Thereafter, 1.5 mg of zeatin was added, and 30 mL each was dispensed into a petri dish.

6) Callus induction When it was confirmed visually that the bacteria had grown around the cotyledon slice, it was transferred to the callus induction medium so that the surface of the leaf was on top, and cultured at 25 ° C for 16 hours. The cotyledon slices were transferred to a new callus induction medium every two weeks.
The callus induction medium was adjusted to pH 5.8 by mixing 1 L of MS medium and 30.0 g of sucrose, added with 3 g of Gellite (registered trademark, Wako Pure Chemical Industries, Ltd.), and autoclaved at 121 ° C. for 15 minutes. After sterilization, zeatin 1.5 mg, kanamycin 100 mg, and augmentin (registered trademark, GlaxoSmithKline Co., Ltd.) 375 mg were added, and each 40 mL was dispensed into a petri dish.

7) Shoot induction When callus is formed from the cotyledon slice and stems and leaves (shoots) begin to appear from the callus, the stem and leaves (shoots) are cut off from the cotyledon slices to accelerate the growth of the stems and leaves (shoots). And transferred to shoot induction medium.
The shoot induction medium was adjusted to pH 5.8 by mixing 1 L of MS medium and 30.0 g of sucrose, added with 3 g of Gellite (registered trademark, Wako Pure Chemical Industries, Ltd.), and autoclaved at 121 ° C., 1
After sterilization for 5 minutes, 1.0 mg of zeatin, 100 mg of kanamycin, and 375 mg of augmentin (registered trademark, GlaxoSmithKline Co., Ltd.) were added, and 40 mL each was dispensed into a petri dish.

8) Rooting induction (rooting selection 1)
When the stem and leaf part had grown to a length of 1 to 2 cm, it was cut off at the root as much as possible and transferred to a rooting medium.
Individuals rooted within 2 weeks in the rooting medium were selected as transformant candidates. Those that did not root in the first rooting medium were cut thinly and transferred to a new rooting medium for rooting induction (rooting selection).
The rooting medium was adjusted to pH 5.8 by mixing 1 L of 1 / 2MS medium and 15 g of sucrose, 3 g of Gellite (registered trademark, Wako Pure Chemical Industries, Ltd.) was added, and the autoclave was 121 ° C., 15 After sterilization for 30 minutes, kanamycin 50 mg and Augmentin (registered trademark, GlaxoSmithKline Co., Ltd.) 375 mg were added, and each 40 mL was dispensed into a petri dish.

9) Rooting guidance (rooting selection 2)
The rooted individuals were transferred to a 300 mL plant box (Tech Jam Co., Ltd.) containing the rooting medium. At that time, roots were cut off, and rooting induction (rooting selection) was performed for the second time.

10) Acclimatization
Plants grown for about one month in a 300 mL plant box (Tech Jam Co., Ltd.) are replanted in pots containing soil and covered with vinyl to avoid sudden drops in humidity and gradually acclimatize to the open air conditions. , Shifted to normal pot planting.

<4> Analysis of target gene ( LeMADS-MC ) expression in plants
1) transgenic plants obtained by total RNA extracted above the plant, a normal-type plants (cultivar: LA2838A), and existing jointless variant (Jointless (j)) (variety: in each plants LA1806) The expression of LeMADS-MC was analyzed.
The SDS / phenol method was used for extraction of total RNA from plants. After homogenizing 1 g of a frozen plant powder sample with liquid mortar and pestle under liquid nitrogen, 10 mL extraction buffer (0.1 M Tris-HCl (pH 8.0), 0.1 M NaCl, 1% SDS), 2 mL 2-mercaptoethanol and 10 mL of phenol chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1) were added and stirred well for 5 minutes. After centrifugation at 8,000 rpm for 10 minutes at room temperature, the supernatant was collected, 10 mL of phenol chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1) was added, and the mixture was stirred well for 5 minutes. After centrifugation again at 8,000 rpm for 10 minutes at room temperature, the supernatant was collected, 25 μL of 5 M NaCl and 0.8 mL of 2-propanol were added per 1 mL of the supernatant, and the mixture was allowed to stand at −80 ° C. for 30 minutes. . Then, after centrifuging at 8,000 rpm for 30 minutes at 4 ° C., the precipitate was dissolved in 900 μL of sterilized water, 300 μL of 10 M LiCl was added, and the mixture was allowed to stand at −80 ° C. for 30 minutes. Further, the mixture was centrifuged at 15,000 rpm for 15 minutes at 4 ° C., and the resulting precipitate was rinsed with 70% ethanol. Dissolve the precipitate after rinsing in 300 μL of sterilized water, add 30 μL of 3M sodium acetate and 750 μL of 100% ethanol, allow to stand at −80 ° C. for 30 minutes, and then centrifuge at 4 ° C. at 15,000 rpm for 15 minutes. The resulting precipitate was rinsed again with 70% ethanol. When the precipitate was completely dried, sterilized water was added to dissolve it, and this was taken as total RNA and stored at −80 ° C.

2) Northern analysis Total RNA was run on a 1.2% agarose gel (1 x MOPS, 5% (w / v) formaldehyde), 10 µg per lane, and transferred to Hybond-N ⁺ membrane (GE Healthcare Bio-Sciences) did. Hybond-N ⁺ membrane is UV cross-linked and pre-hybridized at 68 ° C for 30 minutes with hybridization buffer (5xSSC, 50% formamide, 0.02% SDS, 0.1% N-lauroyl sarcosine, 2% blocking reagent) Hybridization was performed.
DIG RNA Labeling Kit (Roche) based on the cDNA containing the 187th a to 808th g from the start of translation of LeMADS-MC gene (base number 200 to 821 of SEQ ID NO: 1) An RNA probe was synthesized (SEQ ID NO: 7), heat denatured at 68 ° C. for 10 minutes, added to the hybridization buffer, and hybridized overnight at 68 ° C. After that, Hybond-N ⁺ membrane was treated with 2 × SSC / 0.1% SDS solution at room temperature for 10 minutes, 0.2 × SSC / 0.1% SDS solution at room temperature for another 10 minutes, and 0.2 × SSC / 0.1% SDS solution at 68 ° C. Washed twice for 20 minutes. Subsequent operations followed the DIG detection manual (Roche). For detection of the signal, CDP-Star (Roche) was used as a chemiluminescent substrate.

<5> Analysis of introduced DNA in transformant progeny
1) Confirmation of introduced DNA by PCR In order to clarify whether the recombinant gene is actually causing jointless traits, the presence or absence of jointless trait expression in multiple seed-derived plants obtained from recombinant plants At the same time, it was confirmed whether the recombinant gene was inherited.
For the recombinant plants in which the expression of LeMADS-MC was suppressed by the introduction of the recombinant gene, seeds were collected from the grown fruit after selfing.
From the collected seeds, dozens were randomly selected and cultivated by a conventional method in a closed greenhouse to obtain seed-derived plants obtained from recombinant plants.
Total DNA was extracted from the leaves of each plant derived from the seeds obtained from the recombinant plants. GE Healthcare Nucleon Phytopure Genomic DNA Extraction Kits (GE Healthcare Bio-Sciences) were used for extraction. Using ^{GeneAmp (R) PCR System 9700 (} Applied Biosystems , Inc.) with respect to the next resulting DNA, PCR was carried out. After completion of the PCR reaction, electrophoresis was performed on a 1% agarose gel, and the presence or absence of amplification of the introduced DNA was examined. Primers, PCR reaction conditions, etc. are as follows.

(i) Primer sequence for detecting transgene fragments
Primers were designed on the LeMADS-MC gene fragment (MC001 primer) and on the CaBI35S promoter region derived from pBI121 (pBI121-F primer).
MC001 primer: 5'-GAG CTC TTG AAC GAT ATG AAA G-3 '(SEQ ID NO: 5)
PBI121-F primer: 5'-AGA CCC TTC CTC TAT ATA AG-3 '(SEQ ID NO: 8)

(ii) PCR reaction conditions The reaction solution having the composition shown in Table 3 was used per sample, and the reaction was performed according to the schedule shown in Table 4.

<6> Results The results of the expression analysis of the LeMADS-MC gene in <4> are shown in FIG.
In normal plants and existing jointless mutants (j), expression of LeMADS-MC gene is observed, but in the recombinant plant produced this time, expression of LeMADS-MC gene is not observed. This showed that normal tomato was transformed and the expression of LeMADS-MC gene was suppressed. In addition, since the expression of LeMADS-MC gene was observed in the j mutant, it was shown that the recombinant plant produced this time was given a jointless trait by a gene different from j. This is consistent with the fact that the chromosome on which the gene related to the expression of the jointless trait of j is located (No. 11) is different from the chromosome on which the LeMADS-MC gene is located (No. 5). .
FIG. 2 is a photograph of the fruits of the recombinant plant (left side) and normal plant (right side) to which the jointless trait was created this time.
The normal plant on the right side had a delamination, whereas the left recombinant plant had no delamination.
In this study, the expression analysis of the LeMADS-MC gene of an existing jointless mutant ( jointless2 (j-2)) was not performed, but the gene involved in the expression of the jointless trait of j-2 is number 12. Judging from the fact that it sits on a chromosome, the recombinant plant created this time is considered to have been given a jointless trait by a gene different from j-2.

The result of the expression analysis of the transgene in the progeny of the recombinant plant obtained in <5> is shown in FIG.
Table 5 shows the correlation between the presence of the recombinant gene and the phenotype (jointless trait) in the progeny of the recombinant plant to which the jointless trait created this time was given.
In the progeny of the recombinant plant, the line in which the recombinant gene was inherited had no delamination (has a jointless trait), but in the line in which the recombinant gene was not inherited, the delamination was Was formed. This proved that the LeMADS-MC gene imparted a jointless trait.

  According to the present invention, jointless traits can be imparted to various types of tomatoes, and variations can be imparted to processing tomatoes.

Claims (18)

A method for producing a tomato ( Solanum lycopersicum ) having a jointless trait, comprising the step of suppressing the expression of any of the following DNAs (a) to (d):
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.   The method according to claim 1, further comprising the step of suppressing the expression of the DNA by introducing into the cell a DNA encoding an RNA complementary to the transcription product of the DNA of any one of (a) to (d). To make tomatoes.   The DNA encoding the RNA includes a base sequence complementary to a base sequence consisting of at least 30 or more consecutive bases of the base sequence represented by Nos. 182 to 748 of the base sequence shown in SEQ ID NO: 1. Item 3. A method for producing a tomato according to Item 2.   The DNA encoding the RNA comprises a base sequence complementary to the base sequence represented by Nos. 211 to 680, Nos. 182 to 630, or Nos. 296 to 748 of the base sequence shown in SEQ ID NO: 1. 3. A method for producing tomatoes according to 3. Tomato ( Solanum lycopersicum ) having a jointless trait in which the expression of any of the following DNAs (a) to (d) is suppressed;
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.   6. The tomato according to claim 5, wherein DNA encoding RNA complementary to the transcription product of any one of (a) to (d) is retained in the cell.   The DNA encoding the RNA comprises a base sequence complementary to a base sequence consisting of at least 30 consecutive bases of the base sequence represented by Nos. 182 to 748 of the base sequence shown in SEQ ID NO: 1. 6. Tomato according to 6.   The DNA encoding the RNA comprises a base sequence complementary to the base sequence represented by Nos. 211 to 680, Nos. 182 to 630, or Nos. 296 to 748 of the base sequence shown in SEQ ID NO: 1. 7. Tomato according to 7.   Seed obtained from the tomato according to any one of claims 5 to 8.   The processed product which uses the tomato as described in any one of Claims 5-8 as a raw material. A tomato having a jointless character ( So) , comprising cultivating the seed according to claim 9.
lanum lycopersicum ). DNA encoding RNA complementary to the transcription product of DNA of any of the following (a) to (d);
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.   A vector comprising the DNA according to claim 12. A joint comprising: detecting the expression level of any of the following DNAs (a) to (d) in a test tomato; and selecting a test tomato whose expression level of the DNA is smaller than that of a normal tomato A method for selecting a tomato ( Solanum lycopersicum ) having a loess trait;
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp.   A forward primer consisting of a base sequence complementary to a base sequence comprising at least 15 consecutive bases among the complementary sequences of the base sequence represented by Nos. 167 to 633 of the base sequence described in SEQ ID NO: 1, and the sequence described in SEQ ID NO: 1 Prepared from a test tomato using a primer set consisting of a reverse primer consisting of a base sequence complementary to a base sequence containing at least 15 consecutive bases of the base sequence indicated by Nos. 192 to 748 of the base sequence of The method for selecting a tomato having a jointless trait according to claim 14, comprising detecting the expression level of any one of the DNAs (a) to (d) by performing PCR using cDNA as a template. Detecting the presence of any of the following DNAs (a) to (d) in a test tomato, and selecting a test tomato that does not contain the DNA: a tomato having a jointless trait ( Solanum lycopersicum ) Selection method;
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) In the amino acid sequence of SEQ ID NO: 2, encodes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and having an activity of controlling delamination formation DNA,
(C) DNA containing the coding region of the base sequence of SEQ ID NO: 1,
(D) A DNA encoding a protein that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under a stringent condition and has an activity of controlling the delamination formation of fruit pulp. A forward primer consisting of a base sequence complementary to a base sequence comprising at least 15 consecutive bases among the complementary sequences of the base sequences represented by Nos. 14 to 64 of the base sequence shown in SEQ ID NO: 1; Prepared from a test tomato using a primer set consisting of a reverse primer consisting of a base sequence complementary to a base sequence containing at least 15 consecutive bases in the base sequence indicated by Nos. 698 to 748 of the base sequence of The method for selecting a tomato having a jointless trait according to claim 16, comprising detecting the presence of any one of the DNAs (a) to (d) by performing PCR using genomic DNA as a template.   The forward primer is a primer comprising the base sequence shown in SEQ ID NO: 3 or a primer consisting of a base sequence having at least 90% identity with the base sequence, and the reverse primer has the base sequence shown in SEQ ID NO: 4 The selection method of the tomato which has a jointless character of Claim 17 which is a primer which consists of a primer to contain or a base sequence which has at least 90% identity with this base sequence.