WO2011025007A1 - Sorghum purple leaf spot-related gene and use of same - Google Patents

Abstract

As the results of a mapping of a purple leaf spot disease gene ds1 in populations F3 to F5 obtained by mating a purple leaf spot-resistant variety with a disease variety, the gene was successfully identified. Further, it was discovered that resistance to purple leaf spot can be imparted to sorghum by inhibiting the expression of the ds1 gene or mutating the same.

Description

Sorghum purpura-related genes and uses thereof

The present invention relates to a susceptibility gene and resistance gene for sorghum purpura, a method for determining susceptibility and resistance to sorghum purpura, and a method for producing sorghum with purpura resistance.

Purple leaf spot is a spotted disease caused by filamentous fungi, and is a disease that occurs in warm regions south of Kanto. Of the sorghum (sorghum plant) varieties cultivated in Japan, 80% are susceptible to purpura, and in recent years its incidence has been increasing. Individuals infected with purpura generally have reddish-brown spots on the leaves. Depending on the variety, yellowish brown, orange, red to reddish brown spots may occur. These spots spread to form spindle-shaped lesions with a length of 0.5-2 cm and a width of 0.3-1 cm.

So far, sorghum has been mainly cultivated as a livestock feed in Japan, but in recent years, it has been attracting attention as a raw material for biofuel (ethanol). Therefore, preventing the decrease in sorghum sales due to the disease of purple spot disease is an important issue not only in agricultural policy but also in energy policy.

So far, sorghum has been rarely controlled against purpura because its main use was livestock feed.

On the other hand, it was known that there was a difference in resistance to purpura between sorghum varieties. For this reason, attempts have been made to elucidate the mode of inheritance of resistance for the purpose of breeding resistant varieties. As a result, it has been clarified that the resistance to sorghum purpura disease is controlled by a single recessive gene (Non-patent Document 1).

However, the gene responsible for the susceptibility and resistance of sorghum purpura has not been identified yet, and its location and substance on the chromosome remain unknown.

The present invention has been made in view of such circumstances, and its purpose is to identify genes responsible for the susceptibility and resistance of sorghum purpura and elucidate the mechanism of onset of sorghum purpura. There is to do. Another object of the present invention is to provide a method for easily determining the susceptibility and resistance of sorghum purpura based on the elucidated mechanism of sorghum purpura. A further object of the present invention is to provide a method for efficiently producing sorghum with enhanced resistance to purpura based on the elucidated mechanism of the development of sorghum purpura.

In order to solve the above-mentioned problems, the present inventors have established a sorghum SSR marker and insertion / deletion in a large cross population (F3 to F5) of a sorghum resistant variety SLIL-05 and a diseased product type bmr-6. The marker was used to map the purpura disease susceptibility gene (FIGS. 1 and 2).

Specifically, first, rough mapping using an SSR marker was performed on F3 individuals (175 individuals) to elucidate that the purpura disease susceptibility gene is located on chromosome 5. Next, for F4 individuals (640 individuals), individuals with recombination between SSR markers SB3056 to SB3178 (229 individuals) were selected and purpura disease was investigated in the field, and the presence of purpura disease susceptibility genes The region was narrowed down to a region of about 246 kbp. Furthermore, mapping was performed using insertion / deletion markers found based on the nucleotide sequence information of sorghum variety BTx623, and the region of purpura disease susceptibility gene was narrowed down to about 96 kbp. Next, F5 individuals (4235 individuals) were screened using the insertion / deletion marker and SSR marker present in the candidate region of about 96 kbp, and the region where the purpura disease susceptibility gene exists was finally reduced to about 26 kbp. Narrow down to.

The present inventors investigated the expression of genes in the narrowed region by RT-PCR. Among the plurality of candidate genes, only the expression pattern of a specific candidate gene (hereinafter referred to as “ ds1 gene”) It was found that it was consistent with the distinction between susceptibility and resistance to purpura (FIG. 3). In addition, when the tissue-specific gene expression in the resistant variety SLIL-05 and the diseased product species bmr-6 was examined, the diseased variety bmr-6 expressed a large amount of ds1 gene in the foliage. In the cultivar SIL-05, almost no expression in the foliage was observed (FIG. 4). In addition, mRNA expression was observed in susceptible varieties, but mRNA expression was low in resistant varieties (FIG. 4). When the nucleotide sequence around the ds1 gene was determined and the amino acid sequences encoded by the varieties were compared among the cultivars, the resistant cultivar SIL-05 was completely consistent with the susceptible cultivar BTx623 (FIG. 5). However, SIL-05 partially lacked the upstream promoter region of the gene (FIGS. 6 and 7). For this reason, it was speculated that the ds1 gene promoter region deficiency was responsible for the decrease in mRNA expression in SIL-05. A substitution of one amino acid was observed in the susceptible variety BTx623 and the susceptible variety bmr-6. On the other hand, in the ds1 gene in那系MS3B and Greenleaf resistant varieties, there are stop codons in the coding region was estimated to have not combined the normal protein (Figure 5, Figure 6). From these facts, it was found that the ds1 gene is a susceptibility gene for purpura disease, and by suppressing its expression or mutation, the original function is not exerted, thereby imparting purpura disease resistance to an individual.

Furthermore, specific primers for amplifying the coding region and upstream region of the ds1 gene were prepared, and using these, gene amplification of each sorghum variety resulted in the size (genotype) of the amplified product of the gene and Correlations were found between sorghum susceptibility and resistance (Tables 8 and 9).

Based on the above findings, the present inventors can determine the susceptibility and resistance of purpura disease by targeting the identified ds1 gene, and further determine the expression and function of the susceptible ds1 gene. As a result of the suppression, it was found that resistance to purpura can be imparted to sorghum, and the present invention has been completed.

More specifically, the present invention provides the following.
<1> The DNA according to any one of the following (a) to (d), which encodes a protein having an activity of imparting purpura disease susceptibility to sorghum.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 6
(B) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1, 2, 4 or 5
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 6
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4 or 5
<2> The DNA according to any one of the following (a) to (d), which encodes a protein having an activity of imparting purpura resistance to sorghum.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 9 or 12
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 7, 8, 10 or 11
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, or 11
<3> The DNA according to any one of the following (a) to (c), which encodes a protein having an activity of imparting purpura resistance to sorghum.
(A) DNA encoding a double-stranded RNA complementary to the transcription product of DNA according to <1>
(B) DNA encoding an antisense RNA complementary to the transcription product of DNA according to <1>
(C) DNA encoding RNA having ribozyme activity that specifically cleaves the transcription product of DNA according to <1>
<4> A vector comprising the DNA according to any one of <1> to <3>.
<5> A sorghum cell into which the DNA according to any one of <1> to <3> has been introduced.
<6> A sorghum plant comprising the cell according to <5>.
<7> A sorghum plant which is a descendant or clone of the plant according to <6>.
<8> A propagation material for a sorghum plant according to <6> or <7>.
<9> A method for producing sorghum imparted with resistance to purpura disease, characterized by suppressing the expression or function of the DNA according to <1> in sorghum.
<10> A method for producing sorghum in which purpura disease resistance is imparted to sorghum, comprising the step of introducing the DNA according to <2> or <3> into sorghum.
<11> A drug for imparting purpura resistance to sorghum comprising the DNA according to <2> or <3> or a vector into which the DNA is inserted.
<12> A method for determining the susceptibility or resistance to purpura in sorghum, comprising analyzing the base sequence of the DNA or expression control region thereof according to <1> or <2> in a test sorghum A method comprising comparing the nucleotide sequence of
<13> A method for determining the susceptibility or resistance of purpura in sorghum, characterized by detecting the expression of DNA or the molecular weight of the expression product in <1> or <2> in sorghum Method.
<14> A method for determining the susceptibility or resistance of purpura in sorghum, comprising analyzing the nucleotide sequence of a molecular marker linked to the DNA according to <1> or <2> in a test sorghum; A method comprising comparing with a base sequence of a control.
<15> A method for breeding purple spot disease resistant sorghum,
(A) mating a purple scab-resistant sorghum variety with any sorghum variety,
(B) determining the susceptibility or resistance to purpura in the individual obtained by the mating in step (a) by the method according to any one of <12> to <14>, and (c) purpura Selecting a variety determined to have resistance to point disease.

Note that "purpura spot disease" in the present invention, in sorghum, pathogens "Bipolaris sorghicola (Lefebvre & Sherwin) Alcorn , classification: Incomplete Kinmon, incomplete thread Kintsuna" means a spot of diseases caused by. In the present invention, “purpura disease susceptibility” means the property that sorghum infects purpura. In the present invention, “purpura disease resistance” means the resistance of sorghum to purpura infection. This resistance suppresses the onset of purpura or the degree of purpura that has occurred in sorghum (for example, recognized as the number or spread of lesions).

According to the present invention, the purpura disease susceptibility gene ds1 was identified, and the location and structure of the gene on the chromosome and the mechanism of purpura disease onset were elucidated. This makes it possible to easily determine the susceptibility and resistance of sorghum purpura using the ds1 gene and nearby markers. In addition, by suppressing the expression and function of the susceptible ds1 gene, it has become possible to efficiently produce sorghum varieties resistant to purpura. The present invention greatly contributes to prevention of a decrease in sorghum yield due to the disease of purpura.

It is a figure which shows the result of mapping of ds1 candidate area | region using F4 population. In the figure, “A” indicates bmr-6 type, “B” indicates SIL-05 type, and “H” indicates hetero. It is a figure which shows the result of mapping of ds1 candidate area | region using F5 population. It is an electrophoresis photograph showing the result of detecting the expression of a ds1 candidate gene by RT-PCR. In the figure, `` 1 '' is a diseased F4 individual `` # 2-74 A72 '' whose ds1 region is heterozygous of SIL-05 type and bmr-6 type, `` 2 '' is ds1 region is SIL-05 type The resistant F4 individuals “# 2-74 C5” and “3” indicate the resistant variety “SIL-05”, and “4” indicates the susceptible variety “bmr-6”. It is an electrophoretic photograph showing the results of detecting the tissue specificity of the ds1 gene and differences between cultivars by RT-PCR. In the above figure, “R” indicates a root, “L” indicates a leaf, “S” indicates a stem, and “F” indicates a flower. In the figure below, “1” is BTx623, “2” is Greenleaf, “3” is BTx624, “4” is Na MS MS3B, “5” is bmr-6, “6” is SIL-05, “7” Indicates JN43, “8” indicates Senpaku white, “9” indicates JN358, and “10” indicates JN290EE. Odd numbers are susceptible varieties and even numbers are resistant varieties. It is a figure which shows the comparison between the varieties of the amino acid sequence of the protein which ds1 gene codes. FIG. 5 is a continuation diagram of FIG. 5-1. It is a figure which shows the comparison between the varieties of the structure of ds1 gene and its peripheral region. It is a figure which shows the inter-variety comparison of the base sequence of ds1 gene and its expression control region. FIG. 7 is a continuation diagram of FIG. FIG. 7B is a continuation of FIG. 7-2. FIG. 4 is a continuation diagram of FIG. FIG. 4 is a continuation of FIG. 7-4. FIG. 6 is a continuation of FIG. 7-5. FIG. 7 is a continuation of FIG. 7-6. FIG. 8 is a continuation of FIG. 7-7. FIG. 9 is a continuation of FIG. 7-8. FIG. 10 is a continuation of FIG. 7-9. FIG. 11 is a continuation of FIG. 7-10. FIG. 11 is a continuation of FIG. 7-11. It is a figure which shows the target sequence of the primer used for the present Example. FIG. 8 is a continuation of FIG. 8-1. FIG. 8 is a continuation of FIG. 8-2. It is a continuation figure of FIG. 8-3. FIG. 4 is a continuation of FIG. 8-4. FIG. 6 is a continuation of FIG. 8-5. FIG. 7 is a continuation of FIG. 8-6. FIG. 8 is a continuation of FIG. 8-7. FIG. 9 is a continuation of FIG. 8-8. FIG. 10 is a continuation of FIG. 8-9. FIG. 11 is a continuation of FIG. 8-10. FIG. 11 is a continuation of FIG. 8-11. FIG. 13 is a continuation of FIG. 8-12. FIG. 14 is a continuation of FIG. 8-13. FIG. 15 is a continuation of FIG. 8-14. FIG. 16 is a continuation of FIG. 8-15. FIG. 17 is a continuation of FIG. 8-16. FIG. 18 is a continuation of FIG. 8-17. FIG. 19 is a continuation of FIG. 8-18. FIG. 20 is a continuation of FIG. 8-19. FIG. 21 is a continuation of FIG. 8-20. FIG. 22 is a diagram continued from FIG. 8-21. FIG. 23 is a continuation of FIG. 8-22. FIG. 24 is a continuation of FIG. 8-23. FIG. 25 is a continuation of FIG. 8-24. FIG. 26 is a continuation of FIG. 8-25. FIG. 27 is a continuation of FIG. 8-26. FIG. 28 is a continuation of FIG. 8-27. FIG. 29 is a continuation of FIG. 8-28. FIG. 30 is a continuation of FIG. 8-29. FIG. 31 is a continuation of FIG. 8-30. FIG. 32 is a continuation of FIG. 8-31. FIG. 33 is a continuation of FIG. 8-32. FIG. 34 is a continuation of FIG. 8-33. A shows the arrangement of primer sets used for detecting polymorphisms in ds1 candidate genes of sorghum varieties derived from various regions of the world. B is an electrophoresis photograph showing the results of PCR using the primer set shown in A. FIG. As sorghum varieties, lane 1 is Gooseneck (R4 type), lane 2 is Moraba74 (S3 type), lane 3 is BTx623 (S2 type), lane 4 is Greenleaf (R3 type), and lane 5 is Nakei MS3B (R2 type) Lane 6 used bmr-6 (S1 type), and lane 7 used SIL-05 (R1 type). It is a photograph which shows the result (typical example) which performed the test of purpura disease about the sorghum variety derived from each area of the world.

<Susceptible DNA, Resistant DNA>
The present invention provides DNA encoding a protein having an activity conferring purpura disease susceptibility to sorghum (hereinafter referred to as “susceptible DNA”). Identified by the present inventors, the nucleotide sequence sequence of ds1 cDNA derived from susceptible cultivar BTx623 ID NO: 1, SEQ ID NO: The nucleotide sequence of ds1 genomic DNA: 2, the amino acid sequence of the protein they DNA encodes Shown in SEQ ID NO: 3. In addition, the base sequence of the ds1 cDNA derived from the susceptible cultivar bmr-6 is SEQ ID NO: 4, the base sequence of the ds1 genomic DNA is SEQ ID NO: 5, and the amino acid sequence of the protein encoded by these DNAs is SEQ ID NO: 6. Show. One embodiment of the susceptible DNA of the present invention is a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 (typically, the coding region of the base sequence set forth in SEQ ID NO: 1 or 2). Another embodiment is a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 (typically, the coding region of the base sequence set forth in SEQ ID NO: 4 or 5). Containing DNA).

As shown in this example, ds1 cDNA nucleotide sequence from BTx623 (SEQ ID NO: 1) and bmr-6 ds1 cDNA nucleotide sequence from (SEQ ID NO: 4) are compared, and the three locations base And one of them leads to amino acid differences (H and Q at position 851), both of which are susceptible DNA. In the current state of the art, if a person skilled in the art obtains the base sequence information of the susceptible DNA in a specific susceptible sorghum variety (for example, BTx623, bmr-6), the base sequence is modified, Although the encoded amino acid sequence is different, it is also possible to obtain DNA that is also susceptible. Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence. Accordingly, the present invention is, BTx623 and bmr-6 in ds1 protein amino acid sequences: consists of one or more amino acid substitutions in (SEQ ID NO: 3 or 6), deletions, additions, and / or inserted amino acid sequence, It also includes a DNA encoding a protein having an activity of imparting purpura disease susceptibility to sorghum. Here, `` plurality '' is the number of amino acid modifications within the range in which the modified ds1 protein maintains the activity of imparting purpura disease susceptibility to sorghum, usually within 50 amino acids, preferably within 30 amino acids, More preferably, it is within 10 amino acids (for example, within 5 amino acids, within 3 amino acids, 2 amino acids).

Further, in the current state of the art, if a person skilled in the art obtains susceptible DNA from a specific susceptible sorghum variety (for example, BTx623, bmr-6), the nucleotide sequence information of the susceptible DNA Can be used to obtain DNAs encoding homologous genes that are also susceptible from other sorghum varieties. Accordingly, the present invention is, BTx623 and bmr-6 in ds1 DNA (SEQ ID NO: 1, 2, 4 or 5) and a hybridizing DNA under stringent conditions, granted sorghum, purpura spot disease susceptibility It also includes DNA that encodes a protein having the following activity.

Whether the mutant DNA or homologous DNA thus obtained encodes a protein having the activity of conferring purpura disease susceptibility to sorghum is determined by, for example, purpura disease resistant varieties introduced with these DNAs. It can be determined by spraying or inoculating the pathogen of the disease and then examining whether or not to develop purpura or the degree of purpura that has developed (for the test of purpura onset) See Example 1).

The present invention also provides a DNA encoding a protein having an activity of conferring purpura resistance to sorghum (hereinafter referred to as “resistant DNA”). Identified by the present inventors, resistant varieties那系MS3B sequence the base sequence of ds1 cDNA derived NO: 7, SEQ ID NO: The nucleotide sequence of ds1 genomic DNA: 8, amino acids of a protein to which they DNA encodes The sequence is shown in SEQ ID NO: 9. The base sequence of ds1 cDNA derived from the resistant variety Greenleaf is shown in SEQ ID NO: 10, the base sequence of ds1 genomic DNA is shown in SEQ ID NO: 11, and the amino acid sequence of the protein encoded by these DNAs is shown in SEQ ID NO: 12. One embodiment of the resistance-type DNA of the present invention comprises a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 9 (typically comprising a coding region of the base sequence set forth in SEQ ID NO: 10 or 11). And another embodiment includes DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12 (typically, the coding region of the base sequence set forth in SEQ ID NO: 10 or 11) DNA).

As shown in this embodiment,那系MS3b ds1 cDNA nucleotide sequence from (SEQ ID NO: 7) and Greenleaf ds1 cDNA nucleotide sequence from (SEQ ID NO: 10), derived from the susceptible variety BTx623 ds1 Compared with the base sequence of cDNA (SEQ ID NO: 1), a stop codon is generated due to a base mutation. Therefore,那系MS3b ds1 protein and Greenleaf derived ds1 protein from the susceptible variety BTx623 derived ds1 protein (SEQ ID NO: 3) and when compared, C-terminal, regions of the protein are is deficient, the original ds1 Protein function is suppressed. It is considered that suppression of this function imparts purpura resistance to the individual. In the present state of the art, one skilled in the art, certain susceptible sorghum varieties (e.g., BTx623, bmr-6) in the base sequence of ds1 DNA of functional suppression relates purpura spot disease susceptibility protein its encoding It is possible to make such modifications. Further, specific resistance sorghum varieties (e.g.,那系MS3b, Greenleaf) in ds1 DNA nucleotide sequence of, it is possible to perform such modifications as purpura spot disease resistance proteins that code is maintained. Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence. Accordingly, the present invention is, BTx623, bmr-6,那系MS3B or ds1 protein amino acid sequence of the Greenleaf (SEQ ID NO: 3, 6, 9 or 12) one or more amino acids in the substitution, deletion, addition, and It consists of a protein comprising an inserted amino acid sequence, and also contains a DNA encoding a protein having an activity of conferring purpura resistance to sorghum. Here, the term “plurality” refers to the number of amino acid modifications within a range in which the modified ds1 protein has an activity of imparting purpura disease resistance to sorghum. If the ds1 protein in sorghum does not exhibit its original function, it will be resistant to purpura, so the number of amino acid modifications is essentially not limited. Modification is within 500 amino acids, within 400 amino acids, within 300 amino acids, within 200 amino acids, or within 100 amino acids (within 50 amino acids, within 30 amino acids, within 10 amino acids, within 5 amino acids, within 3 amino acids, within 2 amino acids). is there. The modification can be, for example, a deletion on the C-terminal side of the ds1 protein. Indeed, ds1 protein in Greenleaf, as compared to BTx623 or bmr-6 derived ds1 protein, 460 amino acids are deleted.

Furthermore, in the current state of the art, if ds1 DNA is obtained from a specific sorghum variety, those skilled in the art are resistant to other sorghum varieties using the DNA sequence information. DNA encoding a homologous gene can be obtained. Accordingly, the present invention is, BTx623, bmr-6, ds1 DNA in那系MS3B or Greenleaf (SEQ ID NO: 1,2,4,5,7,8,10 or 11) a DNA hybridizing under stringent conditions Thus, DNA encoding a protein having an activity of conferring purpura resistance to sorghum is included.

Whether the mutant DNA or homologous DNA thus obtained encodes a protein having an activity of conferring purpura disease resistance to sorghum is determined by, for example, assembling the ds1 gene of the purpura disease-affected product species with the DNA. Instead, create a sorghum that retains the DNA in a homologous manner, spray or inoculate purpura, and then determine whether purpura develops or the extent of purpura (See Example 1 for a test for the development of purpura disease).

The susceptible DNA of the present invention is an agent for imparting purpura disease susceptibility to sorghum in the sense that introduction thereof can impart purpura susceptibility to sorghum, The resistance-type DNA of the present invention is a drug for imparting purpura resistance to sorghum in the sense that introduction thereof can impart purpura resistance to sorghum.

In addition, artificial mutations introduced into DNA to produce the above-mentioned mutant DNA are, for example, site-directed mutagenesis (Kramer, W. & Fritz, HJ., Methods Enzymol). , 154: 350-367, 1987).

In addition, as a method for isolating the homologous genes described above, for example, hybridization technology (Southern, E. M., Journal of Molecular Biology, 98: 503, 1975) or polymerase chain reaction (PCR) technology (Saiki) , R. K., et al. Science, 230: 1350-1354, 1985, Saiki, R. K. et al. Science, 239: 487-491, 1988). In order to isolate DNA encoding a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5xSSC conditions, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1xSSC. The isolated DNA is at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) at the nucleic acid level or amino acid sequence level. And the like). Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993) by Karlin and Altschul. Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. When analyzing an amino acid sequence by BLASTX, parameters are set to, for example, score = 50 and wordlength = 3. Moreover, when analyzing an amino acid sequence using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known.

The form of the DNA encoding the ds1 protein of the present invention is not particularly limited, and includes cDNA, genomic DNA, and chemically synthesized DNA. Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For genomic DNA, for example, genomic DNA is extracted from sorghum to create a genomic library (as vectors, plasmids, phages, cosmids, BACs, PACs, etc. can be used), expanded, and ds1 gene (for example, By conducting colony hybridization or plaque hybridization using a probe prepared based on the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10 or 11). It is possible to prepare. It is also possible to prepare a primer specific to the ds1 gene and perform PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from sorghum, inserted into a vector such as λZAP to create a cDNA library, developed, and colony hybridization as described above. Alternatively, it can be prepared by performing plaque hybridization or by performing PCR.

<DNA used to suppress expression of sorghum susceptibility ds1 gene>
The present invention also provides DNA used to suppress the expression of the sorghum susceptibility ds1 gene. By introducing these DNAs, purpura resistance can be imparted to sorghum. In this sense, DNA used to suppress the expression of the sorghum susceptibility-type ds1 gene is a drug for conferring purpura resistance to sorghum. Here, “suppression of ds1 gene expression” includes both suppression of gene transcription and suppression of protein translation. Further, “suppression of expression” includes not only complete cessation of expression but also decrease of expression.

One embodiment of the DNA used to suppress the expression of the susceptible ds1 gene of sorghum is a DNA encoding a dsRNA (double-stranded RNA) complementary to the above-described transcript of the susceptible DNA of the present invention. is there. A phenomenon called RNAi (RNA interference), in which the expression of the introduced foreign gene and target endogenous gene is both suppressed by introducing dsRNA having the same or similar sequence to the target gene into the cell. Can cause. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, an RNaseIII-like nuclease called Dicer having a helicase domain is converted to about 21 to 23 base pairs from the 3 ′ end in the presence of ATP. Each one is cut out to generate siRNA (short interference RNA). A specific protein binds to this siRNA to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the transcript (mRNA) of the target gene at the center of the siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

The DNA encoding the dsRNA of the present invention includes an antisense DNA encoding an antisense RNA for any region of a target gene transcription product (mRNA), and a sense DNA encoding a sense RNA for any region of the mRNA And antisense RNA and sense RNA can be expressed from the antisense DNA and the sense DNA, respectively. Moreover, dsRNA can be produced from these antisense RNA and sense RNA.

In the case where the dsRNA expression system of the present invention is held in a vector or the like, there are a case where antisense RNA and sense RNA are expressed from the same vector, and a case where antisense RNA and sense RNA are expressed from different vectors, respectively. is there. The antisense RNA and sense RNA are expressed from the same vector. For example, an antisense RNA expression cassette in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the antisense DNA and the sense DNA. And sense RNA expression cassettes are constructed, and these cassettes are inserted into the vector in the same direction or in the opposite direction.

It is also possible to construct an expression system in which antisense DNA and sense DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA are separated from each strand on both sides. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 'end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Further, in this palindromic style expression system, the two promoter types are preferably different.

In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense RNA expression in which a promoter capable of expressing a short RNA such as polIII is linked upstream of antisense DNA and sense DNA, respectively. A cassette and a sense RNA expression cassette are constructed, and these cassettes are held in different vectors.

The dsRNA used in the present invention is preferably siRNA. “SiRNA” means a double-stranded RNA consisting of short strands in a range that is not toxic in cells. The length of the target ds1 gene is not particularly limited as long as it can suppress the expression of the target ds1 gene and does not exhibit toxicity. The dsRNA chain length is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs.

As the DNA encoding the dsRNA of the present invention, an appropriate sequence (preferably an intron sequence) is inserted between inverted repeats of the target sequence, and a double-stranded RNA having a hairpin structure (self-complementary 'hairpin' RNA (hpRNA )) Construct (Smith, NA, et al. Nature, 407: 319, 2000, Wesley, S. V. et al. Plant J. 27: 581, 2001, Piccin, A. et al. Nucleic Acids Res. 29: E55, 2001) can also be used.

The DNA encoding the dsRNA of the present invention is not necessarily completely identical to the base sequence of the target ds1 gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more). Sequence identity can be determined by the technique described above (BLAST program).

The part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be included in the double-stranded RNA region where RNAs in dsRNA pair with each other.

Another embodiment of the DNA used to suppress the expression of the susceptible ds1 gene of sorghum is as follows: a DNA encoding an antisense RNA complementary to the above-described transcript of the susceptible DNA of the present invention (antisense DNA) It is. Antisense DNA suppresses target gene expression by inhibiting transcription initiation by triplex formation, suppressing transcription by hybridization with a site where an open loop structure is locally created by RNA polymerase, Inhibition of transcription by hybridization with certain RNA, suppression of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, suppression of transition from nucleus to cytoplasm by hybridization with mRNA , Splicing suppression by hybridization with capping site and poly (A) addition site, translation initiation suppression by hybridization with translation initiation factor binding site, translation suppression by hybridization with ribosome binding site near initiation codon, translation of mRNA Area or poly Outgrowth inhibitory peptide chains by the formation of a hybrid with over arm binding sites, and the like gene silencing and the like by hybridization with interaction site between a nucleic acid and protein. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue, "Neurochemistry Experiment Course 2, Nucleic Acid IV Gene Replication and Expression", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin , pp.319-347, 1993). The antisense DNA used in the present invention may suppress the expression of the target ds1 gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the target gene is designed, it will be effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.

Antisense DNA is a phosphorothioate method (Stein, Nucleic Acids Res., 16) based on the sequence information of the disease-susceptible DNA of the present invention (for example, the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 4). : 3209-3221, 1988). The prepared DNA can be introduced into sorghum by a known method described later. The sequence of the antisense DNA is preferably complementary to the endogenous susceptible ds1 gene transcript of sorghum, but may not be completely complementary as long as it can effectively inhibit gene expression. Good. The transcribed RNA preferably has a complementarity of 90% or more (eg, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene. In order to effectively inhibit the expression of the target gene, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

Another embodiment of the DNA used for suppressing the expression of the sorghum susceptibility type ds1 gene is a DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the susceptibility type DNA of the present invention. Some ribozymes have a group I intron type or a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 35: 2191, 1990).

For example, the self-cleaving domain of hammerhead ribozyme cleaves 3 ′ of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, it is shown that it can also be cut by A or U (Koizumi et. Al., FEBS Lett. 228: 225, 1988). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. (Koizumi et.al., FEBS Lett. 239: 285, 1988, Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 35: 2191, 1990, Koizumi et. Al., Nucleic. Acids. Res. 17: 7059, 1989).

Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751, 1992, Hiroshi Kikuchi, Chemistry and Biology 30: 112, 1992). A ribozyme designed to cleave the target is linked to a promoter, such as the cauliflower mosaic virus 35S promoter, and a transcription termination sequence so that it is transcribed in sorghum cells. Such structural units can be arranged in tandem so that multiple sites in the target gene can be cleaved to further increase the effect (Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271, 1992 ). Using such a ribozyme, the transcription product of the target ds1 gene can be specifically cleaved to suppress the expression of the gene.

<Vector, transformed sorghum cell, transformed sorghum plant>
The present invention also introduces a vector containing the DNA of the present invention (susceptible DNA, resistant DNA, DNA for suppressing the expression of the ds1 gene), the DNA of the present invention or a vector containing the same. A sorghum cell, a sorghum plant containing the cell, a sorghum plant that is a descendant or clone of the plant, and a propagation material of the sorghum plant are provided.

As the vector of the present invention, for example, an autonomously replicable vector or a vector capable of homologous recombination in a chromosome can be used. The vector of the present invention usually contains an appropriate expression promoter so that the DNA of the present invention is expressed after introduction into sorghum cells. Examples of promoters used in the present invention include cauliflower mosaic virus-derived 35S promoter and corn-derived ubiquitin promoter. The vector can appropriately include a selection marker, a replication origin, a terminator, a polylinker, an enhancer, a ribosome binding site, and the like. In general, the DNA of the present invention is located downstream of the promoter, and a terminator is located downstream of the DNA. Examples of the terminator include a terminator derived from a cauliflower mosaic virus and a terminator derived from a nopaline synthase gene.

The form of the sorghum cell into which the vector is introduced is not particularly limited, and examples thereof include immature embryos, callus, pollen and the like. As a method for introducing the vector into a sorghum cell and regenerating a sorghum plant, a conventional method in this technical field can be used. As such a method, for example, a method of regenerating a plant body by introducing a gene into an immature embryo or a callus by an Agrobacterium method or a particle gun method, or a method of pollination using pollen that has been gene-transferred by ultrasonic waves is preferable. (J. A. Able et al., In Vitro Cell. Dev. Biol. 37: 341-348, 2001, A. M. Casas et al., Proc. Natl. Acad. Sci. USA 90: 11212 -11216, 1993, V. Girijashankar et al., Plant Cell Rep 24: 513-522, 2005, J. M. JEOUNG et al., Hereditas 137: 20-28, 2002, V Girijashankar et al., Plant Cell Rep 24 (9): 513-522, 2005, Zuo-yu Zhao et al., Plant Molecular Biology 44: 789-798, 2000, S. Guler et al., Plant Cell Rep 28 (3): 429-444, 2009 , ZY Zhao, Methods Mol Biol, 343: 233-244, 2006, AK Shrawat and H Lorz, Plant Biotechnol J, 4 (6): 575-603, 2006, D Syam ala and P Devi Indian J Exp Biol, 41 (12): 1482-1486, 2003, Z Gao et al., Plant Biotechnol J, 3 (6): 591-599, 2005).

The above DNA of the present invention can be introduced into sorghum as exogenous DNA, but can also be introduced into sorghum by crossing with varieties having the DNA of the present invention. “Introduction” in the present invention includes both forms.

Once a sorghum plant having the above-described DNA of the present invention introduced therein is obtained, progeny can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, callus, protoplast, pollen, seed, cut ear, etc.) from the sorghum plant, its progeny or clone, and mass-produce the plant based on them. The present invention includes a plant cell into which the DNA of the present invention is introduced, a plant containing the cell, a progeny and clone of the plant, and a propagation material for the plant, its progeny and clone.

The sorghum plant into which the susceptible DNA of the present invention has been introduced is used for, for example, development (screening) of a drug for imparting purpura resistance to sorghum and for elucidating the mechanism of purpura disease onset. It can be used as a plant. On the other hand, the sorghum plant into which the resistance type DNA of the present invention or the DNA for suppressing the expression of the ds1 gene of the present invention is introduced is compared with a susceptible sorghum plant into which these DNAs are not introduced, The yield can be expected to increase, and it can be used as a more useful crop or biomass.

<Method for producing sorghum with resistance to purpura disease>
The present invention also provides a method for producing a sorghum imparted with resistance to purpura disease, characterized by suppressing the expression or function of the susceptibility type DNA (susceptible type ds1 gene) of the present invention in sorghum. provide. In the present invention, “giving” purpura disease resistance to sorghum is not only to impart purpura resistance to a variety that does not have purpura disease resistance at all, but also to a certain purpura disease. It also includes further increasing the resistance to purpura in varieties having resistance.

One embodiment for suppressing the expression or function of the susceptible DNA of the present invention in sorghum is to introduce the above-described resistant DNA of the present invention into sorghum. Because purpura resistance in sorghum is controlled by a single recessive gene, in order to confer a purpura resistance trait to sorghum, both ds1 alleles in individuals are usually made resistant DNA There is a need. As a result, only resistant DNA is expressed in the individual, and purple spot disease resistance can be imparted to sorghum. Introduction of the resistant DNA of the present invention into the sorghum chromosome can be performed by, for example, mating or homologous recombination. Instead of introducing resistant DNA, a specific DNA sequence may be introduced into the susceptible DNA on the sorghum chromosome to destroy its function.

Another embodiment for suppressing the expression or function of the susceptible DNA of the present invention in sorghum is to introduce DNA for suppressing the expression of the ds1 gene of the present invention into the sorghum. As a result, since a disease-sustainable translation product is not produced from the disease-susceptible DNA in the individual, purple spot disease resistance can be imparted to sorghum.

Other embodiments for suppressing the expression or function of the susceptible DNA of the present invention in sorghum include, for example, binding to a drug that suppresses the expression of the susceptible DNA or a translation product of the susceptible type and The use of a suppressive drug is also considered.

<Method for determining susceptibility or resistance to purpura in sorghum>
The present invention also provides a method of determining susceptibility or resistance to purpura in sorghum. One embodiment of the determination method of the present invention is a method characterized by analyzing the base sequence of the ds1 gene or its expression control region in sorghum and comparing it with the control base sequence. The expression control region of BTx623 is SEQ ID NO: 13, the expression control region of bmr-6 is SEQ ID NO: 14, the expression control region of nasty MS3B is SEQ ID NO: 15, and the expression control region of SIL-05 is SEQ ID NO: : 16 shows the expression control region of Greenleaf in SEQ ID NO: 17.

In analyzing the base sequence of the ds1 gene or its expression control region, an amplification product obtained by amplifying the ds1 gene or its expression control region DNA by PCR can be used. In case of carrying out the PCR, primers used are not limited as long as it can specifically amplify the ds1 gene or its expression control region, ds1 gene or sequence information of the expression control region (e.g., SEQ ID NO: 1,2,4,5,7,8,10,11,13,14,15,16 or 17). Suitable primers include those listed in Tables 3-7. A specific base sequence of the ds1 gene or its expression control region can be amplified by appropriately combining these primers.

The primers described in Table 3 and Table 4 are primers for amplifying the base sequences including the SSR regions described in Table 1 and Table 2. The primers listed in Table 5 and Table 6 are primers for amplifying a base sequence containing insertion / deletion markers (difference in base sequence between varieties due to base insertion / deletion) (# 21 in Table 5). (See FIG. 8 for the nucleotide sequences targeted by the primers shown in Table 6). The primers listed in Table 7 are primers for amplifying the coding region and upstream region of the ds1 gene.

The “control base sequence” to be compared with the base sequence of the ds1 gene in the test sorghum is typically the base sequence of the ds1 gene in the susceptible or resistant variety. The determined base sequence of the ds1 gene and the base sequence in the susceptible strain (eg, SEQ ID NOs: 1, 2, 4, 5) or the base sequence in the resistant strain (eg, SEQ ID NOs: 7, 8, 10, 11) ), It is possible to evaluate whether the ds1 gene in the test sorghum is resistant or susceptible. For example, when there is a large difference in the nucleotide sequence compared to the nucleotide sequence in the susceptible type varieties (for example, SEQ ID NOs: 1, 2, 4, 5) (particularly due to the appearance of a new stop codon or frame shift, When a large change occurs in the molecular weight or amino acid sequence of the encoded protein), the ds1 gene in the test sorghum is determined to have a high probability of being resistant. Moreover, when there is a mutation that suppresses the expression of the ds1 gene in the base sequence of the expression control region of the ds1 gene in the test sorghum (for example, when a part of the expression control region is largely deleted: FIG. 6, FIG. 7), the expression control region of the ds1 gene in the test sorghum is determined to be highly resistant.

Whether the base sequence of the ds1 gene in the test sorghum or its expression control region is different from the base sequence of the control should be analyzed indirectly by various methods other than the determination of the direct base sequence described above. Can do. Examples of such methods include PCR-SSCP (single-strand conformation polymorphism) method, RFLP method using restriction fragment length polymorphism (RFLP), Examples include PCR-RFLP, denaturant gradient gel electrophoresis (DGGE), allele specific oligonucleotide (ASO) hybridization, and ribonuclease A mismatch cleavage.

In the determination method of the present invention, DNA can be prepared from the test sorghum using a conventional method, for example, CTAB method. As sorghum for preparing DNA, not only a plant body in which sorghum has grown, but also sorghum seeds and seedlings can be used.

The base sequence can be determined by a conventional method such as the dideoxy method or the Maxam-Gilbert method. In determining the base sequence, a commercially available sequence kit and sequencer can be used.

Whether a mutation in the base sequence of the expression control region affects the expression of the ds1 gene is determined by constructing a vector in which a reporter gene is linked downstream of the expression control region having the mutation so that the vector can be expressed. It can be determined by introducing into sorghum cells and detecting the reporter activity.

Another embodiment of the determination method of the present invention is a method characterized by detecting the expression of the ds1 gene in sorghum or the molecular weight of the expression product. Here, “detection of gene expression” includes both detection at the transcription level and detection at the translation level. In addition, “detection of expression” is intended to include not only detection of the presence or absence of expression but also detection of the degree of expression.

Detection at the transcription level can be carried out by a conventional method such as RT-PCR (Reverse transcribed-Polymerase chain reaction) or Northern blotting. Primers used in the case of carrying out the PCR is not limited as long as it can specifically amplify the ds1 gene, ds1 gene sequence information (e.g., SEQ ID NO: 1,2,4,5,7,8 , 10 or 11). A suitable example of the primer is a combination of # 21F and # 21R described in Table 7 above.

On the other hand, detection at the translation level can be performed by a conventional method, for example, Western blotting. The antibody used for Western blotting may be a polyclonal antibody or a monoclonal antibody, and methods for preparing these antibodies are well known to those skilled in the art.

Result of the detection of gene expression in a subject, susceptible variety expression level of ds1 gene (e.g., BTx623, bmr-6) if significantly lower than the expression level of, also, morbidity is the molecular weight of the expression products of ds1 gene If the molecular weight is significantly different from that in a sex variety (for example, BTx623, bmr-6), it is determined that the test sorghum has a high probability of being purpura-resistant. In fact, compared to the susceptible variety (BTx623, bmr-6), the expression level of ds1 gene in resistant varieties SIL-05 and Greenleaf significantly lower, also the ds1 proteins in resistant varieties那系MS3B and Greenleaf The molecular weight is significantly small.

Another embodiment of the determination method of the present invention is a method characterized by analyzing the base sequence of a molecular marker linked to the ds1 gene in a test sorghum and comparing it with a base sequence of a control. Here, the “molecular marker” refers to a DNA region that is genetically linked to the ds1 gene and is distinguishable from other DNA regions. The closer the molecular marker is located to the ds1 gene, the easier it is to be inherited simultaneously with the ds1 gene. Therefore , the molecular marker is highly useful in the determination method of the present invention. Molecular markers of the present invention having a high utility is usually one that is present within 50kbp from both end bases of the coding region of ds1 gene, more preferably within 20 kbp, more preferably those present within 10 kbp.

Preferred embodiments of the molecular marker of the present invention are insertion / deletion markers, SSR (simple repetitive sequence) markers, and SNP (single nucleotide polymorphism) markers. An insertion / deletion marker is a DNA polymorphism caused by insertion and / or deletion of a base. The SSR marker is a unit of 2 or 3 bases (for example, “CA”, “CG”, “TA”, “TC”, “AGG”, “CTT”, “CGC”, “GAG”, etc.) several times It is a repeating sequence that repeats several hundred times. Since the number of repetitions varies depending on the individual or strain, this difference in the number of repetitions can be used as a DNA polymorphism. The SNP marker is a DNA polymorphism caused by substitution of one base in the DNA base sequence. A person skilled in the art can appropriately extract a base sequence serving as a molecular marker based on, for example, comparison of base sequences of ds1 gene between varieties (FIGS. 7 and 8).

In addition to the method of directly determining the nucleotide sequence, the nucleotide sequence of the insertion / deletion marker is subjected to PCR using a primer containing the nucleotide sequence containing the marker region, and the obtained amplification product is electrophoresed. The method can be carried out by detecting the difference in the position of the DNA band on the gel. In addition, the analysis of the base sequence in the SSR marker is not limited to the method of directly determining the base sequence and detecting it as a difference in the repeat sequence, but also using PCR using a primer capable of amplifying the base sequence containing the repeat portion. The amplification product thus obtained can be electrophoresed and detected as a difference in the position of the DNA band on the gel. The analysis of the nucleotide sequence in the SNP marker is performed, for example, by performing PCR using a primer that can amplify the nucleotide sequence including the single nucleotide polymorphism part, and the nucleotide sequence incorporated into the single nucleotide polymorphism part in the obtained amplification product. The method can be carried out by specifying the type with a polarization fluorescence analyzer.

As the “control base sequence”, a base sequence of a known molecular marker linked to the ds1 gene can be used. For example, when a base sequence containing the insertion / deletion marker described in Tables 5 and 6 is amplified with the primers described in Tables 5 and 6, the base sequence containing the insertion / deletion marker described in Tables 5 and 6 is susceptible to the disease depending on the sequence or chain length of the amplified DNA fragment Or resistance type (see FIG. 8). Moreover, the primer set of FIG. 9A can be used suitably for such discrimination (Tables 8 and 9).

SSR markers listed in Tables 1 and 2 ds1 gene linkage such susceptible type BTx623. When the nucleotide sequence containing the marker is amplified with the primers described in Tables 3 and 4, it is possible to determine whether it is a susceptible type or a resistant type based on the sequence or chain length of the amplified DNA fragment. it can. As described above, the comparison between the base sequence of the molecular marker in the test sorghum and the base sequence of the control is not limited to the direct base sequence comparison, but other indicators that can evaluate the difference in the base sequence (for example, the PCR described above) By comparison of the molecular weight etc. of the amplification products by

As a result of comparison, if the nucleotide sequence of the molecular marker in the test sorghum is the same type as the susceptibility type molecular marker, it is determined that the test sorghum has a high probability of susceptibility and the same type as the resistance type molecular marker. In some cases, the test sorghum is determined to have a high probability of resistance.

<Method of breeding purple spot disease resistant sorghum>
The present invention also provides a method for breeding purpura-resistant sorghum. The breeding method of the present invention comprises (a) a step of crossing a purple spot disease resistant sorghum variety with any sorghum variety, (b) a susceptibility or resistance to purpura disease in an individual obtained by the crossing, A step of determining by the determination method of the present invention, and (c) a step of selecting a variety which has been determined to be resistant to purpura.

Examples of “arbitrary sorghum varieties” to be bred with purple spot resistant sorghum varieties include, but are not limited to, susceptible varieties and individuals obtained by crossing susceptible varieties and resistant varieties. . By utilizing the breeding method of the present invention, it becomes possible to select purple spot disease-resistant sorghum at the seed or seedling stage, and it is possible to grow varieties having purpura-resistant traits. This can be done in a short period of time.

Hereinafter, the present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples.

Against Example 1] ds1 gene F3 from F5 population obtained by crossing a narrowing resistant variety SLIL-05 in existing areas and susceptible product species bmr-6 of, utilizing a polymorphic marker information Sorghum The purpura disease susceptibility gene ( ds1 ) was mapped (FIGS. 1 and 2). See Tables 1 to 6 for information on polymorphic markers and primers used for mapping. Some of the SSR markers are described in the literature (Yonemaru J, et al., DNA Res 16: 187-193, 2009).

For (1) F3 population (175 individuals) performs mapping using SSR markers (SB3067, SB3146), purpura spot disease susceptibility gene ds1 was found to be in Zajo to chromosome 5.

(2) After screening the F4 population (640 individuals) between SSR markers SB3056 to SB3178, 229 individuals with recombination between these markers were selected, and these individuals were purpura in the field. A test for point disease was performed. As a result, the existence region of ds1 gene was narrowed to about 246Kbp. Insert found based on the nucleotide sequence information of the susceptible variety BTx623 of sorghum / deletion markers (# 12, # 20, #, etc. 17) was used to perform mapping, narrowed the existing area of ds1 gene up to about 96kbp .

(3) For F5 population (4235 individuals), individuals with recombination between these markers were screened using insertion / deletion markers and SSR markers (# 12, SB22274). Further, newly found insertion / deletion markers (# 20, # 21, # 20kara series, and # 12Kara series) using, it narrowed the existing area of ds1 gene. As a result, finally, the presence region of ds1 gene was narrowed to about 26Kbp.

In the susceptible varieties BTx623, two candidate gene sequences (receptor kinase and salt-inducible protein) were found in the narrowed region. Since many of the known plant disease resistance genes are receptors, it was speculated that this receptor kinase gene was a purpura disease resistance gene.

In PCR for amplifying a base sequence containing a polymorphic marker, 5 μl of PCR reagent (GoTaq ^(R) Green Master Mix), 4.4 μl of distilled water, 0.1 μl of DNA (20 ng / μl), primers ( A total of 10 μl of reaction solution containing 0.5 μl of 10p) was prepared. Use 9.5 μl of each reaction, and react at 94 ° C for 2 minutes, 94 ° C for 1 minute → 55 ° C for 1 minute → 72 ° C for 2 minutes, 35 cycles, 72 ° C for 10 minutes. went.

The purpura disease test was carried out by spraying pathogenic bacteria (Bipolaris sorghicola (Lefebvre & Sherwin) Alcorn) on sorghum or by inoculating them by culturing in barley medium. In the method of spraying pathogenic bacteria on sorghum, first, distilled water (0.01% tween20) containing purpura disease bacteria at a concentration of 10 ⁴ to 10 ⁵ cells / ml is sprayed on the leaves of sorghum to such an extent that it gets wet. For 16 hours in a plastic bag. Subsequently, the cells were transferred to a greenhouse at 25 ° C. under bright conditions, and after 7 to 10 days, the disease severity (sickness area / total area) was tested. In the method of inoculating by culturing in barley medium, first, barley with skin was autoclaved for 15 minutes in distilled water, cooled, and then inoculated with purpura. Subsequently, the flask was cultured for about 2 weeks with occasional agitation until the mycelium spread in the barley. Three grains of barley grown with pathogenic bacteria were put into undeveloped leaves of sorghum grown to a total length of about 50 cm, and after 4 to 5 days, the lesions for purpura were examined.

[Example 2] Relationship between expression of ds1 candidate gene and susceptibility / resistance of purpura disease Expression of a gene present in the candidate region was investigated by RT-PCR. As primers, # 21F and # 21R described in Table 7 were used. As a result, only the mRNA expression pattern of the receptor kinase gene was consistent with the distinction between disease susceptibility and resistance (FIG. 3). In leaf RT-PCR using the cross population, heterozygous individuals and bmr-6 type individuals have a large amount of mRNA expression, but SIL-05 type has a very small amount (or none). This is consistent with the report of non-patent homology and resistance (3).

In addition, about the technique of RT-PCR, literature (Kikuchi R. et al, Plant Physiol 149: 1341-1353, 2009, Shimada S. et al, Plant J. 58: 668-681, 2009) was referred.

. [Example 3] creates a Bac library of tissue specificity and the varietal differences resistant varieties SLIL-05 morbidity product species bmr-6 of ds1 gene expression (Ashikawa I. et al, Genetics 180 : 2267- 2276, 2008), Bac clones containing candidate gene regions were screened and their nucleotide sequences were determined. As a result, it was found that bmr-6 has no salt-inducible protein gene. As a result, the salt-inducible protein gene was excluded from the candidate gene candidates, and the receptor kinase gene was designated as a purpura disease susceptibility gene.

Furthermore, when the tissue-specific gene expression in the resistant cultivar SIL-05 and the diseased product species bmr-6 was investigated, the receptor kinase gene was highly expressed in the foliage in the diseased bmr-6. In SIL-05, almost no expression was observed in the foliage (upper figure 4). In addition, expression was observed in susceptible varieties, but the expression level was low in resistant varieties (bottom of FIG. 4). From this, it is considered that sorghum exhibits purpura resistance if there is no gene expression of the receptor kinase gene.

[Example 4] to determine the comparative receptor kinase gene near nucleotide sequence of the sequence of ds1 genes between varieties, BTx623 (susceptible), SIL-05 (resistant), and bmr-6 between (susceptible) Compared. As a result, the amino acid sequence of the coding region of this gene is completely identical between SIL-05 and BTx623, and bmr-6 has three base substitutions compared to BTx623, and one of these mutations has an amino acid sequence. It was accompanied by mutation (851H → Q) (FIG. 5).

In SIL-05, a part of the upstream promoter region of the gene was deleted, and it was speculated that the expression level of mRNA was small (FIGS. 6 and 7). On the other hand, the sorghum cultivar, Nasatsu MS3B, is also resistant to purpura, but when the nucleotide sequence of the receptor kinase gene of this cultivar was analyzed, a stop codon was present in the gene coding region (FIGS. 6 and 7). As a result, it was thought that normal proteins were not synthesized.

For comparison of base sequences between varieties, CrastalW (http://clustalw.ddbj.nig.ac.jp/top-j.html) was used.

[Example 5] Relationship between polymorphisms in ds1 candidate genes of sorghum varieties from various regions of the world and susceptibility and resistance to purpura disease DNA extraction from sorghum plant leaves used the C-TAB method ( MURRAY, MG, and WF THOMPSON, Nucleic Acid Reserach 8: 4321-4325, 1980). PCR was performed using the extracted DNA. The PCR conditions were the same as in Example 1. The primer sets listed in Table 7 were 520F and 141R (primer set # 1), 1141F and 1823R (primer set # 2), # 21GLF and # 21GLR (primer). Set # 3), # 21F and # 21R (primer set # 4) were used (FIG. 9A). When primer set # 4 was used, restriction enzyme treatment was performed after PCR and before electrophoresis. Specifically, after PCR, 5 ul of the PCR sample was separated, and the restriction enzyme MlyI was added to make a total amount of 20 ul. A sample treated with this reaction system at 37 ° C. for 1 hour was electrophoresed.

As a result of testing the genotype of the purple spot disease resistance gene ds1 of sorghum varieties from various regions of the world, amplification products of different sizes were detected by PCR using each primer set. Amplification products were classified according to their size (FIG. 9B).

Tests for purpura of each variety listed in Table 8 and Table 9 were conducted in the same manner as in Example 1. The result (representative example) is shown in FIG. As a result of analyzing the relationship between the size of the amplification product obtained by PCR using each primer set and the susceptibility and resistance to purpura disease, the resistance type for purpura disease susceptibility Could be classified into R1 to R4 (Table 8, Table 9). The classification by genotype determined using DNA was consistent with the results of the actual inoculation test, and it was found that the susceptibility and resistance of purpura disease can be tested by PCR test.

According to the present invention, the susceptibility gene ds1 of sorghum purpura has been identified, and the mechanism of onset of sorghum purpura has been clarified. To determine the susceptibility and resistance of purpura, it has been necessary to conduct infection tests with pathogenic bacteria.However, if the ds1 gene is used as a marker, sorghum is not exposed to the pathogenic bacteria, and seed or Even at the plant stage, it is possible to make a simple determination, and it is possible to efficiently breed purple spot disease resistant varieties. Breeding purpura-resistant sorghum contributes to the reduction of purpura disease damage, and is expected to improve biomass production and produce high-quality feed.

Claims (15)

1. The DNA according to any one of the following (a) to (d), which encodes a protein having an activity of imparting purpura disease susceptibility to sorghum.
   (A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 6
   (B) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1, 2, 4 or 5
   (C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 6
   (D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4 or 5
2. The DNA according to any one of the following (a) to (d), which encodes a protein having an activity of imparting purpura resistance to sorghum.
   (A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 9 or 12
   (B) DNA containing the coding region of the base sequence described in SEQ ID NO: 7, 8, 10 or 11
   (C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12
   (D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, or 11
3. The DNA according to any one of the following (a) to (c), which encodes a protein having an activity of conferring purpura resistance to sorghum.
   (A) DNA encoding a double-stranded RNA complementary to the transcription product of the DNA of claim 1
   (B) DNA encoding an antisense RNA complementary to the transcription product of the DNA of claim 1
   (C) DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the DNA of claim 1
4. A vector comprising the DNA according to any one of claims 1 to 3.
5. A sorghum cell into which the DNA according to any one of claims 1 to 3 has been introduced.
6. A sorghum plant comprising the cell according to claim 5.
7. A sorghum plant which is a descendant or clone of the plant according to claim 6.
8. The propagation material of the sorghum plant according to claim 6 or 7.
9. A method for producing sorghum imparted with resistance to purpura disease, which suppresses the expression or function of the DNA according to claim 1 in sorghum.
10. A method for producing sorghum in which purpura disease resistance is imparted to sorghum, comprising the step of introducing the DNA of claim 2 or 3 into sorghum.
11. A drug for conferring purpura resistance to sorghum comprising the DNA according to claim 2 or 3 or a vector into which the DNA is inserted.
12. A method for determining the susceptibility or resistance of purpura in sorghum, comprising analyzing the base sequence of the DNA or expression control region thereof according to claim 1 or 2 in a test sorghum and comparing it with a control base sequence A method characterized by:
13. A method for determining the susceptibility or resistance of purpura in sorghum, the method comprising detecting the expression of DNA or the molecular weight of the expression product according to claim 1 or 2 in sorghum.
14. A method for determining the susceptibility or resistance of purpura in sorghum, comprising analyzing a base sequence of a molecular marker linked to DNA according to claim 1 or 2 in a test sorghum, and a control base sequence A method characterized by comparing.
15. A method of breeding purple spot disease resistant sorghum,
    (A) mating a purple scab-resistant sorghum variety with any sorghum variety,
    (B) determining the susceptibility or resistance of purpura to the individual obtained by the mating in step (a) by the method according to any one of claims 12 to 14, and (c) purpura Selecting a variety that has been determined to have the resistance.

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