JP2002262877A - New isoamylase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply cloning a gene encoding an isoamylase in an organism, a new isoamylase derived from a blue-green algae belonging to the kingdom Prokaryote, a plant into which a heterogeneous isoamylase is transduced, and a new method for producing a starch produced by the plant. SOLUTION: A method for cloning a gene encoding an isoamylase derived from Synechococcus sp. based on a DNA amplified by PCR from a DNA in a genome or library thereof as primers consisting of specific base sequences, an isoamylase having a specific amino acid sequence derived from Synechococcus sp., and a gene encoding the isoamylase, a higher plant obtained by transducing a gene encoding a heterogeneous isoamylase into an isoamylase- deficient higher plant, and a method for producing a starch by the transgenic plant, are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention provides a novel primer for cloning a gene for isoamylase which plays an important role in the biosynthesis of starch amylopectin. In addition, the present invention relates to Synechococcus PCC7942, a kind of cyanobacteria.
The present invention relates to a novel isoamylase derived therefrom, and a gene encoding the same. Furthermore, the present invention relates to a higher plant in which a gene encoding an isoamylase derived from another organism has been introduced into a higher plant deficient in isoamylase, and a method for producing starch using the transgenic plant.

[0002]

2. Description of the Related Art Starch is a plant energy storage substance and is a kind of polysaccharide composed of α-glucose, which is composed of amylose and aminopectin. Glycogen, like starch, is a polysaccharide composed of α-glucose, which is mainly used as an energy storage substance in animals. Starch is not only used as food and feed as a main component of grains, but also processed into dextrin, oligosaccharides, isomerized sugars, etc., and used in processed foods. It is also used as a raw material. Even if you just say starch,
Rice starch, potato starch, wheat starch, corn starch, etc., have slightly different starch shapes, tastes, and physical properties when gelatinized, depending on their origin. They use starch differently. It has been explained that such differences in starch properties stem from differences in the chemical structure of starch.However, starch is mainly composed of amylose and aminopectin, and the difference in the chemical structure is mainly branched. It is said to be derived from amylopectin having a structure. Starch (Starch)
Is an α-polyglucan that accumulates in storage organs of higher plants. Starch granules are "amyloplast" in the storage tissue
Contained in plastids called. Starch is a mixture of two glucose homopolymers, Amylopectin and Amylose. Amylose makes up 20-30% of the stored starch, but is not essential for the formation of starch granules. Glucose units are linked by α-1,4 glucosidic bonds, and are linear helical molecules containing a small amount of α-1,6 glucosidic bond branches. Amylopectin, on the other hand, accounts for 70-80% of the starch granules, with glucose units extending at α-1,4 glucosidic linkages and α parallel to the main chain.
It has a structure in which branches are connected by -1,6 glucosidic bonds. This characteristic structure is called "cluster" structure. Animal and bacterial storage carbon, glycogen (G
Lycogen), like amylopectin, is composed of glucose homopolymer, but has no cluster structure. Glycogen has been reported to have a "tree like" or "bush like" structure.

FIG. 1 shows the structures of amylose, aminopectin and glycogen. The line shown in FIG. 1 is a chain of α-glucose, and amylose ((C) in FIG. 1) has almost no branching and has a substantially single-chain structure of α-1,4-glucose. Aminopectin ((B) in FIG. 1) has a regular branched structure, and α-1,4
-It has a chain of glucose and a branched structure (cluster) of α-1,6-glucose regularly at regular intervals. Glycogen (FIG. 1A), which is an energy storage substance for animals and the like, has a completely irregular branched structure. Glycogen has smaller molecules and shorter branches than amylopectin, and many of them are water-soluble substances. Amylopectin, on the other hand, has long branches and is filled with glucose at a high density, and is generally a water-insoluble substance.

[0004] Such a cluster structure of amylopectin is advantageous in forming a crystal structure, and starch grains are formed by the crystal structure. The cluster structure of amylopectin is a regular repeating structure of about 9 nm,
This 9 nm size does not show much variation even if the structure or species is different. Looking at the structure of amylopectin in more detail, it has three types of α-1,4-glucoside chains (see FIG. 2). The A chain is the outermost chain and has no branch bond in the chain. B chain is 1 per chain
One or more chains are branched chains, and the B chain is further a B1 chain remaining in one cluster, a B2 chain extending in two clusters, a B3 chain extending in three clusters.
There are chains. The C chain is a chain having a reducing end,
It has one C-chain per amylopectin molecule.

As described above, although the structure of amylopectin is almost constant, the structure of amylopectin is slightly different depending on the type of plant. Recent studies have reported a structural difference between amylopectin in japonica rice with sticky starch and indica rice with fluffy starch. The upper part of FIG. 3 (FIG. 3 (a)) schematically shows the structure of amylopectin of Japonica rice, and the lower part of FIG. 3 ((b) of FIG. 3) schematically shows the structure of amylopectin of Indica rice. Compared to the length of the branches of the clusters, Indica rice is relatively long and its density is relatively dense. It is believed that this makes starch from Indica rice more difficult to gelatinize. It is considered that such a fine structural difference of amylopectin is caused by a difference in a synthesis method when synthesizing amylopectin.

It is believed that amylopectin is synthesized by a continuous reaction of the following four enzymes. (1) ADP glucose pyrophosphorylase (ADPgluco
se pyrophosphorylase (AGPase)), (2) Starch synthase (SS), (3) Starch branching enzyme (SB)
E)) and (4) Starch degrading enzymes (Starch deb
ranching enzyme (DBE)).

[0007] AGPase is an enzyme that synthesizes ADP glucose, which is a raw material of starch polymer. S
S plays a role of connecting ADP glucose to the non-reducing end of amylopectin by an α-1,4 glucoside bond to extend the chain. While SS extends the amylopectin chain, S
BE is an enzyme that forms an α-1,6 glucoside bond of amylopectin, and is an enzyme that forms a branched portion of a branched structure. Conventionally, it has been thought that SS and BE determine the frequency of amylopectin branches and play an important role in forming a cluster structure. That is, the starch-branching degrading enzyme (D)
BE) was thought to be an enzyme not required for cluster formation. However, it has been shown that amylopectin clusters cannot be formed in plants deficient in this degrading enzyme, and it has been reported that DBE is essential for the formation of a cluster structure (MGJa
mes, et al., Plant Physiol., (1995) 7, 417-429; Y.
Nakamura, et al., Plant J., (1997) 12 (1), 143-153;
A. Kubo, et al., Plant Phys., (1999) 121, 399-40
9).

[0008] From these reports on DBE, the formation of a cluster structure in amylopectin indicates that SS and B
It has been found that not only the formation of new bonds by E but also the extra branches formed by BE are decomposed by DBE to maintain a regular cluster structure. α
Two types of DBE which degrade -1,6 glucoside bond branches are known due to differences in substrates. One is isoamylase and the other is pullulanase (also known as R-enzyme or limit dext).
rinase))). Of these two types of DBE, isoamylase can degrade glycogen and the α-1,6 glucosidic bond branch of phytoglycogen, which has a slightly more regularity than glycogen. Does not work. On the other hand, pullulanase acts on pullulan but does not act on glycogen or phytoglycogen.

FIG. 4 summarizes the process of synthesizing amylose, aminopectin and glycogen. The synthesis of glycogen on the left side of FIG. 4 is mainly for animals and bacteria,
UGPase is a synthase of phosphorylated glucose which is a glycogen material, GS is a glycogen synthase, and GBE is a glycogen branching enzyme. The middle part of FIG. 4 shows the synthesis process of amylopectin in the case of a higher plant, and SSS in the figure is water-soluble SS. The right side of FIG. 4 shows the process of synthesizing amylose in a higher plant. GBSS is a granule-bond starch synthase (GBS).
S)). Thus, in higher plants, amylopectin corresponding to the type of plant is produced by the above-mentioned four types of enzymes. The difference in the structure of amylopectin depending on the type of plant is considered to be largely due to the difference in type of these enzymes. For example,
It is presumed that isoamylase, which is important for forming a cluster structure, is slightly different depending on the type of plant.

[0010] By the way, as a method of classifying living things, there are two kingdoms of the "animal kingdom" and "plant kingdom", and a three kingdom theory that includes "protozoan kingdom (Protista kingdom)" such as euglena and diatoms. At present, the five kingdoms have become the mainstream, with the addition of the "fungi kingdom" such as mushrooms and the "prokaryotic kingdom" such as cyanobacteria and bacteria. Among the five classes of living organisms, starch is produced by higher plants in the plant kingdom. As described above, the presence of isoamylase is essential for the biosynthesis of amylopectin, a component of starch. Higher plants have an isoamylase corresponding to each plant. However, organisms that do not produce starch, such as animals,
Does not have isoamylase. Although many bacteria do not have isoamylase, some bacteria do not produce starch, but do so. The reason for the existence of isoamylase in such organisms is not clear at present, and further research is expected.

[0011]

SUMMARY OF THE INVENTION The present invention provides a method for easily cloning a gene encoding isoamylase in an organism. The present invention also provides a novel isoamylase from one of the cyanobacteria, which is an organism belonging to the "Prokaryotes". Further, the present invention provides a plant into which an isoamylase derived from a different species has been introduced, and a method for producing a novel starch produced by the plant.

[0012]

Means for Solving the Problems The present inventors have created a novel primer for cloning a gene encoding isoamylase, and used this primer to synthesize a novel isoamylase from one of cyanobacteria. I found it.

[0013] The present invention relates to a primer comprising an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NOS: 3 and 4 in the sequence listing, ie, 5'-gaaatgcatgtggggggctttac-3 '5'-gccagggtaaagccgtcgtg-3'. From the DNA in the genome or library
The present invention relates to a method for cloning a gene encoding isoamylase based on DNA amplified by CR.
The present invention also relates to an isoamylase having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing, and a gene encoding the same. Furthermore, the present invention relates to a higher plant in which a gene encoding an isoamylase derived from another organism has been introduced into a higher plant deficient in isoamylase, and a method for producing starch using the transgenic plant.

The present inventors first designed primers from sequences (conserved regions) commonly found in isoamylase (see FIG. 5). As shown in FIG. 5, four conserved regions of isoamylase, I, II, III, and IV, are known. The amino acid sequences in these conserved regions are shown in FIG. 5 as maize endosperm,
It is almost common in rice endosperm (rice endosperm) and synechocystis (a kind of cyanobacteria). The present inventors examined the nucleotide sequences of the primers at the positions of the primers 5, 6, and 8 shown in FIG. 5 based on these conserved regions and the nucleotide sequences before and after the conserved regions. An example of the study will be described for the primer 5 and the primer 8.

First, the nucleotide sequence of each organism was compared with respect to the position of the primer 5. Corn (766) cagaaggacc ttgtcatata tgaaatg
cat ttgcgtggat tcaca rice (498) cagaaggatc ttgtaatcta tgagatg
cat ttacgtgggt ttaca synechocystis (469) ctcgaagaca tggtcattta tgaaatg
cat gtgcggggtt tcacc synechocystis (478) tacgccgcca gcattatcta cgaactc
cac gtcgggggct ttacc Pseudomonas (1055) cagaaggatg atgtgatcta cgaggtg
cat gtgcgcggct tcac As a result, the following three types of primers were designed. Primer Ume5-E 5'-ta tgaaatgcat gtgcgggg-3 'Primer Ume5-F 5'-ta cgaactccac gtcggggg-3' Primer Ume5-G 5'-gaaatgcat gtggggggct ttac-3 ' It is provided to match the notation of the sequence.

Similarly, the nucleotide sequences of the primers 6 and 8 were designed as follows. Primer Ume6-B 5'-gcatcccaggcttcagcaat-3 'Primer Ume8-E 5'-gccagggtaaagccgtcgtg-3' Primer Ume8-F 5'-accagggtaaaaccatcatg-3 'These primers 6 and 8 were used as reverse primers. Using these primers, Synechococcus, a kind of cyanobacteria
When PCR was performed using the genomic DNA as a template, the combination of the primers Ume5-G and Ume8-E gave the best results. The combination of the primers substantially matched the length with the rice isoamylase cDNA, and the most clearly amplified band was sequenced by the dye termination (Dye Terminator) method to obtain a DNA probe. Thereby, about 760 obtained from the genomic DNA of Synechococcus
A bp DNA probe could be prepared. In addition,
The nucleotide sequence of the primer Ume5-G was compared to SEQ ID NO: 3 in the sequence listing.
The base sequence of the primer Ume8-E is shown in SEQ ID NO: 4 of the sequence listing, respectively.

Using the obtained DNA probe, ECL
By performing plaque hybridization according to the instructions of the kit (Amersham), Synechococcus (Sy
nechococcus) was obtained. The nucleotide sequence of the isoamylase gene of Synechococcus PCC7942 thus obtained is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 2 in the sequence listing.
Are shown below. The result was Synechococcus
occus PCC7942) has an isoamylase gene of 4745.
bp from 1647 to 3731 were open reading frames. The portion having a homologous sequence with the primer Ume5-G used for the first PCR was 24
It is the sequence of positions 45 to 2467, and the primer Ume8-E
The portion having a homologous sequence to was found to be the sequence of positions 3009 to 3028. Primer Ume5-G 5'-gaaatgcatgtggggggctttac-3 'Genomic DNA 5'-gagctgcatgttggcggcttcac-3' Matched on 18 bases out of 23 bases. As for the primer 8, the primer Ume8-E 5'-gccagggtaaagccgtcgtg-3 'The reverse complementary strand of the primer Ume8-E 5'-cacgacggctttaccctggc-3' Genomic DNA 5'-catgacggctttacgctgcg-3 '16 bases out of 20 bases Was matched.

Using the above DNA fragment of about 760 bp of Synechococcus PCC7942 as a probe, the genome of Synechococcus PCC7942 was subjected to Southern hybridization. The result is shown in FIG. 6 by a photograph replacing a drawing. As a result, the isoamylase gene was considered to be one copy on the genome of Synechococcus PCC7942. Using the obtained DNA fragment as a probe, Synechococcus (Synecoccus)
The isoamylase gene of chococcus PCC7942) was cloned. Synechococcus PCC794
The nucleotide sequence of the isoamylase gene of 2) is shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence according to the one-letter amino acid notation is shown below.

[0019] MTVSSRRPESTVAVDPGQSYPLGATVYPTG 30 VNFSLYTKYATGVELLLFDDPEGAQPQRTV 60 RLDPHLNRTSFYWHVFIPGIRSGQVYAYRV 90 FGPYAPDRGLCFNPNKVLLDPYARGVVGWQ 120 HYSREAAIKPSNNCVQALRSVVVDPSDYDW 150 EGDRHPRTPYARTVIYELHVGGFTKHPNSG 180 VAPEKRGTYAGLIEKIPYLQSLGVTAVELL 210 PVHQFDRQDAPLGRENYWGYSTMAFFAPHA 240 AYSSRHDPLGPVDEFRDLVKALHQAGIEVI 270 LDVVFNHTAEGNEDGPTLSFKGLANSTYYL 300 LDEQAGYRNYT CGNTVKANNSIVRSLILD 330 CLRYWVSEMHVDGFRFDLASVLSRDANGNP 360 LSDPPLLWAIDSDPVLAGTKLIAEAWDAAG 390 LYQVGTFIGDRFGTWNGPFRDDIRRFWRGD 420 QGCTYALSQRLLGSPDVYSTDQWYAGRTIN 450 FITCHDGFTLRDLVSYSQKHNFANGENNRD 480 GTNDNYSWNYGIEGETDDPTILSLRERQQR 510 NLLATLFLAQGTPMLTMGDEVKRSQQGNNN 540 AYCQDNEISWFDWSLCDRHADFLVFSRRLI 570 ELSQSLVMFQQNELLQNEPHPRRPYAIWHG 600 VKLKQPDWALWSHSLAVSLWHPRQQEWL YL 630 AFNAYWEDLRFQLPRPPRGRVWYRLLDTSL 660 PNLEACHLPDEAKPCLRRRDYIVPARSLLLL 690 MARA * 694

Next, from the obtained amino acid sequence, the homology of the amino acid sequence with isoamylase of another plant was examined. The results are shown in Table 1 below.

Table 1 Amino acid sequence of synamococcus (Synechococcus PCC7942) isoamylase (ISA) and homology (%) with various plants and bacteria Plant species Amino acid sequence homology (%) Rice ISA 42.7% Maize ISA 40.9% Cyanobacterium (Synechocystis sp. PCC6803) glgXa 44.1% Cyanobacterium (Synechocystis sp. PCC6803) glgXb 65.2% E. coli glgX 44.5% H. Influenza glgX 40.1% Mycobacterium tuberculosis glgX 45. I% Sulfolobus solfataricus glgX 44.8% Pseudomonas amyloderamosa ISA 30.3% Flavobacterium sp. ISA 33.3%

The Synechococcus of the present invention
The amino acid sequence of the isoamylase of PCC7942 is the same as that of the cyanobacterium Synechocystis sp.
Among the two types of isoamylases in 3), the homology with one of the isoamylases is the highest, at 65.2%, and is the same at 44.1%. The homology with other species was 50% or less in each case, indicating that the isoamylase of the present invention had a unique amino acid sequence different from that of other species. Synechococcus of the present invention
s PCC7942) is shown in FIG. 7.

Next, Synechococcus of the present invention (Synechoccus)
occus PCC7942) was examined for its expression in Escherichia coli. Synechococcus PCC7
Using the genomic DNA of 942) as a template, PCR was carried out for the isoamylase gene to amplify. The resulting PCR
TA cloning of the product (PGEM-TEasy vector: Prolme
ga), and the resulting mixture was transformed with a JM1O9 competent cell. The resulting plate was divided into plates 1 and 2 and blue white selection was performed. The results are shown in Table 2 below.

Table 2 Results of Blue White Selection (Number of Colonies) White Blue White-Blue Total Plate 1 347 193 23 563 Plate 2 442 199 12 453

14 colonies were arbitrarily taken from the white sides of the plates 1 and 2, and the insert was confirmed by colony PCR. The result is shown in FIG. 8 by a photograph replacing a drawing.
Among them, # 2, # 10, # 11, # 20, and # 24 were selected, and these were mass-cultured to extract plasmids. The electrophoretogram after hydrolyzing these with BamHI is shown in FIG. As a result, it was possible to confirm that each plasmid had a 760 bp fragment, and the sequence was performed. From the results of this sequence, all five clones contained Synechococcus.
cus PCC7942). # 10 confirmed in the sequence was subcloned into an E. coli expression vector (PET-15b vector). Transformation was performed and the target clone was selected by colony PCR.
The result of the confirmation is shown in FIG. 10 by a photograph replacing the drawing. Furthermore, the orientation of the insert was confirmed by treatment with the restriction enzyme HindIII. The result is shown in FIG. From these results, it was confirmed that inserts # 2 and # 16 were inserted in the correct direction.

The obtained # 2 and # 16 were used in E. coli (B
L21), and an expression experiment of isoamylase of Synechococcus PCC7942 was performed. However, adding IPTG
Various conditions such as the concentration of (1M isopropyl-β-D-thio-galactopyrenoside), the temperature of E. coli culture, and the treatment before SDS-PAGE were examined. Amylase expression could not be confirmed. Although it was confirmed that the gene was introduced into the expression vector in the correct direction, the reason why the expression could not be confirmed is currently unknown, but generally, isoamylase is a source of α-1, It is an enzyme that cleaves the 6-glucose bond, and the expression of isoamylase unnecessarily in the living body is considered to impair the storage of energy in the organism, and its expression is severely restricted. May be. Therefore, it is fully conceivable that the expression of isoamylase requires an organism-specific promoter for isoamylase expression.

Conventionally, cyanobacteria are said to synthesize glycogen-like α-boliglucan called cyanobacterial starch. However, Synechocystis
sp.PCC6803) is a glycogen-like starch, and nitrogen-fixed Synechococcus (which is different from Synechococcus PCC7942 of the present invention) is a polyglucan having an intermediate structure between glycogen and amylopectin. Has recently been found to be stored (Nakamura et al., Unpublished). The most direct way to understand the function of isoamylase during the synthesis of polyglucan is to create an organism in which the isoamylase gene has been disrupted and observe its phenotype, that is, the structure of polyglucan. It can be said that. Therefore, the present inventors have proposed a homologous recombination (Homologous Recombinati).
on) method by Synechococcus PCC794
An isoamylase-deficient strain of 2) was created. We examined the structure of this mutant polyglucan and tried to observe the function of isoamylase.

FIG. 12 shows an outline of a homologous recombination method using a kanamycin cassette. First, a 1.3 kb kanamycin gene was subcloned into the sequenced # 10 (TA vector containing Synechococcus PCC7942 isoamylase gene) used in the expression experiment in Escherichia coli described above. Restriction enzyme Hind
The insert was confirmed by treatment with III and BamHI. The result is shown in FIG. 13 by a photograph replacing a drawing. FIG.
(A) on the left of Hind from colonies 1A to 1F
FIG. 13B shows the results of the III treatment, and (B) on the right side of FIG. 13 shows HindIII and Ba of colonies 1A and 1B.
It shows the result of the mHI processing. The position of the arrow in FIG. 13 is the position of the target gene. As a result, it was confirmed that the insert was performed. Then
Plates of 1A, 1B, and 1C were prepared at a kanamycin concentration of 3
The cells were inoculated on a 0 μg / ml plate and cultured for 2 weeks. Further, this plate was inoculated on a plate having a kanamycin concentration of 40 μg / ml and cultured for 2 weeks. This strain was subjected to segregation in an A-41 liquid medium having a kanamycin concentration of 25 μg / ml. The results were confirmed by colony PCR. The results of the colony PCR are shown in FIG. The control was a colony having the isoamylase gene of Synechococcus PCC7942. As a result of colony PCR, it was confirmed that the isoamylase gene was replaced and that homologous recombination (Homologous Recombination) was successful.

The resulting Synechococcus (Synechococcus)
PCC7942) is a strain lacking the isoamylase gene. The structure of the starch of this defective strain was analyzed.

Further, the present inventors have proposed a rice expression vector (Sco ISA pGTV v) in order to express the obtained isoamylase of Synechococcus PCC7942.
ector). An outline of the construction is shown in FIGS. The rice SBEI signal peptide (BEsp) was subcloned into PCRII to which the glutelin promoter had been ligated to prepare a plasmid. FIG. 17 shows the results of PCR reaction of this plasmid with a template using BEsp primers as a photograph replacing the drawing. About 20
Subcloning of BEsp was confirmed from the fact that a band of 0 bp could be confirmed. After confirmation by sequencing, from # 6 glutelin promoter, BEsp
And a fragment in which the isoamylase gene of Synechococcus PCC7942 was ligated, and subcloned into a rice expression vector (pGTV-HPT). After transformation, plasmids # 1 and # 2 were taken and PCR (primers were Synechococcus).
occus PCC7942), a chimera primer having an NdeI site added to the base sequence was used for the first and second half primers of the isoamylase gene. ), Restriction enzyme treatment (Hind
III), and confirm these with Sco
ISA PGTV # 1 and # 2. FIG. 18 shows the results.
Is shown in the photograph instead of the drawing. In FIG. 18, lane 2 is the result of # 1 PCR (2 kb is the target band), lane 3 is the result of # 2 PCR (2 kb is the target band), and lane 4 is # 1 HindIII treatment ( 840 bp is the target band), and lane 5 is the result after the treatment with HindIII of # 2 (840 bp is the target band). As a result, it was confirmed that a vector for rice expression (Sco ISA PGTV vector) was constructed.

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention comprises a nucleotide sequence represented by SEQ ID NO: 3 or 4 in the sequence listing, ie, a nucleotide sequence represented by 5'-gaaatgcatgtggggggctttac-3 '5'-gccagggtaaagccgtcgtg-3' Oligonucleotide to PC
The primer of the present invention is used as a primer for R. However, the primer of the present invention is not limited to this nucleotide sequence, and may be used for PCR to clone a gene encoding isoamylase from a library such as a genome or cDNA of an organism. If it can be used as a primer for, a part of this may be replaced with other bases, some other bases may be deleted, or some bases may be deleted. May be added to this sequence, or these modifications may be combined and modified. The degree of modification is preferably within 50%, more preferably within 30%, even more preferably within 25%. When the above-mentioned isoamylase of the present invention was cloned, the matching rate between the base sequence of this primer and the base sequence of the corresponding portion of the actually cloned isoamylase was 18 bases out of 23 bases with the former primer. Therefore, it is about 78.3%, and in the latter case, it is 80% since it is 16 bases out of 20 bases. Therefore, it is as shown in the specific examples that this level of modification is possible.

The Synechococcus of the present invention
The isoamylase derived from PCC7942) has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. If it has isoamylase activity, a part thereof may be substituted with another amino acid, Some other amino acids may be deleted, some more amino acids may be added to this sequence, or these modifications may be combined and modified. The degree of this modification is preferably about 50%, more preferably about 30%, and even more preferably about 20%, provided that the modification does not impair the isoamylase activity. Not something. As described above, the isoamylase of the present invention is one of the two isoamylases of the cyanobacterial synechocystis (Synechocystis sp. PCC6803) even when comparing the amino acid sequence homology with known isoamylases. With highest homology with 65.2%
However, since the homology with other species is 50% or less (see Table 1 above), it can be sufficiently distinguished from known isoamylase even with modification of about 50%. It is.

The Synechococcus of the present invention
The isoamylase gene derived from PCC7942) has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and as described above, with or without modification of the amino acid sequence of the isoamylase, The nucleotide sequence can be modified. For example, a part thereof may be replaced with another base, some other bases may be deleted, and some bases may be added to this sequence. And these modifications may be combined and modified. Furthermore, those having a base sequence capable of hybridizing to these base sequences under stringent conditions may be used. In particular, when the gene of the present invention is to be introduced into an organism other than Synechococcus PCC7942, it is effective to appropriately modify its nucleotide sequence in order to improve the expression efficiency in the introduced species. .

[0034] Isoamylase plays an important role in starch production in higher plants, and it is not an exaggeration to say that the characteristics of starch produced vary depending on the type of isoamylase. Therefore, for example, when the isoamylase gene of the present invention is introduced into a rice deficient in the rice isoamylase gene, the rice is no longer a conventional rice starch, but a new form of starch dependent on the isoamylase of the present invention. Will be produced. This is because isoamylase is involved in the formation of the amylopectin cluster structure, and the rice into which the isoamylase of the present invention has been introduced can produce amylopectin having a different cluster structure from the conventional rice starch. Because it becomes. As described above, since the subtle differences in the cluster structure of amylopectin greatly alter the properties of starch (see, for example, the differences between rice japonica and indica), the isoamylase of the present invention was used. The creation of new varieties plays a major role in the creation of new starches.

The organism into which the isoamylase of the present invention can be introduced is not particularly limited as long as it is an organism producing starch, and can be introduced into various plants such as rice, corn, potato and wheat.

[0036]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (PCR from genome) Primers were designed from sequences (conserved regions) commonly found in isoamylases, and Synechococcus was synthesized using primers consisting of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4. PCR was performed using genomic DNA of (Synechococcus PCC7942) as a template. The PCR reaction was performed as follows. -Hot start method It was held at 92 ° C for 1 minute and at 72 ° C for 1 minute and 10 minutes, and ExTaq was added during this time. 1 minute at 92 ° C, 1 minute at 55 ° C or 60 ° C. After performing these three cycles for 30 cycles, the temperature was kept at 72 ° C. for 3 minutes and cooled to 4 ° C. The band that matched the length of the rice isoamylase cDNA and amplified the most neatly was the dye terminator (Dye Te
rminator) to obtain a DNA probe.

Example 2 (Isolation of Genomic DNA Clones) Plaque hybridization was carried out using the DNA probe obtained in Example 1. Operation is ECL kit (A
mersham). (1) Preparation of probe PC under the PCR reaction conditions where a band of the desired size was obtained.
The reaction was amplified by R until the reaction solution became about 200 μl. Next, it was confirmed by electrophoresis, the target band was cut out with a cutter knife, the DNA was recovered using a gel nebulizer and Micropure 0.22, and then ethanol precipitation was performed. (2) Labeling of probe A probe DNA fragment (10 n
g / μl) and placed in boiling water for 5 minutes.
Then, the mixture was cooled on ice, and 25 μl of the labeling reagent was added. Then, 25 μl of glutaraldehyde was added and the mixture was added at 37 ° C. for 10 hours.
After incubating for minutes, the DNA probe was labeled. (3) Preparation of plate 1 × λ dilution buffer 10 in 14 ml of farcanupe
0 μl, host E. coli (OD600 = 0.5) 600 μl
1 and 10 μl of phage DNA (the amount was determined by performing a pre-experiment), and the mixture was incubated at 37 ° C. for 15 minutes. Thereafter, the LB + 10 mM MgSO ₄ soft top agarose 7ml addition, LB + 10 mM MgS
0 ₄ seeded to the plate and dry in a clean bench. Thereafter, the mixture was incubated at 37 ° C. for about 8 hours and stored at 4 ° C.

(4) Hybridization The plate was cooled on ice, and the membrane was slowly removed from the plate with tweezers. The membrane was soaked in alkaline solution with the DNA side down (2 minutes). Next, the plate was soaked in a neutralizing solution (for 2 minutes) and then washed with 5 × SSC. This is put in a tube dedicated to hybridization, 5 × SSC is added, and the mixture is added at 42 ° C. for 5 minutes, and then the hybridization buffer (42 ° C.) 50
After pre-hybridization was carried out at 42 ° C. for 1 hour, the hybridization buffer was taken out, and the labeled probe prepared in the above (2) was added thereto. Thereafter, the tube was returned to a hybridization-dedicated tube and incubated at 42 ° C. overnight. Then
Discard hybridization buffer, 5 × SS
C and primary wash buffer (+ Urea, 42
C), the membrane was taken out of the tube dedicated for hybridization, and washed with 2 × SSC. Immediately after washing, detection was performed. (5) Detection Take out the membrane from 2 × SSC and use detection reagent 1 (Ame
rsham LiieScience) and Detection Reagent 2 (Amersham Life Scien
ce) was added in 10 ml portions and mixed well. The membrane was immersed in the reagent for 1 minute with the DNA side down. The membrane was put in the vinyl in the cassette, the light was turned off, Kodak Scientific lmagine Film (Kodak) was placed on the vinyl, and the cassette was closed. After 1 hour, development was performed. The plaque hybridization was performed until the second screening. The plaque with the strongest signal was selected. By this operation, Synechococcus (Synech
ococcus PCC7942), a fragment containing the isoamylase gene was isolated. (6) Determination of base sequence of isoamylase gene Among plaque hybridization results, plaques having particularly strong positive signals were isolated. DNA was prepared from the phage and subcloned into a vector such as pUC118, 119 at the EcoRI site. Next, a restriction enzyme map was prepared, the isoamylase gene was divided into short fragments with various restriction enzymes, subcloned, and sequenced by the Dye Primer method.

The nucleotide sequence of the obtained isoamylase of the present invention is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. FIG. 7 shows a restriction enzyme map. Table 1 shows the amino acid sequence homology of the isoamylase of the present invention.

Example 3 (Expression of Isoamylase Gene in Escherichia coli) (1) Construction of Expression Vector Genome D of Synechococcus PCC7942
PC of isoamylase gene using NA as template
An R reaction was performed. The primer at this time was Nd
A chimeric primer to which an eI site was added was used. PC
The R cycle is performed at 92 ° C for 3 minutes, and then at 92 ° C for 30 minutes.
A cycle of 30 seconds at 65 ° C. and 2 minutes at 72 ° C. was repeated 30 times. After the reaction, it was stored at 4 ° C. The PCR product obtained here was subjected to electrophoresis using a low-melting point agarose gel, the target band was cut out, and the DNA was extracted. The obtained PCR product is subjected to TA cloning (PGEM
(Easy Vector: Prolmega) and transformed with a JM109 competent cell. From the blue white selection, only 14 white colonies were selected. These colonies were cultured in large quantities and plasmids were extracted. These Plasmid D
NA restriction enzyme treatment, nested PCR
According to the results of # 2, 10, 11, 20, and 24, it is highly probable that Synomecoccus (Synechococcus PCC7942) isoamylase gene is contained in the insert (see FIGS. 8 and 9), so that sequencing (Sequencin) was performed.
g) was performed. # 2,1 from the sequencing results
It was confirmed that all the clones 0, 11, 20, and 24 contained the isoamylase gene of Synechococcus PCC7942. # 1 out of these
0 was subcloned (NdeI site) into an E. coli expression vector (pET15b). This vector is Hi
It has the s-Tag sequence, and therefore, can be purified in one step by chelation chromatography with His-Bind resin in which the expression product is bound to a metal ion. After transformation to JM109,
The seeds were spread on an ampicillin plate, and colonies were selected by colony PCR. This selection revealed that # 2 and # 3 contained the synamococcus (Synechococcus PCC7942) isoamylase gene. The completed expression plasmid was transformed into host Escherichia coli (BL21) having a T7 RNA polymerase expression system. After confirming the transformat taken here by PCR (see FIG. 10), it was confirmed with a restriction enzyme whether the direction of the gene was correct (see FIG. 11). From this confirmation #
2 and # 16 were used for protein expression experiments.

(2) Synechococcus PC
Expression of isoamylase protein of C7942) An ampicillin (50 μg / ml) was added to 1 ml of LB, and the transformed cells were cultured at 37 ° C. overnight. Super Broth (5 ml, a
mp: 50 μg / ml) with a 25% preculture.
OD600 was checked after 3 hours. After confirming that the OD600 was 0.6 to 1.0, IPTG (1M isopropyl-β-D-thio-galactopyrenoside) was added to a final concentration of 1 mM. 1m after 2.5 hours
Take 1 liter, collect the cells by centrifugation, and add 1M Tris-HCl (pH 8.
0) was added, 50 μl of sample buffer was added, pipetted, and reacted at 100 ° C. for 5 minutes.
S PAGE was performed.

Example 4 (Synechococ (Synechococ)
Homologous Recombinatio of the isoamylase gene of P. cus PCC7942)
n) Gene disruption by the method (1) Construction of vector Figure 12 shows the homologous recombination (Homologous R).
The outline of the construction of the vector by the ecombination method is shown.
Genome D of Synechococcus PCC7942
A PCR reaction of the isoamylase gene was performed using NA as a template. As a primer, a chimeric primer to which an NdeI site was added, which was outsourced in an expression experiment in E. coli, was used. The PCR cycle was performed at 92 ° C for 3 minutes, and then at 92 ° C.
For 30 seconds, at 65 ° C. for 30 seconds, and at 72 ° C. for 2 minutes, 30 cycles were repeated. After the reaction, it was stored at 4 ° C. The PCR product obtained here was subjected to electrophoresis using a low-melting point agarose gel, the target band was cut out, and the DNA was extracted. The obtained PCR product was subjected to TA cloning (PGEM @ Easy Vector: Promega) and transformed with a JM109 competent cell. Only 14 white colonies were selected by colony selection. These colonies were cultured in large quantities and plasmids were extracted. From the results of restriction enzyme treatment of these plasmid DNAs and nested PCR (nested PCR), # 2, 10, 1
1,20,24 are Synechococcus inserts
ococcus PCC7942) is highly likely to contain the isoamylase gene.
g) was performed. # 2,10,
It was confirmed that all of the clones 11, 20, and 24 contained the isoamylase gene of Synechococcus PCC7942. Of these, # 10 was used in the suspected experiment. # 10 and the kanamycin cassette were digested with BamHI. After the treatment with the restriction enzyme, electrophoresis was performed on a low-melting point agarose gel, and the target DNA fragment was recovered. After CIP treatment, ligation (Ligation h
igh: TOYOBO) reaction. Transformation was performed using JM109 competent cells, and selection was performed using a kanamycin medium. After arbitrarily selecting and culturing clones, plasmids were extracted. Two (1A.1B) clones were selected from the results of the restriction enzyme treatment of these clones (see FIG. 13), clone 1C was also added, and these were used in the homologous recombination method.

(2) Homologous recombination (Ho)
mologous Recombination) Synechococcus PCC7942 SPC Cultivate until D730 = 1 (LOW light, air),
Collect 30 ml. Centrifugation (3000 rpm, 5 mi
n, R. T) to obtain a precipitate of cyanobacterial cells. 1 ml of A-41 medium, 4 μl of the vector obtained in the above (1) were added to the precipitate.
g was added. After shaking culture at 30 ° C. for 24 hours in the dark, the cells were seeded on a kanamycin plate (10 μg / ml). These were further subjected to segregation, and the progress was confirmed by PCR. Cyan algae 8 ~
All are mutants because they are known as haploids
Segregation was proceeded until it was replaced (final kanamycin concentration 40 μg / ml). These were spread on an A-41 agar medium, a single colony was picked, and a mutant was confirmed by PCR. FIG. 14 shows the results. The single colony confirmed in this manner was an isoamylase gene deficient mutant of Synechococcus PCC7942.

Example 5 (Structural Analysis of Cyanobacterial Starch) (1) Extraction of Cyanobacterial Starch The culture was carried out to around D730 = 3.5. At this time, an A-41 Fe stave medium was used.
The culture conditions were high CO ₂ concentration in the dark. This culture solution is placed in a 500 ml tube and centrifuged (KUBOTA
KR-20000T) at 3000 rpm for 10 minutes.
The supernatant was discarded and 90% methanol was added. Worship well,
After the cells were broken, centrifugation (3000 rpm, 10 minutes) was performed. A small amount of 90% DMSO (dimethyl sulfoxide) was added to the precipitate, and the mixture was boiled at 90 ° C. for 2 hours with occasional stirring to extract starch. After cooling for 10 minutes, 5
Dispense into 0ml Falcon tube, 1000rpm,
The mixture was centrifuged at 30 ° C for 20 minutes. The supernatant was taken in a 1 L gallon bottle, a small amount of DMSO was added to the remaining precipitate, and this operation was repeated twice to obtain a supernatant. Add 1 to the obtained supernatant.
00% ethanol was made into a 1 L gallon bottle. -
The mixture was cooled at 20 ° C. for 8 hours or more to obtain starch precipitate.

(2) Chain length distribution analysis of polyglucan by capillary electrophoresis A starch (15 to 20 mg) was placed in a glass tube with a lid, 5 ml of 100% methanol was added, and the mixture was boiled for 10 minutes with occasional stirring. After standing to cool, the mixture was centrifuged at 3000 rpm at room temperature for 5 minutes, and the supernatant was removed by suction. 9 for sedimentation
5 m of 0% methanol was added, the mixture was stirred, centrifuged at room temperature for 5 minutes, and the supernatant was removed by suction. This operation was repeated once more.
Add 1 μl of distilled water, stir and add 15 μl of 5N NaO
H was added and boiled for 5 minutes. 100% acetic acid 9.6 after standing to cool
μl, 600 mM sodium acetate buffer (pH 4.
4) 100 μl, 2% NaN ₃ 15 μl, distilled water 1
089.6 μl was added, and 3 μl of isoamylase was added. Incubation was carried out at 37 ° C. with stirring so that the starch was homogeneous. Boil for 20 minutes, allow to cool, replace with a decantation tube
The mixture was centrifuged at room temperature at 15000 rpm for 2 minutes. 0.5 to 1 ml of the supernatant was passed through a deionized column (Bio @ ad AG 501 = 8 (D)) packed in a Köppendorf tube to perform a deionization operation. The sugar content of the deionized sample was quantified by the Modified Park Johnson method (see Biochemistry Experiment 19, Starch and Related Carbohydrate Experiments, page 125). Improved Park Johnson (Modified Par
k Johnson) method is an established method for accurately quantifying the number of reducing ends regardless of the length of the chain. First,
Create a calibration curve and add 198 μl to 2 μl of the sample to be quantified.
1 of distilled water was added. A solution 10 for standard solution and sample
0 μl and 100 μl of solution B were added, and the mixture was boiled for 15 minutes. After boiling, water-cooled with running water for 10 minutes, added 500 μl of solution C,
Left for 20 minutes. Further, add 500 μl of solution C to
After 0 minute, the absorbance at 715 nm was measured with a spectrophotometer, and the number of μl of 5 nmol in each sample was calculated.

Example 6 (Synechococ)
(1) Construction of rice expression vector A signal of an enzyme (Branching Enzyme) branched from a glutelin promoter specifically expressed in endosperm and a synechococcus ( Synechococcus PCC7942) was ligated (see FIG. 15). Branching enzyme (Bran
ching Enzyme) signal is AACCATGGDGTGTCTCACCTCCTCTTC (FOWard): CCATGG = Nco
I site ATACTAGTCACCATAGTTTTGTTTTTTCG (Reverse): ACTAGT = S
It was prepared by PCR using a pe1 site. Synechococcus (Syne
chococcus PCC7942) isoamylase gene is ATACTAGTTCCCGTCGTCGCCCTGAATCG (FoWard): ACTAGT = Sp
e I site ATGAGCTCTTAAGCACGAGCCATCACC (Reverse): GAGCTC = Sa
It was prepared by PCR using the cI site. After subjecting each PCR product to low melting point agarose gel electrophoresis, a target fragment was taken and DNA was extracted. The extracted DNA fragment was stored at 4 ° C. until the next operation. The PCRII vector to which GluB-1 was linked was treated with restriction enzymes NcoI and SpeI. Similarly, the PCR fragment of the signal of the branching enzyme (Branching Enzyme) was treated with the restriction enzymes NcoI and SpeI. Each DNA solution treated with the restriction enzyme was subjected to low-melting point agarose gel electrophoresis, and then the target fragment was taken.
DNA was extracted. After quantifying each DNA concentration, ligation (Ligation High: TOYOBO) was performed.
Spreading on ampicillin plate, enzymatic branching to glutelin promoter by colony PCR (Branching
Enzyme) were selected if they were correctly linked (see FIG. 17). The plasmid of the selected colony was extracted, and this was added to Synechococcus (Cynechococcus PCC794).
The isoamylase gene of 2) is replaced with restriction enzymes Spel and Sa.
cl and the above signal and Synechococcus (Synechococcus) were added to the glutelin promoter in the same manner as above.
Those to which the isoamylase gene of PCC7942) was linked were selected. After confirming the thus selected # 6 by treatment with a restriction enzyme, sequencing was performed.

This was transformed into a rice expression vector (pGTV-H
PT). The outline is shown in FIG. pGTV-HPT was treated with restriction enzymes SalI and SacI. Glutelin Promoter Branched to Enzyme (Br
anching Enzyme signal and Synechococcus (Synech)
ococcus PCC7942) PCRII vector # 6 confirmed to be linked to the isoamylase gene
It was treated with alI and SacI. Each DNA solution treated with a restriction enzyme was subjected to low-melting point agarose gel electrophoresis, and then a target fragment was taken therefrom to extract DNA. After quantifying each DNA concentration, ligation (Ligation High:
TOYOBO). Sow the kanamycin plate,
The insert was confirmed by colony PCR (see FIG. 18). Plasmids of the selected colonies were prepared.

[0049]

The present invention provides a versatile PCR primer for cloning isoamylase from various organisms. The primer of the present invention has specificity and high efficiency. By using this, not only the isoamylase gene can be selectively and simply cloned, but also whether or not the organism has isoamylase can be known by a simple method. . Since isoamylase is deeply involved in the biosynthesis of amylopectin, it is possible to know whether or not the organism is producing amylopectin by confirming the presence of isoamylase. The present invention also provides a novel isoamylase. By introducing the isoamylase of the present invention into various higher plants, a new kind of amylopectin can be produced. Starch containing amylopectin is not only used in foods, but also has industrial uses such as adhesives such as glue and various additives, and is also important for the development of new industrial materials. Furthermore, the present invention provides a plant into which the isoamylase gene of the present invention has been introduced, and is useful for the development of a cereal that produces a new type of starch.

[0050]

[Sequence List] Sequence List SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Novel Isoamylasa <130> PA936544 <160> 4 <210> 1 <211> 4745 <212> DNA <213> Synechococcus sp. Pcc7942 <400 > 1 ggccagcagg cggcgaaaca gacacagcgg ttgaggaagg gaactgttgc gatcgtgcca 60 cgcttaaccc gctcttggta aacggagcaa aaagcagtcc gatagattgg atgaagcgga 120 ttccctgacg taatgctttt tctgcttctc tggttaatcc ttctggcggt tggttcttac 180 tggatgttgc agcgcaatgt gccgcgcctc agccgcacgc cagtctggtt gctctggctg 240 gtgctgatga tgcctgcctt catttggatt ggctggggac tcctactcgg tcagcagggg 300 ggacagctcc cgtttgtgat tttcctgctg ccgctgctga ttagttcact gctctactgg 360 acgttgctgc aatggggacg cctcccaccc agccgatcgc tgcctaccaa tgagaccgaa 420 gcagcagcga tcgcggcagc ctcagagacc ttgcaagtgg ccagtgcccc accggcgcga 480 tcgctgaccg cagaggaaga agcccaggtt cgtcagtgtt ttcccttcga tcgcctgcag 540 cgactggaat atcgctatca agccgtcatt tgtcggggaa atttgcgagg tgatcccgcc 600 acagtttatg agcaagtgca gacggcagtg cagcaacagt ttggcgatcg ctttctggtg 660 atgctgcgag aagacaatgc cggc aagccc ttttttgccc tgattcccaa ccccctgcgc 720 caacaacctc gcctggccct caagcccatg ctagcgctgg tgctgttggg gctgacgcta 780 ctgaccacaa ccttggtggg ctttgtactt agctatgcgg gtcaactggc cgaaatccag 840 ccccagctag caagatcctt agaagacaat ccagcggcac tgttgcgcgg tttaccctac 900 gccctctcgc tactcgcgat tctcggcgta catgaatttg gccatttctg ggctgctcgt 960 aaacaccgcc tgcaagcgag cttgccctac ttcattccgg tgcccgcttt cttaggaacc 1020 tttggcgcct ttgtccgtat ccgcagtccg attcccgatc gcaaggctct gtttgatgtg 1080 ggcgtctcgg gtcccctcgc gggtttggtg atcaccctgc cgctgctgat ctgggggctg 1140 acacaatctc aggtggtgcc gatgccagag cggtccgggc tgctcaactt ctcagccctt 1200 gatccgggcg tctcgatcct gatggggttg atcagtcatc tcagtttggg cgatcgccta 1260 ggactgaatc aggccctgca actgcatccc gtcgcgatcg cgggttactt gggcttgatt 1320 gtcacggcct tgaacctggt gcccgtcggc caactggatg gcggtcacat cgtccatgcc 1380 atgtttggcc agcgccaagg ggcagtcatt ggtcaggtgg cgcgtctctg catcctcgcc 1440 ctttcctttg tgcgatcgga actcctgctc tgggcactgt tgttattgtt gcttccagtc 1500 gccgatgaac cggcgttgaa tgacttgagt gaac tggatg atcgccgcga tggcattggc 1560 ttcttggccc tctttatcct cattttgatt gttttgccct tgccaccagt cttgcagcag 1620 tggctaacgg cgctctaagg actgcc atg act gtt tca tcc cgt cgc cct gag gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc cg cg cg acc 1721 Ser Thr Val Ala Val Asp Pro Gly Gln Ser Tyr Pro Leu Gly Ala Thr 10 15 20 25 gtc tat ccc acc ggc gtc aac ttc tcg ctc tac acc aag tac gcg acg 1769 Val Tyr Pro Thr Gly Val Asn Phe Ser Leu Tyr Thr Lys Tyr Ala Thr 30 35 40 ggc gtt gaa tta ctg ctg ttt gat gac cct gag ggt gcc cag cct caa 1817 Gly Val Glu Leu Leu Leu Phe Asp Asp Pro Glu Gly Ala Gln Pro Gln 45 50 55 cgg aca gtg cgc ctc gat ccg cac ctc aat cgc acc tct ttc tac tgg 1865 Arg Thr Val Arg Leu Asp Pro His Leu Asn Arg Thr Ser Phe Tyr Trp 60 65 70 cat gtt ttt att ccg ggc att cgc tcc ggt cag gtt tat gct tac cgc 1913 His Val Phe Ile Pro Gly Ile Arg Ser Gly Gln Val Tyr Ala Tyr Arg 75 80 85 gtc ttt ggc ccc tac gca cct gat cgc ggc ctc tgt ttt aac ccc aac 1961 Val Phe Gly Pro Tyr Ala Pro Asp Arg Gly Leu Cys Phe Asn Pro Asn 90 95 100 105 aaa gtg ctg ctg gat ccc tac gct cgc ggg gtt gtc ggc tgg cag cac 2009 Lys Val Leu Leu Asp Pro Tyr Ala Arg Gly Val Val Gly Trp Gln His 110 115 120 tac agt cgc gaa gcg gct att aaa ccc agt aat aac tgc gtt caa gcc 2057 Tyr Ser Arg Glu Ala Ala Ile Lys Pro Ser Asn Asn Cys Val Gln Ala 125 130 135 ctg cgt agc gtg gtt gtt gac ccc agc gac tac gac tgg gaa ggc gat 2105 Leu Arg Ser Val Val Val Asp Pro Ser Asp Tyr Asp Trp Glu Gly Asp 140 145 150 cgc cat cca cgc aca ccc tac gct cgc aca gta atc tat gag ctg cat 2153 Arg His Pro Arg Thr Pro Tyr Ala Arg Thr Val Ile Tyr Glu Leu His 155 160 165 gtt ggc ggc ttc acc aag cat ccc aat tcc ggc gtc gcc cct gaa aaa 2201 Val Gly Gly Phe Thr Lys His Pro Asn Ser Gly Val Ala Pro Glu Lys 170 175 180 185 cgt ggc acc tac gct ggt cta atc gaa aaa att ccc tac ctg caa tcc 2249 Arg Gly Thr Tyr Ala Gly Leu Ile Glu Lys Ile Pro Tyr Leu Gln Ser 190 195 200 ctc ggc gtc acg gcc gtt gag ttg ctg ccg gt ttc gat cgc 2297 Leu Gly Val Thr Ala Val Glu Leu Leu Pro Val His Gln Phe Asp Arg 205 210 215 caa gat gcc ccc tta gga cgc gag aac tac tgg ggc tac agc acc atg 2345 Gln Asp Ala Pro Leu Gly Arg Glu Asn Tyr Trp Gly Tyr Ser Thr Met 220 225 230 gct ttt ttt gcg ccc cac gca gcc tac agc tct cgc cat gat cca ctt 2393 Ala Phe Phe Ala Pro His Ala Ala Tla Ser Ser Arg His Asp Pro Leu 235 240 245 ggt cca gtt gat gag ttc cgc gac ctc gtc aag gcg ctc cac caa gca 2441 Gly Pro Val Asp Glu Phe Arg Asp Leu Val Lys Ala Leu His Gln Ala 250 255 260 265 ggg att gag gtg att ctc gac gtg gtg ttc aac gac gct gcta Ile Glu Val Ile Leu Asp Val Val Phe Asn His Thr Ala Glu Gly 270 275 280 aat gaa gac ggt cca acg ctg tct ttc aaa ggt cta gcg aat tca acc 2537 Asn Glu Asp Gly Pro Thr Leu Ser Phe Lys Gly Leu Ala Asn Ser Thr 285 290 295 tac tat ctg ctg gat gaa cag gcg ggc tat cgc aac tac acc ggc tgc 2585 Tyr Tyr Leu Leu Asp Glu Gln Ala Gly Tyr Arg Asn Tyr Thr Gly Cys 300 305 310 ggc aac acc gtc aaa gct aa tcg atc gtg cga tcg ctg att ctc 2633 Gly Asn Thr Val Lys Ala Asn Asn Ser Ile Val Arg Ser Leu Ile Leu 315 320 325 gat tgc ctg cgt tat tgg gtc tcg gaa atg cac gtc gat ggc ttc cyr2 Trp Val Ser Glu Met His Val Asp Gly Phe Arg 330 335 340 345 ttt gac ctt gcg tcg gtg ctg agt cgt gat gcc aat ggc aac ccc cta 2729 Phe Asp Leu Ala Ser Val Leu Ser Arg Asp Ala Asn Gly Asn Pro Leu 350 355 360 tcg gat ccg ccc ttg ctt tgg gcg att gat tcc gat ccg gtt ttg gcc 2777 Ser Asp Pro Pro Leu Leu Trp Ala Ile Asp Ser Asp Pro Val Leu Ala 365 370 375 ggt acg aag ctc att gct gaa gct tgg gac gca gcc tta tat cag 2825 Gly Thr Lys Leu Ile Ala Glu Ala Trp Asp Ala Ala Gly Leu Tyr Gln 380 385 390 gtt ggt acc ttt att ggc gat cgc ttt ggg act tgg aac ggt ccc ttc 2873 Val Gly Thr Phe Ile Gly Asp Arg Phe Thr Trp Asn Gly Pro Phe 395 400 405 cgg gac gat att cgg cgt ttt tgg cgt gga gat cag ggc tgt act tac 2921 Arg Asp Asp Ile Arg Arg Phe Trp Arg Gly Asp Gln Gly Cys Thr Tyr 410 415 420 425 425 gcc ctc a gt caa cgc ctg ctg ggt agc ccc gat gtc tac agc aca gac 2969 Ala Leu Ser Gln Arg Leu Leu Gly Ser Pro Asp Val Tyr Ser Thr Asp 430 435 440 caa tgg tat gcc gga cgc acc att aac ttc atc acc tgc cat gac 3017 Gln Trp Tyr Ala Gly Arg Thr Ile Asn Phe Ile Thr Cys His Asp Gly 445 450 455 ttt acg ctg cga gat cta gtc agc tat agc cag aag cac aac ttt gcc 3065 Phe Thr Leu Arg Asp Leu Val Ser Tyr Ser Gln Lys His Asn Phe Ala 460 465 470 aat gga gag aac aat cgg gac ggg acc aat gac aac tac agc tgg aac 3113 Asn Gly Glu Asn Asn Arg Asp Gly Thr Asn Asp Asn Tyr Ser Trp Asn 475 480 485 tac ggc att gaa gc gag acc gac ccc acg att ctg agc tta cgg 3161 Tyr Gly Ile Glu Gly Glu Thr Asp Asp Pro Thr Ile Leu Ser Leu Arg 490 495 500 505 gaa cgg cag cag cgc aat ttg ctc gcc acg tta ttc ctc gcc cag ggc G209 Glu Arg Asn Leu Leu Ala Thr Leu Phe Leu Ala Gln Gly 510 515 520 aca ccg atg ctg acg atg ggc gat gag gtc aaa cgc agt cag cag ggt 3257 Thr Pro Met Leu Thr Met Gly Asp Glu Val Lys Arg Ser Gln Gln Gly 52 5 530 535 aac aat aac gcc tac tgc caa gac aat gag atc agc tgg ttt gat tgg 3305 Asn Asn Asn Ala Tyr Cys Gln Asp Asn Glu Ile Ser Trp Phe asp Trp 540 545 550 tcg ctg tgc gat cgc cat gtt gat ttc agt cgc cgc ctg 3353 Ser Leu Cys Asp Arg His Ala Asp Phe Leu Val Phe Ser Arg Arg Leu 555 560 565 att gaa ctt tcc cag tcg ctg gtg atg ttc caa cag aac gaa ctg ctg 3401 Ile Glu Leu Met Phe Gln Gln Asn Glu Leu Leu 570 575 580 585 cag aac gaa ccc cat ccg cgt cgt ccc tat gcc atc tgg cat ggc gtc 3449 Gln Asn Glu Pro His Pro Arg Arg Pro Tyr Ala Ile Trp His Gly Val 590 595 600 600 aaa ctc aaa caa ccc gat tgg gcg ctg tgg tcc cac agt ctg gcc gtc 3497 Lys Leu Lys Gln Pro Asp Trp Ala Leu Trp Ser His Ser Leu Ala Val 605 610 615 agt ctc tgg cat cct cgc cag cag gaa tgg ctt tac cat gcc 3545 Ser Leu Trp His Pro Arg Gln Gln Glu Trp Leu Tyr Leu Ala Phe Asn 620 625 630 gct tac tgg gaa gac ctg cgc ttc cag ttg ccg agg cct cct cgc ggc 3593 Ala Tyr Trp Glu Asp Leu Arg Phe Gln Pro Pro Arg Gly 635 640 645 cgc gtt tgg tat cgc ttg ctc gat act tca ctg ccg aat ctt gaa gct 3641 Arg Val Trp Tyr Arg Leu Leu Asp Thr Ser Leu Pro Asn Leu Glu Ala 650 655 660 660 665 tgt cat ctg gg gca aaa ccc tgc cta cgg cgc gat tac atc 3689 Cys His Leu Pro Asp Glu Ala Lys Pro Cys Leu Arg Arg Asp Tyr Ile 670 675 680 gtc cca gcg cga tcg ctc tta ctg ttg atg gct cgt gct Taaaaacaat 37 Val Leu Leu Leu Leu Met Ala Arg Ala 685 690 gcaaacttca ccgtttcagc tggtgatttt cgactgtgat ggtgtgcttg ttgatagcga 3798 acgcatcact aatcgcgtct ttgcagacat gctcaatgaa ctgggtctgt tggtgacttt 3858 ggatgacatg tttgagcagt ttgtgggtca ttccatggct gactgtctca aactaattga 3918 gcgacggtta ggcaatcctc caccccctga ctttgttcag cactatcaac gccgtacccg 3978 tatcgcgtta gaaacgcatc tacaagccgt tcctggggtt gaagaggctt tggatgctct 4038 tgaattgccc tactgtgttg cgtccagtgg tgatcatcaa aagatgcgaa ccacactgag 4098 cctgacgaag ctctggccac gatttgaggg acgaatcttc agcgtgactg aagtacctcg 4158 cggcaagcca tttcccgatg tctttttgtt ggccgccgat cgcttcgg gg ttaatcctac 4218 ggcctgcgct gtgatcgaag acaccccctt gggagtagcg gcaggcgtgg cggcaggaat 4278 gcaagtgttt gNtacgcggg ttccatgccc gcttggcgtc tgcaagaagc cggtgcccat 4338 ctcatttttg acgatatgcg actgctgccc agtctgctcc aatcgtcgcc aaaagataac 4398 tccacagcat tgcccaatcc ctaacccctg ctcgcgccgc aactacacac taaaccgttc 4458 ctgcgcgatc gctcttactg ttgatggctc gtgcttaaaa acaatgcaac cctaaccgtt 4518 tcagctggtg attttcggac gatttggctt acagggataa ctgagagtca acagcctctg 4578 tccgtcattg cacacccatc catgcactgg ggacttgact catgctgaat cacatttccc 4638 ttgtccattg ggcgagaggg gaggggaatc ttctggactc ttcactaagc ggcgatcgca 4698 ggttcttcta cccaagcagt ggcgatcgct tgcttgcagt cttcaat 4745 <210> 2 <211> ger <2> <2> Ser <G> Thr <2> <2> rt <2> 2 <2> rt <2> 2 <2> Ser <2> rt <2) Ala Val Asp Pro 1 5 10 15 Gly Gln Ser Tyr Pro Leu Gly Ala Thr Val Tyr Pro Thr Gly Val Asn 20 25 30 Phe Ser Leu Tyr Thr Lys Tyr Ala Thr Gly Val Glu Leu Leu Leu Phe 35 40 45 Asp Asp Pro Glu Gly Ala Gln Pro Gln Arg Thr Val Arg Leu Asp Pro 50 5 5 60 His Leu Asn Arg Thr Ser Phe Tyr Trp His Val Phe Ile Pro Gly Ile 65 70 75 80 Arg Ser Gly Gln Val Tyr Ala Tyr Arg Val Phe Gly Pro Tyr Ala Pro 85 90 95 Asp Arg Gly Leu Cys Phe Asn Pro Asn Lys Val Leu Leu Asp Pro Tyr 100 105 110 Ala Arg Gly Val Val Gly Trp Gln His Tyr Ser Arg Glu Ala Ala Ile 115 120 125 Lys Pro Ser Asn Asn Cys Val Gln Ala Leu Arg Ser Val Val Val Asp 130 135 140 Pro Ser Asp Tyr Asp Trp Glu Gly Asp Arg His Pro Arg Thr Pro Tyr 145 150 155 160 Ala Arg Thr Val Ile Tyr Glu Leu His Val Gly Gly Phe Thr Lys His 165 170 175 Pro Asn Ser Gly Val Ala Pro Glu Lys Arg Gly Thr Tyr Ala Gly Leu 180 185 190 Ile Glu Lys Ile Pro Tyr Leu Gln Ser Leu Gly Val Thr Ala Val Glu 195 200 205 Leu Leu Pro Val His Gln Phe Asp Arg Gln Asp Ala Pro Leu Gly Arg 210 215 220 Glu Asn Tyr Trp Gly Tyr Ser Thr Met Ala Phe Phe Ala Pro His Ala 225 230 235 240 Ala Tyr Ser Ser Arg His Asp Pro Leu Gly Pro Val Asp Glu Phe Arg 245 250 255 Asp Leu Val Lys Ala Leu His Gln Ala Gly Ile Glu Val Ile Leu Asp 260 265 270 Va l Val Phe Asn His Thr Ala Glu Gly Asn Glu Asp Gly Pro Thr Leu 275 280 285 Ser Phe Lys Gly Leu Ala Asn Ser Thr Tyr Tyr Leu Leu Asp Glu Gln 290 295 300 Ala Gly Tyr Arg Asn Tyr Thr Gly Cys Gly Asn Thr Val Lys Ala Asn 305 310 315 320 Asn Ser Ile Val Arg Ser Leu Ile Leu Asp Cys Leu Arg Tyr Trp Val 325 330 335 Ser Glu Met His Val Asp Gly Phe Arg Phe Asp Leu Ala Ser Val Leu 340 345 345 350 Ser Arg Asp Ala Asn Gly Asn Pro Leu Ser Asp Pro Pro Leu Leu Trp 355 360 365 Ala Ile Asp Ser Asp Pro Val Leu Ala Gly Thr Lys Leu Ile Ala Glu 370 375 380 Ala Trp Asp Ala Ala Gly Leu Tyr Gln Val Gly Thr Phe Ile Gly Asp 385 390 395 400 Arg Phe Gly Thr Trp Asn Gly Pro Phe Arg Asp Asp Ile Arg Arg Phe 405 410 415 Trp Arg Gly Asp Gln Gly Cys Thr Tyr Ala Leu Ser Gln Arg Leu Leu 420 425 430 Gly Ser Pro Asp Val Tyr Ser Thr Asp Gln Trp Tyr Ala Gly Arg Thr 435 440 445 445 Ile Asn Phe Ile Thr Cys His Asp Gly Phe Thr Leu Arg Asp Leu Val 450 455 460 Ser Tyr Ser Gln Lys His Asn Phe Ala Asn Gly Glu Asn Asn Arg Asp 465 470 475 480 480 Gl y Thr Asn Asp Asn Tyr Ser Trp Asn Tyr Gly Ile Glu Gly Glu Thr 485 490 495 Asp Asp Pro Thr Ile Leu Ser Leu Arg Glu Arg Gln Gln Arg Asn Leu 500 505 510 Leu Ala Thr Leu Phe Leu Ala Gln Gly Thr Pro Met Leu Thr Met Gly 515 520 525 Asp Glu Val Lys Arg Ser Gln Gln Gly Asn Asn Asn Ala Tyr Cys Gln 530 535 540 Asp Asn Glu Ile Ser Trp Phe asp Trp Ser Leu Cys Asp Arg His Ala 545 550 555 555 560 Asp Phe Leu Val Phe Ser Arg Arg Leu Ile Glu Leu Ser Gln Ser Leu 565 570 575 Val Met Phe Gln Gln Asn Glu Leu Leu Gln Asn Glu Pro His Pro Arg 580 585 590 Arg Pro Tyr Ala Ile Trp His Gly Val Lys Leu Lys Gln Pro Asp Trp 595 600 605 Ala Leu Trp Ser His Ser Leu Ala Val Ser Leu Trp His Pro Arg Gln 610 615 620 Gln Glu Trp Leu Tyr Leu Ala Phe Asn Ala Tyr Trp Glu Asp Leu Arg 625 630 635 640 Phe Gln Leu Pro Arg Pro Pro Arg Gly Arg Val Trp Tyr Arg Leu Leu 645 650 655 Asp Thr Ser Leu Pro Asn Leu Glu Ala Cys His Leu Pro Asp Glu Ala 660 665 670 Lys Pro Cys Leu Arg Arg Asp Tyr Ile Val Pro Ala Arg Ser Leu Leu 675 680 685 Leu Le u Met Ala Arg Ala 690 <210> 3 <211> 23 <212> DNA <213> Artificial <400> 3 gaaatgcatg tggggggctt tac 23 <210> 4 <211> 20 <212> DNA <213> Artificial <400> 4 gccagggtaa agccgtcgtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial <400> 5 tatgaaatgc atgtgcgggg 20 <210> 6 <211> 20 <212> DNA <213> Artificial <400> 6 tacgaactcc acgtcggggg 20 < 210> 7 <211> 20 <212> DNA <213> Artificial <400> 7 gcatcccagg cttcagcaat 20 <210> 8 <211> 20 <212> DNA <213> Artificial <400> 8 accagggtaa aaccatcatg 20

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing the structure of a storage polyglucan in an organism.

FIG. 2 shows a cluster structure of amylopectin.

FIG. 3 shows the difference in the cluster structure of amylopectin between Japonica and Indica in rice endosperm.

FIG. 4 provides an overview of the biosynthetic pathway of amylose, aminopectin, and glycogen, which are storage polyglucans in organisms.

FIG. 5 shows a conceptual diagram for designing a primer from a sequence (conserved region) commonly found in isoamylase.

FIG. 6 shows Synechococcus PCC7.
942) is a photograph, instead of a drawing, showing the result of Southern hybridization with the genome using a DNA fragment of about 760 bp obtained from the genome as a probe.

FIG. 7 shows Synechococcus of the present invention.
2 shows a restriction map of the isoamylase gene of C. cus PCC7942).

FIG. 8 is a photograph instead of a drawing, showing the results of electrophoresis of a colony nested PCR of 14 colonies obtained from TA cloning (the target clone has a band at the position of the arrow).

FIG. 9 shows # 2, # 10, # 11, # 20, and # of 14 colonies obtained from TA cloning.
24 is a photograph instead of a drawing, showing the result of electrophoresis after BamHI treatment of No. 24.

FIG. 10 shows Synechococcus (Synech coccus) of the present invention.
ococcus PCC7942) was subcloned into an Escherichia coli expression vector (PET-15b vector) and the transformed clone was cloned into a colony PC.
It is a photograph substituted for the drawing which shows the result of R.

FIG. 11 is a photograph instead of a drawing for confirming the result of treating the orientation of the insert in the transformant with the restriction enzyme HindIII.

FIG. 12 shows Synechococcus (Synech coccus) of the present invention.
ococcus PCC7942) in the isoamylase gene,
1 shows an outline of a homologous recombination method using a kanamycin cassette.

FIG. 13 is a photograph instead of a drawing showing the result of confirming inserts by restriction enzyme HindIII and BamHI treatment in the homologous recombination method. (A) on the left side of FIG. 13 shows the results of Hind III treatment on colonies 1A to 1F,
(B) on the right side of FIG. 13 shows Hin of colonies 1A and 1B.
It shows the result of the treatment of dIII and Bam H1. The position of the arrow in FIG. 13 is the position of the target gene.

FIG. 14 shows Synechococcus (Synech coccus) of the present invention.
3 is a photograph instead of a drawing, showing the results of a homologous recombination method of the isoamylase gene of S. ococcus PCC7942). The upper arrow in FIG. 14 is 2.5 kb, and the lower arrow is 2 kb.

FIG. 15 shows Synechococcus (Synech coccus) of the present invention.
1 shows the outline of construction of a rice expression vector (Sco ISA PGTV vector) using the isoamylase gene of S. ococcus PCC7942).

FIG. 16 shows Synechococcus (Synech coccus) of the present invention.
1 shows an outline of construction of a rice expression vector (Sco ISA pGTV vector) using the isoamylase gene of S. ococcus PCC7942).

[FIG. 17] FIG. 17 is a photograph instead of a drawing, showing the result of performing a PCR reaction with a PCRII plasmid to which a glutelin promoter and a rice signal peptide (BEsp) were ligated as a template and using a BEsp primer. The arrow in FIG. 17 indicates 200 bp.

FIG. 18 shows a rice expression vector (Sco ISA pGTV) in which a glutelin promoter, a rice signal peptide (BEsp), and the isoamylase gene of Synechococcus PCC7942 of the present invention are linked.
5 is a photograph instead of a drawing, showing the result of performing a PCR reaction using a template as a template. The upper arrow in FIG. 18 indicates 2 kp, and the lower arrow indicates 840 bp.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 5/10 C12R 1:19) C12R 1:91) C12N 15/00 ZNAA (C12N 9/44 5/00 C C12R 1:19) C12R 1:91) (72) Inventor Kazuhiro Umeda 143-12-1 Horinouchi, Tokyo Pharmaceutical University Tokyo Metropolitan University of Pharmacy (72) Inventor Mikio Tsuzuki 143-12-1 Horinouchi, Hachioji-shi, Tokyo Tokyo Pharmaceutical University Faculty of Life Science F-term (reference) QX07 4B065 AA26X AA83Y AA89X AB01 BA02 CA32 CA41 CA53

Claims (9)

[Claims] 1. A method for cloning a gene encoding isoamylase based on DNA amplified by PCR from DNA in a genome or library using the nucleotide sequences shown in SEQ ID NOs: 3 and 4 in the sequence listing as primers. 2. The method according to claim 1, wherein a part of the base sequence of the primer is substituted with another base and / or a part of the base sequence is deleted or added. 3. The method according to claim 1, wherein the PCR template is genomic DNA. 4. A fragment of a DNA encoding isoamylase comprising a nucleotide sequence represented by SEQ ID NO: 3 or 4 in the sequence listing, or a nucleotide sequence in which a partial base is substituted, deleted and / or added. Primers for amplifying by a PCR method. 5. An isoamylase having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, or an amino acid sequence in which a partial amino acid is deleted, substituted, or added. A gene encoding the isoamylase according to claim 5. 7. A gene comprising the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, or a part of the nucleotide being deleted or substituted;
7. The gene according to claim 6, which has an added base acid sequence or a base sequence capable of hybridizing to those base sequences under stringent conditions. 8. An organism obtained by introducing the gene according to claim 6 or 7 into an organism deficient in isoamylase. 9. The organism according to claim 8, wherein the organism is a higher plant.