JP3088438B2 - Rice waxy sequence and method for improving endosperm component of rice seed using the same - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a rice starch synthase gene and a method for improving the quality of starch in rice grain using the same.

The antisense gene can be effectively expressed in rice using the rice / waxy sequence of the present invention and can suppress the synthesis of starch in grains, so that the amylose content of starch contained in grains can be arbitrarily adjusted. This makes it possible to remarkably improve the palatability of rice or to produce a rice cultivar. In addition, it allows direct modification of the waxy gene using homologous substitution of the gene.

The present invention also relates to the rice transformed in this way.

(Prior art and problems) Starch is the end product of photosynthesis. Therefore, research on starch biosynthesis is one of the important research subjects in plant physiology and biochemistry, but research on this biosynthesis has not progressed much.

Starch is composed of linear amylose having α (1 → 4) linkage of glucose and amylopectin having a branched structure by α (1 → 6) linkage. It is known that the content ratio of amylose and amylopectin in starch granules in plants has a specific value depending on the plant species and cultivar, and the higher-order structure also has a specific form. Starch synthase, which is the main enzyme of starch biosynthesis, synthesizes amylose molecules using ADP glucose or UDP glucose as a substrate. In contrast,
The amylopectin molecule is formed by the cooperative action of a starch synthase that forms an α (1 → 4) bond and a branching enzyme that forms an α (1 → 6) bond. Studies on the mechanism of the formation of the branched structure of amylopectin have been conducted with reference to studies on the mechanism of branching with glycogen. In plants, enzymes having properties similar to those of glycogen branching enzymes have been found.

The mechanism of biosynthesis of stored starch in plants has been considerably elucidated in the field of plant physiology and biochemistry since the discovery of starch synthase. However, the genetic regulatory mechanism remains largely unknown.

The amylose content of rice is thought to be related to the waxy locus gene waxy located on chromosome 6. As this gene product, Wx protein having a molecular weight of about 60,000 daltons is produced in the endosperm of the wurtching variety (Sano; Theor. Appl. Genet., 68 , 467 (1984)). Amylose content in endosperm is proportional to the amount of this protein. That is, the more the Wx protein is present, the higher the amylose content (Sano et al .; Euphytica, 35 , 1 (1986)). In the endosperm of waxy varieties, Wx protein is not found and amylose is not produced. Although there are many other genes that determine motiness, little is known about the molecular biology. The results of genetic analysis in corn, Wx protein is UDP-glucose glycosyl transferase, Wakishii is believed that it would be the structural gene (Kloesgen et al;.. Mol.Gen.Genet , 203, 237 (198
6)).

Cereals are a major source of calories for us and an important source of lipids and proteins. Therefore, qualitative and quantitative improvement of cereal endosperm storage components is a major breeding species. To achieve this goal, it is necessary to search and collect genetic resources for storage components in cereal endosperm and to evaluate their genetic and breeding properties. In maize, various genetic mutations (mutations) related to the endosperm component have been found,
They play an important role in improving corn quality. Furthermore, these mutations provide valuable information for elucidating the genetic control mechanisms in vivo.

Rice is predominantly granulated, and the content ratio of polysaccharide in endosperm starch and the structural change of starch greatly change the taste and cooking characteristics of rice. In particular, it has been pointed out that the amylose content in starch is closely related to taste, and the evaluation of taste of low amylose varieties is high. Therefore, it is effective to reduce endosperm starch amylose to improve the taste of rice.

However, it is extremely difficult to find such low amylose varieties from nature, and it is also very difficult to obtain them by mutation. Moreover, it is not an exaggeration to say that it is impossible to control the amylose content with such conventional breeding techniques.

(Means for Solving the Problems) In view of the current state of the art, the present invention has been made for the purpose of creating a low amylose variety or a variety whose amylose content can be controlled as desired. A major change in thinking without being bound by the law at all, as a result, the gene that determines the amylose content of rice is isolated and identified, and the intended purpose is achieved by breeding using genetic engineering techniques using this. I decided that.

The maize and barley waxy genes have been cloned and sequenced (Kloe
sgen et al .; Mol. Gen. Genet., 203 , 237 (1986), Rohde
et al .; Nucl. Acids Res., 16 , 7185 (1988)), and this nucleotide sequence is available from the EMBL database. However, although the structure of the rice waxy gene has been deduced to some extent from these nucleotide sequences, the exact structure has not yet been clarified. The present inventors have recently developed a PCR (pol
ymerase chain reaction) (Saiki et al .; Science 23
9 , 487 (1988)), and using this technology,
The cloning of the waxy gene was examined.

PCR is a technique in which two opposing primer DNAs are used and the sequence between them is repeatedly synthesized by a DNA polymerase many times to amplify only the sequence of a specific region to a high degree. In PCR, a specific fragment can be amplified by repeatedly performing heat denaturation of a DNA strand, annealing with a primer, and extension reaction with a polymerase. In order to repeat the temperature change, a thermophilic DNA polymerase is used. In a typical reaction system, heat denaturation is performed at 94 ° C., annealing is performed at 37 ° C. to 60 ° C., and polymerase reaction is performed at 72 ° C.

Comparison of the nucleotide sequences of the maize and barley waxy genes showed that both sequences showed good homology in the exon region. In order to amplify the sequence in a certain region using PCR, it is necessary that the primer sequence be successfully hybridized with the target DNA sequence. Even when a DNA having low homology with the primer is targeted, if the primer sequence can be successfully hybridized, the PCR proceeds successfully and the target region is successfully amplified. In order for such a primer to somehow hybridize with the target DNA, the annealing temperature may be adjusted in principle. However, in order for the primer to function normally and to proceed with the DNA polymerase reaction, the sequence of the 3'-end region of the primer must have a structure that is completely adhered to the target DNA.

Therefore, in the present invention, a rice waxy sequence is estimated based on the waxy base sequence of heterologous plants such as corn and barley, and the DNA
Was synthesized and PCR was performed using the same to obtain the desired rice waxy gene very efficiently.

The waxy gene fragment initially obtained according to the present invention does not include the full length of the rice waxy gene. In addition, each of the three isolated fragments had a partial difference in base sequence. This is due to the fact that point mutation is likely to occur during the polymerase reaction during PCR. However, by comparing these sequences, the sequence of the waxy gene existing in the original rice genome was easily deduced.
Therefore, this difference is due to site-specific substitution (Zoller and Smit
h;. Methods in Enzymol, fixes were made by 100.468 (1983)).

When a specific gene can be cloned, it can be used as an antisense gene by binding to an applicable promoter sequence using the reverse DNA base sequence. By introducing this into a plant, it is possible to express an antisense RNA, and thereby it becomes possible to control the expression of a specific gene, and there have been several experimental examples based on this (Smith et al .; Nature). , 334, 724 (1988),
Krol et al .; Gene, 72 , 45 (1988).

According to the present invention, the rice waxy gene successfully cloned according to the present invention is used, and by using the antisense method described above, it is ligated to the promoter sequence in the reverse direction to the original, and the antisense gene It can be. By introducing this antisense gene into a rice plant, antisense RNA is expressed, whereby the expression of the rice waxy gene can be controlled.

Therefore, for the first time according to the present invention, rice breeding with reduced or no amylose content of starch in rice grain has been made possible. Therefore, according to the present invention, the amylose content can be changed to an arbitrary value by changing the promoter sequence to be used.
It is possible to suppress the amylose content of the ultivar variety and produce high-quality rice with a low amylose content.

Of course, since various antisense genes can be prepared, various rice plants having different amylose contents can be bred by this method in combination with the selection of the promoter sequence described above.

After binding the antisense gene prepared according to the present invention to a plasmid, the antisense gene may be introduced into rice protoplasts by a conventional method such as electroporation, and the resulting cells may be regenerated into rice plants. In this way, it is possible to cultivate rice that does not produce amylose, and to freely control the amount of amylose produced.

Conversely, if desired, it is natural that rice having a high amylose content can be obtained by transforming rice using the rice waxy gene of the present invention.

The base sequences obtained in the present invention differ from each other in sequence at some points due to base substitution. This occurred accidentally when PCR was performed,
When an antisense gene to the rice waxy gene was generated, such a change did not pose any problem to this potency. Therefore, it is easily presumed that a partially modified sequence has the same effect. Furthermore, this fragment is very effective as a probe DNA used for cloning the whole rice waxy gene. In addition, by modifying a part of the waxy gene, it is re-introduced into a rice plant to enable the rice waxy gene to be remodeled.

 Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 (1) Synthesis of PCR primers The sequence of the rice waxy gene to be obtained in the present invention has not been reported yet. Therefore, a primer cannot be synthesized based on the sequence of rice waxy itself. However, the nucleotide sequences of the maize and barley waxy genes have already been reported and were available from the EMBL database (Release 20.0). Comparison of the two shows that there is no homology between maize and barley except for the exon portion encoding the protein sequence, and the exon portion has almost no homology in the signal peptide region or untranslated region. Was not seen. In other words, it was found that the portion having homology between both was limited to only the portion encoding the mature protein in the entire gene.

Therefore, a primer sequence was determined based on the barley sequence of a highly homologous portion between the two. The sequence is shown below.

These primer DNAs are DNAs from Applied Biosystems
It was prepared using a synthesizer.

Note that a certain degree of homology is required for normal function as a PCR primer. However, as a result of examining the homology between the corn and barley sequences, it was expected that the degree of matching of the synthesized primers to rice was not high, so in order to stochastically increase the possibility of matching, primers were used. Two types of each were synthesized, and each combination was subjected to a PCR reaction.

(2) Cloning of rice waxy gene The rice nuclear DNA used as material was prepared by the method of Rogers and Bedich (Plant
Mol. Biol., 5 , 69 (1985)). The plasmid pUC13 used in this example was purchased from Pharmacia. PCR (polymerase chain reaction) machine
The reaction system was from Elmer Cetus, and the reaction system was Innis and Gelfand
(PCR Protocols. Innis et al. (Ed.
s.), Academic Press, CA (1990), pp3-12).

PCR was performed using the PCR primers for rice and waxy fishing that were synthesized in the previous section. As a result, the synthesis reaction in the region between the primers proceeded, and amplification of a specific fragment could be recognized.

An attempt was made to insert the obtained fragment into the SmaI site of pUC13. The resulting fragment was subjected to ethanol precipitation twice to remove excess dNTPs and salts, and the ends were blunt-ended using T4 DNA polymerase.
Ligation was performed using the cut fragment and T4 DNA ligase. This reaction solution was introduced into Escherichia coli JM109, and the appearance of transformants that became ampicillin resistant was waited. Transformation of E. coli was carried out according to a standard method (Sambrook et al. (Eds.),
Molecular cloning, Section1.53, Cold Spring Harbor
Laboratory Press). As a result, a large number of transformants were obtained. Of these, three strains selected from among the transformants in which the fragment amplified between primers HVW-2 and HVW-6 were cloned showed an insertion corresponding to the length of the waxy fragment amplified by PCR. Was. The plasmids contained in these clones were named pWX12, pWX15, and pWX27.

In these plasmids, a multicloning site derived from the vector is present on both sides of the rice waxy gene, which is an insert fragment. These clones were subjected to mass culture and plasmid DNA was prepared.

(3) Analysis of nucleotide sequence The nucleotide sequence of pWX12, pWX15, and pWX27 was determined using denatured DNA prepared by alkali denaturation of plasmid DNA as a substrate. The nucleotide sequence was determined by the dideoxy method (Mess
ing, Methods in Enzymol., 101 , 20-78 (1983)). From FIG. 1 to FIG.
Shown in the figure. pWX12 and pWX27 were in the same orientation and pWX15 was in the opposite orientation. These nucleotide sequences differed from each other in several bases. This may be due to a substitution mutation generated during PCR. By combining these base sequences, the sequence of the waxy gene present in the rice genome (without base substitution) was estimated (FIG. 4).

Furthermore, when this sequence was compared with the base sequences of the maize and barley waxy genes, good homology was found between them, and it was confirmed that the obtained fragment was a partial sequence of the rice waxy gene. Was done. As a result of detailed nucleotide sequence comparison, high homology was observed in exons between rice and barley and maize genes, but homology in introns was low. This was consistent with the relationship between barley and corn.

(4) Modification of waxy sequence by site-specific substitution In order to obtain the nucleotide sequence of the rice waxy gene estimated in the preceding section, an attempt was made to correct the portion where the substitution mutation occurred. pWX12
In the case of, a substitution mutation from T to C has occurred in two places. Up to that point, this mutation was designated as the center, and this was designated as T, and 8 bases were synthesized on the 5 'and 3' sides, respectively. In addition, the sequence of pWX12 was
And transferred to the M13 vector. This was subjected to base substitution from C to T using the above-mentioned synthetic primer and a site-specific mutation kit (Mutan-G, manufactured by Takara Shuzo). The obtained sequence was confirmed to be the target sequence by the dideoxy method. The rice waxy gene thus obtained was again transformed into plasmid pUC13
Cloned. This was named pWX00 (fourth
Figure).

(5) Construction of antisense gene pWX12 was cut with EcoRI, and the cut end was blunt-ended using T4 DNA polymerase (DNA bluntin kit, manufactured by Takara Shuzo). Next, this was cut with XbaI to cut out the waxy region of rice. This is called pBl221 (CaMV
With 35S promoter; Jefferson et al .; EMBO j.,
6 , 3901 (1987)) and the fragment cut with XbaI and SmaI. pBI221 was obtained from Clontech. As a result of the ligation, an antisense waxy gene was created in which the waxy gene was reversely connected downstream of the 35S promoter. This was named pWXA23 (FIG. 5).

(6) Transformation of rice pWXA23 plasmid DNA was prepared by the method of Tada et al. (Tada et al .; T
According to heor Appl. Genet., in press (1990)), the cells were introduced into rice (Nipponbare) protoplast cells by electroporation. The experiment was performed twice, and the obtained cells were subjected to the method of Fujimura et al. (Fujimura et al .; Plant Tissue C
ulture Lett., 2 , 74 (1985)). DNA was extracted from these cells and subjected to Southern hybridization. As a result, a DNA fragment presumably derived from pWXA23 was present in 5 strains. Seeds were taken from these transformed plants, and the amylose content of the endosperm starch was determined by an iodine colorimetric method. The results are shown in the table below. Amylose content strain in endosperm starch obtained from transgenic plant Amylose content (%) Transformant strain No.1 10.5 Same No.2 8.8 Same No.3 12.5 Same No.4 10.9 Same No.5 9.6 Nihonbare (Ulchi) 22.5 Koganemochi 0.0 From these results, it was revealed that the transgenic plant into which pWXA23 was introduced had a low amylose content of endosperm starch, and proved to work effectively as an antisense gene.

(Effect of the Invention) For the first time, the present invention has elucidated the structure of the rice waxy gene and succeeded in cloning. In addition, the construction of the antisense gene and the transformation of rice using the same have also been successful.

Therefore, the amylose content can be reduced or eliminated by selecting the antisense gene and, if necessary, the promoter sequence, and the amylose content can be controlled freely. That is, according to the present invention, for example, it is possible to convert a ulti-rich high-yield variety to a stretchy high-yield variety, or to suppress the amylose content of a ulti-yield variety to produce high-quality rice. Thus, it has become possible to advantageously solve the problem described above.

Of course, conversely to the above, it is also possible to breed rice with a high amylose production amount by introducing the rice waxy gene into rice by binding to a promoter sequence without using it as an antisense gene.

[Brief description of the drawings]

1 to 4 show plasmids pWX12, pWX15, pWX27, and pWX0.
It is a rice waxy sequence inserted into 0 respectively. FIG. 5 is a construction diagram of the antisense waxy gene pWXA23.

────────────────────────────────────────────────── ─── Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00 A01H 5/00 C12N 5/00 GenBank / EMBL / DDBJ

Claims (11)

(57) [Claims] 1. A DNA fragment comprising a rice-derived waxy gene DNA fragment comprising a DNA defined by the nucleotide sequence shown below. 2. A DNA fragment comprising a rice-derived waxy gene DNA fragment comprising a DNA defined by the nucleotide sequence shown below, wherein the reverse strand of the DNA fragment is subjected to antisense method to rice plant. A DNA fragment that can control the expression of a rice waxy gene when introduced into the body. 3. A reverse-strand DNA of the DNA fragment according to claim 2.
DNA fragments. 4. A double-stranded DNA having the nucleotide sequence according to claim 2 or 3. 5. A hybrid DNA obtained by incorporating the DNA according to claim 4 into a vector capable of stably existing in plant cells and / or microorganisms. 6. A hybrid DNA obtained by binding the DNA according to claim 4 to a promoter sequence of a gene that can be expressed in a plant cell. 7. A transformed rice cell obtained by introducing the hybrid DNA according to claim 6 into a host rice cell. A transformed rice regenerated from the transformed rice cell according to claim 7. [9] A method for producing RNA derived from the DNA according to [4] in a rice cell using the transformed rice cell according to [7]. 10. A method for controlling the expression level of a rice waxy gene using the method according to claim 9. 11. A method for changing the amylose content in endosperm starch of rice grain using the method according to claim 9.