Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-30
2002-09-17
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S005000, C530S350000, C530S501000
Reexamination Certificate
active
06451537
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a gene capable of encoding the dihydrodipicolinate synthase of rice (
Oryza sativa
), and also to a DNA which relates to said gene. More specifically, this invention relates to novel DNAs for encoding such dihydrodipicolinate synthase of rice which participates in the lysine biosynthetic pathway in rice plant. Additionally, the invention relates to DNAs for encoding novel proteins having the activity of the dihydrodipicolinate synthase. Still additionally, the invention encompasses
Escherichia coli
as transformed by introduction of said novel DNA, as well as such transgenic plants as transformed by said novel DNA, and the seeds of such transgenic plants.
Still more additionally, this invention encompasses novel recombinant vectors in which said novel DNA has been inserted.
BACKGROUND ART
The grain seeds of cereal plants such as rice, corn and wheat are important nutritious sources for humans, cattle and others. However, these grain seeds are nutritionally of a more or less poor value, because of their low contents of lysine as one of the essential amino acids. It has been desired to create a new variety of plant which is capable of generating a cereal seed with a high lysine content and of a high nutritional value.
It has been known that, in a diaminopimelic acid-producing pathway of the lysine biosynthetic pathway in plants, a condensation binding takes place between aspartic acid-&bgr;-semialdehyde and pyruvic acid under the action of the dihydrodipicolinate synthase (sometimes abbreviated as “DHDPS” hereinafter) to produce 2,3-dihydropicolinic acid, from which diaminopimelic acid is then produced by enzymatic reactions through 5 steps, and that lysine is produced from the diaminopimelic acid as produced (Experimental Lecture Series of Biochemistry, Vol. 11, pp. 511-517, Tokyo Chemical Dojin). It is known that DHDPS is an enzyme protein having an activity to produce 2,3-dihydropicolinic acid by involving the condensation binding between aspartic acid-&bgr;-semialdehyde and pyruvic acid.
A report tells that the lysine content in the seed of a useful plant such as corn (
Zea mays
), tobacco (
Nicotiana tabacum
), rape seed (
Brassica campesteris
) and soybean (
Glycine max
) can be enhanced by introduction of the gene for encoding DHDPS, that is, the DHDPS gene, into the plant and then expressing the function of said gene in the transformed plant (PCT International Publication WO 95/15392, JP-W-7-504821, and “Biotechnology”, Vol. 13, pp. 577-582, 1995).
The DHDPS genes have been isolated already from the plants of wheat, corn, soybean and tobacco. The nucleotide sequences of the DNA of these DHDPS genes have been elucidated. For example, the following techniques are reported in a number of literatures.
(1) The analysis of the amino acid sequence of wheat DHDPS, and the isolation of the DHDPS gene from wheat (JP-A-3-127984).
(2) The isolation of DHDPS gene from corn, and the nucleotide sequencing of the DNA of said gene (Molecular & General Genetics, Vol. 228, pp. 287-293, 1991).
(3) Transformation of corn by means of a DNA as obtained by a modification of the DNA of the DHDPS gene of corn, and the over-production of the lysine content in the corn seed (U.S. Pat. No. 5,545,545).
(4) A DNA as obtained by such a modification of the DNA of the tobacco DHDPS gene that a transgenic plant as transformed by the modified DNA is made free from the sensitivity of the DNA-encoded DHDPS to the feed-back inhibition by lysine, so that the lysine content in the transgenic plant can be over-produced (The Plant Journal, Vol. 8, No. 5, pp. 733-743, 1995).
(5) Soybean DHDPS gene (Plant Molecular Biology, Vol. 26, pp. 989-993, 1994).
(6) A method for over-producing the lysine content in a plant seed, which comprises transformation of the plant with using a DHDPS gene as derived from a bacterial species, for example,
Escherichia coli
(European Patent Application Publication No. 0 485 970 A2).
To the best of the knowledge of the present inventors, no literatures have been known, which report an analysis of the amino acid sequence of the rice DHDPS or which report a recovery of the gene of the rice DHDPS or the utilization of the rice DHDPS gene.
It is an object of this invention to provide the rice DHDPS gene which is produced from the rice plant. It is another object of this invention to provide several novel DNAs for encoding a protein having the rice DHDPS activity. It is still another object of this invention to transform useful plants such as corn, rice, soybean, wheat and barley by using said novel DNA for encoding a protein having the DHDPS activity, and to provide such a novel transgenic variety of a useful plant which has gained an ability to generate a seed of a high lysine content.
Any further objects of this invention will be apparent in the following descriptions.
DISCLOSURE OF THE INVENTION
In order to attain the above described various objects, the present inventors have now made a series of investigations. A first investigation has been carried out in order to produce the DHDPS gene from the rice plant. To obtain consequence of the investigations, the present inventors have conducted some experiments wherein the total RNA was extracted from a young rice plant, for example, disrupted green stem and leave, by a method known in the genetic engineering technology; and wherein from the so extracted total RNA was then isolated mRNA by a conventional method; and wherein from said mRNA were successfully produced cDNAs by a commercially available cDNA synthesis kit. It has now been found from the results of a great number of the inventor's empirical experiments that, when the resulting cDNAs mentioned above are conjugated to a such phage vector which had been prepared by treating the EcoRI cleavage end of a DNA fragment of a phage vector &lgr;gt11 (commercially available from STRATAGEN, LTD.) with a calf small intestine-derived alkaline phosphatase, there can be produced replicable recombinant &lgr; phages.
It has now further been found that when
Escherichia coli
Y1088 strain is infected with said recombinant &lgr; phages and then incubated, there can be produced a large number of recombinant &lgr; phages in the forms of numerous plaques comprising the lysogenized bacteria cells; and that the numerous recombinant &lgr; phages present in the resulting numerous plaques are composed of a wide variety of the phages containing therein the aforesaid rice-derived cDNAs and can thus be utilized as a cDNA library of rice.
With reference to the amino acid sequence of the wheat DHDPS protein as described in JP-A-3-127984 and also to one speculative nucleotide sequence of the wheat DHDPS gene, as well as to the nucleotide sequence of the corn DHDPS gene as described in the literature “Molecular & General Genetics”, Vol. 228, pp. 287-293 (1991), the present inventors have now chemically synthesized a first oligonucleotide comprising 25 nucleotides, as well as a second oligonucleotide comprising 24 nucleotides, which are both believed to be appropriate to be used as primers for PCR method.
When a mixture of the aforesaid rice cDNA library (namely, the above-mentioned recombinant &lgr; phages) with the first oligonucleotide and the second oligonucleotide as above has been used to conduct amplification of DNA by PCR method, it has now been found that the first and second oligo-nucleotides can serve as the primers (the complementary DNA) which to be required for the PCR method, and that the cDNAs present in the rice cDNA library can serve as the template, so that a part of the rice DHDPS gene-derived cDNA can be amplified. The present inventors can have then successfully produced an amplified product of said part of the rice DHDPS gene-derived cDNA, as a probe DNA, from the resulting PCR amplification mixture.
The present inventors have used the so produced probe DNA as a screening material to carry out the phage plaque hybridization method, and then there has been got a success fortunately to isolate such four plaques comprising the recombin
Hasegawa Hisakazu
Terakawa Teruhiko
Yamaguchi Masanori
Hokko Chemical Industry Co. Ltd.
Jones W. Gary
Larson & Taylor PLC
Taylor Janell E.
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