JP2810291B2 - Dicotyledonous plants having a peroxidase structural gene derived from horseradish and excellent in growth, and a method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dicotyledon having a trait excellent in growth and having a dicotyledon plant cell into which a chimeric vector having a peroxidase structural gene derived from horseradish and a foreign promoter that can be expressed in a plant cell is introduced. It is intended to provide a plant and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, as a method for genetically improving the growth of a plant, a method by crossing and a method by mutation have been generally performed. Of these, the method by mating is
A method of selecting a good individuals by mating the growth of good pieces body. The mutation method is a method in which the chromosomal DNA of a cell is changed by radiation, ultraviolet light, a chemical substance, or the like.

[0003] However, these methods have a drawback that the probability of obtaining excellent traits in a stable manner is extremely low because all of these methods are derived from chance in nature. In addition, the development of genetic recombination technology in recent years has made it possible to impart traits more directly. That is, a foreign gene is linked under the control of a promoter capable of expressing a trait,
This is a method for obtaining genetically stable plant individuals by directly introducing them into plant cells.

[0004] On the other hand, plant peroxidase is a kind of glycoprotein and has many isozymes (enzymes having the same function and different genes) and isoforms (although the genes are the same, processing after transcription and translation, It is generally known that the enzyme is widely involved in removal of hydrogen peroxide, lignin biosynthesis, auxin metabolism in plant hormones, and the like.

Accordingly, the present inventors have constructed a chimeric vector having a horseradish-derived peroxidase structural gene and a foreign promoter that can be expressed in plant cells by using this gene recombination technique. The present inventors have found that it is possible to obtain a plant having a trait with excellent growth properties by regenerating the obtained dicotyledon plant cells into a plant body, and reached the present invention.

[0006]

Accordingly, an object of the present invention is to construct a chimeric vector having a horseradish-derived peroxidase structural gene and a foreign promoter that can be expressed in plant cells, and introduce the chimeric vector into dicotyledonous plants. Another object of the present invention is to obtain a dicotyledon having excellent growth characteristics by regenerating the obtained dicotyledon plant cells into a plant. That is, an object of the present invention is to impart a useful trait of rapid growth to dicotyledonous plants efficiently transformed by genetic manipulation.

[0007]

SUMMARY OF THE INVENTION The above objects of the present invention are as follows.
Construct a chimeric vector having a peroxidase structural gene derived from horseradish and a foreign promoter that can be expressed in plant cells, introducing the chimeric vector into dicotyledonous plants,
This was achieved by regenerating the resulting dicotyledonous plant cells into plants, thereby producing a plant having genetically modified dicotyledonous plant cells.

The dicotyledon used in the present invention is a dicotyledon having a plant cell which can be genetically modified by introducing and regenerating a horseradish peroxidase prxCla gene under the control of a promoter that can be expressed by genetic manipulation. Any kind of woody plant can be used, but woody plants include evergreen broad-leaved trees such as eucalyptus, acacia, coffee, etc. , Grapes, oysters, and fruit trees such as papaya. Examples of herbaceous plants include tobacco, Arabidopsis, kenaf, potato, sweet potato, morning glory, melon, eggplant, carrot, rapeseed, cotton, and the like.

The peroxidase structural gene derived from horseradish to be introduced into dicotyledonous plant cells is disclosed in Japanese Patent Application Laid-Open No. 63-207386 by one of the applicants of the present invention.
Use the prxCla structural gene. These horseradish-derived peroxidase structural genes are ligated under the control of a promoter that can be expressed in dicotyledonous plants, and introduced into dicotyledonous plant cells by Agrobacterium infection method or particle gun method. By regenerating the above, a dicotyledonous plant, a dicotyledonous plant tissue or a dicotyledonous seed, which is the object of the present invention, can be obtained. Hereinafter, the transformed dicotyledonous plant, dicotyledonous plant tissue or dicotyledonous seed according to the present invention and a method for producing the same will be described in detail.

(Plasmid used in the present invention) The plasmid used in the present invention includes a horseradish peroxidase prxCla structural gene, a promoter that functions in dicotyledonous plants, a foreign gene, and a terminator that functions in dicotyledonous plants. is there. As the promoter, a promoter derived from a cauliflower mosaic virus (hereinafter abbreviated as CaMV) 35S or 19S RNA gene, a horseradish peroxidase prxCla gene promoter, a promoter derived from a nopaline synthase (hereinafter abbreviated as NOS) gene, and the like are used. As the gene, kanamycin resistance enzyme gene (NPTII), β-glucuronidase gene, hygromycin phosphotransferase gene, chloramphenicol acetyltransferase gene and the like can be used. In addition, as the terminator, a terminator derived from a nopaline synthesis gene or the like can be used.

The chimeric gene that can be expressed in dicotyledonous plant cells according to the present invention is composed of two gene expression units, one of which has a horseradish-derived peroxidase gene prxCla in the structural gene portion and the other has One is used as an effective selectable marker in obtaining a desired transformed plant. As a selectable marker gene effective for obtaining a transformed plant, a kanamycin resistance enzyme gene (NPTII), a hygromycin phosphotransferase gene and the like are preferable.

Next, the transformation method of the present invention will be described. (Method of Transformation in the Present Invention) Examples of the method of transformation in the present invention include a method of infecting dicotyledonous plant tissues with Agrobacterium having the above-mentioned plasmid containing the target gene, and a method of subjecting protoplasts to electric pulse treatment to obtain a plasmid. Or a particle gun method in which a plasmid is placed on gold particles and introduced into a dicot plant tissue.

(Culture Method of Transformed Dicotyledon Plant Cells and Selection of Transformants) The culture of the dicotyledon plant cells transformed by the above method is carried out by using a Gamborg B5 medium or Murashige Skoog MS.
As plant hormones in a medium or the like, naphthaleneacetic acid (hereinafter abbreviated as NAA), 2,4-dichlorophenoxyacetic acid (hereinafter abbreviated as 2,4-D), indoleacetic acid (hereinafter abbreviated as IAA), indolebutyric acid (hereinafter referred to as IBA) (Abbreviation) and cytokinins such as benzyladenine (hereinafter abbreviated as BA), kinetin and zeatin. In addition, for the selection of the transformant, an antibiotic such as kanamycin or hygromycin is previously contained in the medium. The culture of the transformed dicotyledonous plant cells was performed at a temperature of 20 to 26 ° C. and an illuminance of 2000 to 3000.
After about 30 days under lux conditions, the resulting transformed dicotyledonous plants are cultured and selected.

As described in the present invention, a chimeric vector having a horseradish-derived peroxidase structural gene and a foreign promoter that can be expressed in plant cells is constructed, and the chimeric vector is introduced into dicotyledonous plant cells. A dicotyledon obtained by regenerating cells into a plant has a characteristic that it is superior in growth to a genetically stable wild type. The mechanism by which horseradish-derived peroxidase gene prxCla contributes to the growth of dicotyledonous plants is not clear.
It is speculated that the prxCla gene product is involved in the auxin metabolism of plant hormones involved in the growth of dicotyledonous plants. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0015]

【Example】

(Test Plant) As a test dicotyledon, tobacco (Nicotianatabacum) whose transformation system was established was used.

[0016] CaMV35S and Western as a promoter (acquisition transformants) horseradish peroxidase p r XCL a structural gene (JP 63-2
No. 07386 discloses a horseradish-derived peroxidase structural gene) and downstream thereof a structural gene of the horseradish peroxidase gene prxCla and a NOS-derived terminator as a terminator (FIG. 1), and a kanamycin-resistant enzyme as a selection marker The gene was used under the control of NOS by the promoter and terminator. These two types of chimeric vectors were used as pBI binary vectors (Jefferson et al. EM).
BOJ. 6: 3901-3907), and the obtained DNA sequence was introduced into aseptically grown tobacco leaf pieces by the Agrobacterium infection method.

The leaf pieces of the tobacco transformed by the above method are kept in an MS medium containing 1 mg / l NAA and 0.01 mg / l IBA for about 3 days, and then 500 mg / l for the purpose of removing Agrobacterium. Was transferred to an MS medium containing 100 mg / l kanamycin for selection of transformants, 100 mg / l kanamycin, the above two antibiotics, and 0.1 mg / l NAA and 1 mg / l IBA, to differentiate foliage. After culturing for about 4 weeks, the differentiated individual of the foliage was transferred to an MS medium containing no plant hormone, and rooted. In addition,
The above culture was carried out at 25 ° C. for 16 hours in a bright place / 8 hours in a dark place. The rooted plant was a No. 5 pot (150 × 160 m) containing peat moss / vermiculite (3/1).
m).

(Confirmation of Transformant) From the transformed tobacco obtained above, DNA was extracted by a conventional method, and the transgene was confirmed by Southern blotting, and the peroxidase activity of the transformant was measured. The results are shown in Table 1. That is, Table 1 below shows the peroxidase activity of the obtained transformants and the copy number of the transgene confirmed by Southern blotting.

[0019]

[Table 1]

As is clear from the results, the transformant was found to exhibit 2 to 10 times the peroxidase activity as compared to the wild type, and to have 1 to 3 copies of the transgene prxCla. In the table, the individual 35S-8, -1
4, -15, -18, -20 are transformants having 35S promoter, individuals C2-10, -12, -20 are:
A transformant having the C2 promoter, individual W-38,
It is wild type.

(Growth of Transformants) Of these transgenic tobaccos, self-pollinated 35S-20 seeds were harvested and grown in a greenhouse for about 3 months. The result of comparing the individual with the wild type is shown in FIG.
As is evident from these results, it was found that tobacco transformed by introducing and regenerating the horseradish peroxidase gene prxCla into cells had significantly increased growth compared to the wild type. did.

(Growth of the transformant and the introduced gene p
(Expression amount of rxCla) Eighteen individuals (Nos. 1, 2, 8, 9, 15, 20, 23, and 48) of the second-generation transformant 35S-20 were randomly selected, and RNA was extracted from each individual. FIG. 3 shows the results of investigating the expression level of the trait by the Northern plotting method.
It was shown to. As is clear from the results, the individual No. Since the expression level was large in 9, 15, and 48, it was found that the introduced horseradish peroxidase gene prxCla greatly contributed to the growth of plants. Note that, in FIG. No band showing the expression level is clearly seen in FIG.
o. 9 or No. Although it is thinner than 48, it appears.

[0023]

As described above, a chimeric vector having a horseradish-derived peroxidase structural gene and a foreign promoter that can be expressed in dicotyledonous plant cells is constructed, and the chimeric vector is introduced into dicotyledonous plant cells. Dicotyledonous plants having genetically modified plant cells obtained by regeneration have excellent growth,
It is extremely useful as a means for promoting the growth of cereals, crops, fruit trees, forest trees, and the like. Needless to say, the dicotyledonous plants transformed by the method of the present invention can also be propagated by a method of propagation such as translucent, cutting and cutting.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing the structure of a chimeric vector containing a horseradish-derived structural gene in the present invention.

FIG. 2 shows a second-generation transformed individual 35S in an example.
It is a figure which compared the growth of -20 and a wild type.

FIG. 3 is a diagram showing the relationship between the growth of tobacco transformed in Examples and the expression level of a horseradish-derived structural gene.

────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Kazuya Yoshida 3-21-11-602 Nishi Awomatanani, Minoh City, Osaka (72) Inventor Masami Sekine 4-8-20-401 (72) Yamada Higashi, Suita City, Osaka Prefecture ) Inventor Mitsuo Takano 3-7A-39-207, Shinsenri-Higashi-cho, Toyonaka-shi, Osaka (72) Inventor Atsushi Niname 7-1-511, Maydai, Takarazuka-shi, Hyogo (56) References SCI ENCE, 117-6! , (1992), P. 1012-1016 PLANT CELL, 2-1! , (1990), p. 7-18 PLANT AND CELL PH YSIOLOGY, 33-8! , (1992), p. 1143-1150 PLANT CELL REPORT S, 13-3-4! , (1994), p. 149 -154 (58) Fields investigated (Int. Cl. ⁶ , DB name) A01H 1/00-5/00 BIOSIS (DIALOG) JICST file (JOIS)

Claims (4)

(57) [Claims] 1. A raw with dicotyledonous cells transfected chimeric vector having an expressible exogenous promoter peroxidase structural gene derived from horseradish and plant cells
Dicotyledonous plant with excellent length . 2. A plant having excellent growth regenerated from a plant tissue having dicotyledonous plant cells into which a chimeric vector having a peroxidase structural gene derived from horseradish and a foreign promoter that can be expressed in plant cells has been introduced. 3. A live with dicotyledonous cells transfected chimeric vector having an expressible exogenous promoter peroxidase structural gene derived from horseradish and plant cells
Plant seeds with excellent length . (4) constructing a chimeric vector having a horseradish-derived peroxidase structural gene and a foreign promoter that can be expressed in plant cells; (b) introducing the chimeric vector into a dicotyledonous plant; A method for producing a dicotyledon having excellent growth and having a horseradish-derived peroxidase structural gene, wherein the dicotyledon cell obtained is regenerated into a plant.