JPH06343473A - Production of plant resistant to bleaching herbicide - Google Patents

Abstract

PURPOSE:To impart a plant with resistance to a herbicide inhibiting zeta-carotene desaturase of a plant as well as to a herbicide inhibiting phytoene desaturase of a plant and to enable the use of these herbicides as a marker in transformation. CONSTITUTION:A plant resistant to a herbicide for inhibiting zeta-carotene desaturase is produced by preparing a DNA sequence coding a polypeptide (phytoene desaturase) having an enzymatic activity to convert phytoene through zeta-carotene to lycopene and introducing the DNA sequence into a plant in a state to enable the manifestation of the genetic information. A marker for the confirmation of the transformation to the herbicide inhibiting zeta-carotene desaturase is composed of a DNA sequence coding a polypeptide having an enzymatic activity to convert phytoene to lycopene.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

The present invention relates to relates to a non-photosynthetic bacteria and is Erwinia (Erwinia) manufacturing technology of bleaching herbicide-resistant plants using the phytoene desaturase (crtI) gene from. More specifically, the present invention provides not only herbicides that inhibit plant phytoene desaturases such as norflurazone and fluridone, but also resistance to herbicides that inhibit plant ζ-carotene desaturases such as SAN 380H and J852. The present invention relates to a method for producing a plant having the same, and use of a phytoene desaturase (crtI) gene derived from Erwinia as a marker gene in plant transformation.

[0002]

2. Description of the Related Art Carotenoid
Is a yellow to orange to red pigment, and is the pigment most widely present in nature. For example, many fish, birds, insects, and other aquatic animals have a beautiful color, but most of these are due to carotenoid pigments (Matsuno, M., Carotenoid physiology in animals). Function and Biological Activity, Chemistry and Biology, Vol.28, No.4
p. 219-227 (1990)). Carotenoids also play important physiological roles in many organisms. In plants and photosynthetic microorganisms, carotenoids are auxiliary pigments for photosynthesis and also have a function of protecting tissues and cells from photooxidative damage (Rau, W. "Functionof caroteno
oids other than in photosynthesis ". Plant Pigment
s. London, Academic Press, 1988, p.231-255. (Goodw
in, TW ed.))). In animals, it has been known that it is a precursor of vitamin A, as well as having a preventive effect and a growth inhibitory effect against various cancers, and is attracting attention as a health food (Kazuo Sueki, β-carotene-Understanding) Bioactivity, Food and Development, Vol.24, No.11, p.6
1-65 (1990)). Some microorganisms that do not perform photosynthesis have carotenoid pigments,
It is presumed that this may have a role of protecting cells from photooxidation reaction. Carotenoids are synthesized from mevalonic acid as a starting material by an isoprenoid biosynthetic pathway that is partially common with steroids and terpenoids. C ₁₅ farnesyl pyrophosphate (EPP) generated by the isoprene-based biosynthesis system is condensed with C ₅ isopentenyl pyrophosphate (IPP) to form C ₂₀ geranylgeranyl pyrophosphate (GGPP).
Next, two molecules of GGPP are condensed to synthesize the first carotenoid, colorless phytoene.
Phytoene undergoes a series of unsaturated reactions to produce phytofluene, ζ-carotene,
Neurosporene, lycopene (lycopen)
e). This lycopene is β-
It is considered that carotenoids that are converted to carotene (β-carotene) and further introduced with hydroxyl groups and keto groups will synthesize various carotenoids called xanthophylls (Br
itton, G. "Biosynthesis of carotenooids". Plant Pi
gments. London, Academic Press, 1988, p.133-182.
(See Goodwin, TW ed.)). As mentioned above, although carotenoids have important biological functions,
Until recently, knowledge of genes and enzymes responsible for carotenoid biosynthesis was very limited. Since the enzyme involved in carotenoid biosynthesis was an unstable membrane enzyme for purification, it was impossible to analyze the function and structure of the enzyme, and as a result, cloning of the gene encoding it was impossible. Because I couldn't. Recently, we have found that the plant-resident bacterium Erwinia uredovo
carotenoid biosynthesis genes of ra, was cloned into E. coli The color tone index, the unique approach of various combinations of these genes were expressed in E. coli, analyzing the carotenoid pigments accumulated in E. coli, E
r. The biosynthetic pathway of uredovora carotenoids was elucidated for the first time in the world at the gene and enzyme level.
As a result, Er. The uredovora carotenoid biosynthetic pathway is phytoene, β-carotene, lycopene,
It was clarified that it is a biosynthetic pathway of basic carotenoids such as zeaxanthin (Misawa, N .; Nakagawa,
M .; Kobayashi, K .; Yamano, S .; Izawa, Y .; Nakamura.
K.; Harashima k. Elucidation of the Erwiniauredovor
a carotenoid biosynthetic pathway by functional an
alysis of gene products expressed in Escherichia c
oli. Journal of Bacteriology, Vol.172, No.12, p.67
04-6712 (1992) and Japanese Patent Application No. 3-58786 (Japanese Patent Application No. 2-53255) by the present inventors: "DNA chain useful for synthesis of carotenoid").
Then Ausch et al., E of the same Erwinia genus
The carotenoid biosynthetic pathway of rwinia herbicola is Er. It has been clarified that it is the same as that of uredovora (see WO91 / 13078). The present inventors have also reported that Er. uredover
By using carotenoid biosynthesis genes of a, E. (Escherichia coli), ethanol producing bacteria Zymomonas Mobili
s , phytopathogenic bacterium Agrobacterium tum
It has made it possible for microorganisms such as efaciens to produce basic carotenoids such as phytoene, β-carotene, lycopene and zeaxanthin (Misawa, N .; Yaman
o, S .; Ikenaga, H. Production of β‐carotene in Z
ymomonas mobilis and Agrobacterium tumefaciens by
introduction of the biosynthesis genes from Erwini
a uredovora. Applied and Environmental Microbiolog
y, Vol.57, No.6, p.1847-1849 (1991), and J above
ournal of Bacteriology, and the above-mentioned patent application by the present inventors). Er. The above-mentioned technique of synthesizing and analyzing these carotenoids in Escherichia coli by utilizing the carotenoid biosynthesis genes of uredovora is also used in the most advanced E. coli gene manipulation experiment system for carotenoid biosynthesis research. Meaning applicable. This is due to the instability of biosynthetic enzymes,
This means that it is possible to carry out a study of carotenoid biosynthesis at the level of genes and enzymes, which has not proceeded so slowly in organisms such as plants. For example, the following is also an example. Er. ur
E. coli carrying the edovora crtE and crtB genes are known to synthesize phytoene,
Photosynthetic bacterium Rhodobacter capsulat
us and cyanobacteria Synechococcu
In s PCC7942, the use of the mutant strain allows the phytoene desaturase (phytoene d)
The gene that is thought to code for the easeurase) has been obtained. The substrate of the gene product of the former microorganism is phytoene, but the reaction product has not been revealed. In the latter microorganism, neither the substrate nor the reaction product was revealed. Linden et al. Introduced these genes into Escherichia coli harboring crtE and crtB and analyzed the synthesized carotenoids by HPLC to analyze the phytoene desaturase (crtI) of photosynthetic bacteria.
It was found that the gene product CrtI) synthesizes neurosporene, and the cyanobacterial phytoene desaturase (Pds) synthesizes ζ-carotene using phytoene as a substrate (Linden, H .; Misawa, N.; Chamovitz,
D .; Pecker, I; Hirschberg, J .; Sandmann, G. Functi
onal complementation in Escherichia coli of differ
ent Phytoene desaturase genes and analysis of accum
ulated carotenes. Z. Naturforsch., Vol. 46c, p. 104
5-1051 (1991)). Also, Synechococc
Using the above system, it was revealed that the phytoene desaturase gene (pds) of tomato fruit obtained by using the phytoene desaturase gene of us PCC7942 as a clue also synthesizes ζ-carotene using phytoene as a substrate (Pecker , I .; Chamovitz, D .; Linde
n, H .; Sandmann, G .; Hirschberg. J. A Single polyp
eptide catalyzing the conversion of phytoene to ζ
‐Carotene is transcriptinally regulated during to
mato fruit ripning. Proc. Natl. Sci. USA, Vol.89,
p.4962-4966 (1992)). Since plant phytoene desaturase can synthesize only ζ-carotene using phytoene as a substrate, there should be another enzyme (ζ-carotene desaturase) that synthesizes lycopene from ζ-carotene in the plant. Linden et al . Have described the gene (zds) encoding this enzyme in Er. ured
The ovora carotenoid biosynthesis gene was used to clone from the cyanobacteria Anabaena PCC7120, and it was revealed that this gene product catalyzes the reaction of lycopene synthesis from ζ-carotene (Linden, H .; Vioque, A. Sandmann, G. Isolation of
a carotenoid biosynthesis gene coding for ζ‐car
otene desaturase from Anabaena PCC7120 by heterolo
gous complementation. FEMS Microbiol. Lett., Vol.
106, p.99-103 (1993)). However, the nucleotide sequence of this gene has not been reported.

[Outline of Invention]

FIG. 1 shows the biosynthetic pathway to β-carotene in plants and cyanobacteria and Erwinia at the gene and enzyme (gene product) level, including the above description. It is a compilation of the findings revealed so far. GGPP synthase and phytoene synthase show exactly the same function between plants (or cyanobacteria) and Erwinia (Sandmann, G .; Misawa, N. New functional assign
ment of the carotenogenic genes crtB and crtE with
constructs of these genes from Erwinia species. F
EMS Microbiology Letters, Vol.90, p.253-258 (1992)
(See also), even at the amino acid sequence level, a significant homology of about 30% identity was shown. Therefore, GGPP synthase and phytoene synthase are
It seems to be well preserved across species. On the other hand, in phytoene desaturase, the functions are different between plants (or cyanobacteria) and Erwinia , that is, the former phytoene desaturase can synthesize only ζ-carotene using phytoene as a substrate, whereas the latter The phytoene desaturase CrtI of can also synthesize lycopene using phytoene as a substrate. Even at the amino acid sequence level, no homology is found in both phytoene desaturases except for some N-terminal regions. In addition, as described above, the functions of both phytoene desaturases are different from those of photosynthetic bacteria, and thus it is considered that the present enzyme has acquired diversity. Plant (or cyanobacteria)
The phytoene desaturase of norflurazone (norf
It is the site of action of herbicides called bleaching herbicides such as lurazon) and fluridon (Sandmann,
G; Boger, P. "Inhibition of carotenoid biosynthesis
by herbicides ". Target Sites of Herbicide Action.
Boca Raton, FL: CRC Press, 1989, p.25-44. (Boger,
P .; Sandmann, G. eds)). Norflurazone and fluridone are non-competitive inhibitors (non-co) that directly attack the enzyme.
It is known to be a mpetitive inhibitor). E
The rwinia phytoene desaturase CrtI is
As described above, it differs from that of plants (or cyanobacteria) in function and structure, and thus it can be expected to be resistant to these bleaching herbicides. In fact, Auschich et al., Erwinia h
erbicola phytoene desaturase gene c
0.8 μ of rtI was introduced into tobacco by adding a transit peptide sequence required for chloroplast translocation.
It is described that a tobacco transformant that grows normally in a medium containing g / ml (2.6 μM) of norflurazone was obtained (see WO 91/13078). However, in this publication, in a tobacco transformant that normally grows in a medium containing norflurazone, Er. herbico
The phytoene desaturase gene crtI of la is integrated into the tobacco chromosome and is transcribed and translated, and the synthesized crtI gene product is transported to the chloroplast of tobacco leaves, and the transit peptide is processed. Since it was not confirmed experimentally, it is possible that the tobacco considered to be a transformant was actually a norflurazone naturally resistant strain by acclimation or acclimation. In fact, the present inventors have experimentally confirmed that a medium containing norflurazone at a concentration of 3 μM may allow tobacco in vitro plants to grow normally. On the other hand, bleaching herbicides such as SAN 380H and J852 are known to be herbicides that inhibit the desaturation (dehydrogenation) reaction from ζ-carotene to lycopene (Boger, P .; Sandmann, G . "Mod
ern herbicides affecting typical plant processes ".
Chemistry of Plant Protection. Vol. 6. SpringerPu
bl., 1990, p.173-216. (Bowers, WS; Ebing, W., Ma
rtin, D., Wegler, R. eds)). in viv
SAN380H or J8 for both o and in vitro
Since plants treated with 52 become accumulating ζ-carotene, they are considered to be herbicides that directly attack the ζ-carotene desaturase enzyme. Erw against herbicides inhibiting ζ-carotene desaturase
Regarding the resistance of the plant into which the nia phytoene desaturase crtI has been introduced, as described above, recently,
Finally, it is a gene encoding ζ- carotene desaturase from the cyanobacterium just been cloned, and that the amino acid sequence has not yet been apparent to, since absolutely unprecedented research example of this resistance is currently the Erwinia It was the present situation that it was completely unknown whether or not a plant could be given resistance to a ζ-carotene desaturase inhibitor using the crtI gene. In view of the above points, it is an object of the present invention to provide a technique for producing a plant having resistance to a bleaching herbicide.

[0004]

The present inventors Means for Solving the Problems], by utilizing the phytoene desaturase of non-photosynthetic bacterium Erwinia (Erwinia) (crtI) gene,
SAN 3 as well as herbicides that inhibit plant phytoene desaturases such as norflurazone and fluridone
It has been found that the plants can be made tolerant to herbicides which inhibit ζ-carotene desaturase in plants such as 80H and J852, and the present invention has been completed based on this finding. That is, the method for producing a resistant plant to a ζ-carotene desaturase-inhibiting herbicide according to the present invention is
It is characterized in that a DNA sequence encoding a polypeptide having an enzymatic activity for converting phytoene into lycopene is introduced into a plant in a state in which its genetic information can be expressed. A plant resistant to a ζ-carotene desaturase-inhibiting herbicide obtained by the above-mentioned production method is introduced with a DNA sequence encoding a polypeptide having an enzyme activity for converting phytoene into lycopene, and has the ability to produce a polypeptide having the enzyme activity. It is characterized by having. Furthermore, the present invention provides the use of the above DNA sequence as a marker gene in plant transformation, that is, a ζ-carotene desaturase-inhibiting herbicide consisting of a DNA sequence encoding a polypeptide having an enzymatic activity for converting phytoene into lycopene. It also relates to a marker for confirming transformation of the agent.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a polypeptide having an enzymatic activity for converting phytoene into lycopene, that is, phytoene desaturase (hereinafter, crt).
Not only a herbicide that inhibits a plant phytoene desaturase but also a herbicide that inhibits a plant ζ-carotene desaturase by utilizing a DNA sequence encoding the same (also referred to as I) (hereinafter, also referred to as a phytoene desaturase gene or a crtI gene) It is intended to provide a technique for producing a plant having resistance to an agent.

For ζ-carotene desaturase inhibitor herbicide
Tolerant plants for the production of resistant plants against ζ-carotene desaturase-inhibiting herbicide in the present invention, a DNA sequence encoding a polypeptide having an enzymatic activity for converting phytoene into lycopene (phytoene desaturase gene) is introduced, The present invention is characterized by having the ability to produce a polypeptide (phytoene desaturase) having such a plant, and such a plant encodes a polypeptide having an enzymatic activity of converting phytoene into lycopene by the production method of the present invention. A DNA sequence (phytoene desaturase gene) which is introduced into the plant in a state in which its genetic information can be expressed can be produced by a method. The plant to which the phytoene desaturase gene is introduced may be any higher plant that performs photosynthesis and is not particularly limited, but specifically, cereals or tobacco, petunia, potato, etc., preferably rice, corn, wheat. , Such as barley. The above-mentioned DNA sequence (phytoene desaturase gene) may correspond to any amino acid sequence as long as the encoded polypeptide has an enzymatic activity for converting phytoene into lycopene, but a typical example thereof is non-photosynthesis. Bacteria Erwinia ( Erw
The DNA sequence of the phytoene desaturase gene (crtI gene) derived from the genus inia ) can be mentioned. The crtI gene derived from Erwinia encodes the amino acid sequence of phytoene desaturase that converts phytoene to lycopene via ζ-carotene. Specific examples of genes derived from Erwinia bacteria include, for example, Er. uredovara crt
The I gene can be used. For details of the nucleotide sequence of this crtI gene, see the above-mentioned Journal of B.
Acteriology, vol.172, p.6704-6712 (1992), the encoded polypeptide (phytoene desaturase (Cr
tI)) amino acid sequence. It goes without saying that the DNA sequence to be used may be degenerate isomers other than those shown in this paper, which encode the same polypeptide and differ in nucleotide sequence only at the degenerate codon.
Furthermore, it has a nucleotide sequence corresponding to a polypeptide maintaining phytoene desaturase activity even if there are amino acid mutations or changes such as substitution, deletion, insertion or addition of some amino acids in the amino acid sequence. If available, it can be used. Further, as a gene derived from a bacterium of the genus Erwinia, Er. herbicola crtI
Gene (see WO 91/13078 publication)
Is also exemplified, and regarding this nucleotide sequence, the above-mentioned Er. u
As in the case of redovora , degenerate isomers or nucleotide sequences corresponding to amino acid mutations can be used.
These DNA sequences can be partially or wholly chemically synthesized, but preferably, they can be obtained by isolating a genomic gene or cDNA encoding phytoene desaturase from a source such as bacteria by a conventional method (above-mentioned publication). (See gazette etc.). The DNA sequence (phytoene desaturase gene) as described above is contained in a vector such as a plasmid, and is introduced into a plant in a state in which its genetic information can be expressed to transform it, thereby tolerating bleaching herbicides. Can produce plants. As the above vector, any binary vector can be used, but preferably pBI 1
21 can be given. In order to express a DNA sequence and produce a polypeptide (phytoene desaturase) encoded by the DNA sequence, an expression control sequence is necessary in addition to the coding region, and such an expression control sequence includes, for example, cauliflower mosaic virus. A derived 35S promoter or the like can be used.
Furthermore, when the produced polypeptide (phytoene desaturase) is desired to be transported to a specific location in the cell (for example, chloroplast), a nucleotide sequence encoding a transit peptide required for transport is further added upstream of the coding region. This enables the transportation. As the sequence encoding the transit peptide, for example, a sequence shown in
As shown in, a DNA sequence encoding the transit peptide of the pea Rubisco small subunit can be utilized. Therefore, the “DNA sequence encoding a polypeptide” in the present invention does not refer to only the coding region of this polypeptide in a limited manner, but also includes an expression control sequence and a sequence encoding a transit peptide together with the coding region. DNA
It also includes sequences. Regarding the basic matters relating to the ligation of an expression regulatory sequence or a sequence encoding a transit peptide, or the production of an expression vector containing these, a generally known method (for example, Toshiyuki Nagata:
"Genetic manipulation of plant cells", proteins / nucleic acids / enzymes (temporary special edition) 1983: 12, Vol.28, No.14, Kyoritsu Shuppan Co., Ltd., p155
1-1568, etc.) can be used.
In addition, as a method for transforming a plant with this DNA sequence (phytoene desaturase gene), that is, a method for introducing a foreign gene into a plant, various methods that have been reported and established, for example, Ti plasmid of agrobaclarium is used as a vector. A method or a method of introducing a gene into a plant protoplast by electroporation can be appropriately used depending on the type of a plant into which the gene is to be introduced (eg, Yoshimi Okada, Shigeru Iida: “Genetic Engineering Molecular Breeding”). Present state and future of technology ”, Plant Biotechnology II, Yasuyuki Yamada / Yoshimi Okada, Modern Chemistry / Supplement 20, Tokyo Kagaku Dojin, p233-264). The foreign gene, that is, the plant into which the phytoene desaturase gene is to be introduced is as described above, but as a plant material for gene introduction, leaves, depending on the introduction method,
It can be appropriately selected and used from arbitrary materials such as stems and roots. The transformant into which the DNA sequence (phytoene desaturase gene) has been introduced can be obtained by a conventional method (for example, Somoto Motoyoshi, Masashi Ugaki: “virus-resistant tomatoes by coat protein gene” plant biotechnology II,
Yasuyuki Yamada / Yoshimi Okada, edited by Hyundai Kagaku / Supplement 20, Tokyo Kagaku Dojin, p153-161) can be used to obtain transformed plants that have grown from young plants to acclimatized plants. This transformed plant has the ability to produce phytoene desaturase that converts phytoene to lycopene via ζ-carotene, and not only herbicides that inhibit plant phytoene desaturase such as norflurazone and fluridone, but also SAN380H, J852, etc. It has acquired resistance to herbicides that inhibit ζ-carotene desaturase of the plant. The resistance test for the above-mentioned various drugs can be carried out by a commonly known method (for example, “Microbiology Experimental Method”: Microbial Research Method Round Table, 1975, Kodansha, I.
V. Physiological property observation method, p199-254)).

The ζ-carotene desaturase gene and
Encoded Polypeptide The gene encoding ζ-carotene desaturase, which is the site of action of bleaching herbicides such as SAN380H or J852 described above, was recently described in cyanobacteria A.
This gene has just been cloned from nabaena (PCC7120) (see FEMS Microbiol.
Lett., Vol.106, p.99-103 (1993)), its amino acid sequence has not been clarified yet, and there are no studies on this resistance to date, Erwi
Utilizing the nia phytoene desaturase gene,
Whether or not plants can be conferred resistance to ζ-carotene desaturase inhibitors has not been known at all. Furthermore, the present inventors determined the nucleotide sequence of this ζ-carotene desaturase gene zds recently cloned from cyanobacteria by the dideoxy method, as shown in Experimental Example 9 described later, and deduced the amino acid deduced therefrom. The sequences were compared to phytoene desaturases from various species previously reported. As a result, surprisingly, this ζ-carotene desaturase zds
The gene product has almost no homology with cyanobacterial or plant phytoene desaturases except for a part of the N-terminal region, and rather, Erwinia or Rh
It showed a significant homology of about 35% with the phytoene desaturase CrtI of Odobacter capsulatus . From this, it is considered that the structure of Erwinia phytoene desaturase is similar to that of plant ζ-carotene desaturase. Therefore, a person skilled in the art would appreciate that Erwinia phytoene desaturase gene inhibits plant ζ-carotene desaturase. One would expect to be unable to confer resistance to herbicides. Figures 3-5 show cyanobacteria ( Anab
The nucleotide sequence of the ζ-carotene desaturase gene derived from aena ) and the amino acid sequence deduced therefrom are shown (see the sequence listing below). The nucleotide sequence of this ζ-carotene desaturase gene and the amino acid sequence of the encoded polypeptide are However, it has not been known so far and is the first disclosed in the present invention.

Use of phytoene desaturase gene In the present invention, a DNA encoding the above-mentioned polypeptide having an enzymatic activity for converting phytoene into lycopene.
Sequence, ie phytoene desaturase (crtI)
Genes (typically from Erwinia)
It can be used as a genetic marker for the transformation confirmation of a bleaching herbicide which inhibits plant ζ-carotene desaturase such as SAN380H or J852 in a plant transformation experiment with various foreign genes (eg drug resistance gene). it can. So far, plant transformation experiments have been carried out using the plant pathogen Agrob.
It is generally carried out using the Ti plasmid system of A. tumefaciens , but the only marker genes that show that a foreign gene has been introduced into plants are kanamycin and hygromycin resistance genes, which are antibiotic resistance genes. There is nothing that can be used, and therefore it is difficult to transform various plant species showing resistance to these agents or to introduce multiple genes into one plant in terms of confirmation of the presence or absence of transformation. Was becoming. In Experimental Example 8 described below, leaf pieces (tobacco plants) were transformed by infection with Acrobacterium ( A. tumefacience ) having a plasmid containing a phytoene desaturase (crtI) gene and a kanamycin resistance gene, and this was transformed with ζ-carotene desaturase inhibition. When cultured on a medium with or without the agent (SAN380H) and kanamycin, it showed abnormal growth on the medium containing SAN380H, that is, a large number of white adventitious shoots with poorer growth than on the control medium containing neither drug. The formation of green adventitious shoots was observed in some of the formations, which were normally grown and were better than those in the medium containing kanamycin, and the green adventitious shoots were normally grown on the medium containing kanamycin. The green adventitious buds were cultured on a regeneration medium to form seedlings, and the chromosomal DNA of the leaves was analyzed. It was confirmed that both the phytoene desaturase gene and the kanamycin resistance gene were incorporated. From this, SA as a selection marker
It was found that N380H enabled the selection of plants into which a kanamycin resistance gene as a foreign gene had been introduced, that is, transformants. Therefore, Erwi
It has been confirmed that the nia phytoene desaturase gene can be used as a marker gene for a ζ-carotene desaturase inhibitor such as SAN380H in a plant transformation experiment with various foreign genes (eg drug resistance gene).

[0009]

The following examples are intended to explain the present invention more specifically, but are not intended to limit the present invention. Experimental Example 1: Construction of plasmid pYPIET4 The conventional gene recombination experiments used here were standard methods (Sambrook, J .; Fr.
itsch, EF; Maniatis, T. Molecular Clonig: A Labo
ratory Manual. Cold Spring Harbor Laboratory Pres
s, 1989). Pea ribulose
The sequence (tp) encoding the transit peptide of the 1,5-2 diphosphate (Rubisco) precursor was obtained from plasmid pSNIF83 (Schreier, PH; Seftor, EA; Sche
ll, J .; Bohnet, HJ The use of nuclear-encoded se
quences to direct the light-regulated synthesis an
d transport ofa foreign protein into plant chlorop
lasts. The EMBO Journal, Vol. 4, p.25-32 (1985)), and isolated as a 204 bp HindIII-SphI fragment. Next, Er. pCRT-I (Fraser, PD; Misawa, N .; Li, which is a plasmid having the uredovora phytoene desaturase gene crtI.
nden, H .; Yamano, S .; Kobayashi, k .; Sandmann, G.
Expression in E. coli, purification and reactivati
on of the recombinant Erwinia uredovora phytoen
e desturase. J. Biol. Chem., Vol. 267, p.19819-198
95 (see 1992)) was double-digested with BamHI and HindIII, and then contained crtI with a partial N-terminal deletion.
A 7 kb BamHI-HindIII fragment was isolated. Next, 7 from the start codon of crtI to the BamHI site
A 6 bp sequence was synthesized. The start codon of crtI was designed to generate a sticky end of SphI.
Then, the three types of fragments obtained above were converted to 204 containing tp
bp HindIII-SphI fragment, synthetic 76 bp
SphI-BamHI fragment, a part of N-terminal c deleted
1.57 kb BamHI-HindIII containing rtI
The fragments were combined in order. Thus obtained 1.84 kb Hi
The ndIII fragment was treated with Klenow enzyme and then the binary vector pBI 121 purchased from Toyobo Co., Ltd.
From which the β-glucuronidase gene had been removed by double digestion with SmaI and SacI, and insertion was performed so as to receive the transcription readthrough of the 35S promoter of cauliflower mosaic virus (CaMV). The plasmid thus obtained was named pYPIET4. pYPIET
1.84 kb containing the tp-crtI gene in 4.
The HindIII fragment portion of E. coli is shown in FIG.

Experimental Example 2: Transformation of plant body via Agrobacterium The plasmid pYPIET4 is a helper plasmid pR.
Junction transfer method using K2013 (Stuy, JH Plasmid t
ransfer in Haemophilus influenzae . Journal of B
Acteriology, Vol. 139, p. 520-529 (1979)), Agrobacterium tumefacie
ns LBA 4404. Plasmid pY
A. with PIET4 The tumefaciens conjugate was prepared by the leaf disk method (Horsch, RB; Fry, JE; H
offmann, NL; Eichholtz, D .; Rogers, SG; Fraley,
RT A simple and general method for transferring
genes into plants. Science, Vol. 227, p.1229-1231
(1985)) and tobacco plants ( Nicotiana
tabacum varieties Samsun)
Was used to transform. Tobacco transformants were selected with 100 μg / ml kanamycin, 500 μg / ml
26 on MS medium containing ml carbenicillin, 1 mg / l benzyladenine, 0.1 mg / l NAA
It was performed under the conditions of light irradiation at 16 ° C. for 16 hours and under darkness for 8 hours. The adventitious buds obtained here were transferred to an MS medium containing no plant hormones and cultured to form roots.
Rooted seedlings were transferred to soil and cultivated in a greenhouse.
Further, when growing and maintaining, root apical seedlings or shoots with petioles were cut off, placed in another MS medium containing no plant hormone, and cultured.

Experimental Example 3 Expression of Phytoene Desaturase Gene crtI in Plants A total of 28 strains of kanamycin-resistant transformed tobacco were isolated by the method described above. crtI gene product (CrtI)
The expression of the crtI gene in the leaves of these transformants was examined by Western blotting using an antibody against Escherichia coli. The expression of the crtI gene was observed in half of the transformed plants, and the transit peptide was cleaved CrtI. The band showed the size of the protein itself. Therefore, it was considered that, in these half of the transformed plants, the tp-crtI gene product synthesized in the leaves was transferred to the chloroplast based on the information of the transit peptide, and then the transit peptide was cleaved. This was also confirmed by immunogold particle detection experiments using antibodies against CrtI. That is, using leaves from one of the tobacco transformants (ET4-1) whose expression was confirmed,
After reaction with gold particles adsorbed to CrtI antibody, ultrathin sections of the leaves were observed with an electron microscope. This experimental method is based on Vivo, A; Andreu, JM; Vina, S.de.la .; Felipe,
MRde. Leghemoglobin in lupin plants (Lupinus al
bus cv Multolupa). Plant Physiol., Vol. 90, p.452-
457 (1989). As a result, it was found that in ET4-1, most of crtI was taken up in the chloroplast and 89% of it was transferred to the thylakoid membrane. The reaction of immunogold particles in the non-transformed tobacco used as a control was 10% or less of that of ET4-1.

Experimental Example 4: Norflurazone resistance of plants expressing the phytoene desaturase gene crtI Transgenic tobacco maintained in MS medium containing no plant hormone (crt by Western blot experiment)
Strains (capable of growing side buds) with petioles were cut out from 14 strains of which I gene expression was confirmed) and 3 μM norflurazone (SAN9789, 4-chloro-5)
-Metylamino-2- (3-trifluor
omethyphenyl) -pyridazin-
3 (2H) one) and no plant hormones
The plate was placed on a medium and cultured at 26 ° C. for 16 hours under irradiation with light and in the dark for 8 hours. As a control, non-transformed tobacco was also treated in the same manner. As a result, ET4-1
Although the partial bleaching (whitening of the leaves) was observed in the 11 strains containing P., the degree of bleaching was smaller than that of the control. No bleaching was observed in the remaining three strains. Therefore, the former was designated as a medium resistant strain and the latter as a strongly resistant strain. As a control, a plurality of petiole-bearing stems obtained from the same in vitro tobacco plant (not transformed) were placed on MS medium containing 3 μM norflurazone and containing no plant hormone, and cultured under the same conditions as above. As a result, some of the controls appeared to have a resistance that was virtually the same as that of the medium-resistant strain. Therefore, in the case of moderately resistant strains, this data alone is weak as evidence of herbicide resistance in transgenic tobacco.
Various experiments were conducted.

Experimental Example 5: Analysis of transgenic plants resistant to bleaching herbicides. Using transgenic tobacco ET4-1, the incorporation of crtI gene product into chloroplasts was confirmed by immunogold particle detection experiments. A bleaching herbicide resistance test was conducted. ET cultured in vitro
The stems with petioles (those capable of growing side buds) were cut from 4-1 and non-transformed tobacco, respectively, and placed on MS medium containing 3 μM norflurazone and containing no plant hormone, and irradiated with light at 26 ° C. for 16 hours. Under the conditions of darkness for 8 hours, the culture was performed for 17 days. Although bleaching was observed in both, the degree of bleaching was stronger in the control non-transformed tobacco than in ET4-1. Table 1 shows the results of quantification of carotenoid content and chlorophyll content using these leaves. Table 1 Content of carotenoid pigment, phytoene, and chlorophyll in tobacco leaves Chromium control plant Control plant ET4-1 transformant In the presence of 3 μM norflurazone Carotenoid pigment 1.3 0.2 0.9 Phytoene <0.1 0.6 0.1 Chlorophyll 8.9 1.5 6.1 In the presence of 3 μM norflurazone, the carotenoid pigment and chlorophyll content in ET4-1 was reduced by only about 30% as compared with the control without norflurazone, but in the non-transformant, both carotenoid pigment and chlorophyll content were 80%. The above decrease was shown, and accumulation of phytoene, a substrate of phytoene desaturase, was observed. Next, ET4-1 and non-transformed tobacco were transplanted to soil, cultivated in normal water for 1 week, and then water containing 3 μM norflurazone was continuously supplied. In the non-transformed tobacco, bleaching began to be observed on the 6th day after the addition of norflurazone, whereas in ET4-1, bleaching began to be observed on the 11th day after the addition of norflurazone. From the above results, it was concluded that ET4-1 acquired some resistance to norflurazone. Similarly, fluridone (EL171, 1-m), which is another phytoene desaturase inhibitor, is used in addition to norflurazone.
Ethyl-3-phenyl-5- (3-trifl
uoromethylphenyl) -4 (1H) -p
γ-done), a ζ-carotene desaturase inhibitor SAN380H (see structural formula below) and J85
It was found that the resistance also increased with respect to 2 (see the following structural formula). Furthermore, it was confirmed that seeds were taken from these plants and seedlings germinated from the seeds were used to pass on the bleaching herbicide resistance to the next generation.

[Chemical 1]

[Chemical 2]

Experimental Example 6: Analysis of bleaching herbicide highly resistant transgenic plants One of the three transgenic tobacco plants, ET4-208, which did not bleach at all in MS medium containing 3 μM norflurazone, was used. Further bleaching herbicide resistance tests were conducted. From ET4-208 and non-transformed tobacco which have been cultured in vitro, stems with petioles (those capable of growing side buds) were cut off, MS medium containing 10 μM norflurazone and containing no plant hormone, and 3 μM Placed in MS medium containing fluridone and not containing plant hormones, at 26 ° C, 16
Culturing was carried out for 28 days under irradiation with light for 8 hours in the dark. As a result, 10 μM norflurazone, 3 μ
With M fluridone, in non-transformed tobacco,
Strong bleaching occurred and the leaves were white. on the other hand,
For ET4-208, 10 μM norflurazone, 3 μ
No bleaching was observed with either M fluridone, and this in vitro plant grew normally to the same extent as the control tobacco in the medium containing no herbicide. Furthermore, ET4 in the presence of 10 μM norflurazone
The carotenoid content and chlorophyll content were quantified using the stem and leaves of -208, and both the chlorophyll and carotenoid content were confirmed to be the same as the control without norflurazone. Next, ET4-208 rooted in MS medium containing no plant hormones and non-transformed tobacco were transplanted to soil, cultivated in normal water for 1 week, and then water containing 3 μM norflurazone was continuously supplied. . In the non-transformed tobacco, bleaching began to be observed on the 6th day after the addition of norflurazone and died within 25 days.
ET4-208 grew normally without any bleaching and formed seeds within 2 months after the addition of norflurazone. From the above results, it was found that ET4-208 acquired strong resistance to bleaching herbicides that inhibit plant-type phytoene desaturases such as norflurazone and fluridone.

Experimental Example 7: ζ-of transgenic plants
Resistance test to carotene desaturase inhibitor Bleaching herbicide that inhibits ζ-carotene desaturase using transgenic tobacco ET4-208 which was found to be resistant to bleaching herbicide that inhibits plant-type phytoene desaturase Resistance test was performed. From ET4-208 and non-transformed tobacco that have been cultivated in vitro, cut stems with petioles (those capable of growing side buds),
Planted on MS medium containing 10 μM SAN380H and no plant hormones and MS medium containing 20 μM J852 and no plant hormones, and exposed to light at 26 ° C. for 16 hours under dark conditions for 8 hours for 14 days. , Culture was performed. As a result, 10 μM SAN380H, 20 μM
No. J852, strong bleaching occurred in the non-transformed tobacco, and the foliage was white. On the other hand, ET
4-208 contains 10 μM SAN380H, 20 μM
No bleaching was observed in any of J. 952 of J. No. J85, and this in vitro plant grew normally to the same extent as the control tobacco in the medium containing no herbicide. Furthermore, ET4-208 in the presence of 10 μM SAN380H
Carotenoid content and chlorophyll content were quantified using the foliage of No. 1, and both chlorophyll and carotenoid content were confirmed to be the same as the control without SAN380H. Next, MS that does not contain plant hormones
ET4-208 rooted in a medium and non-transformed tobacco were transplanted to soil and cultivated in normal water for 5 days, then 10
Water containing μM J852 was kept on. In the non-transformed tobacco, bleaching began to be observed on the 10th day after the addition of J852 and died within 40 days.
Grows normally without any bleaching,
Seeds were formed within 2 months after the addition of J852. From the above results, ET4-208 is not only against bleaching herbicides that inhibit plant-type phytoene desaturases such as norflurazone and fluridone, but also SAN380H.
It was found that it also has a strong resistance to bleaching herbicides that inhibit ζ-carotene desaturase, such as J852 and J852.

Experimental Example 8: Phytoene desaturase gene cr of Erwinia marker gene for ζ-carotene desaturase inhibitor
The following experiment was performed to show that tI can be used as a marker gene for a ζ-carotene desaturase inhibitor. A. containing the plasmid pYPIET4 . tu
The leaf disk method (Horsch, RB; Fry, JE; Hoffmann, NL; Eichhol) using the mefaciens conjugate.
tz, D .; Rogers, SG; Fraley, RT A simple and ge
neral method for transferring genes into plants. S
cience, Vol. 227, p. 1229-1231 (1985)),
Tobacco plant ( Nicotiana tabacum v
arietiesSamsun). After culturing on a feeder plate for 2 days, the infected tobacco leaves were replaced with 20 μm instead of kanamycin.
M SAN380H, 300 μg / ml Claforan, 1 mg / l benzyladenine, 0.1 mg / ml
Transfer to MS plate containing 1 NAA, 26 ° C., 16
Culturing was carried out under conditions of darkness for 8 hours under irradiation with light. As a control, 300 μg / ml Claforan,
1 mg / l benzyladenine, 0.1 mg / l NA
MS plate containing only A and 100 μg / ml
Was also placed on an MS plate containing 300 μg / ml of claforane, 1 mg / l of benzyladenine, and 0.1 mg / l of NAA, and culturing was carried out in the same manner. Then, each of these tobacco leaf discs was placed on the same plate every two weeks. SAN
On the control plate containing neither 380H nor kanamycin, many adventitious buds started to form from the second week of culture, and callus and adventitious buds formed on the entire surface of the leaf disc at the fourth week of culture. In the 4th week of culture, per plate (per plate on which 6 discs were placed),
About 30 green adventitious shoots were formed from the green callus. On the other hand, on the plate for ordinary transformation selection containing kanamycin, only 1-2 adventitious buds were formed per plate at 4 weeks of culture,
The majority of adventitious buds formed on plates without kanamycin were considered non-transformants. On the other hand, on the plate containing SAN380H instead of kanamachiin, about 20 white adventitious buds per plate were observed at 4 weeks of culture, but compared to the control containing neither SAN380H nor kanamycin, The growth of adventitious shoots was poor. Then, mixed with this many white adventitious buds, three normally growing green adventitious buds were formed per plate. The growth of this green adventitious bud was superior to that grown on plates containing 100 μg / ml kanamasin. Further, even when this green adventitious bud was isolated and placed on an MS plate containing kanamycin, it was able to grow normally. Next, SAN38
Green adventitious buds formed on the plate containing OH were isolated, transferred to an MS medium containing no plant hormone and cultured to form a seedling. Chromosomal DNA was obtained from the leaves of these seedlings and subjected to Southern analysis to obtain the crtI gene and the kanamycin resistance gene NPT.
It was confirmed that both II and II were installed. This means that SAN380H as a selection marker was able to select a plant into which a kanamycin resistance gene as a foreign gene was introduced. Therefore, it was revealed that the Erwinia phytoene desaturase gene crtI can be used as a marker gene for a ζ-carotene desaturase inhibitor such as SAN380H.

Experimental Example 9: Determination of the nucleotide sequence of the cyanobacterial ζ-carotene desaturase gene zds The ζ-carotene desaturase gene zds (Linden, H .; Vioque, of the cyanobacteria Anabaena PCC7120 cloned by Linden et al.
A .; Sandmann, G. Isolation of a carotenoid biosynt
hesis gene coding for ζ-carotene desaturase from
Anabaena PCC7120 by heterologous complementation.
The nucleotide sequence of FEMS Microbiol. Lett., Vol. 106, p.99-103 (1993)) was determined by the dideoxy method after a delation reaction combining exonuclease III and mung bean nuclease. An open reading frame of 1497 bp capable of encoding a peptide consisting of 499 amino acids was observed. 3 to 5 (Sequence Listing: SEQ ID NO: 1) show the nucleotide sequence and the amino acid sequence deduced therefrom. This is the first report of the nucleotide sequence and amino acid sequence of the ζ-carotene desaturase gene.
Furthermore, a homology search of the zds gene product (Zds) was carried out using the Genbank and EMBL databases, and all the homologues were found to be carotinodesaturase. This carotenoid desaturase is used in Rhodobacter ca
All were phytoene desaturases, except psulatus crtD. Among them, particularly high homology was recognized by Erwinia and R.M. caps
It was Ultus CrtI. 6-7, Zds
And Er. Uredovora 's CrtI, and
R. The result of having compared the amino acid sequence of CrtI of capsulatus was shown. As a result of the homology analysis considering the gap, Zds was calculated as Er. uredov or
a of CrtI, and, R. It showed capsulatus CrtI and identities of 36% and 34%, respectively. On the other hand, almost no homology was observed with phytoene desaturases of plants and cyanobacteria except some N-terminal regions. This region is presumed to be a binding region for coenzymes such as NAD and FAD. Only in this area, cyanobacterial Syne
Phyto of chococcus PCC7942 Endesa desaturase gene pds (Chamovitz, D; Pecker, I .;
Hirschberg, J. The molecular basis of resistance t
othe herbicide norflurazon. J. Plant Mol. Biol., V
ol. 16, p.967-974 (1991)), and soybean phytoene desaturase pds1 (Bartley, GE; V).
itanen, PV; Pecker, I .; Chamovitz, D .; Hirschber
g. J .; Scolnik, PA Molecular cloning and express
ion in photosynthetic bacteria of a soybean cDNA c
oding for phytoene desaturase, an enzyme of the ca
rotenoid biosynthesis pathway. Proc. Natl. Acad. S
ci.USA, Vol. 88, p.6532-6536 (1991)) are also shown in FIGS.

[0018]

As background above, according to the present invention, phytoene desaturase, within the been carotenoid synthase apparent ever at the genetic level, only an enzyme having diversity between organisms.
Further, as described above, in plants, the desaturation reaction from fetoene through ζ-carotene to lycopene is the site of action of many herbicides called bleaching herbicides. Erwinia phytoene desaturase is an enzyme capable of catalyzing a desaturation reaction from phytoene to lycopene via ζ-carotene (see Journal of Bacteriology, Vol. 17 above).
2, No. 12, p. 6704-6712 (1992), and the above-mentioned patent application filed by the present inventors), an enzyme having a strong dehydrogenation function such as phytoene desaturase of Erwinia has been known so far. Is the only phytoene desaturase of some organisms that has been revealed. Further, as described above, phytoene desaturase of phytoene desaturase and plants Erwinia is the amino acid sequence level, since homology is not found except for a part of N- end region, the phytoene desaturase gene Erwinia, chloroplast It is expected that it is possible to impart resistance to a herbicide that inhibits phytoene desaturase in plants by adding a transit peptide sequence required for transfer to the body and introducing it into plants. On the other hand, regarding the resistance of plants to ζ-carotene desaturase-inhibiting herbicides, recently, the cyanobacteria Anabaena PCC7 has finally been confirmed.
The gene encoding this enzyme has just been cloned from 120 (see above FEMS Microbiol. Lett., Vo.
l. 106, p.99-103 (1993)), the phytoene desaturase gene of Erwinia was used because its amino acid sequence has not yet been clarified and there are no studies on this resistance to date. Thus, it is the present situation that it is not known whether plants can be given resistance to ζ-carotene desaturase inhibitors. Furthermore, the present inventors determined the nucleotide sequence of this ζ-carotene desaturase gene recently cloned from cyanobacteria and compared the deduced amino acid sequence with phytoene desaturases of various species already reported. did. As a result, surprisingly,
This ζ-carotene desaturase gene product shows almost no homology with cyanobacterial or plant phytoene desaturases except for a part of the N-terminal region, and rather, Erwinia or Rhodobacter
It showed a significant homology of about 35% with the phytoene desaturase of capsulatus . From this, it is considered that the structure of Erwinia phytoene desaturase is similar to that of plant ζ-carotene desaturase. Therefore, a person skilled in the art would appreciate that Erwinia
The phytoene desaturase gene of E. coli would not be able to confer plant resistance to herbicides that inhibit ζ-carotene desaturase. On the other hand, the plant transformation experiment was carried out using the plant pathogen Agrobacterium
Currently, the um tumefaciens Ti plasmid system is commonly used, but the only marker gene that indicates that a foreign gene has been introduced into plants is the kanamycin or hygromycin resistance gene positions, which are antibiotic resistance genes. There is nothing actually usable, this is
It has been difficult to transform various plant species that are resistant to these agents and to carry out experiments for introducing a plurality of genes into one plant. Effects of the present invention According to the present invention, utilizing a phytoene desaturase gene having an enzymatic activity to convert phytoene to lycopene, not only herbicides that inhibit plant phytoene desaturases such as norflurazone and fluridone, but also SA
It has become possible to confer on plants resistance to herbicides that inhibit ζ-carotene desaturase in plants such as N380H and J852, and it has become possible to use these herbicides as markers during transformation.

[0019]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2076 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Cyanobacteria Anabaena Strain name: PCC7120 Sequence GATCGCTCTA CAATTTAGTC TTTTGCGAAT TAAAAGAGCA AAATTTTTCT TGGTGAAAAT 60 CAACCGAAAA ATGAAAAGAT GAGAATTTAT GACGCAAATT TATCACCCAA ACTAAACAAA 120 TTTCCTTGAT TGTCTTCAGC AAGTGTTTAA AAAATATAGG CTGCCTAAGT AAACGAGGAG 180 TTCATCT ATG Tla AAA GLA GLA GLA GLA GLA GLA GLA GLA GTC GCT ACA GCT ATC CGT CTT GCT GGA CTT GGG TAT CAA GTA GAA ATC 277 Leu Ala Thr Ala Ile Arg Leu Ala Gly Leu Gly Tyr Gln Val Glu Ile 15 20 25 30 TTT GAG GCG GCT GAA CGT GTT GGT GGA AGA ATG CGC GGT TTT GAA GTT 325 Phe Glu Ala Ala Glu Arg Val Gly Gly Arg Met Arg Gly Phe Glu Val 35 40 45 GAT TCC TAC GCC TTT GAT ACT GGC CCC ACT ATT CTC CAA CTA CCA CAC 373 Asp Ser Tyr Ala Phe Asp Thr Gly Pro Thr Ile Leu Gln Leu Pro His 50 55 60 TTA TAT AAA GAG CTT TTT GAG GAG GCT GGT TTA AAT TTT GCC GAC TAT 421 Leu Tyr Lys Glu Leu Phe Glu Glu Ala Gly Leu Asn Phe Ala Asp Tyr 65 70 75 GTT CAA CTC AAA CGT TTA GAA CCA TAT ACA CGT TTG AAA TTT TGG GAT 469 Val Gln Leu Lys Arg Leu Glu Pro Tyr Thr Arg Leu Lys Phe Trp Asp 80 85 90 GGT ACT CAA CTG GAT ATC ACT TCG GAT CTA CAG TCA TTT AAA ACC CAA 517 Gly Thr Gln Leu Asp Ile Thr Ser Asp Leu Gln Ser Phe Lys Thr Gln 95 100 105 110 CTG GCA ACC TTA CGC TCC GAT TTA CCA TTA GCA TTT GAT CGC TGG TAT 565 Leu Ala Thr Leu Arg Ser Asp Leu Pro Leu Ala Phe Asp Arg Trp Tyr 115 120 125 AGT GAG CAT ATC CGT AAA TAT GAG TTA GGT TAC AAA CCC TAT TTA GCC 613 Ser Glu His Ile Arg Lys Tyr Glu Leu Gly Tyr Lys Pro Tyr Leu Ala 130 135 140 GGC CCT GCA CGC TCT ATT TTT GGT TAC TTG CGC CCA GAT GAC CTG ATG 661 Gly Pro Ala Arg Ser Ile Phe Gly Tyr Leu Arg Pro Asp Asp Leu Met 145 150 155 AAG TTT TTG TCT TTT CGT CCT TGG GAA AAT TTA TAT CAA CAC TTT TGG 709 Lys Phe Leu Ser Phe Arg Pro Trp Glu Asn Leu Tyr Gln His Phe Trp 160 165 170 CG A TTT TTT CAA GAT GAG CGT TTA GTC TAT GAT CTG AGA TAT CCG TCA 757 Arg Phe Phe Gln Asp Glu Arg Leu Val Tyr Asp Leu Arg Tyr Pro Ser 175 180 185 190 AAA TAT TTG GGA ATG CAC CCA ACT GTG GCA TCA AGT GTC TTT AGC CTG 805 Lys Tyr Leu Gly Met His Pro Thr Val Ala Ser Ser Val Phe Ser Leu 195 200 205 ATT CCA TTC TTA GAA TTT TCC CAA GGA GTA TGG CAT CCA GTC GGT GGA 853 Ile Pro Phe Leu Glu Phe Ser Gln Gly Val Trp His Pro Val Gly Gly 210 215 220 TTT CGT GCA TTA GCT CAG GGC TTG GCT AAT GCC GCT CAA GAC TTA GGA 901 Phe Arg Ala Leu Ala Gln Gly Leu Ala Asn Ala Ala Gln Asp Leu Gly 225 230 235 GTG AAA ATT CAT CAT CTG CAT TCG CCT GTT CAC CAA ATC TGG ATT GAC CAA 949 Val Lys Ile His Leu His Ser Pro Val His Gln Ile Trp Ile Asp Gln 240 245 250 GGG CAA GTT CGT GGT TTG GAA CTG GCT GAT GCT TCT CGC CAT CAG TTT 997 Gly Gln Val Arg Gly Leu Glu Leu Ala Asp Ala Ser Arg His Gln Phe 255 260 265 270 GAT ACA GTA GTG ATT AAT GCT GAC TTT GCC TAT GCT GTT CGT CAT CTG 1045 Asp Thr Val Val Ile Asn Ala Asp Phe Ala Tyr Ala Val Arg His L eu 275 280 285 TTA CCA ACT TCA GCA CGC GGT CGT TAC ACT GAT AAC AAG CTT GGG CAA 1093 Leu Pro Thr Ser Ala Arg Gly Arg Tyr Thr Asp Asn Lys Leu Gly Gln 290 295 300 ATG CAA TTC TCA TGC TCT ACC TTC ATG ATC TAT TTA TCA GAC AAT CTT 1141 Met Gln Phe Ser Cys Ser Thr Phe Met Leu Tyr Leu Gly Ile Asn Arg 305 310 315 TAT TTG GGT ATC AAT CGC CGC TAC GAA GAT TTA CCT CAT CAT CAA ATT 1189 Arg Tyr Glu Asp Leu Pro His His Gln Ile Tyr Leu Ser Asp Asn Ile 320 325 330 CGC CGA CTT GAA CGT CCT TGG GTT GAT GAT TCA GCA CTA GAT GAA ACT 1237 Arg Arg Leu Glu Arg Pro Trp Val Asp Asp Ser Ala Leu Asp Glu Thr 335 340 345 350 GAT CCA CCA TTT TAT GTC TGT AAT CCT ACA ATT ATC GAC CCC AGT AAT 1285 Asp Pro Pro Phe Tyr Val Cys Asn Pro Thr Ile Ile Asp Pro Ser Asn 355 360 365 GCA CCT GCC GGC CAC AGC ACT TTA TTT GTT TTA GTA CCA ATT CCC AAC 1333 Ala Pro Ala Gly His Ser Thr Leu Phe Val Leu Val Pro Ile Pro Asn 370 375 380 ACT TCT TAT GCT GTT GAC TGG GAT ATT AAA CAA AAA AGC TAT ACA GAT 1381 Thr Ser Tyr Ala Val Asp Trp Asp Ile Lys Gl n Lys Ser Tyr Thr Asp 385 390 395 TTT ATT CTT AAA CGT TTA CAT TTG CTG GGA TAT CAC AAT ATT GAA CAG 1429 Phe Ile Leu Lys Arg Leu His Leu Leu Gly Tyr His Asn Ile Glu Gln 400 405 410 CAC ATT GTT ACC CAA AGT TGT TAC ACA GCA CAA TCT TGG CTT GAT GAT 1477 His Ile Val Thr Gln Ser Cys Tyr Thr Ala Gln Ser Trp Leu Asp Asp 415 420 425 430 TAT CGC GTT CAT TTG GGT GCT GTG TTT AAT CTC AGC CAC AAT TTG ACT 1525 Tyr Arg Val His Leu Gly Ala Val Phe Asn Leu Ser His Asn Leu Thr 335 340 445 CAG CTT GGG CCC TTT CGT CCA CCC ATC CGT TCG GAA AAT ATT GCC GGA 1573 Gln Leu Gly Pro Phe Arg Pro Pro Ile Arg Ser Glu Asn Ile Ala Gly 450 455 460 CTG TAC TGG ATT GGT GGC GCT GTT CAT CCC GGC AGT GGT TTA CTC ACT 1621 Leu Tyr Trp Ile Gly Gly Ala Val His Pro Gly Ser Gly Leu Leu Thr 465 470 475 ATT TTG GAA GCA TCT CGT AGT GCT GCT GGA TTT ATT CAT CAA GAT TTT 1669 Ile Leu Glu Ala Ser Arg Ser Ala Ala Gly Phe Ile His Gln Asp Phe 480 485 490 GCT TCA ACT GGG TCT TAGCAATCCT GTTCTTTACC TAGCTATTTT TTTTACCCAG 1724 Ala Ser Thr Gly Ser *** 495 AGTCATGTTT AGCTGTCGCA TTCGGATTTT TTAAAATTAA AAATACGAGC TAGTTTAGCA 1784 TTAACAATCA AAAGCTGTTC TGGATTTTTA CTAGGGCAGC TTTGCTCCTG TGGAATTAAG 1844 ATAAACCAGA AACCAATCAC ATACACCCGC TCTGGACACT TGCCACATAT TCAAACAATG 1904 GCAATTGAAT CACTTGACCT TGTTTTCGGT AACGTCAAGA TATGCCTCAT CCAAGGCTAC 1964 CCCTTCTACT ACGTCAGTGT AGCGTTTGAA TATTTCGTGG ATTTGGGCAG ACACTTCTCG 2024 GTAGACATCA AATCTCGGTC TGACAAATAT TAATCGGGGG CAAAGAGCGA TC 2076

[Brief description of drawings]

FIG. 1 is an explanatory view showing the biosynthetic pathway from farnesyl pyrophosphate to β-carotene in Erwinia and plants and cyanobacteria.

[Fig. 2] The produced Erwinia uredovor
explanatory view showing a crtI portion for introducing the plasmid of a to the phytoene desaturase gene crtI plant. To this crtI, a transit peptide sequence of Rubisco small subunit of pea, which is necessary for chloroplast targeting, is added.

Figure 3: Cyanobacteria Anabaena PCC7
Explanatory drawing which shows the base sequence of 120 zeta-carotene desaturase gene zds, and the amino acid sequence estimated from it. The underlined sequence is a putative ribosomal binding site.

FIG. 4 is an explanatory diagram showing the base sequence and amino acid sequence following FIG.

FIG. 5 is an explanatory diagram showing the base sequence and amino acid sequence following FIG. 4.

FIG. 6 is an explanatory diagram showing a comparison of the amino acid sequences of ζ-carotene desaturase gene product Zds of Anabaena PCC7120 and phytoene desaturases obtained from various organisms. Eu-CrtI, Rc-CrtI,
Pds and Pds1 are Erwinia ur , respectively.
Edovora 's CrtI, Rhodobacter
Captulatus CrtI, Synechoco
ccus PCC7942 Pds, soybean Pds1
Is shown. Underlines indicate amino acids common to all of these, * indicates Er . uredovora CrtI and R. Amino acids common to CrtI of capsulatus are shown.

FIG. 7 is an explanatory view showing comparison of amino acid sequences following FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:18)

Claims (4)

[Claims] 1. A ζ-carotene desaturase-inhibiting herbicide, which comprises introducing into a plant a DNA sequence encoding a polypeptide having an enzyme activity for converting phytoene into lycopene in a state in which its genetic information can be expressed. To produce plants resistant to. 2. The method according to claim 1, wherein the polypeptide is phytoene desaturase derived from Erwinia or a mutant thereof. 3. The method according to claim 1, wherein the DNA sequence has a base sequence encoding a transit peptide. 4. A marker for confirming transformation of a ζ-carotene desaturase-inhibiting herbicide, which comprises a DNA sequence encoding a polypeptide having an enzymatic activity of converting phytoene into lycopene.