JPH06181775A - Cinnamyl alcohol dehydrogenase gene of aralia cordata and broad-leaf tree and broad-leaf tree having transduced the same gene - Google Patents

Abstract

PURPOSE:To obtain a new gene useful for controlling content of lignin by arbitrarily changing CAD activity contained in a broad-leaf tree. CONSTITUTION:A gene encoding an amino acid sequence of cinnamyl alcohol dehydrogenase gene of Aralia cordata prescribed by 1-358 of the amino acid sequence of the formula. CAD is purified from young treetop of Aralia cordata, a partial amino acid sequence is determined, an oligonucleotide probe estimated from the amino acid sequence is prepared and a cDNA of CAD is collected from a cDNA library derived from the young treetop of Aralia cordata.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cinnamyl alcohol dehydrogenase (hereinafter sometimes referred to as CAD) gene of Udo and hardwood and a promoter site thereof, and an antisense gene prepared by using all or part of these genes. The present invention relates to a broad-leaved tree in which the lignin content is regulated by introduction.

The genes defining the amino acid sequences of CAD of udo and eucalyptus of the present invention are effectively expressed in broad-leaved trees and are involved in lignin synthesis in broad-leaved trees. Therefore, these ansetins genes were incorporated into broad-leaved trees to be included in the broad-leaved trees. It is possible to control the lignin content by arbitrarily changing the CAD activity carried out. Since lignin decomposition from wood, which is a raw material in paper pulp production, requires a great deal of cost, if the wood with a reduced lignin content according to the present invention is used for paper pulp production, the cost required for lignin removal is significantly increased. It can be reduced and is extremely effective in the field of paper pulp production.

[0003]

2. Description of the Related Art Lignin is a polymer complex universally possessed by higher plants, exists between cells of higher plants, and plays a role of maintaining the strength of the plant. The biosynthesis of lignin was performed by Higuchi et al. (Wood Sci. Technol., 24, 2).
3, 1990). In particular, cinnamyl alcohol dehydrogenase has been well studied for its enzymatic properties. In 1988, C.I. J. Lamb
Reported the isolation of the cDNA for the kidney bean CAD gene (Proc. Natl. Acad. Sc
i. , 85, 5546, 1988). But they are 19
Paper published in 1990 (Plan Mol. Bio
l. , 15, 525, 1990).
Since the protein encoded by A has a high homology with the maize malic enzyme, the question of encoding CAD was suggested. After that, malic enzyme was isolated in various plants, and it has been revealed that the cDNA isolated from common bean does not encode CAD. Such a cloning error is due to the unknown biochemical properties of CAD.

[0004] After that, CAD was conducted by several research groups.
Was reported. Report on the isolation of the tobacco CAD gene in October 1991 (post number: 165
3) The 3rd International Society of Plant Molecular Biology (Tucson, USA)
However, there was no report on the nucleotide sequence and amino acid sequence. In addition, a report on CAD of Taeda pine at the same society (post number: 111
Although 1) was also performed, only a partial amino acid sequence at the amino terminus was shown. 1 for this partial amino acid sequence
It was published in the April 1992 issue of Plant Physiology (Plant Physiology, 9
8, 1364, 1992). However, Teda pine is a gymnosperm (coniferous tree), and when compared with the angiosperms Udo and Eucalyptus of the present invention, there are differences in the enzymatic properties of CAD.

Two types of monolignol (coniferyl alcohol (C
A) and sinapyl alcohol (SA)) are present. In angiosperms, these two types of monolignol form a mixture of lignin, whereas in angiosperms, lignin is formed only with sinapyl alcohol. Report by Kutsuki et al.
hytochemistry, 21, 19, 1982)
In, the CAD was partially purified from a plurality of angiosperms and gymnosperms, and the enzyme activity against CA and SA was measured. The angiosperms CAD act equally on both CA and SA, but the gymnosperm CAD is SA. Was shown to have low activity against. In particular, the enzymatic activity of pine CAD on SA was about 1/5 or less of that of CA. Detailed data on the substrate specificity of other coniferous CAD can be found in a paper on the purification of spruce CAD (European Journal of Bio).
Chemistry, 119, 115, 1984). From this, it is clear that spruce CAD has a 10-fold or more difference in substrate specificity for CA and SA. From the above viewpoints, it can be concluded that CAD of angiosperms (particularly coniferous trees) and CAD of angiosperms are enzymes different from each other with a definite difference in the three-dimensional structure involved in the recognition of substrates.

Lignin is an unnecessary substance in the paper pulp production process, and a great deal of cost is spent in the removal process. Therefore, reducing the lignin content of forest trees is an important breeding goal. With the recent advances in gene recombination technology, it is becoming possible to breed trees using the technology. Therefore, isolation and identification of genes involved in lignin biosynthesis and improvement of forest tree varieties by gene recombination technology using the same have been awaited.

[0007]

In view of the above-mentioned present situation, the present invention aims to produce a forest tree having a low lignin content and control the lignin content in the forest tree.
From the group of enzymes involved in lignin biosynthesis, attention was focused on cinnamyl alcohol dehydrogenase to isolate and elucidate the structure of this gene.

[0008]

[Means for Solving the Problems] As a result of earnest studies from various fields, the present inventors have purified CAD from buds of Udo,
A partial amino acid sequence was determined, an oligonucleotide probe deduced from this amino acid sequence was prepared, and this probe was used to successfully find out the cDNA of CAD from the cDNA library derived from Udo shoots. Furthermore, using the cDNA of Udo CAD as a probe, the CAD gene was successfully found out from the genomic library derived from Eucalyptus. The present invention has been completed as a result of further research based on the above findings. That is, the present invention relates to the Udo and Eucalyptus CAD genes, the base sequences of which are shown in SEQ ID NOS: 1 and 2 of the Sequence Listing.

According to the present invention, the Eucalyptus CAD gene could be detected by using the cDNA of Udo CAD as a probe. This means that the CAD base sequence has high homology between angiosperms such as Udo and Eucalyptus. It is nothing but possession. Therefore, by using the CAD genes of Udo and Eucalyptus and fragments thereof as probes, it is possible to fish out CAD genes from all angiosperms.

CAD gene of Udo and CAD of hardwood
All or part of the gene is artificially constructed as an antisense gene, and this gene is electroporated (Plant Physiol., 92, 22).
6, 1990) and a gene transfer method using Agrobacterium (Plant Physiol., 93, 111).
0,1990) and the like, and can be introduced and expressed in a wide range of broad-leaved trees such as mulberry and poplar as well as udo and eucalyptus.

According to the present invention, the lignin content can be reduced by introducing the CAD antisense gene in a broad-leaved tree in which plant regeneration is established. For example, when wood used as a pulp material is targeted, it is possible to produce a high-quality pulp material having a low lignin content.

Hereinafter, the present invention will be described in detail based on embodiments. Unless otherwise stated, the operating procedure is
Current Protocols in Mole
circular Biology (FM Ausubel
Et al., John Wiley & Sons. 198
The method described in 7) was followed.

[0013]

【Example】

(1) Material Commercially available edible wood was purchased and used as a material. CAD
Coniferyl aldehydes and synap aldehydes (hereinafter, both may be collectively referred to as aldehydes), which are substrates for measuring the activity of, were synthesized according to the method of Kutsuki et al. (Journal of the Wood Research Society 27, 520, 1981). Regarding coniferyl alcohol and sinapiru alcohol (hereinafter, both may be collectively referred to as alcohol), the former was purchased from Aldrich, and the latter was purchased from Gifu University and Dr. Shingo Kawai.

(2) Method of measuring CAD activity The oxidation reaction of alcohol to aldehyde is 200 mM.
0.1m in potassium phosphate buffer (pH 6.25)
MNADP +, 0.07 mM coniferyl alcohol or sinapyl alcohol was added to bring the total volume to 1 mL, the enzyme solution was added, and the reaction was performed at 30 ° C. The reaction is carried out in the measuring cell of the spectrophotometer and
The light absorption at 00 nm was measured, and the alcohol oxidation reaction activity of CAD was calculated.

The reduction reaction from aldehyde to alcohol is carried out by adding 0.1 mM NADPH, 0.05 mM coniferyl aldehyde or synapaldehyde to 100 mL of 100 mM Tris-HCl buffer (pH 8.8).
Then, the enzyme solution was added and the reaction was performed at 30 ° C. The reaction was performed in the same manner as above, and the decreased NADPH was measured by light absorption at 340 nm to calculate the aldehyde reduction reaction activity of CAD.

(3) Purification of CAD When the commercially available udo was allowed to stand for one day while absorbing water, the CAD activity increased about twice, so 4.5 kg of udo after this treatment was used as the starting sample. Cut into about 1 cm square, C
AD extraction buffer (100 mM Tris-HCl buffer (p
H7.5), 20 mM β-mercaptoethanol) 4
After mixing with L, it was finely crushed using a juicer mixer. The sample solution was filtered using gauze, ammonium sulfate was immediately added so as to have a saturation concentration of 40%, centrifugation was performed, and the supernatant was recovered. Ammonium sulfate was added to the supernatant so that the concentration became 70%, and the mixture was centrifuged to collect the precipitate. Precipitate the CAD buffer (10 m
M Tris-HCl buffer (pH 7.5), 10 mM β-
It was then dissolved in mercaptoethanol) to give an enzyme solution, which was desalted by gel filtration. Ammonium sulfate was added to the enzyme solution to adjust the final concentration to 0.5 M, and then hydrophobic chromatography was performed using Butyl Toyopearl manufactured by Tosoh Corporation equilibrated with 0.5 M ammonium sulfate to fractionate the enzyme solution. Elution was performed with a 0.5 M to 0 M linear concentration gradient of ammonium sulfate. The obtained CAD fraction was desalted by gel filtration, and then Mono Q manufactured by Pharmacia
Ion exchange chromatography using a column was performed to further finely fractionate. Elution is 0 for sodium chloride
A linear concentration gradient of M to 0.3 M was used. The resulting CAD fraction was desalted by gel filtration, and then subjected to affinity chromatography using NADP + -agarose manufactured by Pharmacia to give 1 mM NADP.
CAD was eluted with +. This was used as the final CAD CAD sample.

240 μg of purified CAD by the above method
Got When purified CAD was subjected to polyacrylamide gel electrophoresis with a concentration gradient of 8% to 25% using Fast System manufactured by Pharmacia and the protein was detected by the silver staining method, a single band was confirmed. Purified CAD is Supersia 1 made by Pharmacia
As a result of gel filtration using 2 columns and a marker for molecular weight measurement manufactured by the same company, the molecular weight of Udo CAD was 72,
It turned out to be 000. This value is based on previously reported CAD
Was similar to that of. Further, when the same electrophoresis including SDS was performed, the molecular size was 38 kDa.
And two bands estimated to be 39 kDa were confirmed. This suggests that Udo CAD is a dimer composed of two similar subunits. On the other hand, when the Km value for the substrate was measured, it was found that the previously reported CA
Values similar to the Km value of D were obtained. In addition, lower aldehydes (acetaldehyde, propionaldehyde), NA
It was also confirmed that DH and NAD + do not serve as substrates for CAD. From the above results, it was shown that the purified enzyme preparation has a high substrate specificity for aldehyde and alcohol existing only in the lignin biosynthesis system, and it was revealed that this enzyme preparation is wood CAD.

(4) Determination of Amino Acid Sequence of Udo CAD CAD 50 μg of purified CAD was taken, bromine cyanide was added at a molar ratio of 100 times, and the mixture was reacted at 40 ° C. for 20 hours.
Was partially disassembled. After the reaction was completed, the sample solution was freeze-dried, and the obtained peptide was dissolved in 0.1 mL of 0.1% trifluoroacetic acid (hereinafter, abbreviated as TFA). This was fractionated by reverse phase chromatography using a C18 ODS column manufactured by JASCO Corporation. 0% to 80% of acetonitrile containing 0.1% TFA as liquid phase
22 using a linear concentration gradient of
UV absorption at 0 nm was measured. The fractionated peptides were collected by freeze-drying. An automated amino acid sequence analyzer (Model No. 47, manufactured by Applied Biosystems)
0A) was used to determine the amino acid sequence of the resulting peptide. As a result, the partial amino acid sequences of the four CADs shown in FIG. 1 were obtained.

(5) Synthesis of oligonucleotides The amino acid sequence of peptide # 15 was selected from the amino acid sequences shown in FIG.
An oligonucleotide corresponding to this was chemically synthesized using an NA synthesizer (Model No. 381A). The base sequence of the synthesized oligonucleotide is shown in FIG.

(6) Preparation of Udo cDNA Library RNA was extracted from the same wood used for CAD purification as the material to obtain 250 μg of total RNA. Furthermore, affinity chromatography was performed using an oligo dT column manufactured by Pharmacia, and 13.5 μg
Of poly (A) RNA was obtained. 2 μg of poly (A) RN
A, cDNA synthesis kit manufactured by Pharmacia and λgt
11. A cDNA library was prepared using a phage particle packaging kit manufactured by Stratagene.
E. coli (Y1088) prepared cDNA library
When the phages were propagated by infecting Escherichia coli, the cells were eluted with SM buffer and stored. The total number of recombinants in the library was about 1.6 million.

(7) Isolation of cDNA for Udo CAD According to the plaque hybridization method, Udo cDNA was used.
CAD cDNA was isolated from about 300,000 recombinants in the A library. The probe is

The synthetic oligonucleotide indicated in [γ-
Those labeled by a phosphate addition reaction using ³² P] ATP and T4 polynucleotide kinase were used. The temperature during hybridization was set to a relatively mild condition of 42 ° C. As a result, 20 cDNA clones having high homology with the probe were obtained, but it was found that they were all the same cDNA clones. Therefore, the longest cD having about 1.4 kbp (1 bp is 1 base pair) was obtained.
The NA fragment was transferred to a plasmid vector (Bluescript SK +) manufactured by Stratagene, and pUCAD1
It was named 0. This pUCAD10 was used for the subsequent analysis.

(8) cDNA Analysis and Identification of Udo CAD The nucleotide sequence of the cDNA fragment of pUCAD10 was analyzed by the dideoxy method using an M13 sequence kit manufactured by Takara Shuzo. At this time, a total of 5 pUCAD10-derived plasmids were prepared in both directions by shortening the cDNA fragment of pUCAD10 at intervals of about 250 base pairs from one direction by using a Delation Kit manufactured by Promega. Each base sequence was analyzed to about 300 base pairs, and the overlapping base sequence portions were connected.
The base sequence of the obtained pUCAD10 is shown in SEQ ID NO: 1 of the sequence listing shown in Tables 1 to 4.

[0024]

[Table 1]

[0025]

[Table 2]

[0026]

[Table 3]

[0027]

[Table 4]

That is, the sequence of SEQ ID NO: 1 in the sequence listing shown above is

It shows the nucleotide sequence of the cDNA fragment shown in and the amino acid sequence defined thereby.

When the amino acid sequence defined by this cDNA was compared with the amino acid sequence obtained from the amino acid sequence analysis of Udo CAD, it was shown that the two sequences were almost identical. The matched portion is underlined in SEQ ID NO: 1 in the sequence listing. From this, it became clear that this cDNA is cDNA of Udo CAD. Since the amino terminal sequence (peptide N in FIG. 1) was also confirmed, it was shown that this cDNA defines the full length of Udo CAD.

Unlike previous reports on CAD, it is clear that Udo CAD is composed of two similar subunits, and the obtained cDNA has the amino acid sequence of either subunit. Stipulate. The present invention utilizes the CAD gene to finally suppress the CAD activity in plants. That is, the object of the present invention is achieved by suppressing the expression of subunits.

(9) Preparation of Eucalyptus Genome Library Callus derived from Eucalyptus (bottle Oides species) seedling primordia was grown on MS medium. According to the method of Hiono et al. (Abstracts of the 36th Lignin Discussion Group, p. 61, 1991), genomic DNA was extracted from eucalyptus and the restriction enzyme (Sa
u3AI) and partially decomposed with 2% to 25% of sodium chloride by linear density gradient centrifugation to about 16 to 20 k.
A bp genomic DNA fragment was recovered. This genomic DNA
A eucalyptus genomic library was prepared using the fragment and Stratagene's EMBL3 and phage particle packaging kit. The prepared cDNA library was infected with Escherichia coli (SRB) to grow phages, which were then eluted and stored in SM buffer. The total number of recombinants in the library was approximately 2.5 million.

(10) Isolation of Eucalyptus CAD gene From about 500,000 recombinants in the Eucalyptus genomic library, CAD was isolated according to the plaque hybridization method.
The gene was isolated. The probe used was one in which the entire length of the Udo CAD cDNA fragment was labeled with [α- ³² P] dCTP and a random primed DNA labeling kit manufactured by Boehringer. As a result, three CAD gene clones having high homology with the probe were obtained. These restriction enzyme maps were created. As shown in FIG. 3, two of the three clones were clones that overlap each other, and the other one was C on the Eucalyptus gene fragment.
A difference in restriction enzyme sites was observed in only one part in the AD-free part. It is presumed that this is due to a minute difference between homologous chromosomes paired with each other in a forest tree, unlike an artificially pure line such as a crop. The three clones are λECAD74 and λECAD7.
2. It was named λECAD21. In order to confirm that the entire CAD gene was contained in these clones, Southern analysis was performed using the N-terminal side fragment and the C-terminal side fragment of the cDNA of Udo CAD as a probe. As the fragment used as a probe, one having the N-terminal side and the C-terminal side was selected from the deletion clones prepared when the nucleotide sequence of pUCAD10 was analyzed, and the cDNA fragment was used after being cleaved with a restriction enzyme. That is, the N-terminal side probes are from No. 1 to No. 20 shown in SEQ ID NO: 1 in the sequence listing.
The C-terminal side probe is a DNA fragment having the nucleotide sequence shown as No. 3, and the C-terminal side probe is a DNA fragment having the nucleotide sequence shown as Nos. 951 to 1336 shown in SEQ ID NO: 1 in the sequence listing. Eucalyptus CAD clones were digested with a restriction enzyme SalI, separated by agarose electrophoresis, and transferred to a nylon membrane. Southern analysis was performed on the nylon membrane on which this DNA was transferred using each of the N-terminal side probe and the C-terminal side probe, and as a result, λECAD74, λEC
Both probes hybridized with AD21. Therefore, it was concluded that these two clones encode the full-length Eucalyptus CAD. Since λECAD72 did not hybridize only with the C-terminal side probe, it is considered that this clone lacks a part of the C-terminal. Fragments homologous to the probes shown by hatching in FIG. 3 (fragments cleaved by SalI) were ligated to a plasmid vector (pUC19) manufactured by Takara Shuzo, and named pECADS1, pECADS2, and pECADS3, respectively. That is, pECADS1 and pECADS2 are plasmids having a DNA fragment encoding the full-length Eucalyptus CAD. For pECADS1, E. coli containing this plasmid (indication for identification: E.co.
li pECADS1) was deposited at the Ministry of International Trade and Industry, the Institute of Biotechnology, Institute of Biotechnology, and the Patent Microorganism Depositary Center (FERM P-13653). The lambda phage clone λECAD74 cannot be deposited because the above institutions have not accepted the deposit of the phage.
In case of an offer from a person, the phage clone can be distributed.

(12) Analysis of Eucalyptus CAD gene Since pECADS1 has a gene fragment of 8 kb, further fragmentation and its Southern analysis were carried out when the nucleotide sequence of genomic DNA fragment was analyzed. As a result, it was revealed that a fragment of about 3.3 kbp obtained by using the restriction enzymes (SalI and BglI) encodes the full-length CAD, so it was decided to carry out a base sequence analysis for this. After blunting the ends of this 3.3 kbp genomic DNA fragment, the restriction enzyme (Sma
I) digested Takara Shuzo plasmid vector (p
It was ligated to UC119) and named pECADS133. Nucleotide sequence analysis was performed according to the dideoxy method using M13 Sequence Kit manufactured by Takara Shuzo. At this time, a genomic kit of pECADS133 was shortened at intervals of about 250 base pairs from one direction by using a Delation Kit manufactured by Promega to make a total of 9 pECADS.
A 133-derived plasmid was made in both directions. Each base sequence was analyzed to about 300 base pairs, and the overlapping base sequence portions were connected. pECADS2 and p
The same nucleotide sequence analysis was performed for ECADS3, but the nucleotide sequences of these three clones were all the same. Therefore, the following is the genomic DNA of pECADS133
The nucleotide sequence of is shown in SEQ ID NO: 2 of the sequence listing shown in Tables 5 to 11.

[0034]

[Table 5]

[0035]

[Table 6]

[0036]

[Table 7]

[0037]

[Table 8]

[0038]

[Table 9]

[0039]

[Table 10]

[0040]

[Table 11]

That is, the sequence of SEQ ID NO: 2 in the sequence listing shown above is

The following shows the nucleotide sequence of the Eucalyptus genomic DNA fragment described in (1) and the amino acid sequence defined thereby. A promoter site and four intervening sequences were present in this base sequence.

Since both amino acid sequences have a homology of 80.7%, it was revealed that the present genomic DNA is a Eucalyptus CAD gene which defines the full length of Ducal CAD. Also, between Udo and Eucalyptus, C
This is because the base sequence homology of the AD gene is as high as 76.6% (only the coding region). It was estimated that the base sequences of the CAD gene were similar in all angiosperms.

In the nucleotide sequence on the 5'upstream side of the Eucalyptus CAD gene, a transcription initiation signal, TATA box (474 to 481 of SEQ ID NO: 2 in Sequence Listing), CAT
Box (42 to 45, 364 of SEQ ID NO: 2 in Sequence Listing)
To 367 and 23 to 427) and two repeat sequences (123 to 140 and 1 of SEQ ID NO: 2 in the sequence listing)
There were promoter specific sequences such as 59 to 176 and 441 to 450 and 448 to 457). That is, this promoter region is considered to be responsible for the timing- and tissue-specific control in the expression of the CAD gene.

(12) Preparation of antisense gene using Udo CAD cDNA Udo CAD cDNA clone (pUCAD10) was digested with restriction enzyme (NotI) to extract the full length of the cDNA fragment, and blunt-ended with T4 DNA polymerase manufactured by Takara Shuzo. Processed. In addition, two restriction enzymes (AccI and Kpn)
A partial fragment of about 650 bp was taken out by digestion with I) and treated with blunt ends in the same manner. On the other hand, a Ti plasmid vector (pBI12 manufactured by Clontech, which has a 35S promoter derived from cauliflower mosaic virus).
The GUS gene was removed from 1) and blunt-end treatment was performed. The blunt-ended CAD DNA fragment and the vector were each ligated to obtain two types of plasmid DNA having different ligation directions of the cDNA fragments. Of these, the ligation of cDNA is 35
Plasmid D, which is in the opposite direction to the S promoter, that is, in the opposite direction to the direction defining the original amino acid sequence
Regarding NA, pUCAD121 containing the full-length fragment
Named A and containing a partial fragment pUCAD121a
I named it. That is, pUCAD121A is an antisense gene containing the entire Udo CAD gene, pUCAD121.
a is an antisense gene containing a partial fragment of the Udo CAD gene.

(13) Preparation of antisense gene using Eucalyptus CAD gene Eucalyptus CAD gene clones (pECADS1 and pECADS1
The entire length of the insert DNA of ECADS133) was digested with a restriction enzyme to take out a genomic DNA fragment.
Blunt ends were treated with 4 DNA polymerase. On the other hand, the GUS gene was removed from a Ti plasmid vector (pBI121) manufactured by Clontech Co., which had a 35S promoter derived from cauliflower mosaic virus, and blunt-ended. The blunt-ended DNA described above was ligated to obtain pEC
Two kinds of plasmid DNAs having different ligation directions were obtained for the genomic DNA fragments derived from each of ADS1 and pECADS133. Of these, the plasmid DNA in which the ligation of the genomic DNA was in the reverse direction to the 35S promoter, that is, in the direction opposite to the direction defining the original amino acid sequence, was designated as pECAD121A. That is, pE
CAD121A is a Eucalyptus CAD antisense gene. Similarly, the antisense gene derived from pECADS133 was named pECAD121A33.

(14) Introduction of Udo-Cad Antisense Gene into Broadleaf Trees The plasmid DNAs of pUCAD121A and pUCAD121a were prepared by the method of Ohta et al. (Patent Application No. Hei 2-162).
695) and introduced into eucalyptus to regenerate the plant.

Genomic DNA was extracted from all the transformants, and Southern analysis was carried out using the plasmid DNA introduced for each as a probe.
The DNA derived from AD121A was added to pUCAD from the other three strains.
The DNA derived from 121a was confirmed. Proteins were extracted from these 7 transformants and CAD activity was measured. As a result, the CAD activities of the four pUCAD121A-introduced strains were 14% to 27% as compared with the control plants, and these showed an average activity reduction of 78%. On the other hand, the CAD activity of the three pUCAD121a-introduced strains was 16% to 35% of that of the control plant, showing an average decrease of 76% in activity.

The plasmid DNA of pUCAD121A was prepared by the method of Machii et al. (Journal of the Japanese Silkworm Society 59, 105, 199).
According to 0), it was introduced into mulberry and the plant body was regenerated.

Genomic DNA was extracted from all the transformants, and Southern analysis was carried out using the plasmid DNA introduced for each as a probe.
The DNA derived from AD121A could be confirmed. Proteins were extracted from these three transformants and CAD activity was measured. As a result, 4 of pUCAD121A was introduced.
The CAD activity of the strains was 12% to 25% compared to the control plants, which showed an average reduction of 82% activity.

From these results, the antisense gene prepared by using all or a part of the Udo CAD of the present invention was used for the CAD in the transformed plant produced by using the antisense gene.
It was shown that it is possible to reduce the activity. This suggests that it is possible to reduce the lignin content in hardwood.

(15) Introduction of Eucalyptus CAD antisense gene into broad-leaved tree The plasmid DNA of each of pECAD121A and pECAD121A33 was introduced into eucalyptus according to the method of Ohta et al. (Patent application No. Hei2-162695) to regenerate a plant. Let

Genomic DNA was extracted from all the transformants, and Southern analysis was carried out using the plasmid DNA introduced for each as a probe.
DNA derived from AD121A was obtained from 3 strains of pECAD12.
The DNA derived from 1A33 could be confirmed. The CAD activity was measured by extracting proteins from these 7 transformants in total. The CAD activity of the 4 strains into which pECAD121A was introduced was 6% to 15% as compared with the control plant, and these showed an average activity reduction of 89%. On the other hand, pECAD1
Similar results were obtained with the 21A33-introduced 3 strains, showing an average reduction in CAD activity of 90%.

The plasmid DNA of pECAD121A was prepared by the method of Machii et al. (Journal of the Japan Silkworm Society 59, 105, 199).
According to 0), it was introduced into mulberry and the plant body was regenerated.

Genomic DNA was extracted from all the transformants, and Southern analysis was carried out using the plasmid DNA introduced for each as a probe.
The DNA derived from AD121A could be confirmed. The CAD activity was measured by extracting proteins from these four transformants. As a result, the CAD activity of the 4 strains introduced into pECAD121A was 12% to 24% compared to the control plants.
Which showed an average reduction in activity of 82%.

From these results, the eucalyptus CA of the present invention
It was shown that the antisense gene of D can reduce the CAD activity in the transformed plants produced using these genes. This suggests that it is possible to reduce the lignin content in hardwood.

[0056]

INDUSTRIAL APPLICABILITY For the first time, the present invention has succeeded in isolating the CAD gene from udo and eucalyptus. As a result, the present invention can exert various remarkable effects, which will be described below.

It has been shown that CAD is involved in the lignin biosynthesis reaction of plants and that the CAD activity can be regulated in broad-leaved trees by using the CAD gene obtained by the present invention. Therefore, it is possible to reduce the lignin content in forest trees, which greatly saves the cost for lignin removal in the pulp and paper industry.

The present inventors have confirmed that CAD is specifically expressed in tissues that undergo lignification (ie, ligninization). That is, the CAD gene is specifically expressed only in the ligninized tissue. The promoter site of the Eucalyptus CAD gene obtained in the present invention is responsible for this specific expression, and if this is combined with an arbitrary gene, it is possible to prepare an artificial gene that specifically acts on ligninized tissues. Is. That is, by introducing into a plant an artificial gene prepared by ligating the DNA fragment at the promoter site shown in FIG. 3 with an arbitrary structural gene, the protein defined by the structural gene can be specifically expressed in a ligninized tissue. Can be expressed. As a result, it becomes possible to influence the conversion of the constituents of lignin and the contents of cellulose and hemicellulose, which are closely related to lignin, and pave the way for molecular breeding of these. Further, the Eucalyptus promoter site obtained in the present invention is considered to function not only in Eucalyptus but also in other plants, and can be widely used for molecular breeding of plants.

CDNA of Udo CAD obtained in the present invention
Defines the full-length amino acid subunit of Udo CAD. Therefore, if this cDNA is used, it is possible to express CAD in a large amount, purify and isolate it by an artificially produced cell-free translation system and E. coli. If a large amount of CAD can be obtained in this way, by examining the effects of various drugs on it, from all directions,
It becomes possible to develop a drug that acts on CAD and suppresses its activity. That is, if the lignin biosynthesis which accounts for about 25% in the plant body can be stopped and the plant can be killed by the administration of the inhibitor specific to CAD, this can be used as a pesticide. CAD is plant-specific and is considered to have a very low effect on animals without CAD. In addition, such a CAD-specific drug has little adverse effect on the environment and is useful.

[Brief description of drawings]

FIG. 1 shows the amino-terminal portion of purified Udo CAD and the amino acid sequences of four peptides derived from Udo CAD.
Peptide N is a sequence from the amino terminus of CAD.

FIG. 2 shows the nucleotide sequence of an oligonucleotide prepared by referring to the amino acid sequence of peptide # 15 derived from Udo CAD.

FIG. 3 shows a restriction map of Eucalyptus CAD gene clones. The site encoding CAD is indicated by hatching. The region where the base sequence was determined is indicated by an arrow. The promoter site is in the range specified separately.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display area // C12N 5/10

Claims (12)

[Claims] 1. A sequence number 1 to 3 shown in SEQ ID NO: 1 of the sequence listing
A gene encoding the amino acid sequence of Udo cinnamyl alcohol dehydrogenase defined by the number 58. 2. A gene encoding a eucalyptus cinnamyl alcohol dehydrogenase contained in the lambda phage defined by the restriction map shown in FIG. 3 and defined by Nos. 1 to 355 shown in SEQ ID NO: 2 in the sequence listing. Gene encoding the amino acid sequence of eucalyptus cinnamyl alcohol dehydrogenase. 3. A broad-leaved tree-derived cinnamyl alcohol dehydrogenase gene obtained by fishing with all or part of the cinnamyl alcohol dehydrogenase gene according to claim 1 or 2 as a probe. 4. A promoter site contained in the eucalyptus cinnamyl alcohol dehydrogenase gene defined by the restriction enzyme map shown in FIG. 5. A broad-leaved tree having an eucalyptus promoter site according to claim 4 and an antisense gene prepared by using all or part of the cinnamyl alcohol dehydrogenase gene of the woody broad-leaved tree according to claim 3. 6. A broad-leaved tree into which an antisense gene prepared by using the eucalyptus promoter site according to claim 4 and all or part of the cinnamyl alcohol dehydrogenase gene of udo according to claim 1 is introduced. 7. A broad-leaved tree into which an antisense gene prepared by using all or part of the Eucalyptus promoter site according to claim 4 and the Eucalyptus cinnamyl alcohol dehydrogenase gene according to claim 2 is introduced. 8. A broad-leaved tree into which an antisense gene prepared by using all or part of the cinnamyl alcohol dehydrogenase gene of the broad-leaved tree according to claim 3 is introduced. 9. A broad-leaved tree into which an antisense gene prepared by using all or part of the cinnamyl alcohol dehydrogenase gene of Udo according to claim 1 is introduced. 10. A broad-leaved tree into which an antisense gene prepared by using all or part of the eucalyptus cinnamyl alcohol dehydrogenase gene according to claim 2 is introduced. 11. The broad-leaved tree according to any one of claims 5 to 10. 12. The hardwood according to claim 11, wherein the hardwood is eucalyptus or mulberry.