JP3124279B2 - Chloroplast DNA of grasses - Google Patents

Description

The present invention relates to the entire nucleotide sequence of chloroplast DNA of rice and related DNA obtained therefrom.

Elucidation of the entire nucleotide sequence of rice chloroplast DNA according to the present invention provides data on rice chloroplast DNA, and furthermore rice chloroplast DNA
Thus, various DNAs having a clear base sequence can be prepared and provided as reagents by decomposition with various restriction enzymes, thus greatly contributing to the development of genetic engineering.

(Prior art and problems) In general, solar energy is converted into chemical energy by the complex metabolic system of photosynthesis possessed by plants, and all living activities on earth including us are maintained by using this chemical energy. ing. The primary organelles of prokaryotic photosynthetic organisms and plant cells, and chloroplasts, are responsible for photosynthesis.

Chloroplasts, like mitochondria, are equipped with devices necessary for the expression of genetic information such as DNA, and chloroplasts in plant cells have unique genetic information expression devices that are different from the nucleo-cytoplasmic system. It has been revealed that they are conducting their own protein synthesis. Furthermore, in the 1970s, recombinant DNA
Technology and the development of new molecular biology techniques centering on nucleotide sequence determination have enabled the development of molecular biology research on this chloroplast DNA, which has a size of around 150,000 base pairs. Identification and structural analysis were proceeded. As one of them, the entire nucleotide sequence of tobacco chloroplast DNA, a dicotyledonous plant, has been elucidated (K. Shinozaki et al., EMBO Journa).
1, 5 , 2043-2049 (1986)).

The chloroplasts are considered to be composed of hundreds of proteins, of which about 100 are encoded by chloroplast DNA and the rest are encoded by nuclear DNA. Have been. In other words, the organelle, which has a complex structure and function called chloroplast, is composed of chloroplast DNA and cell nucleus D
It is made by the joint work of NA.
Furthermore, it has been found that one of the functional units of a photosynthesis device having various higher-order structures can be produced as a result of the joint work of the two.

The gene organization of chloroplast DNA is 4 rRNA and 30 tRNA genes and 56 other identified protein genes and 38
There are genes that appear to encode an unidentified protein of the species. Not all genes of proteins involved in photosynthesis are present in chloroplast DNA, but it is known that many of them are encoded in chloroplast DNA.

The photosynthesis process is roughly classified into a dark reaction and a bright reaction. The dark reaction involves a group of enzymes that fix CO ₂ , of which ribulose-diphosphate carboxylase / oxygenase (RuBi
Only the large subunit of sCO) is encoded in chloroplast DNA. The protein involved in the light reaction is composed of five complexes, photosystem I, photosystem II, cytochrome b / f complex of electron transport system, proton transporting ATPase and light-harvesting chlorophyll a / b.
Divided into binding protein complexes. Light-harvesting chlorophyll a / b-binding protein among these genes is nuclear DNA
And other complex proteins are also nuclear
It is separately coded as DNA and chloroplast DNA.

By the way, tobacco is an excellent plant as an experimental crop, but cannot be a staple food for human beings. Crops such as wheat, rice, and corn, which are currently staple foods all over the world, are classified as monocots.

Therefore, elucidation of the entire base sequence of chloroplast DNA of a grass plant has been eagerly awaited.

(Solution of Problem) The present inventors have eagerly conceived that elucidating the entire nucleotide sequence of rice chloroplast DNA can contribute to elucidation of genes of monocotyledonous plants such as wheat and corn. As a result of research,
In the present invention, the entire base sequence of rice chloroplast DNA was successfully elucidated.

The DNA base sequence of the present invention has revealed all the genes of rice chloroplasts, which are monocotyledonous plants, and gives great knowledge in knowing the structure of genes of other monocotyledonous plants such as wheat and corn. Be expected. Since chloroplast DNA itself contains a self-contained gene, it is possible to obtain a plant transformant by introducing all or part of the chloroplast DNA into another plant cell. It can be used to introduce a rice chloroplast gene into another plant to obtain a transformed plant having the rice chloroplast gene. In addition, the promoter and terminator regions of genes such as ribosomal RNA genes, ribulose carboxylase large subunit, and photochemical genes encoded by rice chloroplast DNA allow other useful genes to be expressed in chloroplasts. Useful when. The DNA base sequence obtained by the present invention further provides basic data necessary for studying chloroplast DNA. In particular, oligonucleotides (short base pairs) based on the nucleotide sequence of the present invention are extremely useful as markers for studying chloroplast genes.

The present invention relates to a DNA or a rice-derived gene having all or a part of the nucleotide sequence shown in FIG. 1 or a nucleotide sequence equivalent thereto.

Further, the present invention relates to a double-stranded DNA cut out from a DNA having the nucleotide sequence shown in FIG. 1 using a restriction enzyme, and a single-stranded DNA obtained by physical or chemical treatment from the double-stranded DNA. It is about.

The present invention also relates to other biological vector DNAs as described above.
A single-stranded DNA obtained by physical, chemical or physiological treatment from the hybrid DNA, and further capable of self-proliferation of the hybrid DNA in cells. It relates to bacteria containing hybrid DNA.

The entire nucleotide sequence of the rice chloroplast of the present invention is shown in FIG.
FIG. 2 shows a genetic map of the rice chloroplast genome.

 Next, embodiments of the present invention will be described in detail.

Example I. Preparation of rice chloroplast DNA (1) Isolation of chloroplast On the 14th day after seeding of rice (Nipponbare), 200 g of leaves were sufficiently cooled and taken into a homoblender. A sufficient amount of liquid nitrogen is added and crushed several times in pulses. In addition, 15000-180
By crushing twice at 00 rpm for 6 seconds each, the rice leaves are completely powdered and allowed to stand until liquid nitrogen evaporates.

The dried powdered leaves were put in a KA buffer (50 mM Tris-HCl 7 mM EDTA 350 mM sucrose (pH 8.0)) containing 0.1% ESA and 5 mM mercaptoethanol, mixed well, and then mixed with gauze.
Filter sequentially with Miracloth. The filtrate is centrifuged at 2700 rpm for 10 minutes, and the resulting precipitate is dispersed in 10 ml of KA buffer containing 4 µ of mercaptoethanol and stored on ice.

5 ml of the chloroplast suspension was overlaid with a 20%, 45%, and 60% sucrose concentration centrifugation solution prepared using 50 mM Tris-HCl 7 mM EDTA 300 mM sorbitol buffer and 3800 rpm.
And centrifuge for 30 minutes. After completion of the centrifugation, the chloroplast layer was taken out and 3 times the volume of KB buffer (50 mM Tris-HCl 20 mM EDTA (pH
8.0)).

The obtained precipitate is dissolved in 1 ml of KB buffer per 200 g of rice leaves.

(2) Extraction of DNA 30 mg of sarkosyl was added to a chloroplast KB solution (1 ml) and 15
Keep at room temperature for minutes. Further, 500 μg of proteinase E is added, and the mixture is allowed to stand at 37 ° C. overnight.

The solution extracted overnight was added an equivalent amount of water-saturated phenol and added 60 rp.
Mix for 20 minutes and centrifuge at 3000 rpm for 10 minutes to separate. The supernatant is removed, an equal volume of phenol / chloroform (1: 1) is added, mixed at 60 rpm for 30 minutes, and separated by centrifugation to obtain a supernatant.

Add 3M sodium acetate to the supernatant to 0.3M, and add
Add 2.5 volumes of ethanol. After gently stirring, leave at −20 ° C. overnight.

The precipitate obtained is washed three times with 70% ethanol. (The chloroplast DNA was collected at 5 to 10 μg per 200 g of rice leaves.) II. Cloning (1) Preparation of Vector and Insert DNA Fragment pUC19 was used as the vector. pUC19 has a defined multicloning site, which also includes the cleavage site of PstI used in this study.

Approximately 1 μg of pUC19 was digested with PstI, and it was confirmed by electrophoresis whether or not the digestion was completed, and then BAP treatment was performed using alkaline phosphatase A19. After BAP processing,
Phenol extraction was performed to remove the remaining enzyme and used as vector DNA.

About 2 μg of rice chloroplast DNA was prepared, cut with PstI, and then treated with phenol to obtain insert DNA.

(2) Ligation Rice chloroplast DNA has a length of about 120 Kb, and there are 14 or more Pst I cleavage sites, so the insert DNA is a mixture of fragments of various lengths. This time, they were cloned by the shotgun method without isolation.

About 0.5 μg of vector DNA, insert DNA (original chloroplast D
About 1 μg (as NA) was taken and subjected to a ligation reaction at 16 ° C. for 30 minutes using a TAKARA ligation kit.

After the ligation reaction, the reaction mixture was directly subjected to transformation.

(3) Preparation of competent cells Competent cells were prepared using Escherichia coli JM109. JM109 pre-cultured overnight was mixed with 2xYT medium (1 polypeptone per 1
6g, 10g yeast extract, 5g NaCl)
The Klett measurement flask was inoculated and cultured until the Klett became 20.

After cultivation, bacterial cells harvested TFB buffer by centrifugation at 4 ℃ (10mM MES (pH6.3) , 100mM KCl, 45mM MnCl 2 · 4H
₂ O, 10mM CaCl ₂・ 2H ₂ O, 3mM HACOCl ₃ )
Use as a solution. 100 competent cultures yield 25 competent cells.

(4) Transform the cells equivalent to 4 ml of the culture solution for preparing competent cells.
The reaction solution was dispersed in 00 µ of TFB buffer, and 10 µ of the reaction solution after ligation (corresponding to 0.25 µg as vector DNA) was added thereto. The mixture was allowed to stand on ice for 40 minutes. After 40 minutes, it is 1-2 at 42 ℃
Hold for a minute (heat shock) and immediately return to ice.

For 25 ml of YT softagar medium (each containing 8 g of polypeptone, 5 g of yeast extract, 5 g of NaCl, and 6 g of agar), 50 μl of 2
% X-GAL, 20 μm of 100 mM IPTG, and the heat-shock-transformed solution together with 25 μm of a 2.5% ampicillin solution were added to a test tube kept at 46 ° C., followed by thorough stirring.

This solution is spread on a solidified agar plate containing YT agar medium (soft agar medium supplemented with 6 g of agar per addition) and cultured at 37 ° C. overnight.

Plasmid DNA was prepared from the colonies formed on the plate, and the length of the insert DNA was examined one by one by restriction enzyme treatment and electrophoresis to select one having the desired length.

III. Preparation of plasmid DNA (1) Preparation of cloned cells Clones of chloroplast DNA digested with the restriction enzyme PstI were cultured in an LB rich medium (10 g of polypeptone, 1 g of yeast extract, 3 g of NaCl, 3 g of KCl ₂ per culture solution). PO ₄ 3g, N
_{a 2 PO 4 · 12H 2 O14.3g} , was inoculated into pH 7.0) 20 ml containing NH ₄ Cl1g, overnight stationary culture at 37 ° C. (preculture). The preculture was added to the above LB rich medium plus antibiotics plus Klett.
(A device for measuring turbidity of cell growth) is shake-cultured until it shows 100. For Klett 100, chloramphenicol 200mg
And continue shaking culture overnight.

After completion of the culture, the cells are collected by centrifugation and plasmid DNA
Is extracted.

(2) Lysozyme treatment Cells (equivalent to one culture) were treated with 10 mM Tris-HCl (pH 8.0) 1
After washing with a solution consisting of 0.85% NaCl, 50 mM Tr
Disperse in is-HCl (pH 8.0) 1 mM EDTA 25% sucrose solution. Lysozyme solution (5mg / ml-50mM Tris-HCl
(PH 8.0)) and add 6 ml and let stand at 0 ° C for 5 minutes.

After standing, 12 ml of 250 mM EDTA and 2.4 ml of 20% SDS are added and slowly stirred. Next, 1/5 volume of 5M NaCl is added and left at 4 ° C. for 4 hours. The solution after the extraction operation is centrifuged at 4 ° C. for 40 minutes to remove cell residue.

(3) Phenol treatment The extract was treated with TE buffer (10 mM Tris-HCl 1 mM EDTA (pH 8.
Add the same amount of phenol saturated in 0)) and stir well. 1
Centrifuge at 0000 rpm for 1 minute to obtain a supernatant. To this supernatant, 5 M sodium acetate is added to 0.3 M, and a 2-fold amount of ethanol is further added, and the mixture is allowed to stand at -20 ° C for 1 hour.

(4) Fractionation by ultracentrifugation The ethanol solution after the phenol treatment is centrifuged to collect the precipitate. The obtained precipitate is dried under reduced pressure at room temperature.

The dried DNA was mixed with 28 ml of TES buffer (30 mM Tris-HCl 5 mM EDTA).
Dissolve in 50 mM NaCl (pH 8.0), add 28 g of CsCl (1.4 ml) of a 1% ethidium bromide solution, and perform ultracentrifugation at 40,000 rpm for 40 hours.

After completion of the ultracentrifugation, the lower layer fraction is collected from the fractions that emit fluorescence by UV irradiation. The collected fraction is washed with isopropanol and dialyzed overnight. 0.3M in dialysate
Add 5M sodium acetate. Then, two times the amount of ethanol is added, and the mixture is allowed to stand at -20 ° C overnight to obtain a plasmid DNA precipitate.

(5) Analysis of DNA The obtained plasmid DNA was quantified by measuring the absorbance at 254 nm and calculating the amount of DNA at 1 mg / ml as the absorbance of 20.
In addition, the length of the plasmid DNA was determined by comparing the mobility by electrophoresis with the mobility of a standard DNA sample after digestion with a restriction enzyme.

IV. Determination of Nucleotide Sequence (1) Extraction of plasmid by Rapid method and plasmid DNA The plasmid DNA was used for assay of plasmid DNA contained in the transformant. Escherichia coli strain was added to 1 ml of L medium (10 g: polypeptone, 5 g:
After culturing in a yeast extract, 5 g: a nutrient medium containing sodium chloride in 1), the cells are suspended in 300 μm of STET buffer. Add 50μ of lysozyme solution (10mg / ml) to this,
After stirring, heat in boiling water for 40 seconds. After heating, the mixture is rapidly cooled and centrifuged to obtain a supernatant. Then add 350μ to the supernatant
And cooled at -20 ° C for 20 minutes.
The mixture is centrifuged and the precipitate is isolated. Precipitate 50 after drying
It was dissolved in μ of Tris buffer to obtain a crude plasmid extract. The DNA was assayed by examining the cleavage pattern with the restriction enzyme using 10 μl of the crude extract.

(2) Plasmid Preparation by Alkali Method The experiment method was in accordance with the “Gene Manipulation Manual” (edited by Yasutaka Takagi, edited by Kodansha Scientific p.8 (1987)).
The cultivation of the Escherichia coli strain was performed at 37 ° C. for 6 hours in 70 ml of an L medium (containing ampicillin). Plasmid in alkaline solution
After extracting DNA, the crude extract was subjected to phenol extraction and ethanol precipitation to purify.

(3) Purification of plasmid DNA by cesium chloride equilibrium density gradient ultracentrifugation The phenol-treated ethanol solution is centrifuged to collect the precipitate. The obtained precipitate is dried under reduced pressure at room temperature.

The dried DNA was mixed with 28 ml of TES buffer (30 mM Tris-HCl, 5 mM EDT
A, 50 mM NaCl (pH 8.0), 28 g of CsCl (1.4 ml) of 1% ethidium bromide solution, and ultracentrifugation at 40,000 rpm for 40 hours.

After completion of the ultracentrifugation, the lower layer fraction is collected from the fractions that emit fluorescence by UV irradiation. The collected fraction is washed with isopropanol and dialyzed overnight. 0.3M in dialysate
Add 5M sodium acetate. Then, a 2-fold amount of ethanol is added and the mixture is allowed to stand at −20 ° C. overnight to obtain a rasmid DNA precipitate.

(4) Purification of plasmid by Sephacryl S1000 column chromatography Plasmid DNA can be easily purified to a higher purity by performing Sephacryl S-1000 column chromatography instead of the conventional cesium chloride density gradient ultracentrifugation method. Can be obtained. Sephacryl S-1000
Was obtained from Pharmacia. The column used was 1c in diameter
m, length 50 cm, eluent 50 mM Tris-HCl buffer-1 mM E
DTA-50 mM sodium chloride solution was used. Plasmid fractions were identified by electrophoresis.

(5) Transformation of Escherichia coli (introduction of plasmid DNA) A bacterium obtained by cloning a fragment of chloroplast DNA digested with restriction enzyme PstI was transformed into an LB rich medium (10 g of polypeptone, 1 g of yeast extract, 3 g of NaCl, 3 g of NaCl, KH ₂ PO per culture solution). ₄ 3g, N
_{a 2 PO 4 · 12H 2 O14.3g} , was inoculated into pH 7.0) 20 ml containing NH ₄ Cl1g, overnight stationary culture at 37 ° C. (preculture). This preculture was added to Medium 1 containing the above LB rich medium plus antibiotics, and K
Shake and culture with a lett meter (a device for measuring turbidity of cell growth) until it shows 100. Chloramphenicol at Klett100
Add 200 mg and continue shaking culture overnight.

After completion of the culture, the cells are collected by centrifugation and plasmid DNA
Is extracted.

(6) Stepwise deletion method (deletion method) In order to determine the base sequence, the conventional M13 method makes it very difficult to reconstruct (join the base sequences) after reading the base sequence of each sample. there were.

This stepwise deletion method is a method developed for efficiently reading long DNA sequences. If deletions are introduced sequentially (stepwise) from the primer end of the DNA for determining the sequence, and the sequence is determined for each,
The advantage is that the base sequence of long DNA can be determined.

Exonuclease III to introduce stepwise defects
(Exo III) is used. This Exo III is DNA
Is an exonuclease that decomposes in the 3 ′ → 5 ′ direction, but the decomposition does not proceed unless the 3 ′ end is paired with a compensation chain. In other words, if digestion is carried out with a restriction enzyme such that the 5'-end protrudes, Exo III starts to be decomposed at that site, but DNA degradation by Exo III does not occur at the restriction enzyme cleavage site where the 3'-end protrudes. .

Therefore, a restriction enzyme (SphI, PstI, KpnI, SacI) protruding from the 3′-end of pUC118 / 119 and a restriction enzyme protruding from the 5′-end (BamHI, HindIII, etc.) are combined to be deleted. Should be introduced.

After digestion with Exo III, Mung Bean nuclease (Mung Bean nuclease) specifically selects and degrades single-stranded regions.
clease), the end is changed to blunt end, repaired with klenow enzyme, ligated, and returned to double-stranded circular plasmid DNA again. When Escherichia coli is transformed with this plasmid DNA, Escherichia coli having various plasmids can be obtained.

(7) Phage method sequencing and plasmid method sea casing Sequencing was performed by a phage method and a plasmid method by a standard method.

(8) Analysis of base sequence The analysis was mainly performed using a personal computer. The analysis program used was Genetyx from Software Development Company. The results are shown in FIG.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the entire nucleotide sequence of rice chloroplast, and FIG. 2 is a diagram showing a genetic map of rice chloroplast genome.

Continuation of the front page (56) References Japanese Journal of Genetics, Vol. 61 [6] (1986) p. 537-542 Gene, Vol. 50 (1986) p. 271-278 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/29 GenBank / EMBL / DDBJ

Claims (1)

(57) [Claims] 1. A rice chloroplast-derived DNA having the following nucleotide sequence.