CN1055969C - Synthetic gene of rice ribulose-1,5-diphosphocarboxylase/oxygenase major subunit - Google Patents

Abstract

The present invention relates to a structural gene (1466b, p,) of a large subunit of rice ribulose-1, 5-diphosphate carboxylase/oxygenase (Rubisco for short), which is redesigned and completely synthesized according to a high-expression codon distribution coefficient of colibacillus. The synthesized gene containing 45 single restrictive endonuclease sites is effectively expressed (180 mg/l) in colibacillus, and a purified expression product and a small subunit of rice Rubisco generated by gene engineering are recombined and then are analyzed and identified by an immunoblotting method to generate a product with the same molecular weight and the same immunity as natural rice Rubisco.

Description

Paddy rice ribulose 1, the synthetic gene of the big subunit of 5-bisphosphate carboxylase/oxygenase

The present invention relates to molecular biology, relate in particular to and adopt computer aided design (CAD) gene DNA order, genetic engineerings such as the complete synthesis and recombinant technology of DNA and gene expression regulation, make the intestinal bacteria generation and separate the ribulose 1 that obtains from the paddy rice leaf, the big subunit of 5-bisphosphate carboxylase/oxygenase has the polypeptide of same amino acid order.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (ribulose 1,5-bisphosphatecarboxylase/oxygenase, EC 4,1,1,39, abbreviation Rubisco) extensively be present in the ptotoautotroph.Rubisco is present in the chloroplast(id) plastid in higher plant, and content generally accounts for the solubility leaf protein more than 50%, is the abundantest a kind of protein of nature [Ellis, R.J., Trends in Biol.Sci. (1979) 4,241-244].It is a bifunctional enzyme, as carboxylase, and the first step reaction in its catalyzed carbon fixed cycles (light compositing), i.e. ribulose 1,5 bisphosphate (RUBP) and CO ₂Reaction, tellurian according to estimates plant fixes 5 * 10 every year approximately ¹⁴Kilogram CO ₂Make it to become organic carbon; As oxygenase, first reaction of its catalysis photorespiration process, the carbon that photorespiration causes a part to be fixed is released in the air and goes.Rubisco obviously is the key enzyme in the photosynthesis, but its catalytic activity is not high, is not a kind of catalyzer efficiently.Therefore, " improvement " Rubisco promptly improves the relative reactivity of its carboxylation and oxygenation and improves its catalytic efficiency, makes it more effectively to fix limited CO ₂Thereby, accelerate plant production, be the ultimate aim that numerous scientists study Rubisco.

Rubisco has complicated subunit structure, remove outside Rubisco in some purple nonsulfur bacteria only is made up of a kind of subunit, the Rubisco great majority in every other source are made up of 8 big subunits (molecular weight respectively is 50-55kD) and 8 small subunits (molecular weight respectively is 12-18kD), and promptly this proteinic subunit structure is L ₈S ₈Catalytic site on big subunit, but do not have small subunit to be combined on the big subunit, the activity of enzyme extremely faint (be about holoenzyme 0.5%) [Plant Biol. (1989) 8 for Pierce, J., 189].

Paddy rice is the main food crop in area, Asia, selects paddy rice Rubisco as research object, explores its structure and function relationship, improves the photosynthetic efficiency of paddy rice, has realistic meaning.Existing studies show that, as other plant, paddy rice Rubisco is made up of with 8 identical small subunits (abbreviation rbcS) 8 identical big subunits (abbreviation rbcL), gene order is determined by the chloroplast gene coding for its big subunit (being called for short RrbcL), and the RrbcL that derives from DNA sequence has 477 amino-acid residues [referring to document: Nishizawa, Yoko and Hirai, Atsushi, Jpn.J.Genet. (1987) 62,389-395] [Fig. 1].

The application of Protocols in Molecular Biology is an effective way that changes the Rubisco photosynthetic efficiency.Can systematically carry out the research of Rubisco structure-function relationship by recombinant DNA technology (particularly transgenation technology), thereby obtain the gene of high-level efficiency Rubisco.Development along with the DNA synthetic technology, synthetic gene more and more demonstrates its advantage on genetically engineered owing to its ease-to-operate, thereby this is included in and equally distributed single restriction endonuclease sites is set in the synthetic gene makes transgenation become easy, add the controlling elements of transcribing or translating by two ends, saved the trouble of from genome, cloning and express the change protogene that target gene ran at synthetic gene; By in synthetic gene, selecting to be suitable for the codon of given host cell high expression level system, synthetic gene can be expressed efficiently.[referring to document: Ferretti, L,, Karnick, S.S., Khorana, H.G., Nassal, M., and Oprian, D.D., Proc.Natl, Acad.Sci, U.S.A, (1986) 83,599-603.].

For this reason, the objective of the invention is the synthetic big subunit structure gene of paddy rice Rubisco of design, can be directly in intestinal bacteria high expression level go out the polypeptide that has the same amino acid order with the big subunit of natural paddy rice Rubisco, and single restriction endonuclease sites many and that be evenly distributed is set, in structure gene with the genetically engineered system that provides to be convenient to carry out transgenation research.

The present invention be a kind of can be in intestinal bacteria the synthetic gene of the big subunit of paddy rice Rubisco of high expression level, it is codon partition ratio by the colibacillus high expression system, machine aided design and the complete synthesis big subunit structure gene of paddy rice Rubisco (being called for short RrbcL G) as calculated, can give expression to single RrbcL with high yield (180mg/L), the product of this gene has length identical with natural RrbcL (judging from SDS-PAGE) and identical immunity (immunoblotting) on inspection, can assemble with paddy rice Rubisco small subunit (RrbcS), and this gene is convenient to RrbcL is undertaken by sudden change the structure and the functional study of system.

1, the design of gene

Behind the amino-acid sequence input computer with 477 residues, the running relevant procedures, the relative use data of pressing colibacillus high expression gene degenerate codon are [referring to document: Sharp, P.M., et al, Nucl.Acids Res. (1986}14,5134.], design single restriction endonuclease sites commonly used as much as possible and synthetic gene and can in intestinal bacteria, the requirement of effective initial expression design the RrbcL structure gene (Fig. 1) that contains 1466b.p., it contains encoding gene 1437 b, p., ribosome binding site, dual terminator codon, and the sticky end EcoRI and the BamHI at two ends.In this synthetic gene, except the EcoRI and BamHI at two ends, we also are provided with 45 single restriction endonuclease sites commonly used, and their distribution situation is seen Fig. 2, and have only 12 this sites in the corresponding RrbcL natural gene.In addition, contrast RrbcL natural gene has 284 bases different in the coding region, and this is in order to change the codon of 266 amino acid (account for 477 residues 56%), to make RrbcL G be adapted to the colibacillus high expression system.Obviously, RrbcL G more can be effectively in intestinal bacteria high expression level and more help carrying out the positional mutation and the cassette mutagenesis of gene so that systematically be engaged in the research of the 26S Proteasome Structure and Function relation of paddy rice Rubisco.

2, gene is complete synthesis

The present invention with reference to the novel and simple method in this field [referring to document: Chen, H.-B., et al., Nucl.Acids Res. (1990) 18,871-878; Rossi, J.J.et al., J.Biol.Chem., (1932) 257,9226-9229], improved and finished the synthetic of RrbcL G after the innovation to some extent.Become the big fragment of single stranded DNA (ssDNA) with dna ligase through the oligonucleotide fragment that the catalysis of strand method connects chemosynthesis, or two oligonucleotide complementary strands of 3 ' end parts paired are copied into two strands with archaeal dna polymerase, with the DNA recombinant technology strand or the double-stranded large dna fragment cloning of the RrbcL G that connects into advanced a kind of plasmid vector then, and in carrier, be assembled into complete RrbcL G, obtain containing the cloned plasmids of this gene and transform a kind of intestinal bacteria.

Fig. 3 represents that RrbcL G is divided into RL01, RL02, three big fragments of RL03 and they are by A, B, C, D ... or the like 32 oligonucleotide fragments form.Fig. 4, Fig. 5 and Fig. 6 illustrate RL01, RL02, RL03 three big segmental synthetic routes respectively.In each fragment after the DNA sequence analytical proof correctly obtains, they are assembled into (Fig. 7) among the carrier pWR13, the cloned plasmids pC1RrbcL that obtains containing complete RrbcL G (submits Chinese typical culture collection center on November 14th, 1994, China Wuhan, deposit number CCTCC NO:M94069).

3, the expression of complete synthesis RrbcL G

For make complete synthesis RrbcL G can be in intestinal bacteria high expression level, the present invention has selected the expression vector plasmid pPLc2833 by strong promoter control, and (seing before) taken all factors into consideration following 2 requirements when the DNA sequence of design gene: (1). press colibacillus high expression gene degenerate codon partition ratio and select codon, (2). and the ribosome binding site that is complementary at 5 ' of gene-end setting and expression plasmid and gene itself (SD in proper order, ATG and relevant nucleotide sequence).

As shown in Figure 7, RrbcL G is taken out and inserts expression vector pPLc2833 by the DNA reorganization from pC1RrbcL and obtain expression plasmid pE1RrbcL (in the Chinese typical culture collection of submission on November 14th, 1994 center, China Wuhan, deposit number CCTCC NO:M94070), transformed into escherichia coli C600 (pcI857).Transformant through pre-cultivate and abduction delivering after, expression product is through denaturing polyacrylamide gel electrophoresis analysis revealed (Fig. 8): with natural RrbcL G swimming apart from identical position, a protein band that increases gradually with expression time prolongation product quantity appears.

4, the purifying of RrbcL G expression product and evaluation

RrbcL G expression product exists with insolubles form (being so-called inclusion body) in intestinal bacteria, and can obtain purity through the operation of embodiment 4 is the pure product of RrbcL (Fig. 9) 120mg/L more than 95%.The n terminal amino acid sequence analysis shows that the N-end order of purified product is identical with natural RrbcL's; Western blot test shows that purified product and natural Rubisco antibody have specific reaction.

5, behind the RrbcL G expression product purifying with the assembling of synthetic RrbcS

Big subunit has only with small subunit and combines and be assembled into the Rubisco whole molecule, just can show activity.The present invention adopts 5 the method for implementing, big small subunit is dissolved in the denaturant solution with rarer concentration together, progressively denaturing agent is removed in dialysis then, centrifugal clarification dialyzate and ultrafiltration and concentration, the assembling product show through native polyacrylamide gel electrophoresis analysis and western blot test, with natural paddy rice Rubisco same position place have one can with the band of natural Rubisco antibody specific reaction.

6, the structure of RrbcL mutant structure gene and expression

In order to utilize RrbcL G to carry out the RrbcL sudden change, find out the position of amino acid code (one or several) in Fig. 1 that will suddenly change in principle earlier, find near treat that the mutating acid codon is two end restriction enzymes then, metathetical restriction enzyme enzyme fragment is wanted in decision, and synthetic contain the restriction enzyme enzyme fragment of Xinmi City's numeral after, carry out DNA recombination to construct mutant plasmid with similar aforesaid method, and carry out the genetic expression of mutant plasmid.

Here introduce RrbcL V377N mutant: earlier be about to the 377th Xie Ansuan codon GTT and be transformed into the segmental displacement of restriction enzyme of l-asparagine codon AAC in that plasmid pC1RrbcL is enterprising, promptly use new synthetic restriction enzyme enzyme fragment, BsaIII-NgoMI, replace the segmental DNA reorganization of BsaHI-NgoMI original among the pC1RrbcL, obtain containing the plasmid pC1RrbcL-V377N of mutant V377N structure gene, change mutant V377N structure gene over to expression vector pPLc2833 from mutant plasmid pC1RrbcL-V377N with embodiment 3 identical methods then and obtain mutant expression plasmid pE1RrbcL-V377N, and carry out the separation and purification of genetic expression and expression product behind the transformed into escherichia coli C600 (pcI857), Analysis and Identification or the like.With similar step RrbcL G is carried out transgenation and can make up many mutant genes, genetic expression can obtain the mutant of many RrbcL then, obtains the Rubisco holoenzyme by the assembling with small subunit.Study the structure and the function relationship of these enzymes, therefrom can find the Rubisco derivative or the new variety of some excellent propertys.

Below given definition be in order to be illustrated more clearly in the implication that they use in the present invention.

Gene

Be meant that a gene comprises structure gene in the required global DNA part of protein of biosynthesizing, the transcripting starting area of structure gene upstream, it starts and adjustment structure expression of gene and 3 ' end polyadenylic acid order.

Structure gene

Be a part of gene, comprise the part of coded protein and polypeptide, comprise the wherein dna fragmentation of part insertion sometimes.Structure gene can find in cell usually or be common not on its cell position that is introduced into that under latter event, it is referred to as heterologous gene.Heterologous gene can be whole or in part from known any source, this field.Structure gene in the coding region or translation district one or more modifications are arranged, they can influence the biological activity or the chemical structure of expression product, expression rate or express control mode.These modifications include but is not limited to suddenly change, the displacement of insertion, disappearance and one or more Nucleotide.

Synthetic gene

Whole or the major part that is meant coding region in the DNA sequence of a structure gene is chemosynthesis, as cited here, oligonucleotide fragment is that the process chemistry that adopts this field to know is synthetic, be connected with enzyme catalysis and form gene fragment through annealing, further assemble these gene fragments with enzyme catalysis then and become complete gene.The gene suitable with structure with the function of synthetic gene as described herein can adopt positional mutation used in this field or other relevant method to prepare.

The use of degenerate codon

Be meant the selection that a specific host cell is showed when accessing to your password the given amino acid of one of son establishment, in the selection of specific cryptosystem, have certain preferential.The frequency of utilization of specific codon in gene is to remove and measure be encoded identical amino acid whose all passwords (i.e. all degenerate codons) occur in the gene number of times of the number of times that this codon in the gene occurs.Similarly, host cell, the intestinal bacteria of As used herein, the frequency that codon uses can be calculated by the average frequency that codon in a large amount of genes of expressing in host cell uses.It is then better when this analysis is confined to the gene of host cell high expression level.For the purpose of directly perceived, this frequency often is expressed as the relative use of degenerate codon, and promptly the frequency of utilization with codon multiply by the corresponding amino acid whose degenerated code subnumber of this codon.

When synthesizing a gene for the expression in the raising host cell, become this gene design the use of its degenerate codon to meet the relative use of the degenerate codon of host cell cance high-expression gene, can make institute's synthetic gene in host cell, obtain high expression level like this.The present invention adopts Sharp, and the degenerate codon of the colibacillus high expression gene that P.M. etc. deliver is used data [referring to document: Sharp, P.M., et al., Nucl, Acids Res. (1986) 14,5134.] (table 1) relatively.

The clone

Be meant that in a group cell each is all derived from same ancester cell.Cloning most important use is with dna fragmentation, comprises whole gene or some, increases in host cell by carrier (plasmid, phage and coemid).Synthetic RrbcL G of the present invention recombinates into transformed into escherichia coli behind the plasmid and obtains amplification by the ordinary method in this field.

Transform

Be meant that one of a dna fragmentation introducing stably will carrying functional gene did not contain in the organism of this gene in the past.The conversion introducing did not contain in the intestinal bacteria of this gene in the past after above-mentioned RrbcL G advanced plasmid by reorganization.

Genetic expression

Be meant structure gene to transcribe and translate (or translating) and produce coded protein or polypeptide.Synthetic RrbcL G of the present invention can produce in intestinal bacteria and the identical polypeptide of the product of RrbcL natural gene in chloroplast(id), and than natural gene higher expression efficiency is arranged in intestinal bacteria.

The subunit assembling

Being meant that subunit unit unites with specific combination in certain proportion forms a complete unit or a structure.A lot of enzymes all is made up of a plurality of polypeptide subunits, and they assemble the integral body that generation has catalysis activity with special combination.The assembling of subunit is to be finished by a kind of protein of molecular chaperones (Molecular Chaperone) that cries in vivo, then undertaken by sex change～renaturation process often external, also needs the help of molecular chaperones in case of necessity.The assembling of big subunit of paddy rice Rubisco of the present invention and small subunit is carried out external.

The present invention has following advantage:

1. not only designed the nucleotide sequence of the encoding gene of RrbcL, and designed the ribosome bind site that is complementary with its 5 ' end order and expression vector: SD order and initiator codon ATG and relevant nucleotide sequence, can make RrbcL gene of the present invention change expression vector over to like this after synthesizing can express effectively.

2. use System Design corresponding structure gene by the host cell cance high-expression gene codon of expressing, fundamentally overcome the defective of natural gene on codon is selected.We select to have eliminated 42 codons [table 1] that assigned frequency is extremely low in the colibacillus high expression system in the former natural gene at the codon of RrbcL gene, lay a good foundation for realize high expression level in intestinal bacteria.

3. in RrbcL G, by computer aided design (CAD) restriction endonuclease sites just commonly used as far as possible in many ways.Do not comprise the two ends sticky end, be provided with 45 single restriction endonuclease sites commonly used among the RrbcL G, also have a plurality of restriction enzyme positions, two～three sites that have therein commonly used, these sites also are single when occurring in the synthetic gene fragment.Like this, the equispaced is about 30b.p. between two adjacent sites, therefore can utilize RrbcL G to carry out the rite-directed mutagenesis or the cassette mutagenesis research of gene easily.

4. the synthesis step of RrbcL G of the present invention is simple, has adopted the method for two kinds of Nover practicals and promotes and improve.Big segmental synthetic as RL02, after being copied into two strands, the long strand of DNA that connection is obtained is cloned into host cell again, avoided the complicacy of strand clone the unknown, guaranteed the accuracy of synthetic fragment cloning.

5. by after synthetic RrbcL and RrbcS are dissolved in strong denaturant solution, be diluted in the another kind of more weak denaturant solution, the dialysis method of removing denaturing agent is progressively observed the formation of Rubisco molecule again.Though efficiency of assembling is very low, this is the acquisition first of the external assembling of higher plant Rubisco, is worth further research, is structure and the function relationship blaze the trail of in vitro study higher plant Rubisco.

Following embodiment can be used as concrete scheme of the present invention, and they do not limit the scope of the invention.

Materials and methods:

Except that following materials and methods referring to document [Chen, H.-B., et al., Nucl.Acids Res, (1990) 18,871-878.].

Sep-pak C18 reversed-phase column is from Waters company;

Low melting-point agarose gel and Coomassie brilliant blue R-250 are from GIBCO BRL company;

Restriction enzyme, the polynucleotide phosphorylating kinase, the T4 dna ligase is from NewEnglish Biolabs or Boehringer Mannheim company;

DNA sequence is analyzed required primer by this laboratory synthesizing and purifying;

[α- ³⁵S] ATP is from Amersham company;

Sepharose CL 4B and plasmid pUC18 are from Pharmacia-LKB company;

Expression plasmid pPLc2833 and intestinal bacteria C600 (pcI857) are that Massachusetts Institute of Technology professor Khorana gives;

Plasmid pWR13 and intestinal bacteria JM83 are that professor Guo Lihe of Shanghai Inst. of Cytobiology, Chinese Academy of Sciences gives;

Sodium laurylsulfonate (SDS) is from Boehringer Mannheim company;

Nitrocellulose membrane is from U.S. Micro Filtration System company;

4-chloro-naphthols is from Sigma company;

Natural Rubisco antibody helps preparation by the scorching professor of Shanghai Institute of Immunology's period-luminosity;

Other chemical reagent is commercially available home products.

Connection between oligonucleotide fragment is pressed strand method [Chen, H.-B., et al., Nucl.Acids Res. (1990) 18,871-878.] and is carried out.

The complementary strand of two oligonucleotide of 3 '-end parts paired is copied into and carries out after two strands is improved by the method for [Rossi, J.J.et al., J.Biol.Chem., (1982) 257,9226-9229] such as Rossi.Two oligonucleotide fragments of equimolar 3 '-end parts paired add the big fragment of archaeal dna polymerase in the presence of four kinds of dNTP, successively insulation reaction after 30 ℃ of pairing half an hour: 25 ℃, and 5min; 37 ℃, 45min; 25 ℃, 10min.After reaction is finished, go out double-stranded DNA with ethanol sedimentation.

The structure of plasmid:

The plasmid that needs are recombinated carries out double enzymolysis by the restriction enzyme of selecting earlier, zymolyte separates through the low melting-point agarose gel electrophoresis, prepare the big fragment of linear plasmid according to a conventional method, then with the synthetic gene fragment or behind another plasmid double enzymolysis isolating gene fragment be connected, connect product transformed into escherichia coli and with specific antibiotic screening transformant according to a conventional method.From pUC18 or pWR13 deutero-plasmid transformation escherichia coli JM83 37 ℃ of cultivations of penbritin; From pPLc2833 deutero-plasmid transformation escherichia coli C600 (pcI857) penbritin and 30 ℃ of cultivations of kantlex.From the bacterium colony that clones, select the plasmid that single bacterium colony increases according to a conventional method and prepares structure in the LB nutrient solution.[referring to document: Sambrook, J., Fritsch, E.F., and Maniats, T., Molecular Cloning:a laboratorymanual (2nd ed) (1989), Cold Sping Harbor Laboratory Press, New York.]

Document [Chen is pressed in gene DNA sequence analysis in the plasmid, H.-B., et al., Nucl.Acids Res. (1990) 18,871-878.] condition to be used as the paired short oligonucleotide with the universal primer at pUC18 or two ends, pWR13 polylinker district or gene in synthetic be primer.

Genetic expression: introducing here with pPLc2833 is the genetic expression of carrier [referring to document: Remaut, E., Tsao, H., and Fiers, W., Gene (1983) 22,103-113.].

The bacterium liquid that contains expression plasmid of incubated overnight, the dilution back makes A650nm be elevated to 0.5-0.8 30 ℃ of cultivations and changes 41 ℃ of thermal inductions over to, till A650nm no longer increases, induced approximately 4 hours.

The separation and purification of expression product:

Thalline after the above-mentioned expression is collected through the high speed frozen centrifugation, be suspended in the Tris.HCl damping fluid, ultrasonication and separated and collected inclusion body, the latter is after the washing and processing of reagent such as urea, Triton, Guanidinium hydrochloride, be dissolved in the damping fluid of hydrochloric guanidine, and in the presence of Guanidinium hydrochloride and mercaptoethanol,, obtain purity at last and be 95%, natural paddy rice Rubisco antibody had the genetically engineered RrbcL of specific immune reaction through the further separation and purification of gel-filtration.

The external assembling of expression product and RrbcS:

With RrbcL G expression product and gene engineering product RrbcS[Chen, H,-B, andWang, G.-A., Curr.Plant Sci.Biotechnol.Agric. (1993) 15 (Biotechnology in Agriculture), 160-164] be dissolved in together in the damping fluid of hydrochloric guanidine, again with this solution dilution in urea-containing damping fluid, the damping fluid dialysis that successively urea concentration is reduced gradually then, last centrifugal precipitation and the ultrafiltration and concentration removed.Concentrated solution is carried out non-sex change electrophoretic analysis.

The design of embodiment 1:RrbcL G

(A) design of coding DNA order

Under computer VAX/11-780 is auxiliary, carry out the design of RrbcL G, behind known RrbcL amino-acid sequence input computer, software PSQ and NAQ that running American National biomedical research foundation (NBRF) is given, inspection is by the recognition sequence by the restriction enzyme that may occur in the molecular RNA order of degenerated code of computer reverse translation, then according to (1), colibacillus high expression gene codon frequency of utilization (table 1), (2), in selected cloned plasmids pWR13, evenly settle single restriction enzyme in the RrbcL synthetic gene and (3), the segmentation of synthetic gene fragment waits three to require manually selected each amino acid whose codon and the DNA sequence of definite RrbcL encoding gene with eliminating self to match

(B) design of part expression regulation order

As described above.We adopt the expression vector of plasmid pPLC2833 as the RrbcL synthetic gene.In order to make this carrier can actual operation, must between 5 ' end of promotor and synthetic gene, add the startup software of a translation, i.e. ribosome binding site, it comprises that SD is in proper order and initiator codon ATG and relevant nucleotide sequence.200 Nucleotide of the 5 ' end of the mRNA that transcribes out when considering genetic expression can form secondary structure, and the situation of SD order in this status nascendi mRNA 5 ' end secondary structure can have a significant impact [referring to document: Chen the initial sum productive rate of expressing, H.-B.et al., (1995) wait to deliver].Like this computer auxiliary down, trigram by selecting 5 '-end codon and the nucleotide sequence between SD order and the initial code ATG so that SD order and ATG are in the ring district of mRNA secondary structure, thereby have obtained order shown in Figure 1.

At last, add that at 3 ' of the gene order of this design-end two terminator codon TAA and TAG and two ends are the sticky end of needed EcoRI of molecular cloning and BamHI restriction enzyme recognition sequence, obtain the DNA sequence that length shown in Figure 1 is 1466b.p..

Embodiment 2:RrbcL G's is complete synthesis

(A) preparation of oligodeoxynucleotide

RrbcL G is divided into RL01, RL02 and three big fragments of RL03 as shown in Figure 3.RL01 (476b.p.) further is divided into A, B, C, D, E, F and seven underlying stock oligonucleotide fragments of G, two negative burst Segment A of end ' b and fG ', and the bridge-type of the connection usefulness of middle four weak points negative gang of oligonucleotide bc, cd, de and ef.RL02 (496b.p.) is divided into H, I, J, K, L and six underlying stock oligonucleotide fragments of M, and two ends are born gang short-movie section hh and mm, and five bridge-types negative gang of an oligonucleotide fragment hi, ij, jk, kl and lm.The fragment of RL03 (490b.p.) is divided with top different, and it is divided into positive and negative strand three groups and amounts to six fragments, i.e. N (+) and O (-), P (+) and Q (-) and R (+) and S (-).

Amounting to 32 oligonucleotide fragments goes up with the solid phase phosphoramidite triester method synthetic at dna synthesizer (ABI 381A type).After each fragment is synthesized separately, handled 8 hours through 55 ℃ of strong aquas, make it to come off and excise blocking group from solid phase, thick product concentrates afterwards water-soluble, separate with urea-denatured polyacrylamide gel electrophoresis (PAGE), the gel band that contains pure products leaches through hydrogen-carbonate triethylamine (1mol/l), with Sep-pek C18 reversed-phase column press people such as Lo method [referring to document: Lo, K-M., Jones, S.S., Hackett, N.R.﹠amp; Khorana, H.G., Proc.Natl.Acad.Sci.USA. (1984) 81,2285-2289] desalination obtains pure oligonucleotide fragment.

Pure oligonucleotide carries out 5 '-phosphorylation according to a conventional method with T4 polynucleotide phosphorylating kinase and ATP.

(B) the big fragment of connection synthetic gene between oligonucleotide

13 fragments divide three groups to match as shown in Figure 4 for a.RL01 synthetic, i.e. A, B, C, A ' b and bc, D, E and de, F, G and fG ' merge then, add fragment cd and ef, five minutes postcooling to 0 of 30 ℃ of insulations ℃ add the T4 dna ligase, 16 ℃ of reactions 16 hours.Reaction product after low melting-point agarose gel separation preparation, with by EcoRI and ApaI double digestion linear plasmid carrier pUC1802 ^*Link to each other, directly transformed into escherichia coli JM83.Plasmid is extracted in transformant amplification back carry out enzymolysis analysis, and do the DNA sequence analysis to containing the segmental plasmid of RL01 tentatively certainly, with original oligonucleotide fragment the base disappearance that subtracts in the cloned sequence is repaired, again through DNA sequence analytical review, obtain containing the plasmid pRL01 of RL01 gene fragment at last.

The synthetic of b.RL02 with the similar method for preparing RL01, divides three assembly to once connecting (because fragment is longer, connecting temperature should carry out at 30 ℃) as shown in Figure 5, prepares the gene strand.The strand gene fragment links to each other with the pUC1802 linear plasmid that was digested by ApaI and SalI with hh pairing back.After the connection product is settled out by alcohol, with the pairing of mm fragment, in the big fragment of archaeal dna polymerase

* this plasmid is to derive from plasmid pUC18 in the contriver laboratory, and the polylinker district with the latter changes into the DNA recombinant technology: EcoRI-XhoI-ApaI-BamHI-XbaI-SalI-PstI-HindIII.Reaction is one hour under existence and the corresponding condition thereof, adds T4 dna ligase and ATP then, continues reaction six hours, transformed into escherichia coli JM83 at 16 ℃.Transformant amplification extraction plasmid is carried out enzymolysis identify and the DNA sequence analysis, obtain containing the plasmid pRL02 of RL02 gene fragment.

The synthetic method that is different from RL01 and RL02 fully that adopted of c.RL03.As shown in Figure 6, the big fragment of RL03 is divided into three groups and finishes: N (+) and O (-), P (+) and Q (-), R (+) and S (-).With N (+)+O (-) is example, N (+) and O (-) match in 50 ℃ to 25 ℃ process of cooling, add the big fragment of archaeal dna polymerase 30 ℃ of reactions one hour, after being settled out product, it is carried out the double digestion of HindIII and SalI, the product ethanol sedimentation goes out the back and links to each other with the linear plasmid pWR13 of HindIII and SalI double digestion, and transformed into escherichia coli JM83 obtains plasmid pRL0301.Can obtain pRL0302 and pRL0303 respectively from other four fragments similarly.But because these four fragments are grown (the longest 124 nucleotide units that reach), the temperature of polyreaction must be at 37 ℃, and [as R (+)+S (-)] also will add short-movie section (rr) to eliminate the secondary structure of strand in case of necessity.

Three plasmids that obtain are carried out the DNA sequence analysis respectively, and three fragments that as shown in Figure 6 enzymolysis discharged then into pWR13 that recombinates together obtains pRL03, and does all-cis preface analysis.

D. as shown in Figure 7, pRL01, pRL02 and pRL03 are made corresponding enzymolysis respectively, three fragments of emitting are cloned into pWR13 together behind conventional method purifying, transformed into escherichia coli JM83, the final plasmid pC1RrbcL that obtains to contain whole gene.

Embodiment 3: the expression of synthetic RrbcL G in intestinal bacteria

As shown in Figure 7, plasmid pC1RrbcL and pPLc2833 are respectively through the EcoRI/BamHI double enzymolysis, and the former gets the small construction gene fragment, the big fragment of latter's line taking character grain, connection reassembles into expression plasmid pE1RrbcL, and transformed into escherichia coli C600 (pcI857).Single bacterium colony of getting the clone in 20ml LB nutrient solution 30 ℃ sway and spend the night, 50 times of this nutrient solution dilutions are to A then _650nm=0.05-0.06 cultivates about two hours to A for 30 ℃ _650nmDuring=0.5-0.8, changing 41 ℃ of thermal inductions over to expresses, respectively at 15 ', 30 ', 60 ', 120 ' and the 240 ' 1ml that respectively takes a sample, centrifuged deposit partly adds 100ul cellular lysate liquid, suspend and 100 ℃ boiled three minutes, sample on the sample size of optical density(OD) such as get after centrifugal, separate through the PAGE of 15% sodium laurylsulfonate sex change, then with the blue colour developing of coomassie.The result is presented at the electrophoretic migration speed position identical with natural RrbcL swimming speed and occurs one and prolong and the ever-increasing colour band of protein content (Fig. 8) with expression time.

The purifying of embodiment 4:RrbcL G expression product and immunity thereof are identified

Centrifugal collection (7,000xg, 15min, 4 ℃) abduction delivering and at 41 ℃ of 500ml intestinal bacteria (containing pE1RrbcL and pcI857) thalline (about 1.4g) of cultivating four hours, after washing one time with TE damping fluid (pH 8.0), be suspended in 20 milliliters of Tris, (pH 8.0 for the HCl damping fluid, 0.1mol/l) in, ultrasonication cell 5 minutes (ultrasonic 30 seconds, gap 30 seconds) in the ice-water bath, the high speed frozen centrifugation is collected inclusion body (10,000xg, 10min, 4 ℃), after 4 milliliters of aqueous solution that contains urea (2mol/l) and 2 ‰ Triton suspends again and places half an hour, collect insolubles with speed, use above-mentioned identical Tris again, HCl damping fluid flush away urea with previous step, Triton and a spot of foreign protein.The inclusion body that obtains like this with the damping fluid of 10 milliliters of hydrochloric guanidines (8mol/l) [Tris, (pH 7.8,100mmol/l) for HCl, NaCl (50mmol/l), DTT (100mmol/l)] dissolving (100 ℃ 3 minutes), the centrifugal insolubles (26 of removing, 000xg, 15min, 10 ℃).

Above-mentioned clear liquor (5ml) application of sample to Sepharose CL 4B chromatography column is (in 2.2 * 50cm), [(pH 7.8 for Tris.HCl with elutriant in advance for this post, 100mmol/l), NaCl (40mmol/l), BME (1 ‰), NaN3 (0.2 ‰), Guanidinium hydrochloride (6mol/l)] balance, with the speed drip washing product of 3cm/hr, a protein peak, Kav=0.55 only appear in whole elution process.Merge,, obtain the big subunit of about 30mg with the legal protein content of Bradford.Product is on 15% SDS-PAGE separation and electrotransfer to a nitrocellulose membrane, film again by 3% bovine serum albumin saturated after, wash with rabbit antibody and horseradish peroxidase (HRP) the link coupled goat anti-rabbit antibody of anti-paddy rice Rubisco successively, use benzidine (DAB) colour developing (Fig. 9) at last, the result shows that the expression product of synthetic RrbcL G and paddy rice Rubisco antibody have specific reaction.

The assembling of embodiment 5:RrbcL G expression product and gene engineering product RrbcS

Following experiment is all carried out in room temperature except that indicating.Get column chromatography solution two milliliters of (1.5mg/ml) and one milligram of gene engineering product RrbcS (0.5ml) of embodiment 4, with they be diluted to 200 milliliters of damping fluids [urea (4mol/l), Tris, HCl (pH 7.8,40mmol/l), MgCl ₂(10mmol/l), BME (1 ‰), EDTA (1mmol/l), glycerine (10%v/v)] in, successively the same buffer that contains different concns urea to be dialysed respectively 24 hours, urea concentration is followed successively by: 2.5mol/l, 2.0mol/l, 1.0mol/l, 0.1mol/l.To the distilled water dialysis that contains 5% glycerine once, at last to damping fluid [Tris.HCl (and pH 7.8,10mmol/l), MgCl ₂(1mmol/l), BME (5mmol/l), EDTA (0.1mmol/l)] twice of 10 ℃ of dialysis.With dialyzate ultrafiltration and concentration to a milliliter (10 ℃), be concentrated in vacuo to 0.1 milliliter (0 ℃) with the 100KD film again.After sampling used the polyacrylamide gel electrophoresis of sex change and non-sex change to separate respectively, electrotransfer carried out western blot test (a same embodiment) to nitrocellulose membrane.The result shows, has obtained comparing with natural Rubisco, has the assembling molecule (Figure 10) of identical electrophoretic property and immune performance.

The construction and expression of embodiment 6:RrbcL V377N mutant structure gene

(A) RrbcL V377N gene mutation body restriction fragment is selected and design

At first, we find coding the 377th amino acids (be Xie Ansuan, codon GTT V377) select two suitable restriction sites, BsaHI and NgoMI then at its two ends from RrbcL G structure iron (Fig. 1).Codon GTT is made into the codon AAC of l-asparagine, simultaneously also change the 384th Xie Ansuan codon GTA into GTT, the change of nucleotide sequence herein causes the disappearance of original BsaAI restriction site, and this has just made things convenient for the relatively evaluation of mutant gene and protogene.We have synthesized following double-stranded restriction enzyme enzyme fragment like this:

BsaHI

NgoMI

CGTCATTCCGAACGCATCTGGTGGCATC CACGTTTGGCATATG

AGTAAGGCTTGCGTAGACCACCGTAGGTGCAAACCGTATACGGCC

Altered base represented in black matrix, and the line part illustrates the disappearance in BsaAI site.

(B) contain the structure of V377N mutant gene cloned plasmids

At first plasmid pC1RrbcL is carried out the part enzymolysis of BsaHI, with NgoMI the single digested plasmid of the BsaHI for preparing is thoroughly digested again, separate the longest big fragment of linear plasmid of preparation, be connected with the restricted dna fragmentation of above-mentioned new synthetic BsaIII/NgoMI with 1: 20 ratio then, connect product transformed into escherichia coli JM83, transformant confirms to have obtained containing the plasmid pC1RrbcL-V377N of RrbcL V377N mutant gene through the comparison of length and BsaAI enzymolysis situation and former plasmid.

(C) expression and the product that contains RrbcL V377N mutant gene identified

By embodiment 3 identical methods plasmid pCIRrbcL-V377N and pPLc2833 reorganization are obtained expression plasmid pEIRrbcL-V377N, transformed into escherichia coli C600 (pcI857) also carries out genetic expression, then by the identical method purifying expression product of embodiment 4 and example 5, carry out that product is identified and with the assembling test of small subunit.The result is similar, no longer accompanying drawing.

In addition, be similar to embodiment 6, the synthetic gene of the present invention that can progressively suddenly change is changed up to 238 codons wherein, even the mutant gene that obtains like this, its codon still has more than 50% identical with the corresponding codon of the big subunit synthetic gene of paddy rice Rubisco of the present invention.

In table 1, the big subunit synthetic gene of paddy rice Rubisco and the natural gene

The relative usage situation of degenerate codon--------------------------------------------------------------------------------------A. C. S. N. E-H. A. C. S. N. E-H. A. C. S. N. E-H. A. C. S. N. E-H.--------------------------------------------------------------------------------------Phe UUU 6 14 0.46 Ser UCU 96 2.57 Tyr UAU 3 10 0.39 Cys UGU 36 0.67

UUC?16 8?1.54 UCC 4 2?1.91 UAC?14 7?1.61 UGC 7 4?1.33 Leu?UUA 1?11?0.11 UCA 0 6?0.20 ^*** UAA 1 0?---- ^*** UGA 0 0?----

UUG 2?10?0.11 UCG 1 0?0.04 ^*** UAG 1 1?---- Trp?UGG 8 8?1.00 Leu?CUU 1 6?0.22 Pro?CCU 3 8?0.23 His?CAU 3?11?0.45 Arg?CGU?19?10?4.39

CUC 3 0?0.20 CCC 1 3?0.04 CAC?12 4?1.55 CGC 9 7?1.56

CUA 1 7?0.04 CCA 4 6?0.44 Gln?CAA 2 7?0.22 CGA 1 5?0.02

CUG?29 3?5.33 CCG?15 6?3.29 CAG 9 4?1.78 CGG 0 1?0.02 Ile?AUU 3?12?0.47 Thr?ACU?15?17?1.80 Asn?AAU 2 8?0.10 Ser?AGU 1 4?0.22

AUC?20 9?2.53 ACC?13 8?1.87 AAC?13 7?1.90 AGC 4 1?1.05

AUA 0 2?0.01 ACA 1 5?0.14 Lys?AAA?18?20?1.60 Arg?AGA 0 3?0.02 Met?AUG?11?11?1.00 ACG 1 0?0.18 AAG 7 5?0.40 AGG 0 3?0.00 Val?GUU?17?13?2.24 Ala?GCU?20?20?1.88 Asp?GAU 9?21?0.61 Gly?GGU?25?23?2.28

GUC 2 1?0.15 GCC 4 6?0.25 GAC?18 6?1.39 GGC?21 2?1.65

GUA 9?14?1.11 GCA?11?16?1.10 Glu?GAA?23?25?1.59 GGA 0?10?0.02

GUG 3 3?0.50 GCG?10 3?0.80 GAG?10 8?0.41 GGG 0?11?0.04?--------------------------------------------------------------------------------------

A. amino acid symbol C. codon S. synthetic gene N. natural gene ^{* *}. terminator codon

E-H. the relative use of degenerate codon in the colibacillus high expression gene

Claims (2)

1. the paddy rice ribulose 1 of a high expression level in intestinal bacteria, 5-bisphosphate carboxylase/oxygenase (abbreviation Rubisco, EC4,1,1,39) big subunit synthetic gene, the DNA nucleotide sequence that it is characterized in that this gene is following shown-25～1441 Nucleotide.

-25

AATTCTCTAAGGAGGTAAGAACAAA -1ATGTCCCCACAGACTGAAACTAAAGCTAGCGTTGGTTTTAAAGCAGGCGTTAAGGACTACAAGTTGACCTACTACACCCCGGAATATGAA 90?H S P Q T E T K A S V G F K A G V K D Y K L T Y Y T P E Y EACTAAAGACACTGATATCCTGGCGGCTTTCCGTGTAACCCCGCAGCCGGGTGTTCCGCCTGAGGAAGCTGGCGCAGCTGTAGCGGCTGAA 180?T K D T D I L A A F R V T P Q P G V P P K E A G A A V A A ETCTTCCACTGGTACCTGGACCACCGTATGGACTGACGGTCTGACGACCCTGGATCGTTACAAAGGCCGCTGCTACCACATCGAGCCCGTT 270?G G T G T W T T V W T D G L T G L D R Y K G R C T H I E P VGTTGGTGAAGACAACCAGTACATCGCTTACGTTGCATATCCGCTGGACCTGTTCGAGGAAGGTTCGGTAACCAATATGTTCACTTCTATC 360?V G K D N Q T I A T V A Y P L D L F E E G G V T N H F T S IGTTGGTAACGTGTTTGGCTTCAAAGCCCTGCGTGCACTGCGTCTGGAGGATCTGCGCATCCCGCCAACTTACTCTAAAACTTTCCAGGGC 450?V G N V F G F K A L R A L R L E D L R I P P T Y S K T F Q GCCGCCACACGGTATCCAGGTTGAACGTGACAAACTGAACAAATACGGCCGCCCGCTGCTGGGTTGTACCATCAAACCGAAACTGGGCTTA 540?P P H G I Q V E R D K L H K I G R P L L G C T I K P K L G LAGCGCTAAAAACTACGGTCGCGCGTGCTACGAATGTCTACGGGGTGGCCTGGACTTCACCAAGGATGACGAAAACGTTAACTCTCAGCCA 430?G A K N Y G R A C Y E C L R G G L D F T K D D E N V N G Q PTTCATGCGTTGGCGTGATCGATTTGTATTCTGCGCTGAAGCTATCTACAAGTCTCAAGCAGAGACTGGCGAAATCAAGGGCCATTATCTG 720?F H R W R D R F V F C A E A I Y K E Q A E T G E I K G H Y LAATGCTACTGCAGGTACTTGCGAAGAAATGATCAAACGTGCTGTATTCGCGCGTGAGCTCGGTGTTCCGATTGTTATGCACGACTACCTG 810?N A T A G T C E E M I K R A V F A R K L G V P I V H R D Y LACAGGCGGTTTCACCGCTAACACTAGTCTGGCACACTACTGCCGTGACAACGGCCTGCTTCTGCACATCCACCGTGCTATGCATGCTGTA 900?T G G F T A N T S L A H Y C R D N G L L L H I H R A H H A VATCGACCGCCAGAAAAACCACGGTATGCACTTCCGTGTACTGGCAAAAGCTTTGCGTATGTCTGGTGGCGATCACATCCACGCGGGCACC 990?I D R Q K N H G M H F R V L A K A L R H S G G D H I H A G TGTGGTTGGTAAACTCGAGGGTGAACGCGAAATGACTCTGGGTTTTGTCGACCTGCTGCGTGATGACTTCATCGAAAAGGACCGTGCGCGC?1080?V V G K L E G E R E M T L G F V D L L R D D F I K K D R A RGGTATCTTCTTTACCCAGGACTGGGTTTCCATGCCGGGCGTCATTCCGGTTGCATCTGGTGGCATCCACGTATGGCACATGCCGGCGCTG?1170?G I F F T G D W V S M P G V I P V A S G G I G V W H M P A LACTGAGATCTTCGGTGACGACTCTGTTCTCCAATTCGGTGGCGGCACCCTGGGCCATCCTTGGGGCAACGCACCTGGTGCTGCGGCAAAC?1260?T E I F G D D S V L Q F G G G T L G H P W G N A P G A A A NCGTGTGGCCCTGGAGGCCTGCGTTCAGGCTCGTAACGAAGGTCGCGATCTGGCTCGTGAACGCAACGAAATCATTCGTTCCGCTTGCAAA?1350?R V A L E A C V G A R N E G R D L A R E G N E I I R S A C K

1441TGGTCTCCGGAACTGGCCGCGGCTTGTGAAATCTGGAAAGCGATCAAGTTCGAATTTGAGCCGGTTGATAAACTGGACAGCTAATAG?W G P E L A A A C R I W K A I K F E F E P V D K L D S ^* (CCTAG)

2. the purposes of synthetic gene as claimed in claim 1, it is characterized in that can be applicable to produce paddy rice ribulose 1, big subunit of 5-bisphosphate carboxylase/oxygenase and mutant thereof, comprise that synthetic gene is inserted e. coli plasmid vector forms cloned plasmids pC1RrbcL and expression plasmid pE1RrbcL, transformed into escherichia coli host cell then, and the purifying of genetic expression in intestinal bacteria and expression product, and with itself and paddy rice ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit assembles, and forms the Rubisco whole molecule.

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