CN102718849B - Protein related to chlorophyll synthesis and coding gene and application thereof - Google Patents
Protein related to chlorophyll synthesis and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses protein related to chlorophyll synthesis and a coding gene and an application thereof. The protein of the invention is the protein of the following a) or b): a), protein composed of an amino acid sequence shown by SEQ ID NO: 1; b), chlorophyll synthesis-related protein which is derived from a) and is obtained by substitution and/or deletion and/ or addition of one or more than one of amino acid residues in the amino acid sequence shown by SEQ ID NO: 1. The experiment proves that the gene CBR1 of the invention can adjust the chlorophyll synthesis process, and can improve the chlorophyll synthesis capability of a plant; the coding protein of the gene CBR1 can adjust the expression of gene PORA in the chlorophyll synthesis process, and thereby the chlorophyll synthesis is influenced. The gene of the invention can be used for artificial reconstruction or modification of the chlorophyll synthesis.
Description
Technical field
The present invention relates to albumen and encoding gene and the application synthetic relevant to chlorophyll.
Background technology
Photosynthesis is the process that on the earth, maximum-norm utilizes sun power, and it provides organism, energy and oxygen for nearly all vital movement.The 90%-95% of plant dry matter is from photosynthetic product, and photosynthesis is the basic substance that crop yield forms.Photosynthesis mainly carries out in the chloroplast(id) of plant leaf, needs the participation of a series of pigment and protein complexes.Chlorophyll is the main pigment that absorbs luminous energy, and chlorophyllous minimizing or disappearance will make leaf color shoal, and directly affect photosynthetic efficiency and function.Along with molecular biology of plants and biochemical development, people's separating clone synthetic various enzymes and the gene thereof of catalysis chlorophyll.Yet, chlorophyllously synthetic be subject in body and the adjusting of environmental factor, this regulating effect is again the control that is subject to nuclear gene and (or) plastogene, therefore, there is complicated regulated and control network in chlorophyllous synthetic and degraded.Separation and clone's key or important regulatory factor, to disclosing chlorophyllous metabolism, have great importance by artificial reconstructed raising optical energy utilization efficiency.
Summary of the invention
An object of the present invention is to provide a kind of and chlorophyll synthetic proteins and encoding gene thereof.
Albumen provided by the present invention, derives from Arabidopis thaliana, be following a) or b) protein:
A) protein being formed by the aminoacid sequence shown in SEQ ID NO:1;
B) by aminoacid sequence shown in SEQ ID NO:1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant to chlorophyll by a) derivative protein.
Described encoding gene is following 1) or 2) or 3) shown in:
1) its nucleotide sequence is DNA molecular shown in SEQ ID NO:2;
2) under stringent condition with 1) the DNA sequence dna hybridization that limits and the DNA molecular of encoding said proteins;
3) with 1) DNA sequence dna that limits has more than 90% homology and the DNA molecular of encoding said proteins.
Albumen in above-mentioned in order to make (a) is convenient to purifying, can connect label as shown in table 1 at N-terminal or the C-terminal of the protein being comprised of the aminoacid sequence shown in SEQ ID NO:1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (a) or (b) in albumen can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in SEQ ID NO:2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.
Described stringent condition can be at 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, and hybridization at 65 ℃, and wash film with this solution.
The primer pair of above-mentioned arbitrary described encoding gene total length or its any fragment of increasing also belongs to protection scope of the present invention.
Described primer pair is specific as follows: a primer sequence in described primer pair is as shown in SEQ ID NO:3, and another primer sequence in described primer pair is as shown in SEQ ID NO:4.
The recombinant vectors that contains above-mentioned arbitrary described encoding gene, recombinant bacterium, transgenic cell line, recombinant virus or expression cassette also belong to protection scope of the present invention.
In above-mentioned arbitrary described recombinant vectors, described recombinant vectors is the recombinant expression vector obtaining at encoding gene described in the encoding gene of albumen described in the multiple clone site insertion claim 1 of expression vector pJIM19-9Myc or claim 2 or 3.
Another object of the present invention is to provide a kind of method of cultivating the plant of chlorophyll synthesis capability raising.
The method of the plant that cultivation chlorophyll synthesis capability provided by the present invention improves, comprise the steps: to import the encoding gene of above-mentioned arbitrary described albumen in the plant that sets out, obtain comparing with the described plant that sets out the transgenic plant that chlorophyll synthesis capability improves.
In aforesaid method, the encoding gene of described albumen imports by above-mentioned arbitrary described recombinant vectors.
In aforesaid method, described in the plant that sets out be monocotyledons or dicotyledons; Described dicotyledons is Arabidopis thaliana.
Experiment showed, that gene C BR1 of the present invention can regulate chlorophyll building-up process, improve the chlorophyll synthesis capability of plant, its proteins encoded can controlling chlorophyll route of synthesis in the expression of PORA gene, thereby affect chlorophyllous synthetic.Gene of the present invention can be for artificial reconstructed or modify chlorophyllous synthetic.
Accompanying drawing explanation
Fig. 1 is yeast screening assay result.
Fig. 2 is the comparison of PORA gene expression dose.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The preparation of embodiment 1, gene
One, the discovery of gene
Yeast crossbreeding screening library: by DNA fragmentation (5 '-Aattacattaaaatatctataaaatatctataaaatatctactgac-3 ' and 5 '-ctaggtcagtagatattttatagatattttatagatattttaatgt-3 ') 95 ℃ of placements 5 minutes, then naturally cooling makes it be combined into double-stranded DNA gradually, obtains double chain DNA fragment; By EcoRI and XbaI enzyme cutting for pHISi carrier (purchased from Clontech company), after recovery purifying, be connected with double-stranded DNA, obtain yeast vector pHIS-EE; PHIS-EE is transformed in yeast Ym4271 cell, then with cDNA library (purchased from Clontech company) hybridization, screening positive clone on the yeast culture base of disappearance His and Leu then, to cloning and sequencing analysis.
From yeast crossbreeding screening, obtain one of them clone (Fig. 1,1, vehicle Control; 2, CBR1).The nucleotide sequence of the gene containing in this clone is as shown in SEQ ID NO:2, and the long 1164bp of its opening code-reading frame, is denoted as gene C BR1; The aminoacid sequence of the albumen of this genes encoding, as shown in SEQ ID NO:1, is 387 amino acid.
Two, the differential expression of PORA gene in wild-type Arabidopis thaliana and mutant Arabidopis thaliana
The seed of cbr1 mutant is purchased from international Arabidopis thaliana Biological resources center.
Cbr1 mutant is following mutant: compare with Col wild type gene group, only in the cbr1 gene in its genome, there is sudden change in this cbr1 mutant, except mutational site, other sequence and Col wild-type Arabidopis thaliana in this mutant gene group are in full accord.
PORA is very important enzyme in chlorophyll route of synthesis, detects the differential expression of gene PORA in cbr1 mutant and Col wild-type Arabidopis thaliana.
The seed of the seed of cbr1 mutant and Col wild-type Arabidopis thaliana is grown 6 days in incubator, obtain respectively seedling; Extract respectively the total RNA of blade of seedling, take respectively the total RNA of blade of cbr1 mutant and the total RNA of blade of Col wild-type Arabidopis thaliana is template, carry out quantitative RT-PCR analysis, the primer of amplification PORA gene is as follows: (5 '-CCTTCAAGCTGCTTCTTTGG-3 ') and (5 '-CCTTGAGGAAGTCTCTGCAC-3 ').
Result as shown in Figure 2, is found to compare with Col wild-type, and in cbr1 mutant, the expression of PORA gene is suppressed greatly, illustrates that the sudden change of CBR1 gene has affected the expression of PORA gene, i.e. the expression of CBR1 gene regulating PORA gene.
Three, gene overexpression
PJIM19-9Myc carrier document (Yang et al., Plant Cell, 2005, disclosed in 17:804-821), public Ke Cong Institute of Botany, Chinese Academy of Sciences obtains.Agrobacterium GV3101 bacterial strain document (Steven J Clough and Andrew F Bent, Plant Journal, 1998, disclosed in 16:735-743), public Ke Cong Institute of Botany, Chinese Academy of Sciences obtains.
The total RNA of blade of Col wild-type Arabidopis thaliana of take is template, with primer pair p1/p2, carries out RT-PCR amplification, obtains pcr amplification product and is goal gene.
p1：5’-GTCGACCTATGGCGTCGTCTCCGTTGAC-3’(SEQ ID NO：3)
p2：5’-ACTAGTTAAGTGGAGATGAATCTCATGC-3’(SEQ ID NO：4)。
With restriction enzyme XhoI and SpeI enzyme, cut pcr amplification product, reclaim goal gene fragment; With restriction enzyme XhoI and SpeI enzyme, cut pJIM19-9Myc carrier, reclaim carrier large fragment; Goal gene fragment is connected with carrier large fragment; To connect product and transform bacillus coli DH 5 alpha, use LB+Kan Screening of Media, picking positive colony, extract the plasmid of positive colony, plasmid is checked order, result is presented at the gene order inserted between the XhoI of pJIM19-9Myc carrier and SpeI restriction enzyme site as shown in SEQ ID ID:2, and direction of insertion is from XhoI to SpeI.Correct recombinant expression vector is denoted as to pJIM-CBR1.
Plant expression vector pJIM-CBR1 is proceeded to Agrobacterium GV3101 bacterial strain with electric shocking method, and screening positive clone on LB+Spec+Gent resistance culture base, is denoted as GV3101-pJIM-CBR1 by the positive Agrobacterium of recombinating.
The seed of cbr1 mutant, 16 hours illumination/8 hour dark, is cultivated 4 weeks, obtained cbr1 mutant plant for 22 ℃.
With flower infusion method arabidopsis thaliana transformation cbr1 mutant plant.
The screening of transgenic plant and evaluation: after Agrobacterium-mediated Transformation, obtain T1 for seed, on MS+ kantlex 50mg/L substratum, grow 7 to 10 days, screening resistant plant, and forward in compost and grow.After self-fertility, receive T2 for seed, seed is sprouted on kantlex 50mg/L substratum, through self-fertility, obtain T3 for seed again, on MS+ kantlex 50mg/L substratum, screen 100% resistant plant, showing to obtain T3 is the transfer-gen plant that isozygotys for plant.
Using simultaneously proceed to empty carrier pJIM19-9Myc Arabidopis thaliana cbr1 mutant as turning empty carrier contrast.
The isozygoty gene identification of transfer-gen plant: the seed of the seed of the seed of transfer-gen plant, Arabidopis thaliana cbr1 mutant, Col wild-type Arabidopis thaliana, the seed that turns empty carrier contrast are grown 6 days in incubator, obtain respectively seedling; Extract respectively the total RNA of blade of seedling, with primer pair p1/p2, carry out RT-PCR analysis, in transfer-gen plant and the Col wild-type of isozygotying, all can obtain object product shown in SEQ ID NO:2, and cbr1 mutant there is no with turning in empty carrier contrast, shows that mutant has obtained complementation.
By T3 for the seed of transfer-gen plant, the seed of the seed of Arabidopis thaliana cbr1 mutant, Col wild-type Arabidopis thaliana, the seed that turns empty carrier contrast are cultivated under the same conditions, obtain whole plant.Detect respectively isozygoty transfer-gen plant, Arabidopis thaliana cbr1 mutant plant and Col wild-type Arabidopis thaliana plant, turn the blade Determination of Chlorophyll content of the seed of empty carrier contrast.
The detection method of chlorophyll content: extract leaf chlorophyll by 80% acetone extraction method, measure absorption at wavelength 663nm and 645nm, calculate chlorophyll content.
Chlorophyll-a Content calculation formula is 12.7 * A663-2.69 * A645.
Content of chlorophyll b calculation formula is 22.9 * A645-4.48 * A663.
3 repetitions, results averaged ± standard deviation are established in experiment.
Result is specific as follows:
Chlorophyll-a Content is as follows: transfer-gen plant isozygotys: 517.2 ± 39.4 (ng/mg FW); Arabidopis thaliana cbr1 mutant plant: 332.8 ± 35.2 (ng/mg FW); Col wild-type Arabidopis thaliana plant: 499.1 ± 40.6 (ng/mg FW); Turn empty carrier control group result consistent with cbr1 mutant plant.
Content of chlorophyll b is as follows: transfer-gen plant isozygotys: 251.8 ± 24.3 (ng/mg FW); Arabidopis thaliana cbr1 mutant plant: 130.6 ± 18.6 (ng/mg FW); Col wild-type Arabidopis thaliana plant: 227.8 ± 24.4 (ng/mg FW); Turn empty carrier control group result consistent with cbr1 mutant plant.
By comparing the chlorophyll content of cbr1 mutant and Col wild-type seedling, find that cbr1 mutant Determination of Chlorophyll is lower than wild-type, the sudden change that CBR1 gene is described has affected chlorophyllous synthetic, and external source CBR1 transgenosis can be recovered the chlorophyll content of mutant, show that CBR1 brings into play function in the synthetic regulation and control of chlorophyll.
Claims (5)
1. a method of cultivating the plant that chlorophyll synthesis capability improves, comprise the steps: to import the encoding gene of the protein being formed by the aminoacid sequence shown in SEQ ID NO:1 in the plant that sets out, obtain comparing with the described plant that sets out the transgenic plant that chlorophyll synthesis capability improves.
2. method according to claim 1, is characterized in that: the encoding gene of the described protein being comprised of the aminoacid sequence shown in SEQ ID NO:1 imports by following recombinant vectors:
The recombinant expression vector that the encoding gene that described recombinant vectors is the protein that is comprised of the aminoacid sequence shown in SEQ ID NO:1 described in inserting in the multiple clone site of expression vector pJIM19-9Myc obtains.
3. method according to claim 1 and 2, is characterized in that: the encoding gene of the described protein being comprised of the aminoacid sequence shown in SEQ ID NO:1 is DNA molecular shown in SEQ ID NO:2.
4. method according to claim 3, is characterized in that: described in the plant that sets out be monocotyledons or dicotyledons.
5. method according to claim 4, is characterized in that: described dicotyledons is Arabidopis thaliana.
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| CN105566463B (en) * | 2014-10-13 | 2019-04-23 | 中国科学院植物研究所 | One kind albumen relevant to Chlorophyll synthesis and its encoding gene and application |
| CN107287231B (en) * | 2016-04-11 | 2020-05-22 | 中国科学院植物研究所 | RVE1 protein related to plant seed dormancy as well as encoding gene and application thereof |
| CN117106819B (en) * | 2023-08-28 | 2024-06-11 | 西湖大学 | Phaeodactylum tricornutum CHLC gene and application of encoded protein in chlorophyll c synthesis |
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| CN1409962A (en) * | 2002-11-15 | 2003-04-16 | 南京农业大学 | Method of pronoting plant growth and enhancing anti reverse property |
| CN101988073A (en) * | 2009-07-31 | 2011-03-23 | 北京市农林科学院 | Method for culturing drought-enduring plants and recombinant plasmid special for same |
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| CN1409962A (en) * | 2002-11-15 | 2003-04-16 | 南京农业大学 | Method of pronoting plant growth and enhancing anti reverse property |
| CN101988073A (en) * | 2009-07-31 | 2011-03-23 | 北京市农林科学院 | Method for culturing drought-enduring plants and recombinant plasmid special for same |
Non-Patent Citations (6)
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| Arabidopsis thaliana myb family transcription factor;NCBI;《NCBI Genbank:NM_121736.2》;20090821 * |
| NCBI.Arabidopsis thaliana myb family transcription factor.《NCBI Genbank:NM_121736.2》.2009, |
| 转反义GVDE基因烟草的叶绿素荧光参数及抗氧化酶活性的变化;黄金丽等;《中国农业科学》;20081231;第41卷(第1期);308-313 * |
| 过表达OsPSK3基因增加水稻叶片叶绿素含量;黄霁月等;《遗传》;20101231;第32卷(第12期);1281-1289 * |
| 黄金丽等.转反义GVDE基因烟草的叶绿素荧光参数及抗氧化酶活性的变化.《中国农业科学》.2008,第41卷(第1期),308-313. |
| 黄霁月等.过表达OsPSK3基因增加水稻叶片叶绿素含量.《遗传》.2010,第32卷(第12期),1281-1289. |
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