CN104098661B - The albumen relevant to rice stress-tolerance and chlorophyll content of rice and encoding gene thereof and application - Google Patents
The albumen relevant to rice stress-tolerance and chlorophyll content of rice and encoding gene thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of albumen relevant to rice stress-tolerance and chlorophyll content of rice and encoding gene thereof and application.The protein that the present invention provides, named WSL albumen, from rice varieties Asominori, is following (a) or (b) or (c): the protein that (a) is made up of the aminoacid sequence shown in sequence in sequence table 1；(b) by the aminoacid sequence shown in sequence in sequence table 1 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derives relevant to plant stress tolerance；(c) by the aminoacid sequence shown in sequence in sequence table 1 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derives relevant to chlorophyll content of plant.The present invention is for being further elucidated with the resistance to inverse molecule mechanism of plant and having important theory significance and realistic meaning by engineered technology and means cultivation high-quality, resistance to inverse New Crop Varieties.
Description
Technical field
The present invention relates to a kind of albumen relevant to rice stress-tolerance and chlorophyll content of rice and encoding gene thereof and answer
With.
Background technology
Oryza sativa L. is one of most important cereal crops, and more than half population is with it as staple food in the world.Oryza sativa L. Leaf color mutant
Body is the ideal material of the processes such as research plant chloroplast growth, photomorphogenesis and photosynthesis.Base at mutant
On plinth, the molecular mechanism disclosing Development of Chloroplasts and morphogenesis is to improve light energy of rice utilization ratio, increases yield
Premise.It addition, chloroplast is in addition to carrying out photosynthesis, it also also exists complexity with nucleus and mitochondrion
Signal communication, in order to synergism maintains the normal growth of plant to grow.
PPR albumen is one of protein family maximum in a class plant, has 480 members, this kind of egg at rice seed
White half all comprises multiple PPR repetitive sequence.Most of member of this family is positioned chloroplast or mitochondrion
In, but also there is location the unknown of minority albumen.Rice seed it has been reported that up to the present only 5, PPR albumen,
Function and the mechanism of action of other member all do not know about.Having been reported display, PPR albumen can be directly incorporated in some
On the particular sequence of precursor RNA, participate in the shearing of RNA, editor, stability regulation and control and translation process, at Cytoplasm
The bioprocesss such as male fertile regulates and controls, the signal transmission between embry ogenesis, nucleus and chloroplast, environment-stress response
In play a role.
Summary of the invention
It is an object of the invention to provide a kind of albumen relevant to rice stress-tolerance and chlorophyll content of rice and coding base thereof
Cause and application.
The protein that the present invention provides, named WSL albumen, from rice varieties Asominori, is following (a)
Or (b) or (c):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1；
(b) by shown in sequence in sequence table 1 aminoacid sequence through one or several amino acid residue replacement and/
Or lack and/or add and the protein that by sequence 1 derives relevant to plant stress tolerance；
(c) by shown in sequence in sequence table 1 aminoacid sequence through one or several amino acid residue replacement and/
Or lack and/or add and the protein that by sequence 1 derives relevant to chlorophyll content of plant.
Protein in above-mentioned (b) or (c) can synthetic, it is possible to first synthesizes its encoding gene, then carries out biology
Expression obtains.The encoding gene of the protein in above-mentioned (b) or (c) can be by by shown in sequence in sequence table 2
DNA sequence lacks the codon of one or several amino acid residue, and/or carries out the missense of one or several base pair
Sudden change, and/or hold the coded sequence connecting label to obtain at its 5 ' end and/or 3 '.
The gene (WSL gene) encoding described WSL albumen falls within protection scope of the present invention.
Described WSL gene is following (1) or (2) or (3) or (4) or the DNA molecular of (5):
(1) coding region DNA molecular as shown in sequence 2 in sequence table；
(2) albumen relevant with plant stress tolerance to the DNA sequence hybridization that (1) limits and coding under strict conditions
DNA molecular；
(3) relevant with chlorophyll content of plant to the DNA sequence hybridization that (1) limits and coding under strict conditions
The DNA molecular of albumen；
(4) DNA sequence limited with (1) at least has 70%, at least has 75%, at least has 80%, at least
Have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98%
Or at least there is 99% homology and coding and the DNA molecular with plant stress tolerance correlative protein；
(5) DNA sequence limited with (1) at least has 70%, at least has 75%, at least has 80%, at least
Have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98%
Or at least there is 99% homology and coding and the DNA molecular with chlorophyll content of plant associated protein.
Described stringent condition is at 0.1 × SSPE(or 0.1 × SSC), in the solution of 0.1%SDS, under the conditions of 65 DEG C
Hybridize and wash film.
The present invention also protects a kind of method cultivating transgenic plant, for encoding described WSL albumen in suppression purpose plant
The expression of gene, obtain the transgenic plant that resistance of reverse reduces.Described resistance of reverse concretely salt tolerant or resistance to abscisic acid.
The present invention also protects a kind of method cultivating transgenic plant, for encoding described WSL albumen in suppression purpose plant
The expression of gene, obtain the transgenic plant that chlorophyll content reduces.
Described " encoding the expression of the gene of described WSL albumen in suppression purpose plant " is by described purpose plant
Middle importing interference carrier realizes；Described interference carrier is for insert specific DNA fragment first and specific DNA fragment second respectively
Enter the recombiant plasmid that the different multiple clone site of expression vector obtain；The sequence 2 of described DNA fragmentation first such as sequence table is certainly
Shown in 5 ' end the 1st to 629 nucleotide；Described DNA fragmentation first and described DNA fragmentation second reverse complemental.
Described expression vector concretely pCUbi1390-△ FAD2 carrier.
Described interference carrier concretely following recombiant plasmid: skeleton carrier is pCUbi1390-△ FAD2 carrier,
Its Sac I restriction enzyme site forward insert sequence table double chain DNA fragment, insert at its SnaB I restriction enzyme site
With double from the double chain DNA fragment reverse complemental shown in the 1st to 629 nucleotide of 5 ' end of the sequence 2 of sequence table
Chain DNA fragment.
Described interference carrier can be by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, showing
Microinjection, conductance, the conventional biology methods such as agriculture bacillus mediated convert plant cell or tissue, and the plant that will convert
Tissue cultivating becomes plant.Described purpose plant is monocotyledon or dicotyledon.Described monocotyledon can be water
Rice, such as rice varieties Asominori.
The present invention also protects a kind of method cultivating transgenic plant, for being gone out by the channel genes encoding described WSL albumen
Send out plant, obtain the transgenic plant that resistance of reverse increases.Described resistance of reverse concretely salt tolerant or resistance to abscisic acid.Described
The plant that sets out can be monocotyledon or dicotyledon, concretely Oryza sativa L., such as Oryza sativa L. Asominori.
The present invention also protects a kind of method cultivating transgenic plant, for being gone out by the channel genes encoding described WSL albumen
Send out plant, obtain the transgenic plant that chlorophyll content increases.The described plant that sets out can be monocotyledon or dicotyledonous
Plant, concretely Oryza sativa L., such as Oryza sativa L. Asominori.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing described WSL gene belong to the present invention's
Protection domain.
The application in regulation plant stress tolerance of the described WSL albumen falls within protection scope of the present invention.Described resistance of reverse
Concretely salt tolerant or resistance to abscisic acid.Described plant can be monocotyledon or dicotyledon, concretely Oryza sativa L.,
Such as Oryza sativa L. Asominori.
The application in regulation plant chlorophyll synthesis and/or degraded of the described WSL albumen falls within the protection model of the present invention
Enclose.Described plant can be monocotyledon or dicotyledon, concretely Oryza sativa L., such as Oryza sativa L. Asominori.
The experiment proves that ABA sensitivity is strengthened, to coming off by the Plant Leaf yellow skin of disappearance WSL gene
The resistance of reverse of acid lowers, and reduces the resistance of reverse of salt stress, and chlorophyll content lowers.I.e. WSL albumen participates in conciliation and plants
Thing resistance of reverse, participation regulation photosynthetic response efficiency are relevant to chlorophyll content.The present invention is for being further elucidated with plant
Resistance to inverse molecule mechanism also has important by engineered technology and means cultivation high-quality, resistance to inverse New Crop Varieties
Theory significance and realistic meaning.
Accompanying drawing explanation
Fig. 1 is pCUbi1390-△ FAD2 Vector map.
The phenotype of plant when Fig. 2 is normal condition cultivation.
The chlorophyll content of plant when Fig. 3 is normal condition cultivation.
Fig. 4 is the phenotype that plant grows under ABA or NaCl stress conditions.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions,
It is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats in fact
Test, results averaged.N6 culture medium: purchased from U.S. PhytoTechnology Laboratories, article No. is C167.
By programs such as screening, sequence alignment, functional verifications, from rice varieties Asominori(Wu W, Zheng XM,
Lu G,Zhong Z,Gao H,Chen L,Wu C,Wang HJ,Wang Q,Zhou K,Wang JL,Wu F,
Zhang X, Guo X, Cheng Z, Lei C, Lin Q, Jiang L, Wang H, Ge S, Wan J(2013)
Association of functional nucleotide polymorphisms at DTH2with the northward
expansion of rice cultivation in Asia.Proc Natl Acad Sci U S A.19;110
(8): 2775-80, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science) find a new albumen, will
Its named WSL albumen.WSL albumen is as shown in the sequence 1 of sequence table.By the unnamed gene of coding WSL albumen it is
WSL gene, the open reading frame of its cDNA is as shown in the sequence 2 of sequence table.
Agrobacterium tumefaciems EHA105(Agrobacterium tumefaciens EHA105): list of references: New
Agrobacterium helper plasmids for gene transfer to plants.Hood,Elizabeth E;
Gelvin,Stanton B;Melchers,Leo S;Hoekema,Andre.Transgenic Research,2(4):
P.208-218-218(1993).；The public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
PCUbi1390-△ FAD2 carrier (seeing Fig. 1), records in the following literature: Wax crystal-sparse
leaf2,a rice homologue of WAX2/GL1,is involved in synthesis of leaf cuticular
wax.Mao B.,et al.(2012).Planta235:39–52.；The public can be from Chinese Academy of Agricultural Sciences crop
Science Institute obtains.
Embodiment 1, WSL gene inhibition express acquisition and the qualification of plant
One, the structure of WSL gene RNA interference carrier (recombiant plasmid pCUbi1390-△ FAD2-WSL)
1, the acquisition of WSL gene interference fragment
(1) RNAprep pure plant total RNA extraction reagent box is used (to have purchased from sky root biochemical technology (Beijing)
Limit company), extract Oryza sativa L. Asominori(Oryza sativa) the total serum IgE of 14 days seedling, reverse transcription obtains
cDNA。
(2) cDNA obtained with step (1) is as template, with drawing of WSL-sense-F and WSL-sense-R composition
Thing, to carrying out PCR amplification, obtains pcr amplification product.
WSL-sense-F:5’-TTCTGCACTAGGTACCAGGCCTG ATGGAGGCCGCCGCGTCGCTG-3’；
WSL-sense-R:5’-CTGACGTAGGGGCGATAGAGCTC GTGTAACCCTGCTTCAACTTCATGC-3’。
(3) cDNA obtained with step (1) is as template, with WSL-antisense-F and WSL-antisense-R
The primer of composition, to carrying out PCR amplification, obtains pcr amplification product.
WSL-antisense-F:5’-CGGGGATCCGTCGACTAC ATGGAGGCCGCCGCGTCGCTG-3’；
WSL-antisense-R:5’-AGGTGGAAGACGCGTTAC GTGTAACCCTGCTTCAACTTCATGC-3’。
2, the structure of WSL gene RNA interference carrier (recombiant plasmid pCUbi1390-△ FAD2-WSL)
(1) with restricted enzyme Sac I enzyme action pCUbi1390-△ FAD2 carrier, the line of about 12060bp is reclaimed
Character grain.
(2) using clontech infusion kit(Dalian treasured biotech firm, by specification operates) by step 1
(2) pcr amplification product of obtaining be integrated in the linear plasmid that step (1) obtains by homologous recombination, obtain
Recombiant plasmid pCUbi1390-△ FAD2-sense-WSL.
(3) with restricted enzyme SnaB I enzyme action recombiant plasmid pCUbi1390-△ FAD2-sense-WSL, reclaim
The linear plasmid of about 12689bp.
(4) using clontech infusion kit(Dalian treasured biotech firm, by specification operates) by step 1
(3) pcr amplification product of obtaining be integrated in the linear plasmid that step (3) obtains by homologous recombination, obtain
Recombiant plasmid pCUbi1390-△ FAD2-WSL.
According to sequencing result, recombiant plasmid pCUbi1390-△ FAD2-WSL is carried out structure and is described as follows: skeleton carrier
For pCUbi1390-△ FAD2 carrier, insert the sequence 2 of sequence table from 5 ' ends at its Sac I restriction enzyme site forward
Hold the double chain DNA fragment shown in the 1st to 629 nucleotide, insert and sequence table at its SnaB I restriction enzyme site
Sequence 2 is from the double chain DNA fragment of the double chain DNA fragment reverse complemental shown in the 1st to 629 nucleotide of 5 ' end.
Two, WSL gene inhibition expresses the acquisition of plant
1, recombiant plasmid pCUbi1390-△ FAD2-WSL is imported Agrobacterium tumefaciems EHA105, obtain recombinational agrobacterium
EHA105/pCUbi1390-△FAD2-WSL。
2, WSL gene inhibition expresses the acquisition of plant
EHA105/pCUbi1390-△ FAD2-WSL is proceeded to Oryza sativa L. Asominori(hereinafter referred to as wild rice)
In the callus of embryo, specifically comprise the following steps that
(1) with the LB liquid medium suspension recombinational agrobacterium containing 50 μm ol/L kanamycin
EHA105/pCUbi1390-△ FAD2-WSL, obtains OD600nmThe bacteria suspension of ≈ 0.5.
(2) bacteria suspension that the mature embryo callus of water intaking rice Asominori obtains with step (1) mixes, and infects
30min, is positioned over callus after blotting bacterium solution with filter paper and co-cultures culture medium (containing 0.03924mg/L acetyl Flos Caryophylli
The solid N6 culture medium of ketone) on, cultivate 3 days for 24 DEG C.
(3) wound healing that step (2) obtains is seeded in the solid N6 culture medium containing 150mg/L G418,24 DEG C of trainings
Support 16 days.
(4) take the healthy wound healing that step (3) obtains, be seeded in the solid N6 culture medium containing 200mg/L G418,
24 DEG C of cultivations, every 15 days subcultures are once.
(5) take the healthy wound healing that step (4) obtains, be seeded to division culture medium (containing 150mg/L G418,2mg/L
Kinetins, the solid N6 culture medium of 0.05mg/L naphthalene acetic acid) on, cultivate 45 days (now above-ground plant parts height for 24 DEG C
Degree is about 15cm), open bottleneck seedling exercising 3 days, then transplant to greenhouse production, be T0For plant.
(6) by T0It is plant for plant selfing, results seed cultivation, is T1For plant.
(7) by T0For plant and T1Extract genomic DNA for plant and use what 1390-F and FAD2-R formed to draw
Thing is to carrying out PCR qualification.1390-F corresponds in Fig. 1 corresponding to 10707-10730bp, the FAD2-R primer of Fig. 1
10827-10846bp, if amplifying the band that size is about 728bp, then explanation is for positive plant.
1390-F:5'-TGCCTTCATACGCTATTTATTTGC-3'；
FAD-R:5'-GAAGCGACGGACCTGGAGAT-3'.
For a certain T0For plant, if this plant and T thereof1Identifying for plant PCR and be the positive, this plant is for isozygotying
WSL gene inhibition express plant, this plant and self progeny thereof are that WSL gene inhibition expresses strain.There are
Strain is expressed to 41 WSL gene inhibition.
Three, the acquisition of empty carrier Oryza sativa L. is turned
Recombiant plasmid pCUbi1390-△ FAD2-WSL is replaced to carry out step 2 with pCUbi1390-△ FAD2 carrier,
To turning empty carrier plant.
Four, the phenotype under conditions of normal cultivation and chlorophyll content
By T1(the WSL gene inhibition taken at random expresses strain, named to express plant for WSL gene inhibition
RNAi-3), T1In generation, turns empty carrier plant and rice varieties Asominori(WT represents) seed (each strain
30 seeds) soak 3 days after be seeded in compost, grow and take pictures and measure chlorophyll content for 10 days.
Chlorophyll measuring method reference (Wu Z, Zhang X, He B, Diao L, Sheng S, Wang J, Guo X,
Su N,Wang L,Jiang L,Wang C,Zhai H,Wan J.A chlorophyll-deficient rice mutant
with impaired chlorophyllide esterification in chlorophyll biosynthesis.Plant
Physiol.2007Sep;145 (1): 29-40).
The partial enlarged drawing that Fig. 2, B are A is shown in by phenotype photo.Rice varieties Asominori and the table turning empty carrier plant
Type is consistent.Compared with rice varieties Asominori, WSL gene inhibition expresses the blade edge albefaction of plant.
The testing result of chlorophyll (Chl) content is shown in that Fig. 3 C(FW represents fresh weight).Containing of chlorophyll a (Chl a)
The ratio of the content of amount and chlorophyll b (Chl b) is shown in Fig. 3 D.Rice varieties Asominori with turn empty carrier plant
Chlorophyll content is consistent, and the content of chlorophyll a is the most consistent with the ratio of the content of chlorophyll b.WSL gene inhibition table
Reach the chlorophyll content of plant and be substantially less than rice varieties Asominori, but the content of chlorophyll a and chlorophyll b
The ratio of content is not significantly different from.
Five, the toleration to ABA
By T1(the WSL gene inhibition taken at random expresses strain, named to express plant for WSL gene inhibition
RNAi-3), T1In generation, turns empty carrier plant and rice varieties Asominori(WT represents) seed (each strain
50 seeds) soak 3 days after be seeded in MS culture medium, start timing from seed germination, by children after cultivating 4 days
Seedling is transferred to the MS culture medium containing 1.5 μMs of ABA and continues to cultivate 10 days, observes phenotype and takes pictures.Experiment repeats
3 times.
Fig. 4 A is shown in by photo.Rice varieties Asominori is consistent with the phenotype turning empty carrier plant.Rice varieties
The growing way of Asominori is significantly better than WSL gene inhibition and expresses plant.Result shows, after suppression WSL gene expression
The toleration of ABA is reduced by plant.
Five, the toleration to NaCl
By T1(the WSL gene inhibition taken at random expresses strain, named to express plant for WSL gene inhibition
RNAi-3), T1In generation, turns empty carrier plant and rice varieties Asominori(WT represents) seed (each strain
50 seeds) soak 3 days after be seeded in the MS culture medium containing 150mM NaCl and cultivate 10 days, observe phenotype
And take pictures.Experiment is repeated 3 times.
Fig. 4 B is shown in by photo.Rice varieties Asominori is consistent with the phenotype turning empty carrier plant.Rice varieties
The growing way of Asominori is significantly better than WSL gene inhibition and expresses plant.Result shows, after suppression WSL gene expression
The toleration of NaCl is reduced by plant.
Claims (7)
1. the method cultivating transgenic plant, it is characterised in that: for suppression purpose plant encodes by sequence table
The expression of the gene of the protein of the aminoacid sequence composition shown in sequence 1, obtains the transgenic plant that resistance of reverse reduces；
Described resistance of reverse is salt tolerant or resistance to abscisic acid；Described plant is Oryza sativa L..
2. the method for claim 1, it is characterised in that: described " suppression purpose plant in encode by sequence table
The expression of the gene of the protein of the aminoacid sequence composition shown in middle sequence 1 " it is by leading in described purpose plant
Enter what interference carrier realized；Described interference carrier is for insert table respectively by specific DNA fragment first and specific DNA fragment second
Reach the recombiant plasmid that the different multiple clone site of carrier obtain；The sequence 2 of described DNA fragmentation first such as sequence table is from 5 '
Shown in the 1st to 629 nucleotide of end；Described DNA fragmentation first and described DNA fragmentation second reverse complemental.
3. the method cultivating transgenic plant, it is characterised in that: for suppression purpose plant encodes by sequence table
The expression of the gene of the protein of the aminoacid sequence composition shown in sequence 1, obtains the transgenic that chlorophyll content reduces
Plant；Described plant is Oryza sativa L..
4. method as claimed in claim 3, it is characterised in that: described " suppression purpose plant encodes by sequence table
The expression of the gene of the protein of the aminoacid sequence composition shown in middle sequence 1 " it is by leading in described purpose plant
Enter what interference carrier realized；Described interference carrier is for insert table respectively by specific DNA fragment first and specific DNA fragment second
Reach the recombiant plasmid that the different multiple clone site of carrier obtain；The sequence 2 of described DNA fragmentation first such as sequence table is from 5 '
Shown in the 1st to 629 nucleotide of end；Described DNA fragmentation first and described DNA fragmentation second reverse complemental.
5. the method cultivating transgenic plant, it is characterised in that: for encoding by shown in sequence in sequence table 1
The channel genes of protein of aminoacid sequence composition sets out plant, and obtaining resistance of reverse increases and/or chlorophyll content increases
Transgenic plant；Described resistance of reverse is salt tolerant or resistance to abscisic acid；Described plant is Oryza sativa L..
6. the protein being made up of the aminoacid sequence shown in sequence in sequence table 1 answering in regulation plant stress tolerance
With；Described resistance of reverse is salt tolerant or resistance to abscisic acid；Described plant is Oryza sativa L..
7. the protein being made up of the aminoacid sequence shown in sequence in sequence table 1 regulation plant chlorophyll synthesis and/
Or the application in degraded；Described plant is Oryza sativa L..
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| CN102234318A (en) * | 2010-04-27 | 2011-11-09 | 中国农业科学院作物科学研究所 | Plant stress tolerance related protein TaTPRPK1, encoding gene thereof, and application thereof |
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| BAD87953;Sasaki T等;《Genbank》;20080216 * |
| 水稻功能基因图位克隆研究进展;童继平等;《中国生物工程杂志》;20121231;第32卷(第3期);第115-124页 * |
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