CN100465274C - Rice leaf intersection angle related gene and its coded protein and use - Google Patents
Rice leaf intersection angle related gene and its coded protein and use Download PDFInfo
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Abstract
The rice leaf intersection angle related gene of the present invention is protein with one of the following amino acid residue sequences: 1. SEQ ID No. 2 in the sequence list; and 2. the amino acid residue sequence of SEQ ID No. 2 through substitution, deletion and/or addition of 1-10 amino acid residues and coding protein related to rice leaf intersection angle. The coding gene of the rice leaf intersection angle related protein has antisense transgenic plant line with leaf intersection angle in the later vegetation growth phase and generative growth phase of the T0 generation, T1 generation and T2 generation obviously greater than that of the contrast. The present invention provides one important way for culturing rice variety with increased or decreased leaf intersection angle and raising the yield and disease resistance of rice.
Description
Technical field
The present invention relates to rice leaf intersection angle related gene and proteins encoded thereof and application, particularly rice leaf intersection angle related gene and proteins encoded thereof and this gene application in the control rice leaf intersection angle.
Background technology
The morphogenesis of paddy rice is the growth course of a complexity, and the plant type of paddy rice in final decision.And the plant type of paddy rice is and closely-related Main Agronomic Characters such as output and disease resistance, its constituent element is except that comprising effective tillering number, tillering angle, fringe portion form and plant height, the angle that also comprises simultaneously rice leaf and paddy rice stem, the i.e. factors such as size of leaf angle.Reasonably plant type can make certain Rice Population improve per unit area yield to greatest extent.
At present, some genes relevant with morphogenesis are come out by the clone successively in the paddy rice.As the foreign laboratory clone's of Chinese scholar Lee family MOC1 gene directly influence paddy rice tillering number (Li X, Qian Q, Fu Z, et al., Nature422,618-621).The research of relevant tillering angle, the mutant that people such as present Japanese scientist Takashi just have an extreme tillering angle two of map based clonings are the genes involved of the mutant er of the mutant 1a of the loose flat growth of crouching and compact vertical growth.But aspect the research of leaf angle, up to now, still do not have the separated evaluation of the affected mutant of leaf angle come out or the relevant report of the clone of genes involved, Function Identification.
Interested gene provides possibility in the paddy rice for people study for the genomics of paddy rice and the work of information biology.Up to now, the Huada Gene Research Center, Beijing adopted the Shotgun method to the sequence of paddy rice more than 90% carried out checking order (Science296,79-92).South, China Shanghai cara gene has carried out accurate order-checking (Feng Q, Zhang Y, Hao P, et al., Nature420,259) to No. 4 karyomit(e)s of paddy rice.Japan has also carried out accurate order-checking (Sasaki T, Matsumpto T, Yamamoto K, etal., Nature400,259) to No. 1 karyomit(e) of paddy rice.Other each bar karyomit(e) examining orders are all underway, will finish in the near future.Simultaneously, the cDNA clone of 28,000 paddy rice cloned in Japan in 2003, and its relevant information biology has been carried out analyzing (Wyrwitcz et al., Science303,168).
Antisense RNA Technique is a technology that has developed the research gene function of comparative maturity.It is that gene is inserted into the downstream of promotor in reverse mode, makes the mRNA of expression and the mRNA reverse complemental of endogenous this gene, thereby can combine with endogenous mRNA, and then can check native gene and translate into proteinic process.When we carry out Function Identification to some interested genes, adopt Antisense RNA Technique that we are understood under the repressed situation of this expression of gene, whether processes such as growth and development of plant are affected, thereby can infer what kind of growth and development process this gene has participated in.
Summary of the invention
The purpose of this invention is to provide a kind of rice leaf intersection angle associated protein and encoding gene thereof.
Rice leaf intersection angle associated protein provided by the present invention, name is called OsLJB1, is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 2;
2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or interpolation and the protein relevant with rice leaf intersection angle of one to ten amino-acid residue.
SEQ ID № in the sequence table: 2 are made up of 320 amino-acid residues.
The encoding gene of above-mentioned rice leaf intersection angle associated protein also belongs to protection scope of the present invention.
The cDNA sequence of above-mentioned rice leaf intersection angle associated protein encoding gene can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 95% above homology, and the identical function protein DNA sequence of encoding;
3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the sequence 1 in the sequence table.
Wherein, the rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.
SEQ ID № in the sequence table: 1 by 963 based compositions, and its encoding sequence is from 5 ' end the 1st to the 963rd bit base.
Contain expression carrier of the present invention and clone and all belong to protection scope of the present invention, as pUNANTILJB and the intestinal bacteria that contain pUNANTILJB.
Increase arbitrary segmental primer in the above-mentioned rice leaf intersection angle associated protein encoding gene to also within protection scope of the present invention.
Rice leaf intersection angle associated protein encoding gene of the present invention can be used for controlling the size of rice leaf intersection angle.
In actual applications, the antisense expression vector rice transformation of the encoding gene of above-mentioned rice leaf intersection angle associated protein can be obtained the paddy rice that the leaf angle increases.
The carrier that sets out of antisense expression vector that is used to make up the encoding gene of above-mentioned rice leaf intersection angle associated protein can be Ti class plasmid vector and virus vector, is preferably Ti class plasmid vector.
Described method for transformation can be agrobacterium mediation converted method, particle gun mediated transformation method or pollen tube passage method, is preferably the agrobacterium mediation converted method.
Above-mentioned recombinant expression vector all can make up according to ordinary method.
Rice leaf intersection angle associated protein of the present invention and encoding gene thereof are by the pulvinus adaxial and its surface of control paddy rice and the growth of abaxial side, thus the size of control rice leaf intersection angle.This is that first that find up to now participates in the albumen and the gene of rice leaf intersection angle regulation process.The leaf angle of the antisense transgene plant strain of this gene system T0 generation, T1 generation and T2 generation at later stage and the reproductive stage of nourishing and growing, significantly greater than contrast.And the size of the leaf angle in this period has significant effects for the supply of the filling stage energy substance of paddy rice, and directly determining the output of paddy rice.The present invention provides an important approach for the output and the disease resistance of cultivating increase of leaf angle or the paddy rice that reduces and raising paddy rice.
Description of drawings
Fig. 1 is the RT-PCR product electrophoretogram of OsLJB1
Fig. 2 is the physical map of OsLJB1 gene antisense expression vector
Fig. 3 is for spending the phenotypic map of No. 10 reproductive stage in antisense gene plant T2 generation and the wild-type
Fig. 4 is the Southern results of hybridization
Embodiment
The screening of embodiment 1, OsLJB1 and clone
By (54:471-487) est database carries out bioinformatic analysis for Lan et al.2004, Plant Mol Biol, therefrom screens the zinc-finger protein transcription factor gene of a C-x8-C-x5-C-x3-H type of coding to 10K cDNA chip.The sequence total length is about 2.5kb in the genome of this gene, 963 bases of maximum encoder block (ORF) total length among its cDNA, one 320 the amino acid whose albumen of encoding.
The sequence data that provides according to information biology designs following primer:
5 ' end primer: ATGA GTC GGC GGC AGG AGA TT (sequence of wherein ruling is the EcoRI site); 3 ' end primer: TAAAAG TTA AAT AGC AAC CCA ACA (sequence of wherein ruling is the XhoI site) obtains the cDNA sequence of OsLJB1 by the RT-PCR amplification, specifically (TaKaRa, user manual Japan) carries out with reference to Plant RT-PCR Kit 2.01.
Get the various reverse transcription reagent mix (MgCl of total RNA of 1-2 μ g rice seedling (approximately 1-2 μ l) and Kit
24 μ l; 10 * RNA PCR Buffer, 2 μ l; RNase Inhibitor 0.5 μ l; RNase free Water 8.5 μ l; DNTP Mixture 2 μ l; Reverse Transcriptase 1 μ l; Oligo dT-Adaptor 1 μ l).Behind the mixing, 42 ℃ of 30min; 99 ℃ of 5min; 5 ℃ of 5min finish reverse transcription reaction.
Draw 2 μ l reverse transcription products, carry out the PCR reaction as template: enter amplification program behind 94 ℃ of 2min: 94 ℃ of 1min, 62 ℃ of 1min, 72 ℃ of 1min, after 30 circulations, 72 ℃ of 7min.
The PCR product separates through conventional agarose electrophoresis, obtains being about the DNA band (Fig. 1) of 1kb.Reclaim this fragment and clone on T-easy carrier (Promega) and obtain recombinant vectors T-easy-OsLJB1, order-checking, obtain the cDNA sequence of the OsLJB1 shown in sequence in the sequence table 1, its coding has the protein of the amino acid residue sequence of sequence 2 in the sequence table.
The paddy rice that embodiment 2, cultivation leaf angle increase
1, the structure of the reverse expression plasmid of OsLJB1
Extract corn seedling DNA: treat that milpa grows to 5-6 sheet true leaf, one section of clip true leaf, about 0.2g places liquid nitrogen to grind; Extraction damping fluid (the 0.1M Tris-HCl (pH8.0) that adds the new preparation of 800 μ l then; 50mM EDTA (pH8.0); 0.5M NaCl; 1%SDS; 1% beta-mercaptoethanol), thermal agitation makes its whole suspensions; 65 ℃ of water-bath incubation 30min, every 5min puts upside down mixing once; Add the 5M potassium acetate of 250 μ l precoolings then, put upside down mixing immediately, on ice 5min; Add equal amounts of phenolic/chloroform, extracting once, the centrifugal 5min of 12000rpm; Collect the isopropanol precipitating DNA that supernatant adds 0.6 times of volume, room temperature is placed 40min; The centrifugal 15min of 12000rpm (4 ℃), supernatant discarded; Precipitation is respectively washed once with 70%, 100% ethanol; After the drying, be dissolved among the ddH2O that 20 μ l contain 100 μ g/ml RNase.Get 2uL and be diluted to 100ul, therefrom take out 2uL and make template, carry out pcr amplification.The PCR primer sequence is as follows: 5 ' end primer: GG
A AGC TTC TGC AGT GCA GCG TGA CCCGG (sequence of wherein ruling is the HindIII site), 3 ' end primer: CG
G GAT CCA AGT AAC ACC AAA CAACAG GG (sequence of wherein ruling is the BamHI site).Reaction system is 50 μ l, and the PCR response procedures is: enter amplification program behind 94 ℃ of 3min: 94 ℃ of 1min, 60 ℃ of 2min, 72 ℃ of 1min, and after 32 circulations, 72 ℃ of 10min.The total length that amplification obtains is the corn ubiquitin promoter sequence of 2003bp.Hind III and BamH I double digestion PCR product obtain having the corn ubiquitin promoter fragment of sticky end, and be standby.
With Sac I and EcoR I the Noster terminator sequence is downcut from the pBI221 plasmid, be connected between the SacI and EcoR I site of pUC19, obtain pUC19-Noster.Hind III and BamH I double digestion pUC19-Noster reclaim big fragment, are connected with the corn ubiquitin promoter fragment that has sticky end, obtain pUN19.
Utilize the partially digested and Hind III complete degestion pUN19 of EcoR I then, downcut the fragment that is about 2.3kb that comprises corn ubiquitin promoter and Noster from pUN19, be connected in plasmid pCAMBIA1301 (Center forthe Application of Molecular Biology to International Agriclture, www.cambia.org) between EcoR I and the Hind III site, obtain plasmid pUN1301.
Read frame two ends design primer at the OsLJB1 gene, 5 ' end primer is GG
G GTA CCA TGA GTC GGC GGCAGG AGA TT (sequence of wherein ruling is the KpnI site), 3 ' end primer is C
GA GCT CC TAA AAG TTA AATAGC AAC CCA ACA (sequence of wherein ruling is the SacI site), is the fragment of template amplification to 963bp by PCR (its response procedures is with embodiment 1) with the cloning vector T-easy-OsLJB1 that builds, utilize KpnI and SaI enzyme that this fragment and plasmid pUN1301 are carried out double digestion respectively, reclaim the big fragment of OsLJB1 and plasmid pUN1301, and according to mol ratio 3:1 (fragment: plasmid) ratio connects, and the ligation system is 20 μ l:
T
4Dna ligase 2 μ l
10 * damping fluid, 2 μ l
PCR reclaims product 12 μ l
Plasmid reclaims product 4 μ l
Ligation 12h transforms DH5 α competent cell, obtains positive strain through screening, with this recombinant plasmid called after pUNANTILJB (its physical map as shown in Figure 2).This recombinant plasmid adopts from the Ubiquitin promotor startup OsLJB1 gene of corn and expresses with reverse manner, thereby can suppress the expression of native gene.
With reference to electric exciter (EasyJecT Plus electric exciter, Britain EquiBio company limited) operational guidance, plasmid pUNANTILJB is changed among the agrobacterium tumefaciens EHA105 by electrization, obtain positive colony through screening.
2, pUNANTILJB is to conversion and the positive seedling GUS dyeing of paddy rice
With reference to (Plant Mol Biol. such as Hiei, 1997, method rice transformation 35:205-218): the Agrobacterium EHA105 that will carry plasmid pUNANTILJB expands and to be inoculated into 20ml and to contain in the YEB liquid nutrient medium of Km (kantlex) 50mg/L 28 ℃ and shake bacterium and be cultured to logarithmic growth late period; Therefrom get 0.5ml again and be forwarded in the same YEB substratum of 50ml, be cultured to OD under the similarity condition
600Be about 0.5.After centrifugal 10 minutes, precipitate resuspended cultured agrobacterium tumefaciens 4000g with isopyknic MS substratum.Infect according to ordinary method and to spend No. 10 callus in the paddy rice, differentiation obtains the positive seedling of hygromycin resistance on coculture infection, the screening of resistance substratum and division culture medium.
The hardening while, the young root segment 2-3 millimeter of getting the positive seedling of hygromycin resistance carries out GUS dyeing to be identified.The GUS staining fluid consists of 100mmol/L phosphoric acid salt pH7.0,0.1%Triton X-100,10mmol/L EDTA, the 0.5mmol/L Tripotassium iron hexacyanide, X-Gluc 1mg/mL).37 ℃ of incubations 2 hours are observed blue reaction, and blue reaction appears in result about 30% transgenosis strain, illustrate that foreign gene expresses.
When treating that seedling grows to 10 centimetres of left and right sides, open the container closure film, hardening 2-3 days, then seedling is moved into sun glasshouse, receive and plant, obtain the T1 seed.
In order further to eliminate the poor environment influence that in the greenhouse, may exist, determine the genetic stability of this transgenosis proterties simultaneously, to obtain the T2 seed later on plants in outdoor solarium, the result as shown in Figure 3, the plant that shows antisense OsLJB1 gene shows tangible changeable leaf angle, and it is big that the leaf angle becomes.Among Fig. 3, spend in the wild-type No. 10 for contrast on a last left side; The last right side is antisense gene plant T2 generation.Figure below is that the pulvinus of transfer-gen plant and wild-type is compared.
3, Southern hybridization
The extraction of paddy DNA: T2 grows to 5-6 sheet true leaf for plant in the greenhouse by the time, one section of clip transgenic paddy rice true leaf, and about 0.2g places liquid nitrogen to grind as far as possible; Extraction damping fluid (the 0.1MTris-HCl (pH8.0) that adds the new preparation of 800 μ l then; 50mM EDTA (pH8.0); 0.5M NaCl; 1%SDS; 1% beta-mercaptoethanol), thermal agitation makes its whole suspensions; 65 ℃ of water-bath incubation 30min, every 5min puts upside down mixing once; Add the 5M potassium acetate of 250 μ l precoolings then, put upside down mixing immediately, on ice 5min; Add equal amounts of phenolic/chloroform, extracting once, the centrifugal 5min of 12000rpm; Collect the isopropanol precipitating DNA that supernatant adds 0.6 times of volume, room temperature is placed 40min; The centrifugal 15min of 12000rpm (4 ℃), supernatant discarded; Precipitation is respectively washed once with 70%, 100% ethanol; After the drying, be dissolved among the ddH20 that 20 μ l contain 100 μ g/ml RNase.
The rice total dna of getting 30 μ g with an amount of restriction enzyme (1 μ g/5U) adopt EcoRI, BamHI, HindIII respectively enzyme cut 18h.The enzyme that takes a morsel is cut product and is checked that enzyme cuts effect, and enzyme is cut completely DNA on 0.7% sepharose electrophoresis 4-5 hour.After electrophoresis finishes, gel at 1.5M NaCl and 0.5M NaOH sex change 45min, is cut the redundance of gel, by wicking action DNA is transferred on the nylon membrane, transfering buffering liquid is 20 * SSC.Nylon membrane dries the back in 80 ℃ of baking 0.5-2hr, and vacuum is preserved.
Hybridization probe is synthetic: with [
32P] d-CTP is marker, pcr amplification method label probe fragment, the PCR primer is: 5 ' end primer: ATG AGT CGG CGG CAG GAG ATT; 3 ' end primer: TCC CAA ATG GTT GAC CTGAA.Template is a rice total dna.Reaction system is 50 μ l, and the PCR response procedures is: enter amplification program behind 94 ℃ of 2min: 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 1min, and after 32 circulations, 72 ℃ of 7min.The cDNA OsLJB sequence of the total length that obtains of amplification, from initiator codon to terminator codon length overall 963 bases.The probe that mark is good reclaims the test kit purifying through the PCR product.With the PCR product thermally denature 10min that reclaims, ice bath cools off rapidly, promptly can be used for the dna probe of hybridization.
Before the hybridization, after nylon membrane soaked in 5 * SSC, change in the prehybridization solution, 65 ℃ of following prehybridization 1-2hr after prehybridization finishes, add
32The dna probe of P mark is hybridized 16-20hr down for 65 ℃.After hybridization finishes, wash film damping fluid (2 * SSC and 0.5%SDS, room temperature 5min) and less salt is washed under the film damping fluid (0.1 * SSC and 0.5%SDS, 65 ℃ of 30min) at high salt respectively, respectively wash twice.After drying, carry out radioautograph with X-ray film.-70 ℃ of exposures are developed a film after 1-4 days according to a conventional method, and the result shows that three kinds of restriction endonuclease products all show a tangible hybrid belt as shown in Figure 4, illustrate that this gene is single copy in genome.Among Fig. 4, three swimming lanes are respectively the results of hybridization that EcoRI, BamHI, HindIII enzyme are cut product from left to right.
Sequence table
<160>2
<210>1
<211>963
<212>DNA
＜213〉Oryza paddy rice (Oryza sativa var.Lansheng)
<400>1
<210>2
<211>320
<212>PRT
＜213〉Oryza paddy rice (Oryza sativa var.Lansheng)
<400>1
Claims (10)
1, rice leaf intersection angle associated protein, its amino acid residue sequence is shown in SEQ ID NO:2.
2, the encoding gene of the described rice leaf intersection angle associated protein of claim 1.
3, gene according to claim 2 is characterized in that: the cDNA sequence of described rice leaf intersection angle associated protein encoding gene is shown in SEQ ID NO:1.
4, the expression vector that contains the encoding gene of claim 2 or 3 described rice leaf intersection angle associated protein.
5, the clone that contains the external source conversion encoding gene of claim 2 or 3 described rice leaf intersection angle associated protein.
6, the primer of amplification claim 2 or 3 described rice leaf intersection angle associated protein encoding genes is right.
7, the application of the encoding gene of claim 2 or 3 described rice leaf intersection angle associated protein in control rice leaf intersection angle size.
8, application according to claim 7 is characterized in that: the method that obtains the paddy rice of leaf angle increase is the antisense expression vector rice transformation with the encoding gene of claim 2 or 3 described rice leaf intersection angle associated protein.
9, application according to claim 8 is characterized in that: the carrier that sets out of antisense expression vector that is used to make up the encoding gene of described rice leaf intersection angle associated protein is Ti class plasmid vector or virus vector.
10, according to Claim 8 or 9 described application, it is characterized in that: described method for transformation is agrobacterium mediation converted method, particle gun mediated transformation method or pollen tube passage method.
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| CN101659965B (en) * | 2009-08-25 | 2011-11-16 | 中国科学院植物研究所 | Method for breeding transgenic paddy rice with changeable leaf angle and special recombinant carrier thereof |
| CN101921777B (en) * | 2010-08-31 | 2012-01-25 | 浙江省农业科学院 | Application of rice leaf inclination control gene SAL1 |
| CN102952809B (en) * | 2011-12-13 | 2014-10-08 | 华中农业大学 | Application of MAPKKK (Mitogen-activated Protein Kinase Kinase)-family ILA1 gene in controlling included angle of rice leaves |
| CN109182372B (en) * | 2018-09-11 | 2020-07-24 | 中国农业科学院烟草研究所 | Application of tobacco NtPEED gene in regulation and control of tobacco petiole included angle |
| CN116121442B (en) * | 2023-02-07 | 2024-07-05 | 宝清北方水稻研究中心 | InDel molecular marker SG2-InDel of rice grain type QTL, reagent, kit and application thereof |
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| CN1406282A (en) * | 2000-03-01 | 2003-03-26 | 研究与发展研究院公司 | Transgenic plants with increased seed yield, biomass and harvest index |
| CN1412323A (en) * | 2002-11-25 | 2003-04-23 | 中山大学 | Method for screening transgenic plant and its used bivalent plant expression vector |
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| CN1406282A (en) * | 2000-03-01 | 2003-03-26 | 研究与发展研究院公司 | Transgenic plants with increased seed yield, biomass and harvest index |
| CN1412323A (en) * | 2002-11-25 | 2003-04-23 | 中山大学 | Method for screening transgenic plant and its used bivalent plant expression vector |
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