CN102505016B - Transmembrane protein gene triticum asetivum leucine rich repeat 3 (TaLRR3) with leucine rich repeat (LRR) structure domain as well as expression vector and application thereof - Google Patents
Transmembrane protein gene triticum asetivum leucine rich repeat 3 (TaLRR3) with leucine rich repeat (LRR) structure domain as well as expression vector and application thereof Download PDFInfo
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- CN102505016B CN102505016B CN 201110448452 CN201110448452A CN102505016B CN 102505016 B CN102505016 B CN 102505016B CN 201110448452 CN201110448452 CN 201110448452 CN 201110448452 A CN201110448452 A CN 201110448452A CN 102505016 B CN102505016 B CN 102505016B
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Abstract
The invention discloses a transmembrane protein gene triticum asetivum leucine rich repeat 3 (TaLRR3) with a leucine rich repeat (LRR) structure domain as well as an expression vector and an application thereof and belongs to the field of gene engineering. The complementary deoxyribonucleic acid (cDNA) sequence of the transmembrane protein gene TaLRR3 with the LRR structure domain is SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO.2. The gene is from ordinary wheat (Triticum asetivum L.) 92R137 and is reported in the wheat for the first time. The TaLRR3 is induced by stripe rust in a stripe rust resistant wheat variety 92R137, the expression is enhanced, and in addition, the expression level is much higher than the expression level in the disease susceptible variety Yangmai 158. The gene is inserted into pBI220 to obtain an over-expression vector, in addition, the susceptible stripe rust wheat variety Yangmai 158 is converted, and T0-generation stripe rust qualification results show that the over-expression of the TaLRR3 can improve the stripe rust resistance of the susceptible stripe rust wheat variety.
Description
Technical field
The invention belongs to the genetically engineered field, disclose Genes Encoding Transmembrane Proteins TaLRR3 and expression vector and the application of a tool LRR structural domain.
Background technology
The harm that is subject to the Various Diseases worm whole breeding time of wheat (Triticum aestivum), wherein infecting the stripe rust happening part that causes by wheat stripe rust (Puccinia striiformis f.sp.tritic) mainly is blade, secondly be on leaf sheath and the stem, also can occur on fringe section grain husk shell and the awns.Stripe rust is one of most important disease that threatens in NORTHWEST CHINA, southwest, varieties in Huang-Huai-Hai Plain and northwest spring wheat district, and is particularly serious in the area that cools of high latitude or high height above sea level.This disease often causes the wheat Severe Reduction, and such as medium popular time underproduction 10-20%, the then underproduction 30% of time of being very popular, but especially big popular time underproduction 50-60% are in the minority plot even No kernels or seeds are gathered, as in a year of scarcity.Since the establishment of the nation, medium-scale, 10 fairly large Epidemics of Wheat Strip Rusts once occured 16 times in China, involved especially 14 provinces and regions that affect the whole country in 2005, area occurs reach more than 5,700 ten thousand mu.Because stripe rust of wheat has the characteristics such as popular frequency is high, explosive by force, velocity of propagation is fast, occurrence scope is wide, the prevention difficulty is large, improving yield of wheat and stable yields in serious threat, therefore strengthen the disease-resistant research of stripe rust of wheat, thereby it is popular very urgent effectively to prevent and treat the stripe rust of wheat big area.
The Comprehensive Control Strategy of " to plant anti-rust kind as main, chemical control and cultural control are auxiliary " is taked in the at present control of stripe rust of wheat at present.Seed selection also rationally utilizes anti-, anti-rust kind, and carry out reasonable kind layout, can effectively reduce the summer bacterium source of surviving the winter, and is to prevent and treat stripe rust of wheat the most economy, safety and effective measures.China has cultivated large quantities of rust-proofing wheat breeds, and these kinds have played vital role in the epidemiological process of control China stripe rust of wheat.Physiologic Races of Wheat Stripe Rust has variability, at present the disease-resistant gene in the Major Wheat Cultivars is overcome by the physiological strain of 29,31, No. 32 and new variation in the bar gradually, also extremely lack new anti-source, in case condition is suitable, stripe rust is great outburst again, thereby causes heavy economic losses.Therefore, based on good Resistant sources to stripe rust, accelerating excavation, clone and the utilization of novel Stripe Rust Resistance Gene, and utilize transgenic technology to breed good disease-resistant variety, is the quick approach of accelerating the disease-resistant gene utilization and improving the breeding for disease resistance process.
Cytogenetics institute of Agricultural University Of Nanjing utilizes tetraploid duckbill wheat and Dasypyrum villosum hybridization, backcross after many generations with common wheat again, selected Triticum aestivum-Haynaldia villosa translocation line 92R137, since 1994 enter domestic breeding utilization, in Sichuan, the stripe rust Chang Faqu such as Yunnan, Shaanxi have carried out for many years plantation, stripe rust has been shown stable highly resistant, especially to 29,30,32 performance high resistance-immunity in the wheat stripe rust dominant races bar.Genetic analysis shows, the resistance among the 92R137 is by Dominant gene, and is dominant inheritance, and this gene is from duckbill wheat, by the international wheat cdna called after Yr26 of NK.
Yr26 is a Stripe Rust Resistance Gene resource with significant application value, successfully clones some key gene in this gene or its disease-resistant approach, then can cultivate stripe rust resisting transgenic wheat new variety establish a firm foundation for utilizing the transgenosis means.
Summary of the invention
The objective of the invention is the defects for prior art, a kind of Genes Encoding Transmembrane Proteins TaLRR3 of tool LRR structural domain is provided.
Another object of the present invention provides the expression vector of this gene.
Another purpose of the present invention provides the application of this gene and expression vector.
Purpose of the present invention can be achieved through the following technical solutions:
The Genes Encoding Transmembrane Proteins TaLRR3 of tool LRR structural domain, from common wheat (Triticum asetivum L.) 92R137, its nucleotides sequence is classified SEQ ID NO.1 as.
The protein TaLRR3 of the Genes Encoding Transmembrane Proteins coding of this tool LRR structural domain, its aminoacid sequence is SEQ ID NO.2.
The expression vector that contains the Genes Encoding Transmembrane Proteins TaLRR3 of described tool LRR structural domain.
The expression vector of the described Genes Encoding Transmembrane Proteins TaLRR3 that contains described tool LRR structural domain preferably with pBI220 for the carrier that sets out, described TaLRR3 gene is inserted gained between the BamH I of pBI220 and Sac I restriction enzyme site.
The application of the Genes Encoding Transmembrane Proteins TaLRR3 of described tool LRR structural domain in cultivating the stripe rust resisting wheat kind.
The described application of expression vector in cultivating the stripe rust resisting wheat kind that contains the Genes Encoding Transmembrane Proteins TaLRR3 of tool LRR structural domain.
Beneficial effect:
The present invention clones the Genes Encoding Transmembrane Proteins TaLRR3 that obtained a tool LRR structural domain and coded protein TaLRR3 thereof from wheat, be reported first in the wheat.TaLRR3 can be used for genetic engineering breeding, is inserted into expression vector pBI220, and the Overexpression vector of this gene that obtains imports in the susceptible wheat breed, can improve sense stripe rust wheat breed to the resistance of stripe rust.
Description of drawings
Fig. 1 utilizes Q-PCR to analyze the expression X-coordinate 0,6,12,24 of TaLRR3 in stripe rust resisting 92R137 and sense stripe rust Yangmai No.158, the wheat leaf blade sample that 72 expression strip rust bacterias are induced 0 hour, 6 hours, 12 hours, 24 hours, 72 hours.
Fig. 2 TaLRR3 overexpression Vector construction
A: plant expression vector pBI220; The B:pBI220:TaLRR3 expression vector.
Fig. 3 TaLRR3 transforms the T of Yangmai No.158
0PCR Molecular Identification for positive plant
M:DL2000; 1: the water negative control; 2: the Yangmai No.158 negative control; 3: the plasmid positive control; 6,13: positive plant; 4-5,7-12,14: negative plant.
Fig. 4 TaLRR3 transforms the T of Yangmai No.158
0Stripe rust resistance for positive plant is identified
1: disease-resistant contrast 92R137; 2: susceptible contrast Yangmai No.158; 3-11: the disease-resistant plant in the positive plant.
Embodiment
Be subjected to the clone of the tool LRR structural domain Genes Encoding Transmembrane Proteins Ta-LRR of strip rust bacteria abduction delivering among embodiment 1 92R137
92R137 is the translocation line (Chen that creates after the double diploid after Agricultural University Of Nanjing hybridizes with tetraploid duckbill wheat (Triticum turgitum) and Dasypyrum villosum (Haynaldia villosa) repeatedly backcrosses with the hexaploid common wheat again, P.D., Qi, L.L., Zhou, B., S.Z.Zhang, D.J.Liu.1995Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew.TAG, (91): 1125-1128.).Contain Stripe rust resistance gene Yr26 on the 1B karyomit(e) of 92R137, the resistance of the dominant races such as 29,31,32 reaches high resistance-immune level (Chunmei Wang in the strong toxicity microspecies of this gene pairs bar, Yiping Zhang, Dejun Han, Zhensheng Kang, Guiping Li, Aizhong Cao, Peidu Chen.SSR and STS markers for wheat stripe rust resistance gene Yr26.Euphytica, 2008,159:359-366.).
In order to clone the disease-resistant related gene in the disease-resistant approach of Yr26, gene chip hybridization method screening stripe rust resisting wheat 92R137 and the difference expression gene of feeling in the stripe rust wheat Yangmai No.158 are adopted in this laboratory in early-stage Study.Idiographic flow is as follows: the seed of the seed of stripe rust resisting wheat 92R137 and sense stripe rust wheat Yangmai No.158 is sowed in the culture dish germinates, be transplanted to the basin alms bowl after showing money or valuables one carries unintentionally, one leaf phase induced the strip rust bacteria spore inoculating of collecting from sense stripe rust wheat lines to seedling, and sampling in 12 and 36 hours after inoculation, extract respectively RNA (the Trizol reagent with Invitrogen company extracts), form two experimental group R12 (92R137 induces 12 hours sample), R36 (92R137 induces 36 hours sample) and two control group S12 (Yangmai No.158 is induced 12 hours sample), S36 (Yangmai No.158 is induced 36 hours sample).The RNA sample of two experimental group and two control groups is used respectively Superscript
TMThe II reverse transcription becomes cDNA (test kit available from Gibco/BRL, Gaithersburg, MD, USA), and further becomes cRNA in in-vitro transcription.The cRNA sample of 2 experimental group and 2 control groups respectively with four wheat chip of expression spectrum GeneChip (Affymetrix wheat cdna chip of expression spectrum, part number 900515) hybridization, the chip hybridization experiment is finished in " Shanghai biochip engineering center of country ", and experimental procedure is with reference to Affymetrix company specification sheets " Expressed Analysis Technical Manual " (http://www.affymetrix.com/support/technical/manual/expression_m anual.affx).The ratio of experimental group hybridization signal and control group hybridization signal is greater than 2 as standard on the chip, the up-regulated expression gene among the screening stripe rust resisting material 92R137.One of them difference expression gene is Ta.22666, this gene is a disease-resistant gene analogue, and comparing and comparing with control group S36 at experimental group R36 at experimental group R12 and control group S12, this gene all there are differences expression, has Close relation so tentatively judge this gene and stripe rust resistance.
The RNA reverse transcription that the blade of inducing 12 hours through strip rust bacteria take 92R137 extracts becomes cDNA as template, with the primer P1 (CAGCTGACATCTCTGGATCT according to chip probe Ta.22666 design, SEQ ID N0.3) and P2 (GAAGCAGTGCGGAAGTAG, SEQ ID N0.4) carries out RT-PCR for primer, obtain the homologous fragment of Ta.22666 gene among the 92R137, and further utilized the RACE method to obtain the full length sequence of this gene.The sequence of this full length gene 1154bp, sequence is shown in SEQ ID NO.1, sequential analysis shows that this sequence comprises a total length ORF, 5 '-UTR (non-translational region) 99bp, 3 '-UTR 254bp, ORF (open reading frame) 801bp wherein, 266 amino acid of encoding, sequence is shown in SEQ ID NO.2.Analyze through SMART software (http://smart.embl-heidelberg.de/), this genes encoding a kind of transmembrane protein, the N end comprises the LRR structural domain, nearly C end is by a hydrophobic transmembrane zone that is comprised of 24 amino acid, so this gene is a transmembrane protein (LRR containing transmembrane protein) with LRR structural domain, is TaLRR3 with this unnamed gene.
The expression characteristic that embodiment 2TaLRR3 gene is induced by strip rust bacteria
In order to study the expression pattern of TaLRR3 in anti-sense stripe rust material, the RNA reverse transcription cDNA that utilizes disease-resistant material 92R137 and susceptible material Yangmai No.158 to induce 0,6,12,24,72 hour through strip rust bacteria is template, utilizes P1 and P2 to carry out real-time fluorescence quantitative PCR (Q-PCR) analysis for primer.The PCR program is: the PCR reaction is in the upper amplification of real-time fluorescence quantitative PCR instrument (MyIQ, Bio-Rad company, the U.S.) and detect fluorescence.Contain 2 * SYBR Green PCR Master Mix 10uL in the 20uL PCR reaction system, 0.5 μ M primer P1 and P2, reverse transcription cDNA template 2uL, water is supplemented to 20uL.Amplification is: 95 ℃ 10 minutes, then 95 ℃ 15 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minute, totally 40 circulations.After reaction finishes, carry out the mensuration of melting curve.The gene expression detection level is analyzed with the MyiQ system software.The result shows that in 92R137, TaLRR3 is subjected to strip rust bacteria to induce up-regulated expression, raises significantly in 12 hours, and expression level is the highest behind the 24h, and expression in 72 hours descends; In Yangmai No.158, the expression amount of each time period of TaLRR3 all is lower than the expression amount in 92R137, especially in inoculation 12 hours and 24 hours these two material time section differences the most remarkable (Fig. 1).
Embodiment 3TaLRR3 overexpression vector makes up and transforms common wheat Yangmai No.158 and stripe rust resisting evaluation
Take from the cDNA of 92R137 as template, with the full length gene sequences Design of utilizing the TaLRR3 that the RACE method the obtains primer P3 (CGCGGATCCATGGCTGATGATACCAAG across ORF, SEQ ID NO.5) and P4 (CGAGCTCTCATATCCGGACGACGTA, SEQ ID NO.6), and P3 is with the restriction enzyme site of BamHI, and P4 is with the restriction enzyme site of SacI.Use primer that P3 and P4 are carried out pcr amplification, reclaim amplified fragments.With BamHI and SacI amplified production is carried out double digestion, enzyme is cut carrier pBI220 (Jefferson RA after product is inserted into BamHI and SacI double digestion, Kavanagh TA, Bevan MW.GUS fusions:beta-glucuronidase as a sensitive andversatile gene fusion marker in higher plants.EMBO J.1987,6:3901-3907.) in, TaLRR3 is placed the multiple clone site place of 35S promoter back, replace carrier itself with gus gene.Thus target gene TaLRR3 is cloned into the downstream of strong promoter 35S, obtains expression vector pBI220:TaLRR3 (Fig. 2).
It is as follows to utilize gene gun conversion method TaLRR3 to change the method for transformation of feeling the stripe rust acceptor over to: 1,7 days about 2000 Yangmai No.158 rataria callus of picking preculture, ooze substratum (MS+ABA0.5mg/L+ caseinhydrolysate 500mg/L+2 at height, 4-D2mg/L+ glucose 30g/L+0.4mol/L N.F,USP MANNITOL, pH5.8) upper pre-treatment 4-5 hour; The Overexpression vector pBI220:TaLRR3 that 2, will carry goal gene TaLRR3 is transformed into the Yangmai No.158 callus by particle bombardment, oozes substratum at height after the bombardment and continues to cultivate 16 hours.3, callus is transferred to upper dark the cultivation for 2 weeks of recovery media (1/2MS+ caseinhydrolysate 500mg/L+2,4-D2mg/L+ sucrose 30g/L, pH5.8); 4, callus is transferred to (1/2MS+ABA0.5mg/L+ caseinhydrolysate 500mg/L+2,4-D1mg/L+ sucrose 30g/L+4mg/LBialaphos, pH5.8) 2 weeks of screening and culturing on the screening culture medium that contains weedicide; The callus that 5, will have Herbicid resistant is transferred to (1/2MS+L-paddy ammonia phthalein amine 1mmol/L+ caseinhydrolysate 200mg/L+KT 1mg/L+IAA 0.5mg/L+ sucrose 30g/L+ agar 0.8% in the division culture medium, pH5.8) break up, treat to transfer them to when Bud Differentiation grows to 2-4cm root media (1/2MS+KT1mg/L+ sucrose 30g/L+ agar 0.8%, pH5.8) in.6, be about 8cm, root system when healthy and strong to regrowth, can the open pipe hardening 1-2 days, the substratum residue that last flush away root system carries just can be transplanted the engagement alms bowl.Obtain totally 307 of regeneration plants.
Extract all regeneration plant genomic dnas, transformed plant is utilized the inner primer P5 of promotor (GTTATGCTGGAGGTTGAGGAGA, SEQ ID NO.7) and the inner primer P6 of gene (GACCAAAGGGCAATTGAGAC, SEQ ID NO.8) carries out pcr amplification, to identify positive plant.PCR program: 10-50ng genomic dna template, each 0.5 μ l of the P5 of 10 μ M and P6; 2.5 μ l 10 * buffer; 2.5 the dNTP of μ l 2.5mM; 1.5 the Mg of μ l 25mM
2+0.25 μ l (5U/ μ l) Taqpolymerase (TaKaRa) adds water to 25 μ l.The PCR reaction conditions is: 94 ℃ of denaturations 3 minutes; 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 2 minutes, 33 circulations; 72 ℃ were extended 10 minutes.The PCR product detects through 1% agarose gel electrophoresis.Wherein the 26 strains purpose band of about 1200bp that can increase is accredited as positive plant.Figure 3 shows that T
0For the PCR Molecular Identification of positive plant, from 11 strain transfer-gen plants, identify 2 strain positive plants.
To the positive plant of Partial Characterization, negative plant, disease-resistant adjoining tree 92R137 and susceptible adjoining tree Yangmai No.158, at leaf phase inoculation strip rust bacteria, carry out phenotype statistics and Disease Resistance Identification after 15 days.Disease-resistant adjoining tree 92R137 shows high resistance to strip rust bacteria, produces the hypersensitive necrosis spot on the blade, grows without spore; Susceptible adjoining tree Yangmai No.158 without the hypersensitive necrosis spot, is covered with the strip rust bacteria spore to the high sense of strip rust bacteria performance on the blade; Transgenosis T
0Compare with unconverted Yangmai No.158 for positive plant, the strip rust bacteria resistance level is significantly improved, only have a small amount of spore on the blade, and produced the hypersensitive necrosis spot similar to disease-resistant 92R137 on the blade, this is blade produces resistance to strip rust bacteria sign (Fig. 4).
Claims (6)
1. the Genes Encoding Transmembrane Proteins of a tool LRR structural domain
TaLRR3, it is characterized in that its nucleotides sequence classifies SEQ ID NO.1 as.
2. the protein TaLRR3 of the Genes Encoding Transmembrane Proteins of the described tool LRR of claim 1 structural domain coding is characterized in that its aminoacid sequence is SEQ ID NO.2.
3. the Genes Encoding Transmembrane Proteins that contains tool LRR structural domain claimed in claim 1
TaLRR3Expression vector.
4. expression vector according to claim 3 is characterized in that the described Genes Encoding Transmembrane Proteins that contains tool LRR structural domain
TaLRR3Expression vector be with
PBI220Be the carrier that sets out, with claimed in claim 1
TaLRR3Gene inserts
PBI220 BamH IWith
Sac IGained between restriction enzyme site.
5. the Genes Encoding Transmembrane Proteins of tool LRR structural domain claimed in claim 1
TaLRR3Application in cultivating the stripe rust resisting wheat kind, wherein, described wheat breed is Yangmai No.158.
6. claim 3 or the 4 described Genes Encoding Transmembrane Proteins that contain tool LRR structural domain
TaLRR3The application of expression vector in cultivating the stripe rust resisting wheat kind, wherein, described wheat breed is Yangmai No.158.
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| US7214786B2 (en) * | 2000-12-14 | 2007-05-08 | Kovalic David K | Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement |
| CN101760547A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Evaluation and utilization of wheat stripe rust resistance genes |
| WO2010095138A1 (en) * | 2009-02-19 | 2010-08-26 | Carmel-Haifa University Economic Corporation Ltd | Novel kinase-start gene conferring resistance to plant disease and transgenic plants comprising it |
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