CN1952144A - Promoting growth of root and/or improving drought resistance of plant by using paddy gene OsRRG1 - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering technologies. The invention specifically involves the separation and cloning of a rice DNA fragment, functional verification and application. The said DNA fragment contains the growth of rice root related genes OsRRG1 which gives the rice root elongation growth and / or increases anti-drought ability of rice. Combines the DNA fraction of the gene with exogenous regulating gene and introduces promoter into the plant, the length of transgenic plants root and capability of anti-drought increases significantly.

Description

Utilize paddy gene OsRRG1 to promote the growth and/or the raising plant drought ability of roots of plants

Technical field

The present invention relates to plant biotechnology field.Be specifically related to separating clone, functional verification and the application of a kind of paddy DNA fragment (gene).Described gene is relevant with the elongation growth of roots of plants.With the complete translation district (Coding sequence) of this gene with directly change general plant materials over to after cauliflower mosaic virus promoter (CaMV35S) combines, the root length of transfer-gen plant significantly increases, drought-resistant ability strengthens.

Background technology

Plant can be subjected to many Effect of Environmental in the process of growth, arid often causes the extensive underproduction of farm crop, is bottlenecks of agricultural development in many areas.Cultivate the drought resistance crop varieties is one of major objective of agricultural cience and farming techniques research always.When being subjected to arid or lack of water and coercing, plant is resisted by a series of mechanism or adapts to the unfavorable factor that arid causes, wherein drought tolerance (Drought tolerance) and to keep away drought (Drought avoidance) be topmost mechanism.

Drought tolerance is meant the variation of plant materials recipient cell external environment condition and by number of ways it is delivered in the cell, some response genes of meeting abduction delivering, producing some makes cell avoid the transcription factor that arid, high salt, low temperature etc. are coerced the functional protein of injury, osmoregulation material and transmitted signal and regulate gene expression, thereby corresponding reaction (Xiong etc. are made in variation to external world, Cell signaling during cold, drought and salt stress.Plant Cell.14 (suppl), S165-S183,2002).And can correctly express the meticulous adjusting that is subjected to regulatory factor in the process that those functional genes are made a response to environment.Transcription factor when organism experiences environment stress, can be regulated and control the expression of a series of downstream genes as a kind of regulatory gene, thereby strengthens the tolerance of plant materials to adverse circumstance, reaches the effect that the opposing unsuitable environmental condition is coerced.Kawasaki etc. (2001) utilize expression chip to analyze the early expression spectrum of paddy rice under high-salt stress, discovery has a large amount of genes to be induced or to suppress, the adjusting that the abduction delivering of these genes has been subjected to transcription factor participates in (Kawasaki S, Borchert C, DeyholosM, Wang H, Brazille S, Kawai K, Galbraith D and Bohnert H J.Gene expression profiles duringthe initial phase of salt stress in rice.Plant Cell.2001,13:889-905.).And in Arabidopis thaliana, find AP2/EREBP, Zinc finger, Myb, bZIP class transcription factor family is under different environment stresses, but abduction delivering or be suppressed (.Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stressesby Using a Full-Length cDNA Microarray.Plant Cell.2001 such as ShinozakiK, 13:61-72.), thereby think that these transcription factor families play very important regulating and controlling effect in the answering of plant to adverse circumstance.Information according to existing Arabidopis thaliana transcription factor, people are doing many trials aspect the drought resistance in plants improvement, the transgenic arabidopsis plant that for example utilizes DREB1A and DREB2A to cultivate, its low temperature patience and arid, high salt patience is all than strong (the Two transcription factors such as Liu Q of wild-type, DREB1 and DREB2, with an EREBP/AP2DNA domains separate two cellular signal thansduction pathways in drought-andlow-temperature-responsive gene expression, respectively, in Arabidopsis.Plant Cell.1998,10:1391-1406.).In recent years, it is quite a few that transcription factor by other type of genetic transformation improves the report of resistance of plant, as Arabidopis thaliana NAC gene (Tran etc., Isolation and functional analysis of Arabidopsis stress-inducible NACtranscription factors that bind to a drought-responsive cis-element in the early responsive todehydration stress 1 promoter.Plant Cell, 16:2481-2498,2004), paddy rice zinc finger protein gene OSISAP1 (Mukhopadhyay etc., Overexpression of a zinc-finger protein gene from rice confers tolerance tocold, dehydration, and salt stress in transgenic tobacco.Proc Natl Acad Sci USA, 101:6309-6314,2004).

Keep away drought and be meant that plant avoids the disadvantageous effect that lack of water causes by various Different Strategies, as adjusting breeding time, plant size, close pore, the physiological leaf roll, increasing the root degree of depth etc.Wherein increase the root degree of depth and be considered to strengthen a kind of effective measures the most (Blum A.Crop responses to drought and the interpretation of adaptation.Plant GrowthRegulation 20:135-148,1996.) that plant is kept away non-irrigated ability and don't influences plant biomass.Because root length is the quantitative character of a controlled by multiple genes, at present the research of its hereditary basis is also mainly rested on mapping aspect, the location (Champoux etc. of quantitative trait locus (QTL), Locating genes associated with rootmorphology and drought avoidance in rice via linkage to molecular markers.Theor.Appl.Genet.90:969-981,1995; Yadav etc., Mapping genes controlling root morphology and root distributionin a doubled-haploid population of rice.Theor.Appl.Genet.94:619-632,1997; Kamoshita etc., Mapping QTLs for root morphology of a rice population adapted to rainfed lowland conditions.Theor.Apppl.Genet.104:880-893,2002; Zheng etc., Mapping QTLs and candidate genes for riceroot traits under different water-supply conditions and comparative analysis across threepopulations.Theor.Appl.Genet.107:1505-1515,2003).Clone and the functional analysis (Mouchel etc. of an example only in Arabidopis thaliana, have been reported with root long correlation QTL gene, Natural genetic variation in Arabidopsis identifies BREVISRADIX, a novel regulator of cell proliferation and elongation in the root.Genes Dev., 18:700-714,2004).

Paddy rice is one of most important food crop, and increase maximum root length is kept away non-irrigated ability with raising and had great importance for the drought resistance that improves paddy rice.But in paddy rice, also do not clone and isolate the gene and the transfer-gen plant thereof that promote the root elongation growth at present.The present invention has identified a candidate gene on a QTL basis relevant with rice root length, utilize this gene to carry out the long and drought-resistant ability of root that rice genetic transformed and identified transfer-gen plant.

Summary of the invention

The dna fragmentation that the objective of the invention is the complete coding region section of a root growth genes involved of separating clone from paddy rice utilizes this gene to promote the elongation growth of rice root and/or the drought-resistant ability of raising plant.This gene is carried out structural analysis, and it belongs to the special unknown gene family of plant, because therefore the adjustable root growth of this gene is named as OsRRG1 (Oryza sativa Regulator of Root Growth 1).

The present invention relates to separate and use a kind of dna fragmentation of the OsRRG1 of comprising gene, this fragment is given the elongation growth of rice root and/or is improved the drought-resistant ability of plant.Wherein, described de dna fragmentation perhaps is equivalent to the height homologous DNA sequence shown in the SEQ ID NO:1 basically shown in sequence table SEQ ID NO:1, and perhaps its function is equivalent to the subfragment of sequence shown in the SEQ ID NO:1.

Can adopt the OsRRG1 gene of having cloned to make probe, screening obtains gene of the present invention or homologous gene from cDNA and genomic library.Equally, also can adopt PCR (polymerase chain reaction) technology, from genome, mRNA and cDNA amplification obtain OsRRG1 gene of the present invention and any interested section of DNA or with its homologous section of DNA.Adopt above technology, can separate the sequence that obtains comprising the OsRRG1 gene, with this sequence and any expression vector transformed plant that can guide foreign gene in plant, to express, can obtain that root length increases and (or) arid ability enhanced transfer-gen plant.Gene of the present invention adds any strong promoter or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.Gene of the present invention also can use enhanser in being building up to plant expression vector the time, and these enhanser zones can be ATG initiator codon and neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the translation of whole sequence.

Carrying OsRRG1 expression carrier of the present invention can be by using Ti-plasmids, plant viral vector, directly DNA transforms, microinjection, conventional biotechnological means such as electroporation imports vegetable cell (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2 ^NdEdition).

Can use to comprise that OsRRG1 expression carrier of the present invention transforms the host and comprises the paddy rice various plants, cultivate the plant variety of dark root and drought resisting.

The present invention will be further described below in conjunction with drawings and Examples.

Description of drawings

What sequence table SEQ ID No:1 showed is the sequence dna fragment that includes the OsRRG1 gene coding region of separating clone of the present invention.

Fig. 1: OsRRG1 gene isolation of the present invention and evaluation schema.

Fig. 2: adopt ClustalW software (public use software) with homologous gene sequence comparative result in OsRRG1 gene and the plant.

Fig. 3: with RT-PCR detect the OsRRG1 gene in different tissues expression level.1. the root in tillering phase; 2. sword-like leave; 3. stem; 4. be shorter than the young fringe of 1cm; 5.3-5cm young fringe; 6. be longer than the young fringe of 10cm; 7. stamen; 8. gynoecium; 9.3 the spire in week; 10.3 the etiolated seedling in week.

Fig. 4: use the expression of Northern hybridization analysis OsRRG1 gene in transfer-gen plant, first road is contrast, and all the other are transgenosis independence transfer-gen plant.(A) express (root of choosing tillering phase is a material) for the inhibition of RNAi mediation; (B) be overexpression (blade of choosing seedling stage is a material).

Fig. 5: representative OsRRG1 suppresses express transgenic plant (T ₀Generation) root in seedling stage (after taking root 40 days) is long.

Fig. 6: representative OsRRG1 overexpression transfer-gen plant (T ₀Generation) root in seedling stage (after taking root 30 days) is long.

Fig. 7: representative OsRRG1 overexpression transfer-gen plant becomes the long and drought resisting performance of the root of strain phase (being planted in the pvc pipe).Left side figure is leaf roll situation after cutting off the water supply 5 days, and middle two strains are contrast (CK), and both sides are transfer-gen plant (TG); Right figure is the long comparison of root after the plant maturation.

Fig. 8: the physical map of overexpression carrier pCAMBIA1301 of the present invention.The promotor CaMV 35S that uses in the invention process example is inserted multiple clone site EcoRI and SacI place, promptly constitute the conversion carrier pC1301S that the present invention uses.

Fig. 9: the physical map of inhibition expression vector pHellsgate2 of the present invention.

Embodiment

Previous work of the present invention is (to see Yue etc. according to the QTL mapping of rice root form shape, Genetic analysis for droughtresistance of rice at reproductive stage in field with different types of soil.Theor Appl Genet, 2005).Used colony is being parent's RIL (RIL) colony in rice varieties " precious Shan 97 " (the hybrid rice parent that a kind of China generally applies) and " middle non-irrigated No. 5 " (upland rice variety of a public use that is provided by the Chinese Shanghai academy of agricultural sciences).By the QTL mapping analysis, detect the long QTL (qMRDV2-1) of control root in the interval of the 2nd chromosomal RM573 and RM318 two marks.The gene that the genomic dna in the interval of RM573 and RM318 two marks is predicted is analyzed, found to have the root growth related gene B REVIS RADIX that reports in a candidate gene (being OsRRG1 of the present invention) and the Arabidopis thaliana that certain sequence similarity is arranged.In view of the above, cDNA with this gene separates by the separation process of Fig. 1, and the step of going forward side by side is carried out functional verification, and the result shows: this cDNA is the cDNA fragment of OsRRG1 gene, be a BREVIS RADIX homologous gene that influences plant-root growth, and can be used for improving the drought-resistant ability of plant.Main according to the following aspects is arranged: check order to OsRRG1 (1), analyzes and find that its coded product and people's protein B REVIS RADIX homology reach 62.1%, and contain conservative structural domain (Fig. 2).(2) to its expression analysis (Fig. 3) in different tissues, finding all has certain expression amount at OsRRG1 in different tissues, and the highest with the expression amount in the root.(3) by RNAi technology (Wesley etc., Construct design for efficient, effectiveand high-throughput gene silencing in plants.Plant J, 27:581-590,2001) expression (Fig. 4) of inhibition paddy rice native gene OsRRG1, transfer-gen plant is compared with adjoining tree, and its root length reduces (Fig. 5); And with its full-length gene in plant behind the overexpression (Fig. 4), transfer-gen plant is compared its root length with adjoining tree significantly increase (Fig. 6).(4) the overexpression transfer-gen plant of OsRRG1 gene is compared with the wild-type adjoining tree, and under identical drought stress condition, its leaf roll time significantly postpones, and the long phenomenal growth (Fig. 7) of maximum root demonstrates and keeps away non-irrigated ability enhancing.

Following examples further define the present invention, and have described the method that the present invention separating clone on above-mentioned previous work basis includes the dna fragmentation and the checking OsRRG1 gene function of OsRRG1 gene complete coding section.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.

Embodiment 1: separating clone includes the dna fragmentation and the OsRRG1 gene of OsRRG1 constant gene segment C

Is that the QTL mapping analysis of parent's RIL (RIL) colony (is seen Yue etc. according to Yue etc. with rice varieties " precious Shan 97 " and " middle non-irrigated No. 5 ", Genetic analysis for drought resistance of rice at reproductive stage in field withdifferent types of soil.Theor Appl Genet, 2005) result has a long QTL (qMRDV2-1) of control root in the interval of the 2nd chromosomal RM573 and RM318 two marks.The gene that we predict the genomic dna in the interval of RM573 and RM318 two marks anatomizes, and the root growth related gene B REVIS RADIX sequence similarity of reporting in the gene (being gene OsRRG1 of the present invention) of finding to have a prediction and the Arabidopis thaliana reaches 62.1%.By searching Japanese paddy rice total length database (http://cdna01.dna.affrc.go.jp), find its pairing cDNA clone J033051H11, the genome sequence of this gene is positioned at the position that is positioned at 37924bp-44818bp on the 2nd karyomit(e) BAC clone AP003994.Genome sequence according to OsRRG1 correspondence among the BAC clone AP003994, predict its promoter region, and design primer PF (5-TAAGGATCCGAGAGGCTAGGAAATGCTTG, the sequence specific primer adds joint BamHI site) and FR (5-TAATCTAGAAGCGGCAAAGTAGTAGT, the sequence specific primer adds joint XbaI), the full-length cDNA of this gene is increased from total RNA of " bright extensive 63 " (a hybrid rice parent that China generally applies).Amplified production is exactly sequence 1-2103bp of the present invention.Concrete steps are: adopt the total RNA (extracting method is according to above-mentioned TRIZOL reagent specification sheets) in TRIZOL reagent (available from Invitrogen company) extraction rice varieties " the bright extensive 63 " blade, utilize ThermoScript II (available from Invitrogen company) with synthetic cDNA first chain of its reverse transcription, reaction conditions is: 65 ℃ of 5min, 42 ℃ of 50min, 70 ℃ of 10min.Amplify the full-length cDNA of OsRRG1 gene with above-mentioned primer (PF and PR).The RT-PCR reaction conditions is: 94 ℃ of pre-sex change 2min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 2min, 40 circulations; 72 ℃ are extended 5min.The PCR product that amplification is obtained is connected into pGEM-T carrier (available from Promega company), and screening positive clone and order-checking (ABI3730 sequenator) obtain required full-length gene cDNA.This clone is named as pGEM-OsRRGlc.

Embodiment 2, the structure and the conversion of the expression of OsRRG1 gene inhibition, overexpression and promoter expression vector

In order to illustrate the function of this gene better, the applicant suppresses it to express and overexpression in paddy rice, verify from the phenotype of transfer-gen plant.

It is as follows suppress to express (RNAi) carrier construction method: (5 '-CTAGATGGAGACGAAGGTGATCTG) is the specific fragment (401bp) that template amplification goes out the OsRRG1 gene with the clone pGEM-OsRRG1c from embodiment 1 acquisition at first to use RNAi primer R2RF:(5 '-GCAGGCACCAATACAAGTCC) and R2RR.Reaction conditions is: 94 ℃ of pre-sex change 2min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 2min, 30 circulations; 72 ℃ are extended 5 and report min.With this fragment subclone to the pGEM-T carrier.Use again primer ALLF (5 '-GGGGAC AAG TTT GTA CAA AAA AGC AGG CTT AAT ACG ACT CAC TAT AGG G) and ALLR (5 '-GGGGAC CAC TTT GTA CAA GAA AGC TGG GTA TTT AGG TGA CAC TAT AG) from have this segmental pGEM-T carrier amplify one can be for the fragment (451 bp) of recombinating on the pHellsgate2 carrier.The PCR condition is the same.Recombining reaction is undertaken by the recombinant clone test kit specification sheets that Invitrogen company provides.PHellsgate2 is the carrier (Wesley etc. that are specifically designed to the RNAi vector construction that published, Construct design for efficient, effective and high-throughput gene silencing inplants.Plant J, 27:581-590,2001).

The overexpression carrier construction method is as follows: at first with the positive colony pGEM-OsRRG1 plasmid BamHI and the XbaI double digestion that obtain among the embodiment 1, reclaim the external source fragment; Simultaneously, the enzyme that uses the same method is cut the genetic transformation carrier pC1301S that carries different promoters.Enzyme cuts complete, uses chloroform: primary isoamyl alcohol (pressing and ratio: 24: 1) extracting, purifying enzyme is cut product.Carrier after cutting with endonuclease bamhi that comprises the OsRRG1 gene and enzyme is done ligation, transformed into escherichia coli DH10 β (bacterial strain is available from Invitrogen company).Cut screening positive clone by enzyme, obtain conversion carrier.The carrier pC1301S of genetic transformation is plant genetic conversion carrier pCAMBIA1301 (Fig. 7 commonly used in the world, from Australian CAMBIA[Center for the Application of Molecular Biology to International Agriculture] laboratory) on the basis, the tobacco mosaic virus (TMV) CaMV 35S promoter of introducing widely used constitutive expression at restriction enzyme site EcoRI and SacI place obtains.

By agriculture bacillus mediated rice genetic method for transformation it is imported in the rice varieties " in spend 11 " (rice varieties of the public use that China Paddy Rice Inst provides), through the callus of cultivating in advance, infecting, cultivating altogether, screening having hygromycin resistance, break up, take root, practice transplantation of seedlings, obtain transfer-gen plant.Agriculture bacillus mediated paddy rice (japonica rice subspecies) genetic transforming method is used people's reported method (Hiei etc. such as Hiei, Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequenceanalysis of the boundaries of the T-DNA, Plant J, 6:271-282,1994) carry out.

The substratum of above-mentioned agriculture bacillus mediated genetic transformation concrete steps is as described below:

Agent prescription: (1) reagent and solution abbreviation: the abbreviation of the used plant hormone of substratum is expressed as follows among the present invention: 6-BA (6-benzyladenine); CN (Pyocianil); KT (kinetin or title phytokinin); NAA (naphthylacetic acid); IAA (indolylacetic acid); 2,4-D (2,4-dichlorphenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (dimethyl sulfoxide (DMSO)); N6max (a large amount of composition solution of N6); N6mix (N6 trace ingredients solution); MSmax (a large amount of composition solution of MS); The main solution formula of Msmix (MS trace ingredients solution) (2):

1) preparation of N6 substratum macroelement mother liquor [10 times of concentrated solutions (10X)]:

Saltpetre (KNO ₃) 28.3g

Potassium primary phosphate (KH ₂PO ₄) 4.0g

Ammonium sulfate ((NH ₄) ₂SO ₄) 4.63g

Sal epsom (MgSO ₄7H ₂O) 1.85g

Calcium chloride (CaCl ₂2H ₂O) 1.66g

Dissolving is settled to 1000ml under the room temperature then one by one.

2) preparation of N6 substratum trace element mother liquor [100 times of concentrated solutions (100X)]

Potassiumiodide (KI) 0.08g

Boric acid (H ₃BO ₃) 0.16g

Manganous sulfate (MnSO ₄4H ₂O) 0.44g

Zinc sulfate (ZnSO ₄7H ₂O) 0.15g

Dissolving and be settled to 1000ml under the room temperature.

3) molysite (Fe ₂EDTA) preparation of stock solution (100X)

Prepare the 800ml distilled water and be heated to 70 ℃, add b diammonium disodium edta (Na ₂EDTA2H ₂O) 3.73 grams, fully the dissolving back kept 2 hours in 70 ℃ of water-baths, was settled to 1000ml, and 4 ℃ of preservations are standby.

4) VITAMIN stock solution (100X) preparation

Nicotinic acid (Nicotinic acid) 0.1g

VITMAIN B1 (Thiamine HCl) 0.1g

Vitamin B6 (Pyridoxine HCl) 0.1g

Glycine (Glycine) 0.2g

Inositol (Inositol) 10g

Add water and be settled to 1000ml, 4 ℃ of preservations are standby.

5) preparation of MS substratum macroelement mother liquor (10X)

Ammonium nitrate (NH ₄NO ₃) 16.5g

Saltpetre 19.0g

Potassium primary phosphate 1.7g

Sal epsom 3.7g

Calcium chloride 4.4g

Dissolving and be settled to 1000ml under the room temperature.

6) preparation of MS substratum trace element mother liquor (100X)

Potassiumiodide 0.083g

Boric acid 0.62g

Manganous sulfate 0.86g

Sodium orthomolybdate (Na ₂MoO ₄2H ₂O) 0.025g

Copper sulfate (CuSO ₄5H ₂O) 0.0025g

Dissolving and be settled to 1000ml under the room temperature.

7) 2, the 4-D stock solution, the 6-BA stock solution, naphthylacetic acid (NAA) stock solution, indolylacetic acid (IAA) stock solution: 1 is mg/ml.

8) glucose stock solution: 0.5g/ml.

9) preparation of AS stock solution: weigh AS 0.392g, DMSO 10ml.

(3) be used for the culture medium prescription that rice genetic transforms

1) inducing culture

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

2,4-D stock solution 2.5ml

Proline(Pro) (Proline) 0.3g

CH 0.6g

Sucrose (Sucrose) 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.

2) subculture medium

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

2,4-D stock solution 2.0ml

Proline(Pro) 0.5g

CH 0.6g

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.

3) pre-culture medium

N6max mother liquor (10X) 12.5ml

N6mix mother liquor (100X) 1.25ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.15g

Sucrose 5g

Agar powder (Agarose) 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, seals sterilization.Use preceding heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in the culture dish poured in packing into.

4) be total to substratum

N6max mother liquor (10X) 12.5ml

N6mix mother liquor (100X) 1.25ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.2g

Sucrose 5g

Agar powder 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, seals sterilization.Use preceding heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (the every ware of 25ml/) in the culture dish poured in packing into.

5) suspension culture base

N6max mother liquor (10X) 5ml

N6mix mother liquor (100X) 0.5ml

Fe ²⁺EDTA stock solution (100X) 0.5ml

VITAMIN stock solution (100X) 1ml

2,4-D stock solution 0.2ml

CH 0.08g

Sucrose 2g

Adding distil water is regulated pH value to 5.4 to 100ml, divides to install in the triangular flask of two 100ml, seals sterilization.Add 1ml glucose stock solution and 100 μ l AS stock solutions before using.

6) select substratum

N6max mother liquor (10X) 25ml

N6mix mother liquor (100X) 2.5ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.625ml

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Adding distil water is regulated pH value to 6.0 to 250ml, seals sterilization.The dissolving substratum adds 250 μ l HN and 400ppmCN before using, and (25ml/ ware) in the culture dish poured in packing into.

7) break up substratum in advance

N6max mother liquor (10X) 25ml

N6mix mother liquor (100X) 2.5ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

6-BA stock solution 0.5ml

KT stock solution 0.5ml

NAA stock solution 50 μ l

IAA stock solution 50 μ l

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.9, seals sterilization.The dissolving substratum adds 250 μ lHN and 200ppm CN before using, and (25ml/ ware) in the culture dish poured in packing into.

8) division culture medium

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

6-BA stock solution 2ml

KT stock solution 2ml

NAA stock solution 0.2ml

IAA stock solution 0.2ml

CH 1g

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 6.0.Boil and be settled to 1000ml, divide to install to 50ml triangular flask (50ml/ bottle), seal sterilization.

9) root media

MSmax mother liquor (10X) 50ml

MSmix mother liquor (100X) 5ml

Fe ²⁺EDTA stock solution (100X) 5ml

VITAMIN stock solution (100X) 5ml

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.8.Boil and be settled to 1000ml, divide to install to (25ml/ pipe) in the pipe of taking root, seal sterilization

The transgenic rice plant difference called after R2R-N (wherein R2R is the pHellsgate2-OsRRG1 carrier, contains constitutive promoter CaMV 35S, the numbering of N independence transformed plant) that OsRRG1 is suppressed expression; Transgenic rice plant difference called after R2S-N (R2S is the pC1301S-OsRRG1 carrier, contains constitutive promoter CaMV 35S) with the OsRRG1 overexpression.Each conversion carrier has obtained independently transgenic rice plant of at least 30 strains, and the present invention obtains independent transgenic rice plant 70 strains altogether.

Embodiment 3: the expression level that detects paddy rice native gene OsRRG1

With rice varieties " bright extensive 63 " is material, and the RNA that extracts the tissue of different growing stage detects OsRRG1 expression of gene level with RT-PCR.Organize respectively for 10 kinds that choose and be: 1. the root in tillering phase; 2. sword-like leave; 3. stem; 4. be shorter than the young fringe of 1cm; 5.3-5 young fringe; 6. be longer than the young fringe of 10cm; 7. stamen; 8. gynoecium; 9.3 the spire in week; 10.3 the etiolated seedling in week.Total RNA adopts TRIZOL reagent (available from Invitrogen company) to extract (extracting method is according to above-mentioned TRIZOL reagent specification sheets), utilize ThermoScript II (available from Invitrogen company) with synthetic cDNA first chain (method is according to ThermoScript II reagent specification sheets) of its reverse transcription, reaction conditions is: 65 ℃ of 5min, 42 ℃ of 50min, 70 ℃ of 10min.With cDNA first chain is template, adopt primer R2RF (5 '-GCAGGCACCAATACAAGTCC) and R2RR (5 '-CTAGATGGAGACGAAGGTGATCTG) pcr amplification goes out the specific fragment (401bp) of OsRRG1 gene.Adopt primer (AF:5 '-specific fragment (610bp) of the paddy rice Actin1 gene of GCGTCGACTCCACTCTCGC and AR:5 '-CCATGAAACAAATCCAACAACA) amplify to be to carry out quantitative analysis as internal reference.Reaction conditions is: 94 ℃ of pre-sex change 2min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 2min, 30 circulations; 72 ℃ are extended 5min.The OsRRG1 gene that amplifies and the special band of Actin1 gene are handled with GelImager software (GenRes company), with the brightness quantification of band, and be that standard is carried out after the homogenization relatively OsRRG1 expression of gene amount in the different tissues with the brightness of the special band of Actin1 gene.Result (Fig. 3) shows: the OsRRG1 gene all has a certain amount of expression in selected tissue, but relative higher with expression amount in the young fringe with root.

Embodiment 4:OsRRG1 suppresses express transgenic T ₁The growth of family root in seedling stage

Whether the expression that the present invention adopts Northern hybridization to detect OsRRG1 in the RNAi transgenic rice plant root of test section is suppressed (Fig. 4 A).The result shows that the expression of OsRRG1 in the RNAi transgenic rice plant more than 60% is subjected to inhibition in various degree.The present invention has chosen 5 OsRRG1 and has expressed the T that is subjected to obviously to suppress ₁Family is carried out the mensuration of the speed of growth.Concrete steps are as follows: each T ₁Choose 30-40 grain seed contain seed soaking in G418 (a kind of commercial microbiotic that uses is available from the Promega company) aqueous solution (50mg/ml) in the plant transgene screening for family, remove not chitting piece (not containing genetically modified seed).Transgenosis and the back 5 days seedling water planting of wild-type contrast (spending 11 in the rice varieties) germination are planted in 90 * 60 * 20cm ³The cultivation box in, cultivating box cover has 92 * 62cm ²The wooden cover of size is drilled with equally distributed 96 holes on the wooden cover, kind seedling in every hole is with the fixing plant of sponge.The nutritive medium composition of water planting is as follows: by the scale of the inorganic nutritive element of chemical reagent or commodity chemical fertilizer: N:40ppm; P:10ppm; K:40ppm; Ca:40ppm; Mg:40ppm; Mn:0.5ppm; Mo:0.05ppm; B:0.2ppm; Zn:0.01ppm; Cu:0.01ppm; Fe:2ppm.The mother liquor prescription is as follows:

1、NH ₄NO ₃：914g/10L；2、NaH ₂PO ₄-2H ₂O：403g/10L；3、K ₂SO ₄：714g/10L；4、CaCl ₂：886g/10L；5、MgSO ₄-7H ₂O：3240g/10L；6、MnCl ₂-4H ₂O：15g，(NH ₄)6Mo ₇O ₂₄-4H ₂O：0.74g，H ₃BO ₃：9.34g，ZnSO ₄-7H ₂O：0.35g，CuSO ₄-5H ₂O：0.31g，FeCl ₃-6H ₂O：77g，(CH ₂O) _n：119g。Mix with the 500ml vitriol oil dissolving back respectively, and it is 10L that the said components water is settled to volume.

During water planting, add each 5mL of above-mentioned mother liquor (making the pH5.8-6.0 of nutrient solution) in every 3970mL water.

The every family of the transgenic paddy rice that is used to test is planted 8 individual plants, block design immediately, three repetitions (contrast is set in each box).The careful rice shoot that takes out is measured root length during after germination the 10th, 20,45 days.Fig. 5 is that the root length in seedling stage of representative transgenosis family and contrast compares.The result shows: the short 3.8-7.4cm (table 1) of root length of the root length comparison photograph of most of transfer-gen plant.

Root length and plant height that table 1 gene OsRRG1 of the present invention suppresses transfer-gen plant and contrast compare (unit: cm)

T 1Family	The 10th day average root of germinateing is long	The 20th day average root of germinateing is long	The 45th day average root of germinateing is long	The 45th day average plant height germinates
					R2R-1 R2R-2 R2R-3 R2R-4 R2R-5 R2R-6 R2R-7 R2R-8 R2R-9	11.3 * 10.2 * 10.8 * 11.7 * 12.1 * 13.6 9.1 ** 10.9 * 11.3 *	21.3 *19.7 *21.5 *18.4 **22.4 23.1 17.9 **20.8 *21.2 *	32.1 30.4 *31.9 *32.1 34.0 34.2 28.4 **32.1 *30.7 *	34.6 32.5 33.7 33.9 34.3 36.5 31.7 35.4 34.2
CK	14.7	25.2	35.4	36.5

Annotate: ^*Expression has marked difference with contrast.

The growth of embodiment 5:OsRRG1 overexpression transgenosis T1 family root in seedling stage

The present invention adopts Northern hybridization, and (method is referring to J. Sa nurse Brooker etc., molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating),, 2002, Science Press, Beijing) expression amount (Fig. 4 B) of OsRRG1 in the transgenic rice plant of test section overexpression.The result shows that the expression amount of OsRRG1 in the transgenic rice plant more than 70% is significantly higher than contrast, illustrates that the OsRRG1 transgenosis has obtained expression in these plant.Fig. 6 is that representative overexpression transgenosis family T0 compares with the root length in seedling stage that contrasts in rooting process for plant.In order further to compare the promoter action of this gene pairs rice root growth of overexpression, the present invention has chosen 7 OsRRG1 overexpression T ₁Family has been carried out the mensuration of root growth speed under the different condition.Concrete steps are as follows: each T ₁Choose 20-30 grain seed for family and in the aqueous solution of the Totomycin that contains 50mg/ml, soak seed, remove not chitting piece (not containing genetically modified seed).With transgenosis and the back 5 days seedling water planting plantation of wild-type contrast (spending 11 in the rice varieties) germination.Water planting kind method for planting is with embodiment 4.Block design immediately, three repetitions (contrast is set in each box), during each repeated, each transgenic paddy rice family was planted 8 individual plants.The careful rice shoot that takes out is measured root length during after germination the 10th, 20,45 days.The result shows: the long 6.8-16.4cm of root length (table 2) of the root length comparison photograph of transfer-gen plant.

The root length of table 2 gene OsRRG1 of the present invention overexpression transfer-gen plant and contrast and plant height be (unit: cm) relatively

T 1Family	The 10th day average root of germinateing is long	The 20th day average root of germinateing is long	The 45th day average root of germinateing is long	The 45th day average plant height germinates
					R2S-1	17.2 *	29.5 *	39.8 *	36.2

R2S-2 R2S-3 R2S-6 R2S-7 R2S-8 R2S-9	17.3 * 14.4 * 15.8 * 14.1 13.9 16.2 *	29.7 * 26.1 28.5 * 26.7 27.3 30.0 *	38.4 * 36.2 37.9 * 36.5 34.8 40.7 **	37.1 35.9 36.2 34.7 35.1 36.3
					CK	13.2	26.3	35.7	35.4

Annotate: ^*Expression has marked difference with contrast.

Embodiment 6:OsRRG1 overexpression transgenosis T ₁Family is tied to form the growth of strain phase root under drought condition and keeps away drought

Thereby in order to verify keeping away drought and whether strengthening and cause drought resistance to strengthen of transgenic rice plant more exactly, the present invention further utilizes pvc pipe to carry out potted plant arid Processing Test.Concrete steps are as follows: T1 soaks seed at the aqueous solution that contains the 50mg/ml Totomycin for the seed of each family, removes not chitting piece, and the back 1-2cm seedling that will germinate directly is seeded in the pvc pipe carefully; Pvc pipe is high 1 meter, and diameter 20cm places an end closure on ground to make it water-tight, and the pipe side has three apertures, is respectively 10cm, 40cm and 70cm from the distance of sealing end; Clog with soft rubber ball when not discharging water; Use in the pipe with the plastics bag of the identical size of caliber and fill the sand soil (river sand and fine clay were by 1: 1 uniform mixing) that mixes; Every pipe is planted a strain rice plant, 10 individual plants of every T1 family plantation.Cutting off the water supply (extract soft rubber ball and poke plastics bag) in the time of about 15 days from heading, observing the time that the record blade begins to roll up, Fig. 6 is representative transgenosis family and the performance to impinging upon after cutting off the water supply 5 days.After the adjoining tree blade was rolled up (promptly the 5-6 point is observed in the afternoon, and all blades of individual plant wind up fully) entirely, allowed it grow to ripe back to all plant rehydrations and investigate setting percentage morning next day.Because the heading stage of transfer-gen plant and contrast is identical, plant size basically identical, the influence also less (judging) that the different plants present position is different by their leaf roll fate of identical controlled observation is set at different positions, therefore the edaphic condition unanimity can be used for the leaf roll speed to weigh and keep away drought.Setting percentage (each family is examined or check 10 strains, and all effective fringes are examined or check in every strain), tiller number and the maximum root of ripe back examination transgenosis family are long.Data show, the appearance comparison of transfer-gen plant leaf roll symptom was according to late 1-5 days, the setting percentage comparison of transfer-gen plant is according to exceeding 6.1% to 25.9%, and the long comparison of maximum root is according to long 6.4-22.7cm (table 3), and tiller number, heading stage and biological yield be no significant difference then.The drought resistance that transgenic rice plant is described strengthens mainly because it is kept away non-irrigated ability and strengthens and cause behind the overexpression OsRRG1 gene.

Table 3 gene OsRRG1 of the present invention overexpression transfer-gen plant is kept away drought relatively with contrast boot stage

T 1Family	The leaf roll time (my god)	Maximum root long (cm)	Setting percentage (%)	Plant height (cm)	Tiller number
						R2S-1 R2S-2 R2S-3 R2S-6 R2S-7 R2S-8 R2S-9	8.2 ** 7.5 ** 5.1 * 7.2 ** 4.0 5.2 * 6.4 *	93.8 ** 88.2 ** 78.3 * 84.6 ** 77.5 * 78.3 * 82.7 *	96.0 ** 91.2 ** 80.2 * 84.3 ** 76.2 79.2 * 86.7 **	127 131 121 128 119 122 134	14.2 13.7 13.8 15.1 11.9 13.2 14.6
CK	3.2	71.1	70.1	133	15.1

Annotate: the leaf roll time is meant the fate that begins to roll up from about 20% the blade of cutting off the water supply. ^*Expression has marked difference with contrast.

Sequence table

Sequence table (SEQUENCE LISTING)

＜110〉Hua Zhong Agriculture University

＜120〉utilize paddy gene OsRRG1 to promote the growth and the raising plant drought ability of roots of plants

<130>

<141>2005-10-13

<160>2

<170>PatentIn version 3.1

<210>1

<211>2100

<212>DNA

＜213〉paddy rice (Oryza sativa)

<220>

<221>gene

<222>(1)..(2100)

<223>

<220>

<221>primer_bind

<222>(1956)..(1978)

<223>

<220>

<221>primer_bind

<222>(93)..(112)

<223>

<220>

<221>CDS

<222>(558)..(1793)

<223>

<400> 1

gctgctttcg ctcgatttcg ggggcggatc agacggccgc gagcgaatcc ggggatctcc 60

cccctccctc ccgctctgtt cctgcctctg ctccccgccc gtttcctctc aatccttcct 120

tctccctgct tctctccctc gcggaaaaaa aaaagaaaag aaaaggaaaa gggggagtgg 180

aaaagatctc ctcttcgtcc tgtttctgtt ggcgtcgctg ctcggcagct cgttcgctag 240

ctaccaccca aaaggcggca gtcgatcgcc tgatagctca gccgctctcc tctgtcgctt 300

ccgagctctt gtgccgccgc gcgccggcga ggatctgtcg accgagagag gagccacccg 360

ccgtccaaga agaggaggaa gaagatcatc tttgcgtcag aaggagcagt gacgagatac 420

cactgtccgc gtggtcacaa gcattgctgc gagactgtga ttggcgggag ttgctttgga 480

tgagcaatag cctgatggtg gcttgctaac tgggaaggag gatttgcgcg gttctggact 540

actcgagagg ctaggaa atg ctt gcg tgc atc gcg tgt tcg acc aag gac 590

Met Leu Ala Cys Ile Ala Cys Ser Thr Lys Asp

1 5 10

ggc ggg gag ggc ggg cac cgg tcc gcc acc gcc acg ccc aac tcc ggc 638

Gly Gly Glu Gly Gly His Arg Ser Ala Thr Ala Thr Pro Asn Ser Gly

15 20 25

aag tct ctg act tca cag ttg aag gac atg gtg ctc aag ttc tcc ggc 686

Lys Ser Leu Thr Ser Gln Leu Lys Asp Met Val Leu Lys Phe Ser Gly

30 35 40

agc ggc agg cac caa tac aag tcc ggc ggg agc ccg tcg ttg agg acc 734

Ser Gly Arg His Gln Tyr Lys Ser Gly Gly Ser Pro Ser Leu Arg Thr

45 50 55

agc cgc ttc cac cgc tcc agc cgc ctc gcc gcg tac ccg ggc atc atc 782

Ser Arg Phe His Arg Ser Ser Arg Leu Ala Ala Tyr Pro Gly Ile Ile

60 65 70 75

gac gag tcg ggc ttc acg tcg gac ggg gcc ggc gag gcc tat act tac 830

Asp Glu Ser Gly Phe Thr Ser Asp Gly Ala Gly Glu Ala Tyr Thr Tyr

80 85 90

atg agg acg acg acg gcg agc gcc ggc gcc cgg gcc gcg ccg tcg acg 878

Met Arg Thr Thr Thr Ala Ser Ala Gly Ala Arg Ala Ala Pro Ser Thr

95 100 105

tgg gac ttg ccg ccc aag gtg aac cac cgc agc ttc cag ccg cgc gtg 926

Trp Asp Leu Pro Pro Lys Val Asn His Arg Ser Phe Gln Pro Arg Val

110 115 120

atc agg agc ccg agc gcg agc ggg gta ccg agc atc ggg gag gaa gac 974

Ile Arg Ser Pro Ser Ala Ser Gly Val Pro Ser Ile Gly Glu Glu Asp

125 130 135

tac gac gac gac gac gac gac gat gac gag gag aca gtc ctc ctg gag 1022

Tyr Asp Asp Asp Asp Asp Asp Asp Asp Glu Glu Thr Val Leu Leu Glu

140 145 150 155

gag gac cgc gtg ccg cgg gag tgg acg gcg cag gtg gag ccc ggc gtg 1070

Glu Asp Arg Val Pro Arg Glu Trp Thr Ala Gln Val Glu Pro Gly Val

160 165 170

cag atc acc ttc gtc tcc atc ccc ggc ggc gcc ggc aac gat ctg aag 1118

Gln Ile Thr Phe Val Ser Ile Pro Gly Gly Ala Gly Asn Asp Leu Lys

175 180 185

cgc atc cgt ttc agc cgg gag atg ttt aac aag tgg gag gcg cag cgg 1166

Arg Ile Arg Phe Ser Arg Glu Met Phe Asn Lys Trp Glu Ala Gln Arg

190 195 200

tgg tgg ggg gag aac tac gac cgc gtg gtg gag ctc tac aac gtg cag 1214

Trp Trp Gly Glu Asn Tyr Asp Arg Val Val Glu Leu Tyr Asn Val Gln

205 210 215

acg ttc agc cgg cag cag ggc ttc tcg acg ccg acg tcc tcc gtc gac 1262

Thr Phe Ser Arg Gln Gln Gly Phe Ser Thr Pro Thr Ser Ser Val Asp

220 225 230 235

gaa gcc atg cag aga gat tcg ttc tac tcc cgc gtc ggc tcg acg agg 1310

Glu Ala Met Gln Arg Asp Ser Phe Tyr Ser Arg Val Gly Ser Thr Arg

240 245 250

gag agc ccg gcg atg atg atg ccg ccg ccg ccg ccg ctg ccg tcg tcg 1358

Glu Ser Pro Ala Met Met Met Pro Pro Pro Pro Pro Leu Pro Ser Ser

255 260 265

ggt gct ggc agg gag cac ccg atc agc cgg aca gcg tcg agc aag gcg 1406

Gly Ala Gly Arg Glu His Pro Ile Ser Arg Thr Ala Ser Ser Lys Ala

270 275 280

cag ctg tcg tcg tcg tcg tcg gtg gcg gcg gct cgg ccg ccg ttc tac 1454

Gln Leu Ser Ser Ser Ser Ser Val Ala Ala Ala Arg Pro Pro Phe Tyr

285 290 295

ccg tcc acg gcc gtg ccg gac ccg tcc gac cac gtg tgg gcg cac cac 1502

Pro Ser Thr Ala Val Pro Asp Pro Ser Asp His Val Trp Ala His His

300 305 310 315

ttc aac ctc ctc aac tcc gcg gcc gcc gga cca gcg gcg ccg tac gac 1550

Phe Asn Leu Leu Asn Ser Ala Ala Ala Gly Pro Ala Ala Pro Tyr Asp

320 325 330

ccg tcg cgc ggc acg acg tcg tcc cgg gac gag gcg tcc gtg tcc atc 1598

Pro Ser Arg Gly Thr Thr Ser Ser Arg Asp Glu Ala Ser Val Ser Ile

335 340 345

agc aac gcg agc gac ctg gag gcc acg gag tgg gtg gag cag gac gag 1646

Ser Asn Ala Ser Asp Leu Glu Ala Thr Glu Trp Val Glu Gln Asp Glu

350 355 360

cct ggg gtg tcc atc acc atc cgc gag ttc ggc gac ggc acc cgc gag 1694

Pro Gly Val Ser Ile Thr Ile Arg Glu Phe Gly Asp Gly Thr Arg Glu

365 370 375

ctc cgc cgc gtc cgg ttc agc cgc gag aga ttt ggc gag gag cgg gcc 1742

Leu Arg Arg Val Arg Phe Ser Arg Glu Arg Phe Gly Glu Glu Arg Ala

380 385 390 395

aag gtg tgg tgg gag cag aac cgg gac cgg ata cac gcg cag tat ctg 1790

Lys Val Trp Trp Glu Gln Asn Arg Asp Arg Ile His Ala Gln Tyr Leu

400 405 410

tga gctaacgcga gacccatcgt tgatgagagt gaagtgatac tgtgacagaa 1843

gctaacgcgt cgccgggaag aatagttaag agtgtgatga tactactact ttgccgctgc 1903

tacggttagt gctactaggt tttttttaca gggtgaatat aggatgaggg ggaaggatgc 1963

tgtggtctga gaagcagcag gctcagtcaa gctgctactc ttgttaactg accactttgg 2023

tcttccgttt gtaaccttta tggacagggt gttcgtttaa ttcgtttgtt ttagcatggt 2083

ttcatgtgat cctcgac 2100

<210>2

<211>411

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>2

Met Leu Ala Cys Ile Ala Cys Ser Thr Lys Asp Gly Gly Glu Gly Gly

1 5 10 15

His Arg Ser Ala Thr Ala Thr Pro Asn Ser Gly Lys Ser Leu Thr Ser

20 25 30

Gln Leu Lys Asp Met Val Leu Lys Phe Ser Gly Ser Gly Arg His Gln

35 40 45

Tyr Lys Ser Gly Gly Ser Pro Ser Leu Arg Thr Ser Arg Phe His Arg

50 55 60

Ser Ser Arg Leu Ala Ala Tyr Pro Gly Ile Ile Asp Glu Ser Gly Phe

65 70 75 80

Thr Ser Asp Gly Ala Gly Glu Ala Tyr Thr Tyr Met Arg Thr Thr Thr

85 90 95

Ala Ser Ala Gly Ala Arg Ala Ala Pro Ser Thr Trp Asp Leu Pro Pro

100 105 110

Lys Val Asn His Arg Ser Phe Gln Pro Arg Val Ile Arg Ser Pro Ser

115 120 125

Ala Ser Gly Val Pro Ser Ile Gly Glu Glu Asp Tyr Asp Asp Asp Asp

130 135 140

Asp Asp Asp Asp Glu Glu Thr Val Leu Leu Glu Glu Asp Arg Val Pro

145 150 155 160

Arg Glu Trp Thr Ala Gln Val Glu Pro Gly Val Gln Ile Thr Phe Val

165 170 175

Ser Ile Pro Gly Gly Ala Gly Asn Asp Leu Lys Arg Ile Arg Phe Ser

180 185 190

Arg Glu Met Phe Asn Lys Trp Glu Ala Gln Arg Trp Trp Gly Glu Asn

195 200 205

Tyr Asp Arg Val Val Glu Leu Tyr Asn Val Gln Thr Phe Ser Arg Gln

210 215 220

Gln Gly Phe Ser Thr Pro Thr Ser Ser Val Asp Glu Ala Met Gln Arg

225 230 235 240

Asp Ser Phe Tyr Ser Arg Val Gly Ser Thr Arg Glu Ser Pro Ala Met

245 250 255

Met Met Pro Pro Pro Pro Pro Leu Pro Ser Ser Gly Ala Gly Arg Glu

260 265 270

His Pro Ile Ser Arg Thr Ala Ser Ser Lys Ala Gln Leu Ser Ser Ser

275 280 285

Ser Ser Val Ala Ala Ala Arg Pro Pro Phe Tyr Pro Ser Thr Ala Val

290 295 300

Pro Asp Pro Ser Asp His Val Trp Ala His His Phe Asn Leu Leu Asn

305 310 315 320

Ser Ala Ala Ala Gly Pro Ala Ala Pro Tyr Asp Pro Ser Arg Gly Thr

325 330 335

Thr Ser Ser Arg Asp Glu Ala Ser Val Ser Ile Ser Asn Ala Ser Asp

340 345 350

Leu Glu Ala Thr Glu Trp Val Glu Gln Asp Glu Pro Gly Val Ser Ile

355 360 365

Thr Ile Arg Glu Phe Gly Asp Gly Thr Arg Glu Leu Arg Arg Val Arg

370 375 380

Phe Ser Arg Glu Arg Phe Gly Glu Glu Arg Ala Lys Val Trp Trp Glu

385 390 395 400

Gln Asn Arg Asp Arg Ile His Ala Gln Tyr Leu

405 410

Claims (5)

1, the dna sequence dna of the elongation growth of giving rice root of OsRRG1 gene mediated and/or raising paddy rice drought-resistant ability, it is the dna sequence dna shown in the 1-2103 position among (a) SEQ ID NO:1, or (b) coding and the identical protein DNA sequence of (a) encoded protein matter.

2, the described dna sequence dna of claim 1 of suitable promotor connection.

3, the described dna sequence dna of claim 2, it is the dna sequence dna shown in the SEQ ID NO:1

4, give the elongation growth of roots of plants and improve the sequence of drought-resistant ability, it be with SEQ ID NO:1 in dna sequence dna similarity shown in the 1-2103 position reach the dna sequence dna more than 90% or reach protein sequence more than 75% with the coded protein sequence similarity of SEQ ID NO:1.

5, the application of each described dna sequence dna of claim 1-4 in elongation growth that increases rice root and/or raising paddy rice drought-resistant ability.

Images