CN103710354B - Dry-rice drought-resistant gene, function markers and application - Google Patents
Dry-rice drought-resistant gene, function markers and application Download PDFInfo
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Abstract
The invention discloses a dry-rice drought-resistant gene, function markers and application, and the dry-rice drought-resistant gene OsbHLH120 belongs to a bHLH (basic helix-loop-helix) transcription factor, and is located between ninth rice chromosome RM24271 and RM566; compared with a normal rice sequence, the dry-rice drought-resistant gene OsbHLH120 has 15 bases of deletion in a coding region, and the deleted bases are located at 628th-642th bp (base point) of the downstream of a gene transcription initiation codon. At the same time, dry-rice specific gene function markers are designed according to rice and dry-rice sequence difference characteristics of the transcription factor, dry-rice resources containing the characteristic base sequence of the gene can be purposefully selected by the function markers, the dry-rice resources are used in drought-resistant rice breeding, and the function markers are used for marker assisted selection for more rapid and accurate drought-resistant rice variety breeding.
Description
Technical field
The present invention relates to a kind of upland rice anti-drought gene, also relate to functional label and the application of anti-drought gene simultaneously, belong to biological technical field.
Background technology
Upland rice, also known as dryland rice, the dry farming ecotype of Asian Cultivated Rice (Oryza sativa L.), is suitable for the plantation of broad variety nonirrigated farmland.Having the features such as drought tolerance is strong, barren tolerance good, wide adaptability due to it, is the valuable germ plasm resource of paddy drought resistance improvement breeding.Along with the worsening shortages of water resources, excavate and utilize upland rice favorable genes, the rice varieties cultivating strong drought resistance also seems more and more important.
Transcription factor can activate as trans-acting factor or the transcribing of suppressor gene, and finely regulating downstream functional gene is expressed, and realizes the response to various adverse circumstance.The network system of multiple transcription factor composition plays very important effect in the response and regulation and control of the abiotic stress of plant; Have been reported the transcription factors such as SNAC1, OsSKIPa, DST and can strengthen Rice Drought Resistence ability.There are some researches show that bHLH family transcription factor also participates in paddy rice stress response process in recent years, the motif in bHLH family structure territory contains about 60 amino acid, be made up of a basic aminoacids district (basic region) and Helix (HLH region) district, basic region is about containing 15 amino acid, wherein containing more basic aminoacids, the activity in this region mainly with transcription factor and DNA particular sequence in conjunction with relevant, the Main Function in Helix district forms dimer with other protein binding, collaborative functionating.In paddy rice, at least 165 members are contained in bHLH family, and wherein OsbHLH148 and OsJAZ interactions between protein participates in JA signal pathway and regulates rice drought tolerance, and high expression level OsbHLH148 can cause rice plant to the patience of drought stress; OrbHLH001 is also a kind of bHLH class transcription factor, the expression of AKT1 can be induced to raise with the E-box specific combination of OsAKT1 promoter region, regulate and maintain ionic equilibrium under salt stress.
LOC_Os09g28210(OsbHLH120) bHLH transcription factor family is belonged to, between the 9th karyomit(e) RM24271 and RM566, coding region intronless.There are some researches show, this gene can by drought-induced expression.3 leaf 1 heart stages, carried out 20%PEG6000 Stress treatment, and by the display of paddy rice Affymetrix chip analysis, PEG coerces and causes OsbHLH120 transcription factor expression level in drought-enduring variety Xiang Feng 119 root systems morning to raise, and raises 2.2 times.Paddy rice Japan is fine carries out 20%PEG6000 Stress treatment in 5 leaf phases, and quantitative fluorescent PCR analysis finds that in Stress treatment inducer blade, this genetic expression is raised, and 8h expression amount reaches maximum after treatment; Simultaneously rice leaf full-length genome chip of expression spectrum interpretation of result also demonstrates Drought at seedling stage and coerces OsbHLH120 expression amount can be caused to raise, and rise multiple reaches 3.79, and this shows the relation of this transcription factor and drought resistance.
Functional label is that the polymorphism motif causing phenotypic character to make a variation according to functional gene inside develops.Once hereditary effect is positioned on specific functional motif, the functional label developed based on this dependency does not then need checking further just can determine the presence or absence of target alleles under different genetic background.In application, functional label can avoid the selection mistake produced because restructuring exchanges, more effective when the target gene of colony detects, owing to deriving from gene internal, the performance of direct reflection objective trait, can detect accurately, tracking target gene, and its genetic effect value has universality, and reliability is high, to biological engineering colony and natural population all effective, utilizing backcross transformation drought resisting, during the transfer of the proterties such as high yield, the application of functional label better can avoid Linkage drag, so the exploitation of functional label contributes to the research based on candidate gene strategy in the application of the molecular marker assisted selection promoted in breeding and association analysis, and be conducive to excavating beneficial gene in germ plasm resource.The exploitation of upland rice specific transcription factor OsbHLH120 functional label, excavation and the research of Upland rice germplasm will be promoted, contribute to the various allelotrope fully excavating this site in upland rice, and advantage drought resisting allelotrope in allelotrope and corresponding Drought Resistance Germplasm material can be obtained in conjunction with drought resisting phenotype analytical, for the selection of parent in the cross-breeding of Drought-resistant rice varieties, the transformation of drought-resistant character lays the foundation.
Summary of the invention
The object of this invention is to provide a kind of upland rice anti-drought gene.
In order to realize above object, the technical solution adopted in the present invention is to provide a kind of upland rice anti-drought gene, and described upland rice anti-drought gene OsbHLH120 belongs to bHLH class transcription factor, between paddy rice the 9th karyomit(e) RM24271 and RM566; This upland rice anti-drought gene OsbHLH120 has lacked 15 bases compared with the normal sequence of paddy rice in coding region, described disappearance base is positioned at genetic transcription initiator codon downstream 628-642bp.
15 bases of described disappearance are as shown in SEQ ID NO.1.
The present invention also aims to provide a kind of anti-drought gene functional label InDel-bHLH120 according to the design of upland rice anti-drought gene distinguished sequence.
The technical solution adopted in the present invention is also to provide a kind of anti-drought gene functional label InDel-bHLH120 according to the design of Rice Drought Resistence gene specific sequence, and primer sequence is as follows:
Forward primer sequence: 5 '-GGCCATCCACTACGTCAAGT-3 '
Reverse primer sequences: 5 '-GGGAAACGATTGTCTCATCACC-3 '.
The theoretical amplification fragment of this anti-drought gene functional label in paddy rice is 201bp, and the theoretical amplification fragment in upland rice is 186bp.
The present invention also aims to provide the application of a kind of upland rice anti-drought gene in the molecular marker assisted selection breeding of drought resisting new rice variety.
The technical solution adopted in the present invention is also to provide the application of a kind of upland rice anti-drought gene in the molecular mark selection of drought resisting new rice variety.
Upland rice anti-drought gene OsbHLH120 of the present invention is the bHLH transcription factor between paddy rice the 9th karyomit(e) RM24271 and RM566, compared with rice sequences, is being positioned at genetic transcription initiator codon downstream 628-642bp, is lacking 15 bases.Simultaneously, the present invention is according to this transcription factor floods and droughts rice sequence differences feature design upland rice specific gene functional label, autotelic selection the upland rice resource of this gene expression characteristics base sequence can be contained by this functional label, and these upland rice resources are used for Drought-resistant rice breeding, and apply this functional label and carry out molecular marker assisted selection breeding, with more fast, accurately carry out the seed selection of Drought-resistant rice varieties.
Accompanying drawing explanation
Fig. 1 is the part sequencing result of OsbHLH120 (3-2) to 4 representative water, the representative kind amplified production of upland rice;
Fig. 2 is the detected result of InDel-bHLH120 to 34 water, upland rice material;
1-21 is rice varieties, is followed successively by: fine, richer, the new rice 18 of Japan, Zheng rice 18, Xinfeng No.2, glad No. 1 of side, glad No. 4 of side, town rice 88, Zheng rice No. 4,9311, osmanthus towards No. 2, special blue or green, should 1577, II-32B, CBB23, Chinese Fragrant Rice, Peiai 64S, short pin Nan Te, two or nine No. 1, south, Zhenshan 97B, the precious B of bamboo;
22-34 is upland rice variety, is followed successively by: IRAT109, IAPAR9, milli lattice labor, Dandong dryland rice, platform east dryland rice, upland rice 277, upland rice 297, No. 2, Zheng's drought, Qin Ai, red upland rice, white hair rice, the purple skin in Funing, Tianjin upland rice;
Fig. 3 is InDel-bHLH120 to the detected result of (paddy rice richer/upland rice IRAT109 introgressive line IL392) F2 colony;
M is DNA Marker, P1 is that rice varieties is richer, and P2 is upland rice IRAT109, F1 is richer and heterozygous genotypes that is IRAT109, and other bands are all the individual sample of F2 colony.
Embodiment
The acquisition of embodiment 1 upland rice anti-drought gene and distinguished sequence thereof
1, experiment material
Rice varieties: fine, richer, the new rice 18 of Japan, Zheng rice 18, Xinfeng No.2, glad No. 1 of side, glad No. 4 of side, town rice 88, Zheng rice No. 4,9311, osmanthus towards No. 2, special blue or green, publicly occupy 73.
Upland rice variety: IRAT109, IAPAR9, milli lattice labor, Dandong dryland rice, platform east dryland rice, upland rice 277, upland rice 297, No. 2, Zheng's drought, Qin Ai, No. 6, Zheng's drought, No. 9, Zheng's drought.
2, DNA extraction
Get blade in rice seedling, adopt SDS method to extract DNA, concrete steps are as follows:
Get fresh blade in the mortar of precooling, by liquid nitrogen grinding to Powdered, be then transferred in 2mL centrifuge tube; Add 600 μ L SDS extracting solutions (65 DEG C of preheatings), 65 DEG C of water-bath 30min, put upside down mixing for several times every 5min; Then add 400 μ L chloroforms: primary isoamyl alcohol (24:1), soft mixing shakes 10min, the centrifugal 10min of 12000g; Be transferred to by supernatant liquor in another 1.5mL sterile centrifugation tube, add 400 μ L chloroforms: primary isoamyl alcohol (24:1), soft mixing shakes 10min, the centrifugal 10min of 12000g; Supernatant liquor is transferred in another 1.5mL sterile centrifugation tube, adds the Virahol of equal-volume precooling (-20 DEG C), in-20 DEG C of standing 30min after mixing; Abandon supernatant after the centrifugal 10min of 12000g, precipitate 2 times with 70% ethanol rinse, dehydrated alcohol rinsing 1 time, centrifugal rear removing residual ethanol, dries; Finally add 100 μ L TE buffer solution DNA, be stored in-20 DEG C for subsequent use.
3, PCR order-checking and sequencing analysis
OsbHLH120 sequence (Genbank accession number: NM_001069892.1) in fine with rice varieties Japan is for template, and design primer, the DNA fragmentation of the OsbHLH120 in each floods and droughts rice varieties that increases, primer is designed by software Primer5.0, and sequence is as follows:
OsbHLH120(3-2)F：5′-CGTGCACCAGCTGGACTT-3′，
OsbHLH120(3-2)R：5′-AGGTACGGGAAACGTACTGC-3′；
Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd., uses sterilizing ddH
2o is diluted to 10 μm of ol/L, and-20 DEG C save backup.
PCR reaction system is 20.0 μ L, comprises 10 × Taq Buffer2.0 μ L, DNA1.5 μ L, 10 μm of ol/L primer (OsbHLH120 (3-2) F/R) each 1.0 μ L, 10mmol/L dNTP0.5 μ L and 2.5U/uL Taq DNA polymerase 0.2 μ L, ddH2O13.8 μ L.
Pcr amplification program is: denaturation 94 DEG C of 4min; Sex change 94 DEG C of 30s, anneal 55 DEG C of 30s, extends 72 DEG C of 30s, 35 circulations; Extend 72 DEG C of 10min, 4 DEG C of preservations.Detect amplified production with 1% agarose gel electrophoresis, checked order by Hua Da Gene science Services Co., Ltd after DNA Purification Kit.
Sequence alignment analysis (Fig. 1) is carried out by BioEdit software, find upland rice IRAT109, IAPAR9, milli lattice labor, Dandong dryland rice, platform east dryland rice, upland rice 277, upland rice 297, No. 2, Zheng's drought, the Qin likes that 9 upland rice are all lacking 15 bases apart from genetic transcription initiator codon downstream 628-642bp, and paddy rice Japan is fine, richer, town rice 88, 9311, new rice 18, Zheng rice 18, Xinfeng No.2, glad No. 1 of side, glad No. 4 of side, Zheng rice No. 4, osmanthus is towards No. 2, special blue or green, but there is not the disappearance of this base fragment in public residence 73, No. 6, Zheng's drought, although No. 9, Zheng's drought is also upland rice, but it is identical with rice sequences, there is not above-mentioned deletion fragment.Its reason is these two upland rice varieties is all that paddy rice and upland rice selection cross form, and in the hybridization assembly chosen process of paddy rice and upland rice, this gene fragment is replaced by the genotype of paddy rice.So there is the base sequence identical with paddy rice at this gene locus two upland rice varieties.Can learn thus, the OsbHLH120 containing this deletion sequence, specific to typical upland rice, has direct relation with the drought resistance of cultivated rice.
Embodiment 2 anti-drought gene functional label InDel-bHLH120 and the gene type assay to floods and droughts rice resource thereof
1, primer is designed
Choose the sequence of deleted segment both sides in upland rice OsbHLH120 gene, according to the base sequence information within the scope of the 300-500bp of its deletion sites both sides, utilize software Primer5.0 to design primer, sequence is
InDel-bHLH120F：5′-GGCCATCCACTACGTCAAGT-3′（SEQ ID NO.2），
InDel-bHLH120R：5′-GGGAAACGATTGTCTCATCACC-3′（SEQ ID NO.3）；
Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd., and be diluted to 10 μm of ol/L with sterilizing ddH2O ,-20 DEG C save backup.
2, experiment material and Drought Resistance Analysis thereof
Paddy rice: fine, richer, the new rice 18 of Japan, Zheng rice 18, Xinfeng No.2, glad No. 1 of side, glad No. 4 of side, town rice 88, Zheng rice No. 4,9311, osmanthus towards No. 2, special blue or green, should 1577, II-32B, CBB23, Chinese Fragrant Rice, Peiai 64S, short pin Nan Te, two or nine No. 1, south, Zhenshan 97B, the precious B of bamboo.
Upland rice: IRAT109, IAPAR9, milli lattice labor, Dandong dryland rice, platform east dryland rice, upland rice 277, upland rice 297, No. 2, Zheng's drought, Qin Ai, red upland rice, white hair rice, the purple skin in Funing, Tianjin upland rice.
The Identification of Drought experimental cultivation of material is in Agricultural University Of He'nan's scientific and educational park drought resisting booth, if Osmotic treatment and normal water divisional processing two kinds of planting types.Normal water divisional processing, at water conservation 10cm in the time of infertility, is undertaken by paddy field Routine Management pattern.The rainfall of Osmotic treatment Artificial Control.Material in June 5 dry land live, October 9 gather in the crops.Simple grain program request, test adopts randomized block design, repeats, often repeats plantation 3 row for 2 times, the long 1.5m of row, line-spacing 0.25m, spacing in the rows 0.08m.Water, dry land bulk application pure N150kg/ha, P before sowing
2o
5150kg/ha and K
2o150kg/ha.Tillering phase, dry land imposed urea 300kg/ha.Drought canopy controls after rainfall, and dry land plant carries out Severe drought to early stage of tillering and coerces emerging, and Post flowering carries out moderate water stress.Upland rice time of infertility irrigates totally 5 times, and about 350mm, is respectively the 80mm that after planting pours water immediately, and pour water middle of tillering 60mm, and jointing-booting stage is poured water 70mm, and pour water flowering period 80mm, irrigation at seed filling stage 60mm.Time ripe, the 2nd interline 5 strain got continuously by every material, investigates single plant yield, obtains the mean value of each proterties, as the measured value of single plant yield.Using the output relative value of Osmotic treatment and normal moisture as the overall target of evaluating material drought resistance, by drought resistance by weak, material being divided drought resisting I grade, drought resisting II grade and quick non-irrigated 3 ranks by force.13 upland rice material drought resistances are all comparatively strong, and all show as drought resisting I grade or drought resisting II grade, and the drought resistance of 21 rice varieties is all poor, drought resisting rank is all quick drought, as table 1.
The drought resisting rank of table 1 34 floods and droughts rice varieties
3, the genotype detection of InDel-bHLH120 mark to water, upland rice variety
DNA extraction, PCR amplification system and program are with embodiment 1.Amplified production 6% polyacrylamide denaturing gel electrophoresis is separated.After electrophoresis terminates, with stationary liquid (10% ethanol, 0.5% glacial acetic acid) more than immobilized gel 30min; Distilled water rinsing 5min; With 2g/L AgNO
3solution infiltration 30min; The rapid rinsing 10s of distilled water; Develop the color 30min to use nitrite ion (15g/L NaOH, 0.4% formaldehyde solution) again.Take out after last distilled water rinsing 3min and dry, analyze banding pattern.
Utilize InDel-bHLH120 to the marker gene type analysis of floods and droughts rice material, amplify 2 kinds of banding patterns, the paddy rice of quick drought is a kind of banding pattern, and the upland rice of strong drought resistance is another kind of banding pattern (containing disappearance).Japan is fine, 9311, special blue or green wait 12 rice varieties (see embodiment 1) to learn through order-checking not lack 15 bases in coding region, suitable 1577, II-32B, CBB23, Chinese Fragrant Rice, Peiai 64S, short pin Nan Te, two or nine No. 1, south, Zhenshan 97B, the genotype of precious B10 the paddy rice of bamboo is all identical with above-mentioned Rice Genotypes, and 4 red upland rice of upland rice variety, white hair rice, the purple skin in Funing, the OsbHLH120 genotype of Tianjin upland rice then with IRAT109, IAPAR9, 9 kinds that checked order (see embodiment 1) identical (Fig. 2) such as milli lattice labor, this illustrate OsbHLH120 deletion sequence by upland rice peculiar.InDel-bHLH120 can detect the existence of this specific deletion sequence in upland rice well, also can well distinguish the drought resistance of material, is conducive to the drought resisting material that autotelic selection in Drought-resistant Breeding contains the gene locus of deletion fragment.
Embodiment 3 anti-drought gene functional label InDel-bHLH120 is to the gene type assay of F2 for colony
Experimental population is that the introgressive line IL392(of upland rice IRAT109 is richer for background parent with rice varieties, and upland rice IRAT109 is the BC that donor parents obtains
5f
5introgressive line material, OsbHLH120 is included in the introgressed segment of upland rice IRAT109) with the richer F2 of paddy rice for colony.
Utilize InDel-bHLH120 to the marker gene type analysis of F2 colony, concrete implementation step, with embodiment 2, amplifies 3 kinds of banding patterns (Fig. 3), two kinds of parent's banding patterns that isozygoty (with richer and IRAT109), a kind of heterozygous.Isozygoty to part OsbHLH120 genotype the Identification of Drought of offspring individuals, and also display is better than the richer genotypic offspring individuals of paddy rice containing OsbHLH120 genotype offspring individuals drought resistance of isozygotying.Show that this functional label can well be used for the screening of gene OsbHLH120 upland rice idiotype and drought resisting material.
Claims (1)
1., according to an anti-drought gene functional label InDel-bHLH120 for upland rice anti-drought gene distinguished sequence design, it is characterized in that, primer sequence is as follows:
Forward primer sequence: 5 '-GGCCATCCACTACGTCAAGT-3 '
Reverse primer sequences: 5 '-GGGAAACGATTGTCTCATCACC-3 ';
Described upland rice anti-drought gene OsbHLH120 belongs to bHLH class transcription factor, between paddy rice the 9th karyomit(e) RM24271 and RM566; This upland rice anti-drought gene OsbHLH120 has lacked 15 bases compared with the normal sequence of paddy rice in coding region, described disappearance base is positioned at genetic transcription initiator codon downstream 628-642bp.
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