CN109112140A

CN109112140A - Application of the OsSN28 gene in control rice high temperature resistant

Info

Publication number: CN109112140A
Application number: CN201810248055.2A
Authority: CN
Inventors: 熊立仲; 常煜
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-03-24
Filing date: 2018-03-24
Publication date: 2019-01-01

Abstract

The invention belongs to field of plant genetic project technology.More particularly to application of the OsSN28 gene in control rice high temperature resistant.The present invention uses candidate gene screening method, pass through reverse genetics means, clone obtains a control rice high temperature resistant response gene OsSN28, sequence shown in 1-1862 bit base of the nucleotide sequence of the gene as described in SEQ NO:1, the protein sequence of gene coding is as shown in SEQ NO:2.Heading stage high temperature stress phenotypic evaluation the result shows that, overexpression OsSN28 gene cause transgenic paddy rice to high temperature resistance promoted, and the deletion mutation of OsSN28 gene then cause rice high-temperature resistance significantly reduce.Present invention demonstrates the biological function of the gene and its approaches and methods of application.

Description

Application of the OsSN28 gene in control rice high temperature resistant

Technical field

The present invention relates to technical field of rice gene engineering.One is obtained more particularly to separation, clone and by functional verification Kind can be improved application of the rice Os SN28 gene of high temperature tolerance ability in rice high temperature resistant genetic improvement.The present invention uses The method of candidate gene screening is cloned into control rice high temperature resistant response gene OsSN28, high temperature by reverse genetics means Coerce phenotypic evaluation the result shows that, overexpression OsSN28 gene can be improved pollen fertility of the rice under thermal extremes environment with Setting percentage, and the missing of OsSN28 gene then will lead to rice flower organ to high temperature hypersensitization, it was confirmed that the function of the gene and Application approach.

Background technique

Abiotic stress (such as arid, damage or crop failure caused by waterlogging, high temperature, low temperature, saline and alkaline) seriously restricts growth and development and the work of plant The production of object increases income, and rice is one of main cereal crops in the world, adaptation and resistance of the Study On Rice to abiotic stress Mechanism, it will help carry out degeneration-resistant genetic improvement to it, expand planting range, improve yield.In order to adapt to various environmental changes, It survives in adverse circumstance, plant forms the Stress response regulatory mechanism of a series of complex in long-term evolutionary process, leads to The perception to environment-stress signal, transmitting and responsing reaction are crossed, a system occurs in molecular level, cellular level and plant level Column variation, generates corresponding tolerance mechanism or adaptability, to reduce stress bring injury to the maximum extent, remains basic Physiological activity (Xiong etc., Cell signaling during cold, drought and salt stress.Plant Cell.14 (suppl), S165-S183,2002).Can functional gene correctly express during making a response to environment, by Fine-tuning for regulatory factor is arrived.Transcription factor is as a kind of controlling gene, when organism experiences environment stress, can regulate and control A series of expression of downstream genes reaches to enhance plant to the tolerance of adverse circumstance and resists unsuitable environmental condition stress Effect.Most types of transcription factor has been involved in the abiotic stress responsing reaction of plant, including AP2/EREBP, BZip, HD-ZIP, MYB, MYC, NAC and Zinc finger class transcription factor (Yamaguchi-Shinozaki K, Shinozaki K.Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses.Annu Rev Plant Biol,2006,57:781- 803).By genetic engineering, part adverse circumstance response transcription factor has been successfully applied to paddy rice anti contravariance genetic breeding.Wherein, NAC (NAM, ATAF and CUC) is distinctive one kind transcription factor, part of member, such as SNAC1 and SNAC3 in a plant Deng transgenic plant can be significantly improved to resistance (Hu etc. of the abiotic stresses such as high temperature, arid after overexpression in rice .Overexpressing a NAM,ATAF,and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice.Proc Natl Acad Sci USA,2006,103:12987- The .A stress-responsive NAC transcription factor SNAC3confers such as 12992. and Fang heat and drought tolerance through modulation of reactive oxygen species in rice.J Exp Bot,2015,21:6803-6817)。

Rice is important cereal crops and model plant, and the development and flowering of floral organ and pollinating process are to environment Temperature is extremely sensitive, and in global warming phenomenon increasingly significant today, the rice for cultivating high-temperature resistance enhancing has weight The meaning wanted.The rice sensitive to hot environment at heading stage is screened by reverse genetics means in our early-stage study Mutant, successful clone identify a NAC transcription factor base to rice ear sprouting period high temperature response with significant regulating and controlling effect Because of OsSN28.In view of the transgenic rice plant of overexpression OsSN28, setting percentage and yield are relatively negative under high temperature environment Control significantly improves, and carries out further functional study to OsSN28, identifies what it was played in terms of improving paddy rice anti contravariance Function will have very important significance for cultivating degeneration-resistant new rice variety.

Summary of the invention

The purpose of the present invention is the high temperature resistances using rice NAC transcription factor gene OsSN28 rice.Present invention separation With apply a kind of DNA fragmentation comprising OsSN28 gene, when lacking the segment, Rice Resistance high temperature stress reduced capability, and utilizing After constitutive promoter drives the segment excess to transcribe OsSN28 gene, rice enhances high temperature stress tolerance.

The nucleotide sequence of the OsSN28 gene is as shown in SEQ ID NO:1, sequence length 1862bp, the gene The sequence of the protein of coding encodes 252 amino acid residues as shown in SEQ ID NO:2.

Round pcr can be used, amplification obtains this hair from oryza sativa genomic dna and the cDNA as obtained by mRNA reverse transcription Bright OsSN28 gene can pass through conversion water on the genes overexpression carrier such as this sequence construct to pCAMBIA1031U Rice plants, can be by improving the expression quantity of the gene, to obtain the transgenic rice plant enhanced high temperature tolerance ability.

The expression vector for carrying OsSN28 gene of the present invention can be by using Ti-plasmids, plant viral vector, direct DNA Conversion, microinjection, the standard biologics technical method such as electroporation import plant cell (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press,New York,pp.411-463；Geiserson and Corey,1998,Plant Molecular Biology(2nd Edition)。

Present invention will be further explained below with reference to the attached drawings and examples.

Detailed description of the invention

Sequence table SEQ ID NO:1 is the complete sequence (1-1862bp) for the OsSN28 gene that the present invention separates, wherein 66- 239 base positions and 972-1556 base position are exon (exon) sequences.

Sequence table SEQ ID NO:2 be code area (CDS) sequence (1-759bp) of OsSN28 gene that the present invention separates and Its corresponding amino acid sequence (1-759bp), encodes 252 amino acid residues.

Sequence table SEQ ID NO:3 is the sequence of the protein of OsSN28 gene coding of the invention, encodes 252 amino The protein sequence of sour residue.

Fig. 1: using Clustal Ω software (open use software) by the protein sequence of OsSN28 predictive genes and rice and The result that stress response relevant NAC transcription factor protein sequence in part is compared in arabidopsis.Description of symbols:

SN28:XP_015625739.1Sequence source:Oryza sativa rice；

SNAC1:XP_015630558.1, Sequence source:Oryza sativa rice；

SNAC2:XP_015620920.1, Sequence source:Oryza sativa rice；

RD26:NP_001078452, Sequence source:Arabidopsis thaliana arabidopsis；

ATAF1:NP_171677, Sequence source:Arabidopsis thaliana arabidopsis；

TaNAC69:AAU08785, Sequence source:Triticum aestivum wheat.

Fig. 2: using Real-time PCR detection OsSN28 in a variety of adverse circumstances (arid, high temperature, low temperature, abscisic acid, that is, ABA Deng) stress after expression quantity variation, in addition to applied at elevated temperature fringe portion, other samples are four leaf stage blade.Description of symbols: OsSN28 gene relative expression quantity with (0 time) before handling for referring to (being set as 1), result is to repeat three times, error line generation Table standard deviation (SD).

The tissue expression spectrum analysis of Fig. 3: OsSN28 gene.Description of symbols: Calli is the callus group of squamous subculture It knits；Embryo is the embryo organ of the seed of current year harvest；Root and Shoot is respectively the four leaf stage children grown in root media The root and ground green portion of seedling；Nternode is tillering stage stalk；Leaf L2 and Flag leaf are respectively falling for heading stage Two leaves and sword-like leave, Collar are the pulvinus of sword-like leave；Flag leaf sheath is the leaf sheath of sword-like leave；Three Panicle samples point Not Wei different length (developmental stage) tassel；Huff, Pistil, Anther are respectively the glume of the grain husk flower after earing；Gynoecium And stamen.Testing result is based on repeating three times, and error line indicates standard deviation (SD).

Fig. 4: OsSN28 gene overexpresses in material (SN28-OE) and ossn28T-DNA insertion mutation material (sn28m) The detection of OsSN28 gene transcription level.Description of symbols: the A figure in Fig. 4 is real-time quantitative PCR (Real-time PCR) inspection The transgene negative material surveying two independent SN28-OE familys (OE-25 and OE-27) and being isolated by its T0 generation (OECK) in OsSN28 gene transcription level as a result, rice ubiquitin1 gene as reference gene, Y value table Show multiple of the expression quantity of OsSN28 in SN28-OE relative to the expression quantity of OsSN28 in OECK, be as a result based on repeating three times, Error line represents standard deviation (SD).B figure in Fig. 4 is to detect sn28m Mutants homozygous using reverse transcription PCR (RT-PCR) And in the negative control material (mCK) being isolated by its heterozygous individual OsSN28 gene transcription level as a result, rice Actin1 gene is as reference gene.

Fig. 5: sn28 mutant plants floral organ reduces high temperature stress tolerance.Description of symbols: the A figure in Fig. 5 Show that the blooming stage anther form under normal circumstances with sn28m Mutants homozygous after high-temperature process and negative control mCK (pays attention to Sn28m anther bending deformation after high-temperature process, and mCK is normal column and energy normal crack loose powder).B figure in Fig. 5 is Fertility testing result is contaminated with the pollen iodine of sn28m after high-temperature process and mCK under normal circumstances, it is seen that after high-temperature process in sn28m The ratio of fertile pollen is not significantly higher than mCK.C figure in Fig. 5 is sn28m and mCK pollinates after normal condition and high-temperature process Effect detection, it is seen that after high-temperature process, the pollen of sprouting on mCK column cap is significantly more than sn28m.D figure in Fig. 5 shows pole End high-temperature process boot stage material fringe extraction and complete it is solid after phenotype, sn28m tassel completely infertility and lower part grain husk flower (place Opposite upper parts developmental stage is more early when reason) whitening dead situation occur, although and mCK setting percentage declines to a great extent, remain to tie Real and grain husk spends not no dead situation substantially.

Fig. 6: SN28-OE plant pair high temperature stress tolerance improves.Description of symbols: the A figure in Fig. 6 and the B in Fig. 6 Figure is respectively that the pollen iodine dye fertility detection of SN28-OE family OE-25 and negative control OECK under high-temperature process is pollinated with column cap Effect observation is as a result, the fertile pollen of SN28-OE material is more under high temperature.In Fig. 6 C figure show high-temperature process after OE-25, The solid situation of OE-27 and OECK, it is seen that the setting percentage of SN28-OE material is significantly higher than negative control.D figure in Fig. 6 is the summer The explanation situation of OE-25, OE-27 and OECK after season natural high-temperature stress.E figure in Fig. 6 be in Augusts, 2016 (high temperature) and In August, 2014 (normal) temperature measurement result, abscissa is the date since August 1st, No. * heading for being labeled as material Date.F figure in Fig. 6 is that (i.e. bear fruit grains are big with effective fringe rate ratio for the setting percentages of associated materials after natural high-temperature stress in paddy field In ratio of 5 spike numbers in total spike number) statistical result, box traction substation error line indicates extreme value range, and nowel line represents 1/4 Quantile, middle line represent median, and top box line represents 3/4 quantile, and each measured value is marked by dot, OE-25, OE-27, OECK is respectively red, blue, black.R1-R4 is that the independent of 4 cells repeats, each 6~10 plants of Materials Measurement of each repetition.** Represent extremely significant difference (student t-test, p < 0.01) of each family of SN28-OE relative to OECK in each repetition.

Specific embodiment

Following embodiment defines the present invention, and describing the present invention in separation clone includes that OsSN28 gene is completely compiled The DNA fragmentation of code section, and the method for verifying OsSN28 gene function.According to description below and these embodiments, ability Field technique personnel can determine essential characteristic of the invention, and without departing from the spirit and scope of the invention, can be with Various changes and modifications are made to the present invention, so that it is applicable in different purposes and condition.

Embodiment 1: separation clone's OsSN28 gene

Applicant is using TRIZOL reagent (the being purchased from Invitrogen company) flower from the rice varieties of 43 DEG C of high-temperature process Fringe is extracted in 11 (also known as ZH11, original material come from Institute of Crop Science, Chinese Academy of Agricultural Science, are routine experiment material) Portion's total serum IgE (extracting method is according to above-mentioned TRIZOL reagent specification), it is (public purchased from Invitrogen using reverse transcriptase SSIII Take charge of) its reverse transcription is synthesized into cDNA, reaction condition: 65 DEG C of 5min, 42 DEG C of 120min, 70 DEG C of 10min.Using this cDNA as template, With primer SN28-F:5 '-ATGGCGATGACACCGCAGCTAGC-3 ' and SN28-R:5 '- CTAGCCACCATGGTTTCTTTGCA-3 ' amplifies the full-length cDNA of OsSN28 gene, and (759bp is shown in sequence table SEQ ID NO:2).PCR reaction condition: 95 DEG C of initial denaturation 3min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 35 circulations；72℃ Extend 10min.The PCR product that amplification obtains is connected into pGEM-T Easy carrier (purchased from Promega company), positive gram of screening It is grand and confirmation is sequenced, OsSN28 full length gene cDNA is obtained, this clone designation is pGEM-OsSN28 by applicant.

Embodiment 2: the expression of the detection endogenous OsSN28 gene of rice

Applicant selects in rice varieties and spends material of 11 (ZH11) as expression pattern analysis.Specific step is as follows:

(1) expression detection of the OsSN28 gene under different environment stresses.After presprouting of seeds, in normal growth item It is cultivated 18-20 days under part, until carrying out various adverse circumstances and Plant hormone treatment when four leaf stage.Osmotic treatment be by seedling directly from It takes out and is exposed in air in water planting liquid (common prescription), respectively 0.5 hour, 1 hour, 3 hours, 5.5 before stress, after stress Hour sampling；High-salt stress is to move into seedling in the water planting liquid containing 200mmol/L NaCl by water planting liquid, is being coerced respectively Before, stress after 0.5 hour, 1 hour, 3 hours, 5.5 hours sample；Low temperature stress is rice seedling to be put into 4 DEG C of artificial climates Room, before stress, stress after 0.5 hour, 1 hour, 3 hours, 5.5 hours sample.HORMONE TREATMENT is with 100 μM of abscisic acids (ABA) uniformly behind sprinkling rice plant surface, respectively before stress, 0.5 hour, 1 hour, 3 hours, 5.5 hours after stress Sampling.High temperature stress is to move it into about 45 DEG C of temperature of growth case when the fringe of material will be extracted out and placing 0.5 hour Entire fringe portion is sampled afterwards.

(2) expression detection of the OsSN28 gene in rice different growing stage, different tissues.In spend 11 seeds After vernalization, growth is cultivated under normal growing conditions, after germination 10 days, moves into and cultivates native potting, it is to be grown to four leaf stage when, point Ground chlorenchyma and root are not taken；In tillering stage, stalk is taken；In boot stage, the young fringe of different length is taken respectively；After heading, point Glume, stamen, gynoecium, blade, leaf sheath and pulvinus are not taken；In addition also the callus in squamous subculture is sampled.It extracts The method of total serum IgE is that (extracting method is according to the use of TRIZOL reagent using TRIZOL reagent (purchased from Invitrogen company) Specification), using reverse transcriptase SSII (be purchased from Invitrogen company) by its reverse transcription synthesize cDNA (method according to The operation of Invitrogen company reverse transcriptase reagent operation instructions), reaction condition: 65 DEG C of 5min, 42 DEG C of 120min, 70 DEG C 10min.Using the cDNA of above-mentioned reverse transcription synthesis as template, with primer OsSN28-qF (5 '-GAACGACACCGTCGGGTTT- 3 ') and OsSN28-qR:(5 '-CCGGCGACGACGAAGAT-3 ') to OsSN28 gene carry out specific PCR amplification.It uses simultaneously Primer (uF:AACCAGCTGAGGCCCAAGA-3 ' and uR:5 '-ACGATTGATTTAACCAGTCCATGA) is to rice Ubiquitin1 gene (LOC_Os03g13170) does specific amplified, to carry out quantitative analysis as internal reference.Reaction condition are as follows: 95℃5min；95 DEG C of 10sec, 60 DEG C of 5sec, 72 DEG C of 34sec, 40 circulations.Fluorescence detection real-time quantitative is carried out in reaction process It analyzes (according to a conventional method).

The result shows that induction of the expression of OsSN28 gene by with high salt, low temperature, drought stress, in fringe portion also by height The induction of temperature stress, while (3~5.5 hours) also can up-regulated expression (Fig. 2) after ABA handles the long period.In addition, normal In the case of, OsSN28 gene is mainly less than 0.3cm in seedling root and two leaves, sword-like leave blade and the pulvinus at heading stage, length Seedling in have higher expression, and expression quantity is lower (Fig. 3) in stamen and the aerial part of seedling.

The separation of embodiment 3:ossn28 mutant is identified

The picking OsSN28 gene position from South Korea's Postech Rice mutant pool (http://www.postech.ac.kr) The corresponding T-DNA insertion mutation body 2D-30113 (sn28m, Dongjin background) of point.The wherein institute in above-mentioned website mutant library The flanking sequence (sequence length is 850bp) of the sn28m mutant 2D-30113 of login, sequence is as follows:

AGTGTTAGCGTTCAACACACCGCAGCTAGNATTTTCTCGCATGCNTCCAGGGTTTCGGTTCCAGCCG ACGNACGAGCAGCTTGTCGTCGACTACTTGCAGAGGCGTACCGNTGCGCAGCNCNTGCATNNCTCC NGACNTCACNGATNTCNACGTTNACAACGTCGAACCCGNGNNAGCNTNCCAGNGGCAANATANATG CNCGCTTNAATTGGCATGNGCCTCCNNCCGTACGNGGNCCCCNACNGGNNGNAGAANTCATTCNAA NCNGTNAACNTNATTTNCCCGAATGTNTCGANGNNCNATTNNTATNATGNAAGNAAAANCGTGAAT GGNCGTTNCCNTCAGAAAAAAAAANNGGGNNNAGGGGAANAGCCGCCNTTTNGCGGGGGTTAANC NCTTTTTCCNCCTTTTTGGGCTTTTNCGANNAAAAANANGCGATCCCCGCGCAATANNGGANCCTTN GATCATTTTNCNTGANAAAAANAAAAACNGANAAATGAATTCCAATTCGNTGNNTAAGCAAATCAC TAATTNACCTAAANAATCCGGANCTTGGTGGGATANTNCTNCCGGTAGAGAGCNCCNGNGCGTCGA TTTGCATCCAGGNNNGGCNGAANNAGNCTTAGGACNGGGAGGCTTAAGCCTTGGCNTGTGCNCCNA ACNGGGNNAAANCCCCNTAAAANCCCNTTAAATTTGGGAAAAGAATGAATACTATTAAAACCCNTA CNTTGCCTCTCTAGAGCCGCCTGCTACNCGAGTCTNTGTAGGCGAAAAATAAAATTTACCTGATGCG ACCGGAAAGNAAATTTTGGTGGNCTGNAANNGACTAGCNAATTGCNGNTGGCNNGG

According to T-DNA insertion point, design primer SN28-mF:5 '-CATAAACGGATGAGTGTGCG-3 ' and SN28- MR:5 '-GCTGGCATCGCTCATATTTC-3 ', and cooperate T-DNA special primer 2772-L2:5 '- CTAGCTAGAGTCGAGAAT TCAGT-3 ' identifies sn28m Mutants homozygous.Simultaneously reverse transcription, PCR the results show that In sn28m Mutants homozygous, the transcription of OsSN28 gene can not be detected (the B figure in Fig. 4), that is, show sn28m mutant Middle OsSN28 gene is suppressed significantly.Reverse transcription PCR uses primer SN28-RTF:5 '-ACTACTTGCAGAGGCGTACC-3 ' And SN28-RTR:5 '-GCTATCATCAATCTC CTCATC-3 ' expands OsSN28 gene, amplification condition: 95 DEG C of initial denaturations 3min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations；72 DEG C of extension 7min, while using primer Actin1- F:5 '-CTCAACCCCAAGGCTAACAG-3 ' and Actin1-R:5 '-ACCTCAGGGCATCGGAAC-3 ' amplifying rice Actin1 gene is as internal reference, reaction condition: 95 DEG C of initial denaturation 3min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 30sec, 26 A circulation；72 DEG C of extension 7min.

By the sn28m Mutants homozygous for having identified genotype and the negative control material mCK separated by its heterozygous individual Live streaming is into cask after vernalization.It is to be grown to four leaf stage when transplant into big drum, every group of material plants eight barrels, every barrel one Strain.The soil of test is southern china rice cropping soil, and every drum soil loading amount is identical, and the same time uses after transplanting The tap water of amount pours, when plant strain growth to fringe development later stage, fringe will be extracted out, by four plants of same time shifts in every group of material Enter 45 DEG C of temperature or so, relative humidity 90% and look after in sufficient growth case, carries out 1 hour high-temperature process, remaining material is set In normal habitat.Place the material in normal environment continued growths for after treatment, and all materials are observed after fringe is fully drawn out Expect the form and clever flower development situation of fringe portion, while taking 5 grain husk flowers on fringe top, detects its pollen using conventional iodine dye method and educate Property, and to 3 hours after its flowering, its column cap pollination situation is observed using aniline blue decoration method (conventional method).The results show that Under normal circumstances, i.e., the stamen form of the sn28m mutant bloomed and negative control mCK is not significantly different, but in high temperature When reblooming after processing, the stamen of mCK is normal column and the loose powder that can crack, and sn28m mutant obtains stamen as bending It fills and cracking degree is lower (the A figure in Fig. 5).Meanwhile sn28m mutant iodine dye pollen fertility and column cap flower after high-temperature process Powder germination rate is substantially less than control mCK (the B figure in Fig. 5 and the C figure in Fig. 5).In addition, sn28m mutant fringe leans on lower part Grain husk flower (in stress in developmental stage relatively early) occur the withered situation that bleaches after high temperature stress, and mCK base This occurs without this situation, and sn28m mutant setting percentage of whole fringe after high temperature stress is also significantly lower than control material mCK (Fig. 5 In D figure).

The building and conversion of embodiment 4:OsSN28 gene overexpression carrier

Overexpression carrier construction method is as follows: first by positive colony pGEM-OsSN28 plasmid obtained in embodiment 1 (by obtaining in embodiment 1) uses primer SN28-OEF (5 '-GGTACC ATGGCGATGACACCGCAGCTAGC-3 ') and SN28- OER (5 '-GGATCC CTAGCCACCATGGTTTCTTTGCA-3 '), amplifies the DNA fragmentation comprising OsSN28 overall length, reaction Condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 35 circulations；72 DEG C of extension 10min.It should Segment is connected to T4DNA ligase again after being digested with restriction enzyme KpnI and BamHI equally to disappear through KpnI and BamHI On the pCAMBIA1301U carrier of change, it can be used for converting after carrying out sequencing confirmation to carrier.

Be conducted into rice varieties ZH11 by the rice transformation system of mediated by agriculture bacillus, by preculture, It infects, co-culture, screen the callus with hygromycin resistance, break up, take root, practicing seedling, transplanting, obtaining transgenic plant.Agriculture bar Method (Hiei etc., Efficient that rice (japonica rice subspecies) genetic conversion system that bacterium mediates is reported in Hiei et al. Transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence Analysis of the boundaries of the T-DNA, Plant J, 6:271-282,1994) on the basis of improve into Row.20 plants of independent transgenic rice plants are obtained in conversion carrier altogether.

Specific steps: (1) callus induction: by mature rice (in spend 11) seed decladding, then successively with 70% Alcohol treatment 1 minute, 0.15% mercury chloride (HgCl₂) the surface of the seed disinfection 15 minutes；With sterilizing washing seed 4-5 times；It will Seed puts (ingredient is seen below) on the induction medium；Culture medium after inoculation is placed at dark and is cultivated 4 weeks, temperature 25 ± 1 ℃.(2) callus subculture: selecting the embryo callus subculture of glassy yellow, consolidation and relatively dry, is put on subculture medium (ingredient is seen below) The lower culture 2 weeks, 25 ± 1 DEG C of temperature of dark.(3) preculture: the embryo callus subculture of consolidation and relatively dry is selected, pre-culture medium is put in Upper dark lower culture 2 weeks, 25 ± 1 DEG C of temperature.(4) Agrobacterium is cultivated: training in advance on the LA culture medium with corresponding resistance selection Support Agrobacterium EHA105 (deriving from the Australia laboratory CAMBIA, commercial strains) two days, 28 DEG C of cultivation temperature；It will be described It is inner that Agrobacterium is transferred to suspension medium (ingredient is seen below), cultivates 2-3 hours on 28 DEG C of shaking tables.(5) Agrobacterium is infected: will be trained in advance Feeding callus is transferred in the bottle to have sterilized；The suspension of Agrobacterium is adjusted to OD₆₀₀0.8-1.0；By callus in agriculture It is impregnated 30 minutes in bacillus suspension；It is blotted in transfer callus to the filter paper to have sterilized；It is then placed within to co-culture and cultivates 3 on base It, 19-20 DEG C of temperature.(6) callus washing and selection culture: sterilizing water washing callus to invisible Agrobacterium；It is immersed in and contains 30 minutes in the aqua sterilisa of 400ppm carbenicillin (CN)；It is blotted in transfer callus to the filter paper to have sterilized；Shift callus extremely Select 2-3 times on Selective agar medium (ingredient is seen below), 2 weeks every time (screening carbenicillin concentration is 400ppm for the first time, the It is carbenicillin concentration 250ppm, hygromycin concentration 250ppm after secondary).(7) break up: kanamycin-resistant callus tissue is transferred to pre- point Change and is cultivated 5-7 weeks at dark on culture medium (ingredient is seen below)；In the callus to differential medium of the pre- differentiation culture of transfer, illumination It is cultivated under (by rice tissue culture conventional manual illumination condition), 26 DEG C of temperature.(8) it takes root: cutting the root generated when differentiation；So After transfer them in root media and cultivated 2-3 weeks under illumination, 26 DEG C of temperature.(9) it transplants: washing off the residual culture on root Seedling with good root system is transferred to greenhouse by base, while wet in several days initial holding moisture.

The preparation of agents useful for same and culture medium in conversion: (1) reagent and solution abbreviation: in the present invention used in culture medium The abbreviation of plant hormone is expressed as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine)；CN (Carbenicillin, carbenicillin)；KT (Kinetin, kinetin)；NAA (Napthalene acetic acid, naphthalene second Acid)；IAA (Indole-3-acetic acid, heteroauxin)；2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid)；AS (Acetosringone, acetosyringone)；CH(Casein Enzymatic Hydrolysate, caseinhydrolysate)；HN (Hygromycin B, hygromycin)；DMSO (Dimethyl Sulfoxide, diformazan Base sulfoxide)；N6max (a large amount of ingredient solutions of N6)；N6mix (N6 micro constitutent solution)；MSmax (a large amount of ingredient solutions of MS)； MSmix (MS micro constitutent solution).

(2) main solution formula:

1) preparation of N6 culture medium a great number of elements mother liquor [10 times of concentrates (10X)]:

It dissolves one by one, is then settled to 1000ml at room temperature.

2) preparation of N6 culture medium microelement mother liquor [100 times of concentrates (100X)]

It dissolves at room temperature and is settled to 1000ml.

3) molysite (Fe₂EDTA the preparation of liquid (100X)) is stored

Prepare 800ml distilled water and is heated to 70 DEG C, addition b diammonium disodium edta (Na₂EDTA·2H₂O) 3.73 grams, It is kept for 2 hours in 70 DEG C of water-baths after completely dissolution, is settled to 1000ml, 4 DEG C save backup.

4) vitamins stock liquid (100X) is prepared

Water is added to be settled to 1000ml, 4 DEG C save backup.

5) preparation of MS culture medium a great number of elements mother liquor (10X)

It dissolves at room temperature and is settled to 1000ml.

6) preparation of MS culture medium microelement mother liquor (100X)

It dissolves at room temperature and is settled to 1000ml.

7) 2,4-D stores liquid, and 6-BA stores liquid, and methyl α-naphthyl acetate (NAA) stores liquid, and heteroauxin (IAA) stores liquid: 1 is mg/ml。

8) glucose storage liquid: 0.5g/ml.

9) AS stores the preparation of liquid: weighing AS 0.392g, DMSO 10ml.

(3) it is used for the culture medium prescription of rice transformation

1) induced medium

Add distilled water to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and is settled to 1000ml, be dispensed into 50ml Triangular flask (25 ml/ bottles), sealing sterilizing.

2) subculture medium

3) pre-culture medium

Add distilled water to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, and sealing sterilizes.Use preceding heating for dissolving culture medium And 5ml glucose storage liquid and 250 μ l AS storage liquid is added, (25ml/ ware) is poured into culture dish in packing.

4) base is co-cultured

Add distilled water to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, and sealing sterilizes.Use preceding heating for dissolving culture medium And 5ml glucose storage liquid and 250 μ l AS storage liquid is added, (the every ware of 25ml/) is poured into culture dish in packing.

5) suspension medium

Add distilled water to 100ml, adjusts pH value to 5.4, be dispensed into the triangular flask of two 100ml, sealing sterilizes.It uses Preceding addition 1ml glucose storage liquid and 100 μ l AS store liquid.

6) Selective agar medium

Add distilled water to 250ml, adjusts pH value to 6.0, sealing sterilizes.Using preceding dissolution culture medium, 250 μ l HN are added With 400ppm CN, (25ml/ ware) is poured into culture dish in packing.

7) pre- differential medium

Add distilled water to 250ml, 1N potassium hydroxide adjusts pH value to 5.9, and sealing sterilizes.Using preceding dissolution culture medium, add Enter 250 μ l HN and 200ppm CN, (25ml/ ware) is poured into culture dish in packing.

8) differential medium

Add distilled water to 900ml, 1N potassium hydroxide adjusts pH value to 6.0.1000ml is boiled and be settled to, 50ml is dispensed into Triangular flask (50 ml/ bottles), sealing sterilizing.

9) root media

Add distilled water to 900ml, 1N potassium hydroxide adjusts pH value to 5.8, boils and is settled to 1000ml, be dispensed into pipe of taking root (25ml/ pipe), sealing sterilizing.

Embodiment 5:OsSN28 gene overexpression transgenic lines high temperature stress phenotypic evaluation

The present embodiment has chosen two SN28-OE familys (OE-25 and OE-27) and has carried out high temperature stress experiment.Specific step It is rapid as follows: overexpression transgenic lines rice paddy seed decladding is sterilized into (75% ethanol postincubation 3min, 0.15% mercuric chloride solution 30min is handled, for several times with sterile water wash), it germinates on the 1/2MS minimal medium containing 50mg/L hygromycin, it will be by T0 The 1/2MS without hygromycin is sowed within one day for the transgene negative control family OECK evening that SN28-OE is isolated to cultivate substantially On base, about after a week when seedling it is long to third piece leaf when select that growing way is consistent and good seedling kind enters in keg.It is to be grown extremely It is transplanted when four leaf stage into big drum, every group of material plants eight barrels, one plant every barrel.The soil of test is that southern china rice is planted Ridging, every drum soil loading amount is identical, and is poured with the time using equivalent amount of water after transplanting, to plant strain growth to fringe development later stage, When will extract out, four plants in every group of material are moved into 45 DEG C of temperature or so simultaneously, relative humidity 90% and are looked after sufficient Case is grown, carries out 1 hour high-temperature process, remaining material is placed in normal habitat.Place the material in normal rings for after treatment Border continued growth, observes the form and clever flower development situation of all material fringe portion after fringe is fully drawn out, while taking the 5 of fringe top Piece grain husk flower, detects its pollen fertility using iodine dye method, and to 3 hours after its flowering, observes its column using aniline blue decoration method Head pollination situation.In addition, summer in 2016 associated materials are grown into four leaf stage using the above method after be transplanted into normal irrigation Paddy field, plant 4 cells altogether, each 10 plants of the alternate plantation of each cell OE-25, OE-27 and OECK records the pumping of each material The real-time temperature in ear period (average value of all single plants exposing number of days used from germination to the first fringe in family) and field, to material The tassel of the normal solid rear harvest yellow maturity of material simultaneously investigates setting percentage (ratio of the real grain of single plant in total grain number) and effective fringe Rate ratio (accounting of real tassel of the grain greater than 5 in all tassels in single plant).The results show that in the case where growing case high-temperature process, The fertile pollen amount and column cap pollination rate of SN28 overexpression family OE-25 and OE-27 are significantly higher than negative control OECK (Fig. 6 In A figure and Fig. 6 in B figure), and the setting percentage of OE-25 and OE-27 is also significantly greater than OECK (C figure) in Fig. 6.2016 The field temperature of August is as shown in the D figure in Fig. 6, wherein the heading period section of each material marks on transverse axis, it is seen that each material Material is in heading in natural high-temperature stress state (highest temperature is greater than 40 DEG C in the daytime, and night minimum temperature is greater than 30 DEG C).In Fig. 6 E figure be under above-mentioned field conditions overexpress material OE-25, OE-27 and negative control OECK solid situation, it is seen that OE- 25, the ability of OE-27 is significantly stronger than OECK.The setting percentage of above-mentioned field material and effective fringe rate are than statistical result such as figure 6F, R1-R4 respectively represent 4 hillslope processes, it is seen that the setting percentage of the OE-25 and OE-27 of each cell and effective fringe rate are than equal It is significantly higher than OECK, illustrates that SN28 overexpresses the high-temperature resistance of material with respect to negative control under heading stage natural high-temperature environment It is obviously improved.

Sequence table

<110>Hua Zhong Agriculture University

<120>application of the OsSN28 gene in control rice high temperature resistant

<141> 2018-03-21

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1862

<212> DNA

<213>rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(1862)

<220>

<221> exon

<222> (972)..(1556)

<220>

<221> exon

<222> (66)..(239)

<400> 1

catcacaaaa cacataaaac tatatcatca tcttccatag ctgtacacat cctatatttt 60

cattcatggc gatgacaccg cagctagcat tttctcgcat gcctccaggg tttcggttcc 120

agccgacgga cgagcagctt gtcgtcgact acttgcagag gcgtaccgct gcgcagccat 180

gcgttactcc cgacatcacc gatatcgacg tttacaacgt cgacccgtgg cagcttccag 240

gtgctatata tatgcgcact ttaatttgca tgagcctcac tacatacggt gctctctact 300

ggtagtagaa atcaatctaa accgttaatc ttatttgacc gaatggttca gagatctatt 360

tatattatgg tagtaaaatc gtggatgggt gttatcgtca gaaaaaaaaa taggggtgaa 420

gggtatagtc gtcatttagc agtggctaaa ctcttttttc tccttttttg gctttttcga 480

tgaaatatga gcgatgccag cgcaatagat gatcatagat caatttagct gagaaaaaac 540

ataaatgaca aaataatttc taatcattgt attagtaaat tactaattta ccttaataat 600

actgatacta gtagtataat actaccagta gagagcacca gagcgtcgat ttgcatacga 660

ggcagggcgg aggcaggcct aggacaggta gggcttaagc cctaggcttg ttgcctccaa 720

aatcatggtt aaactctctt aaatcatcat ttaaatttgg gaaaagaaat gaataactaa 780

ttaataacac cctacctttg tcctactctt agagtccgtc actgcatacg ccgaagttct 840

attgatagag cagaaaaaaa ttacaaaatt ataaccctga aatgtcgaaa ccaggaaaaa 900

ggaaaaaata tatattgatt gtgaccctga tagatgcgat actaatgcaa catatattga 960

ctgtgtttgt agccatggcg atgtatggat cggatcatga ccggtacttc ttcacgatgg 1020

cggcccgaga ggcgcaggcc agacgaacga caccgtcggg tttctggaag cccaccggca 1080

caaagaagac gatcttcgtc gtcgccggtg ggcatgaggt gcccaccgcc gtcaagagga 1140

ggttcgtctt ctacctcggc caccaccaac catcgggcag caacaacaac aacaaaacat 1200

catggatcat gcatgagtac cgtctcatga actctccaag agcggcagtg ccgtcgtctt 1260

cttcggtgaa tcgtcttccc actgatgatc tcacggaaga gatggtgctg tgtaggatct 1320

ccaacaagga cctgcctaaa ccacccttca tccacaacag cttgttgcag ttctcttcag 1380

tggggttgaa tggtgatggg tataattact tgatccttga tcaccttgag cctccagcaa 1440

tggagtatcc taatgttggc attggtaatg ttgatgatgc tgctgctggt actgatgatc 1500

cgggtgacct tgatgaggag attgatgata gcatgcaaag aaaccatggt ggctagaatt 1560

aaccagtagc atcatgagtg aatggttttg cgggagattt gagttaaata tcagataata 1620

aaataataat acaaagaata aaaagaaaaa caaacctgta tagttagcag gttttgattc 1680

atttcgttga actttgtgtg cttgttagct agcctatata ccaaacatgc cttggcgagg 1740

ctttctattt gtatatagct ttttttcttt tttctattgt aaggttcgaa ctctattgag 1800

ttgattgaat aaacattatg caatttatgt aacatattgt ctataaagtt ctattggcat 1860

gc 1862

<210> 2

<211> 759

<212> DNA

<213>rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(759)

<220>

<221> CDS

<222> (1)..(759)

<400> 2

atg gcg atg aca ccg cag cta gca ttt tct cgc atg cct cca ggg ttt 48

Met Ala Met Thr Pro Gln Leu Ala Phe Ser Arg Met Pro Pro Gly Phe

1 5 10 15

cgg ttc cag ccg acg gac gag cag ctt gtc gtc gac tac ttg cag agg 96

Arg Phe Gln Pro Thr Asp Glu Gln Leu Val Val Asp Tyr Leu Gln Arg

20 25 30

cgt acc gct gcg cag cca tgc gtt act ccc gac atc acc gat atc gac 144

Arg Thr Ala Ala Gln Pro Cys Val Thr Pro Asp Ile Thr Asp Ile Asp

35 40 45

gtt tac aac gtc gac ccg tgg cag ctt cca gcc atg gcg atg tat gga 192

Val Tyr Asn Val Asp Pro Trp Gln Leu Pro Ala Met Ala Met Tyr Gly

50 55 60

tcg gat cat gac cgg tac ttc ttc acg atg gcg gcc cga gag gcg cag 240

Ser Asp His Asp Arg Tyr Phe Phe Thr Met Ala Ala Arg Glu Ala Gln

65 70 75 80

gcc aga cga acg aca ccg tcg ggt ttc tgg aag ccc acc ggc aca aag 288

Ala Arg Arg Thr Thr Pro Ser Gly Phe Trp Lys Pro Thr Gly Thr Lys

85 90 95

aag acg atc ttc gtc gtc gcc ggt ggg cat gag gtg ccc acc gcc gtc 336

Lys Thr Ile Phe Val Val Ala Gly Gly His Glu Val Pro Thr Ala Val

100 105 110

aag agg agg ttc gtc ttc tac ctc ggc cac cac caa cca tcg ggc agc 384

Lys Arg Arg Phe Val Phe Tyr Leu Gly His His Gln Pro Ser Gly Ser

115 120 125

aac aac aac aac aaa aca tca tgg atc atg cat gag tac cgt ctc atg 432

Asn Asn Asn Asn Lys Thr Ser Trp Ile Met His Glu Tyr Arg Leu Met

130 135 140

aac tct cca aga gcg gca gtg ccg tcg tct tct tcg gtg aat cgt ctt 480

Asn Ser Pro Arg Ala Ala Val Pro Ser Ser Ser Ser Val Asn Arg Leu

145 150 155 160

ccc act gat gat ctc acg gaa gag atg gtg ctg tgt agg atc tcc aac 528

Pro Thr Asp Asp Leu Thr Glu Glu Met Val Leu Cys Arg Ile Ser Asn

165 170 175

aag gac ctg cct aaa cca ccc ttc atc cac aac agc ttg ttg cag ttc 576

Lys Asp Leu Pro Lys Pro Pro Phe Ile His Asn Ser Leu Leu Gln Phe

180 185 190

tct tca gtg ggg ttg aat ggt gat ggg tat aat tac ttg atc ctt gat 624

Ser Ser Val Gly Leu Asn Gly Asp Gly Tyr Asn Tyr Leu Ile Leu Asp

195 200 205

cac ctt gag cct cca gca atg gag tat cct aat gtt ggc att ggt aat 672

His Leu Glu Pro Pro Ala Met Glu Tyr Pro Asn Val Gly Ile Gly Asn

210 215 220

gtt gat gat gct gct gct ggt act gat gat ccg ggt gac ctt gat gag 720

Val Asp Asp Ala Ala Ala Gly Thr Asp Asp Pro Gly Asp Leu Asp Glu

225 230 235 240

gag att gat gat agc atg caa aga aac cat ggt ggc tag 759

Glu Ile Asp Asp Ser Met Gln Arg Asn His Gly Gly

245 250

<210> 3

<211> 252

<212> PRT

<213>rice (Oryza sativa)

<400> 3

Met Ala Met Thr Pro Gln Leu Ala Phe Ser Arg Met Pro Pro Gly Phe

1 5 10 15

Arg Phe Gln Pro Thr Asp Glu Gln Leu Val Val Asp Tyr Leu Gln Arg

20 25 30

Arg Thr Ala Ala Gln Pro Cys Val Thr Pro Asp Ile Thr Asp Ile Asp

35 40 45

Val Tyr Asn Val Asp Pro Trp Gln Leu Pro Ala Met Ala Met Tyr Gly

50 55 60

Ser Asp His Asp Arg Tyr Phe Phe Thr Met Ala Ala Arg Glu Ala Gln

65 70 75 80

Ala Arg Arg Thr Thr Pro Ser Gly Phe Trp Lys Pro Thr Gly Thr Lys

85 90 95

Lys Thr Ile Phe Val Val Ala Gly Gly His Glu Val Pro Thr Ala Val

100 105 110

Lys Arg Arg Phe Val Phe Tyr Leu Gly His His Gln Pro Ser Gly Ser

115 120 125

Asn Asn Asn Asn Lys Thr Ser Trp Ile Met His Glu Tyr Arg Leu Met

130 135 140

Asn Ser Pro Arg Ala Ala Val Pro Ser Ser Ser Ser Val Asn Arg Leu

145 150 155 160

Pro Thr Asp Asp Leu Thr Glu Glu Met Val Leu Cys Arg Ile Ser Asn

165 170 175

Lys Asp Leu Pro Lys Pro Pro Phe Ile His Asn Ser Leu Leu Gln Phe

180 185 190

Ser Ser Val Gly Leu Asn Gly Asp Gly Tyr Asn Tyr Leu Ile Leu Asp

195 200 205

His Leu Glu Pro Pro Ala Met Glu Tyr Pro Asn Val Gly Ile Gly Asn

210 215 220

Val Asp Asp Ala Ala Ala Gly Thr Asp Asp Pro Gly Asp Leu Asp Glu

225 230 235 240

Glu Ile Asp Asp Ser Met Gln Arg Asn His Gly Gly

245 250

Claims

1. a kind of isolated OsSN28 gene is improving rice to the application in high temperature stress tolerance, which is characterized in that should The nucleotide sequence of gene is as described in SEQ NO:1 shown in the 1-1862 bit base of sequence.

2. a kind of isolated OsSN28 gene is improving rice to the application in high temperature stress tolerance, which is characterized in that should The protein sequence of gene coding is as shown in SEQ NO:2.

3. OsSN28 gene described in claim 1 is improving rice to the application in high temperature stress tolerance, feature exists In the nucleotide sequence of the gene is as described in SEQ NO:1 shown in the 1-1862 bit base of sequence.

4. OsSN28 gene as claimed in claim 2 is improving rice to the application in high temperature stress tolerance, feature exists In the protein sequence of gene gene coding is as shown in SEQ NO:2.

5. the application of claim 3 or 4, which is characterized in that by transgenosis, overexpress OsSN28 gene in rice, mention High rice is to high temperature stress tolerance.