CN108409846A

CN108409846A - A kind of soybean salt-tolerance correlation myb transcription factor and its encoding gene and application

Info

Publication number: CN108409846A
Application number: CN201810519791.7A
Authority: CN
Inventors: 王庆钰; 何禹璇; 闫帆; 王英; 李景文; 王天亮; 尹智超; 徐杨; 张鑫生; 杨旭光
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2018-05-27
Filing date: 2018-05-27
Publication date: 2018-08-17
Anticipated expiration: 2038-05-27
Also published as: CN108409846B

Abstract

The present invention relates to a kind of soybean salt-tolerance correlation myb transcription factor and its encoding gene and applications, belong to plant genetic engineering field.The base sequence of soybean myb transcription factor gene is SEQ ID N0.1, and amino acid sequence is SEQ ID N0.2.The present invention has cloned one and the relevant soybean GmMYB68 transcription factor genes of salt tolerance, is analyzed expression pattern of the gene in wild type and genetically engineered soybean and drought resistance, is of great significance to cultivating incultured soybean kind.

Description

A kind of soybean salt-tolerance correlation myb transcription factor and its encoding gene and application

Technical field

The present invention relates to plant genetic engineering fields, and in particular to a kind of soybean salt-tolerance associated transcription factor MYB and its volume Code gene and application.

Background technology

Soybean (Glycine max L.) provides vegetable protein and fat, also the raw material of industry and herding can be used as to raise Material.One side China is as the maximum Soybean import state in the whole world, on the other hand, including pest and disease damage, low temperature, arid, soil alkaline Biology and abiotic stress including change cause serious threat to domestic Soybean production.Therefore, enhance adverse circumstance defence capability, It is extremely urgent to cultivate the germ plasm resource with more preferable resistance on economical character.Although traditional breeding method obtains a large amount of Achievement, but study, the limitation of the complexity and quality germplasm shortage of degeneration-resistant mechanism long by breeding cycle.By modern molecular biology Technology and traditional breeding method mode combine, and study the degeneration-resistant mechanism of plant, become the inevitable choice of numerous breeders.

The abiotic stress of plant stress network it is extremely complex, the transcriptional control containing lots of genes level.Plant passes through To the transcriptional control of related adverse circumstance stress gene, achieve the purpose that adjust physiological development.Ionic stress, pathogen, moisture, temperature Deng the environmental stimulus factor for influencing plant signal conduction, in order to avoid the damage of environment stress, plant has had a set of self adjust Section mechanism is protected from the unfavorable environmental pressure such as low temperature, arid, with high salt.Myb gene is important in transcription factor family A member participates in plant growth metabolism, the response of environmental factor, and builds up the various Mechanism of Physiological and Biochemical of plant with important Adjustment effect.The amino acid sequence N-terminal of MYB class transcription factors in plant has what 1-3 was made of 51-53 amino acid Helically-turnover-helical conformation sequence (R1, R2 and R3), i.e. MYB structural domains.The effect of hydrophobic core is mainly derived from MYB knots 3 conservative trp residues in domain are closed, to maintaining spiral-turnover-helical conformation to play a major role.Most MYB albumen are all Transcription activator, can activate the expression of target gene, also have small part to inhibit the expression of target gene, play negative regulation.Turn The factor is recorded mainly by being specifically bound with target sequence, to play the regulating and controlling effect to downstream gene.Romero and Urao Deng research shows that the same MYBSI (conserved sequence T/CAACG/TGA/C/TA/C/T) of myb transcription factor, II (conserved sequences of MYBS For TAACTAAC), CNGTTR, GKTWGTTR, GKTWGGTR (N A, G, C or T；K, G or T；R, A or G；W, A or T), The elements such as TAACTG combine.

Eukaryotic gene function expression network is extremely complex, is related to multiple modifications, translation, transcription, wherein turn Regulation and control in record level are particularly critical, all have important shadow to growth and development of plants, Response to stress, signal transduction and disease resistance etc. It rings.

There may be a large amount of myb transcription factor in monocotyledon and dicotyledon, due to MYB in plant inverse Play an important roll in border, therefore, Function Identification is carried out to other members in soybean MYB families, can be to take plant gene Breeding technique enhances the resistance to inverse quality of soybean, creates outstanding degeneration-resistant material and provides effective genetic resources.

Invention content

A kind of soybean salt-tolerance correlation myb transcription factor of present invention offer and its encoding gene and application.

Soybean salt-tolerance correlation myb transcription factor provided by the present invention is from soybean varieties Jilin 32 (by Jilin Province Academy of agricultural sciences Fu Jian researcher presents), it is named as GmMYB68.

The base sequence of the soybean myb transcription factor gene is SEQ ID N0.1.

The protein of the soybean myb transcription factor gene code, amino acid sequence are SEQ ID N0.2.

Sequence SEQ ID N0.1 are by 780 base compositions；SEQ ID N0.2 are made of 259 amino acid residues, contain two A SANT structural domains, i.e. MYB structural domains：Positioned at 4-53,56-104 amino acid, belong to typical R2R3-MYB transcription factors.

The carrier that foreign gene is expressed in plant can be guided using any type, it will be provided by the present invention The encoding gene of GmMYB68 imports plant cell, the transfer-gen plant with salt-resistance.Use the gene constructed plant of the present invention When expression vector, any type enhancing promoter or inducible promoter can be added before its transcription initiation nucleotide.In order to Convenient for transgenic plant cells or plant are identified and screened, used carrier can be processed, plant is such as added (gentamicin, to block that mould for alternative label (gus gene, luciferase genes etc.) or resistant antibiotic marker Element etc.).Carry GmMYB68 of the present invention expression vector can by using Ti-plasmids, Ri plasmids, plant viral vector, directly The conventional biology methods such as DNA conversions, microinjection, conductance, agriculture bacillus mediated convert plant cell or tissue, and by the plant of conversion Object tissue cultivating is at plant.The host being converted is either monocotyledon, can also be dicotyledon.

The present invention has cloned one and the relevant soybean GmMYB68 transcription factor genes of salt tolerance, to the gene wild Expression pattern and drought resistance in type and genetically engineered soybean are analyzed, and are of great significance to cultivating incultured soybean kind.

Description of the drawings

Fig. 1 is the PCR amplification result figure of GmMYB68 genes, wherein M：DNA Marker(DL2000)；1,2,3,4： GmMYB68 post transcription cloning results；

Fig. 2 is GmMYB68 gene adverse circumstance induced expression ideographs；

Fig. 3 is GmMYB68 gene organizations expression pattern figure；

Fig. 4 is plant expression vector structural schematic diagram；

Fig. 5 .1 are that GmMYB68 genes are applied in part T2 for the herbicide (glyphosate) in the qualification result of genetically engineered soybean Screening figure is smeared, in figure：Arrow logo is to smear herbicide position, and WT lines are withered after smearing turns yellow, transfer-gen plant without Significant change；

Fig. 5 .2 are GmMYB68 genes in part T2 for the Bar genetic test figures in the qualification result of genetically engineered soybean, figure In：M：DNA Marker(DL2000)；1-22：PTF101.1-GmMYB68 transfer-gen plants；+：Recombinate matter Grain；-：Negative control (template is water)；

Fig. 5 .3 are that GmMYB68 genes hybridize in part T2 for the Southern blot in the qualification result of genetically engineered soybean Analysis chart, M：DNA Marker；1,2,3,4：The T2 of Hind III digestions is for transfer-gen plant Southern Blot is identified；+：Recombinant plasmid；-：Negative control (WT lines)；

Fig. 6 .1 are GmMYB68 transfer-gen plant Salt-Tolerance Identification figures, are that Wild-type soy and T3 are saline and alkaline for genetically engineered soybean Handling result, wherein WT are Wild-type soy, and OE is T3 for genetically engineered soybean；

Fig. 6 .2 are GmMYB68 genetically engineered soybean germination rate measurement charts, and wherein WT is Wild-type soy, and OE is to turn base in T3 generations Because of soybean,

Fig. 7 is the change of GmMYB68 transfer-gen plants soluble sugar content (A) and proline content (B) under Saline Alkali Stress Change figure, Wild-type：Wild-type soy；GmMYB68-OE：The accumulation of GmMYB68 transgenic lines, soluble sugar and proline It is related to Response to stress in plant, after Saline Alkali Stress is handled, the soluble sugar and proline of wild type and transfer-gen plant Content significantly rises, and the wherein ascensional range of GmMYB68 transfer-gen plants rises notable compared with wild type；

Fig. 8 is T3 for GmMYB68 anti contravariance related genes fluorescence real-time quantitative PCR result figure in genetically engineered soybean, wherein：A： ABA signal pathway key regulators GmABI5；B：With high salt to plant, arid and low temperature stress molecule response regulatory gene DREB2；C：Na⁺/ H transporter genes GmNHX1；D：The key enzyme phenylalanine lyase of phenylpropyl alcohol alkane metabolic pathway；E：With plant The object course of disease reacts relevant gene GmNPR1-1；F：Relevant gene GmNPR1-2 is reacted with the plant course of disease, above-mentioned 5 genes are equal Be confirmed in pertinent literature with the relevant important gene of plant stress-resistance.

Specific implementation mode

Embodiment 1：The Cloning and sequence analysis of GmMYB68 genes

The extraction of soybean RNA and the synthesis of cDNA：With reference to Quan Shi King CompaniesPlant RNA Kit explanations Book extracts 32 blade total serum IgE of soybean Jilin, usesFirst-Strand cDNA Synthesis Super Mix (purchased from full formula gold) carries out reverse transcription, synthesizes cDNA；

Using the immature embryo express spectra sequencing gained sequence (20d, 30d, 50d) in soybean varieties Jilin 32, NCBI is utilized Sequence analysis tools (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis obtain two with MYB transcribe because Son has the unknown cDNA sequence of high homology, and using Primer5 software Design primers, immature embryo cDNA carries out for template PCR amplification.Reaction system is as follows：

PCR programs are：

PCR product electrophoresis detection on 1% Ago-Gel, ultraviolet gel imager shooting preserve；

Above-mentioned amplified fragments are used into Ago-Gel QIAquick Gel Extraction KitQuick Gel Extraction It is attached with cloning vector pMD18-T (being purchased from TaKaRa) after Kit (Quan Shi gold) recycling, reaction system is as follows：

Carrier T 0.5ul

PCR recovery products 2ul

Solution I 2.5ul

16 DEG C are ligated and transformed into E. coli competent overnight, and send Hua Da gene sequencing, and verification sequence is correct.

Obtained through PCR amplification include GmMYB68 gene open reading frames sequence, encode 259 amino acid.Prediction The molecular weight of GmMYB68 albumen is 145.636kDa, isoelectric point pI=5.0.There are two amino acid structure predicts that the albumen contains SANT structural domains, i.e. MYB structural domains are located at 4-53,56-104 amino acid, belong to typical R2R3-MYB transcription factors..

Embodiment 2：Expression pattern under the induction of GmMYB68 gene adverse circumstances

When plant grows to four leaf stage, seedling is transferred to after cultivating 3d in Hogland nutrient solutions and is handled as follows：

Salt stress processing：Soybean seedling is placed in the Hogland nutrient solutions of the NaCl containing 200mM；

Low-temperature treatment：Soybean seedling is placed in 4 DEG C of illumination box；

Osmotic treatment：Soybean seedling is placed in the Hogland nutrient solutions containing 20%PEG8000；

Mechanical wounding processing：It is rowed dry wound at 3-5 to soybean seedling blade with the scalpel blade of sterilizing；

ABA processing：Soybean seedling is sprayed with the ABA containing 200uM；

Above-mentioned processing respectively after treatment 0h, 1h, 2h, 5h, 10h, sample for 24 hours, vegetable material is immediately placed in liquid nitrogen ,- 80 DEG C save backup；

The extraction of total serum IgE and the synthetic method of cDNA are glimmering in real time according to the design of the cDNA sequence of GmMYB68 with embodiment 1 Fluorescent Quantitative PCR primer (F:5’-TCGGTCTGGGAAATCGTG-3’；R: 5’-CTCACCACTCGCAGCATCT-3’)；With soybean Constitutive expression gene GmACTII (GeneBank searching numbers：U60500)(F:5’-GAGCTATGAATTGCCTGATGG-3’； R:5 '-CGTTTCATGAATTCCAGTAGC-3 ') it is internal reference gene, using ABI PRISM7500 real-time PCRs, The cDNA of each tissue site of soybean and each Stress treatment sample point is that template carries out fluorescence real-time quantitative PCR；Specific steps reference Tip Green qPCR SuperMix specifications are operated；

Reaction system：

PCR programs are：

94℃ 30s

55℃ 5s

72℃ 34s

40 cycles, using 2^-△△CTMethod analyzes data, determines that the relative expression quantity of gene, each sample point set 3 technologies It repeats, experiment sets the repetition of 3 secondary pollutants altogether.

The relative expression quantity of the lower GmMYB68 genes of 1 adverse circumstance of table processing；

The result shows that there is up-regulated expression trend under the processing of various adverse circumstances in GmMYB68 genes.Wherein at salt stress After managing 5h, the expression quantity of GmMYB68 reaches highest, and overall expression level is higher than other Stress treatments, therefore, infers GmMYB68 can be by a variety of environment stress induced expressions, wherein the expression response to salt stress is most apparent.

Embodiment 3：Expression pattern of the GmMYB68 genes in soyabean tissue

Total serum IgE and the reverse transcription of the root, stem, flower, leaf, cotyledonary node, hypocotyl and 20 days embryos in extraction soybean Jilin 32 respectively At cDNA, method is the same as embodiment 1.Real-time fluorescence quantitative PCR is carried out by masterplate of the cDNA of above-mentioned tissue, method is the same as embodiment 2.

Expression of the table 2GmMYB68 genes in soybean different tissues:

Stem	Leaf	Flower	Rataria	Cotyledonary node	Hypocotyl
						1.413027	1.512265	24.98434	4.243418	2.049935	13.57257

GmMYB68 has expression in each tissue site of soybean, wherein it spends, the expression quantity highest of hypocotyl and rataria, Although his position also has expression, expression quantity relatively low；Expression quantity in root is minimum.

Embodiment 4：The structure of GmMYB68 gene plant expression vectors and the genetic transformation of soybean cotyledon node

With reference to the small extraction reagent kit specification of plasmid of Quan Shijin biotech firms, takes and be incubated overnight bacterium solution pMD18T- GmMYB68 extracts plasmid as template, and XbaI and SacI restriction enzyme sites, (F are added in upstream and downstream primer:5’- TGCTCTAGACAAAGGACATGGATCGGATAAAAGG-3 ', R:5’-CGAGCTCTTATTCAACCCTACCAATTCCCATCC - 3 ') PCR amplification is carried out, amplified production and plant expression vector pTF101.1-35S are subjected to digestion with restriction enzyme respectively Reaction, reaction system are carried out with reference to NEB companies reagent specification：

37 DEG C of digestion 2h, digestion products are detected into row agarose gel electrophoresis, recycle target fragment.And use T4 ligases PCR fragment after digestion is attached with carrier, after sequencing identification is correct, correct recombinant plasmid will be sequenced and be named as PTF101.1-GmMYB68, conversion Agrobacterium EHA101 (are purchased from Biovector), it is spare to preserve strain.

With reference to the method for Guo east congruence, Agrobacterium-mediated transformation soybean cotyledon node tissue.Take epidermis smooth, disease-free spot, Soya seeds without mould, the complete mature of flawless, are put into closed container, are produced with 100ml NaClO and 3.5ml concentrated hydrochloric acids Raw chlorine suffocating sterilization 10-12h.By the soya seeds after disinfection after dispelling remaining chlorine on superclean bench, hilum to It sows after sprouting about 16h overnight in germination medium, is cut two panels cotyledon along central axes with sterile scalpel, removal is original Leaf bud, and it is about the cut of 3mm in cotyledon and the manufacture of hypocotyl junction.The explant prepared is put into and is invaded equipped with engineering bacteria In the sterile ware of dye liquor, 80 explants are infected every about 70ml bacterium solutions, sterile petri dish can be sealed to be placed on when infecting and shaken On bed, 100rpm gently vibrates 20-30min, so that explant is come into full contact with and infects liquid；Co-culturing, one layer of culture medium upper berth is sterile Paraxial face upward of cotyledonary node after infecting is placed on filter paper by filter paper, per 8 explants of ware or so, 24 ± 1 DEG C, and light culture 4d. After co-culturing 4d, the hypocotyl of cotyledonary node elongation is cut, retains 3mm or so, by 45 ° of upward oblique cuttings of adaxial and its surface of cotyledonary node explant Adventitious bud inducing is carried out in differential medium.26 DEG C or so of cultivation temperature, illumination for 24 hours, every 14 days subcultures are primary, replace fresh Culture medium.Extra young shoot is cut during subculture, and manufactures new wound in explant and culture medium contact surface to promote adventitious bud to lure It leads.It after adventitious bud inducing 28d, is transferred in Elongation of adventitious bud culture medium, cuts off cotyledon, it is primary per 14d subcultures, replace fresh cultured Base, until Elongation of adventitious bud to 3-5cm.Elongation of adventitious bud can be cut to when 3-5cm, after the IBA for dipping suitable 1mg/L It is transferred to root media, it is 20d or so to go out the root time, and being transferred to 26 DEG C of phjytotrons when growing healthy and strong root system is refined Seedling, transplanting harvest T₀It is further analyzed for seed and to filial generation.

Embodiment 5：The screening and identification of GmMYB68 genetically engineered soybeans

ELISA test strip：Regrowth about 0.2g tender leafs are taken, are placed in clean centrifuge tube, the grinding of 100ul sterile waters is added.It willTest strips have one end of arrow mark to immerse in lapping liquid several minutes, and colour developing end is the positive in two red lines Testing result, colour developing end are negative result in a red line.

Herbicide screening：When the T0 in greenhouse is fully deployed compound leaf for transgenic seedling second, choose in trifoliolate leaf Between leaflet smear glyphosate (1 ‰ m/v) solution, and mark, after smearing 3d, observe and photograph to record.Arrow logo is Smear herbicide position, WT lines are withered after smearing turns yellow, and transfer-gen plant is without significant change.Illustrate that transfer-gen plant is taken With the riddled basins Bar genes on plant expression vector, contain Herbicid resistant.

SouthernBlot hybridizes：The offspring through ELISA test strip and herbicide screening positive plant, i.e. T2 generations is selected to turn base Because of the mature leaf 4-5 pieces of plant, total DNA is extracted, DNA digestion, recombinant plasmid are carried out with restriction enzyme Hind III PTF101.1-GmMYB68 is positive control, and the DNA single endonuclease digestion products of WT lines are as negative control.In transfer-gen plant Band is copied containing 1-2 target gene, illustration purpose gene is integrated into the form of singly copying in soybean genome.

Embodiment 6：Saline and alkaline Analysis of tolerance on thirties of the T3 for GmMYB68 genetically engineered soybeans

With the Na of the NaCl solution of 200mM and 100mM₂CO₃Solution is handled respectively as salt stress treatment fluid and alkaline stress Liquid takes uniform 9 basin of genetically engineered soybean seedling of growing way to be divided into three groups, and when having just enter into four leaf stage, salt alkali process is carried out to soybean, right Same treatment is carried out according to group watering pours water, while to the Wild-type soy of same breeding time, as wild type control group.At total Reason 20 days.

After Saline Alkali Stress is handled, the blade of WT lines and transfer-gen plant generates yellow spotting and curling is existing As.The phenomenon that apparent hypoevolutism is presented in WT lines, and symptoms are mild for transfer-gen plant, and transfer-gen plant is to Na₂CO₃ Tolerance be better than NaCl.

In addition, with the NaCl solution and Na of above-mentioned concentration₂CO₃Solution pours T3 for transgenic seed and Wild-type soy kind Son, handles 4 days, counts the germination rate of seed by every group 100.

Genetically engineered soybean and Wild-type soy seed are under the treatment conditions of watering pours water, germination rate no significant difference, but After Saline Alkali Stress processing, the germination rate of transfer-gen plant is better than Wild-type soy, in germination rate experiment, transfer-gen plant table It is now to the stronger tolerances of NaCl.

Embodiment 7：T3 is for physiological index determining after genetically engineered soybean salt alkali process

1) measuring chlorophyll content

Fresh blade 0.2g is taken, shreds and is placed on clean 50ml centrifuge tubes, 10ml absolute ethyl alcohols and 80% acetone is added Isometric mixed liquor.48h is stood at dark, until chlorophyll extraction is complete, OD is measured using ultraviolet specrophotometer₆₆₃With OD₆₄₅, with formula C_T=20.29OD₆₄₅+8.05OD₆₆₃Calculate chlorophyll content；

2) soluble sugar content measures

Fresh leaf agreement that contracts a film or TV play to an actor or actress 1.0g is weighed, shreds and is placed in Boiling tube, 10mlH is added₂O boils 20min in boiling water bath, It is cooled to room temperature, filters in volumetric flask, be settled to 100ml, take extracting solution 1ml to be measured, 5ml anthrone reagents are added, it is quickly mixed After even, 10min is boiled in boiling water bath, takes out cooling, measures the OD values under 620nm wavelength, and reference standard curve calculates soluble sugar Content；

3) proline content measures

It weighing 0.05g samples and is put into centrifuge tube, the sulfosalicylic acid of 10ml 3% is added, boiling water bath boils 30min after sealing, After being cooled to room temperature, 3000rpm centrifuges 10min, takes supernatant to be measured；

It takes 1ml extracting solutions in test tube, 1ml ice vinegar, 1ml3% sulfosalicylic acids, 2ml2.5% acid ninhydrines is added to mix 4ml toluene is added after cooling in boiling water bath chromogenic reaction 60min after even, and oscillation extraction red material takes upper phase after static, The OD values measured under 520nm wavelength find corresponding concentration of proline according to standard curve, and reference formula is calculated：Dried meat ammonia Acid content (ugg^-1FW)=C × V/2/W/115；Wherein C is the proline content (ug/ml) looked by standard curve, V is to carry Liquid total volume (ml), W is taken to be sample quality (g), 115 be proline relative molecular mass；

4) photosynthetic measurement

Using the portable photosynthesis measurement system of LI-6400 types, using the red blue-light source of fixation, setting intensity of illumination is 1200umol·m^-2·s^-1.In the 10th day morning 10 of processing:00 to 12:00 is measured, and measurement site is number the on plant Leaflet among 2 groups of trifoliolate leaves.Net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular gas concentration lwevel (Ci) and steaming Rate (Tr) is risen by being directly read on instrument；

Data processing and variance analysis are completed using statistics program SPSS21.0.In text data be all provided with repeat three times it is flat Mean value ± standard error.The analysis of physical signs is compared using LSD Multiple range tests；

The results show that after salt alkali process, the chlorophyll content of WT lines and transfer-gen plant is compared with normal growth Condition is declined, and Different stress processing fall is different.By NaCl and Na₂CO₃After processing, WT lines leaf is green Cellulose content has dropped 32.9% and 22.5% respectively, and transfer-gen plant chlorophyll content has dropped 36.1% and 0.11% respectively；

Under normal growth state, the Photosynthetic physiological indexes and no significant difference of wild type and transfer-gen plant, the saline and alkaline side of body After compeling, net photosynthetic rate (Pn) is on a declining curve, and the Net Photosynthetic Rate of wild type has dropped 30.2% He respectively 42.1%, transfer-gen plant then has dropped 27% and 21.8% respectively.In alkaline stress level, the net photosynthesis speed of transfer-gen plant There are significant differences compared with wild type for rate.In addition, stomatal conductance (Gs), transpiration rate (Tr) and Decrease in Net Photosynthetic Rate trend Unanimously, and intercellular gas concentration lwevel (Ci) illustrates wild type and transfer-gen plant then on the contrary, there is increase trend after Stress treatment The main reason for causing photosynthetic rate to reduce under Saline Alkali Stress is not the non-limiting effect of stomata, thus it is speculated that is mesophyll cell light Closing activity reduces caused C N metabolism imbalance.Compared with the control, salt stress has the physical signs of plant with alkaline stress and cuts So different influence.Existing pertinent literature proves, when by environment stress, the stronger plant photosynthetic capacity of resistance by To influence it is smaller, this experiment prove, the conversion of GmMYB68 genes, be conducive to alleviate Saline Alkali Stress to plant chlorophyll content with Photosynthetic influence.

Influences of the table 3GmMYB68 to Photosynthetic physiological indexes under Saline Alkali Stress：

Note：* significant difference P ＜ 0.05 are labeled as, * * are labeled as significant difference P ＜ 0.01

Having relative literature confirms, the accumulation of soluble sugar and proline is conducive to plant and is tieed up under adverse environmental factor Eucaryotic cell structure and viability are held, the resistance of reverse of plant is improved.After salt alkali process, in wild type and GmMYB68 plant can Dissolubility sugar and proline largely accumulate.Under normal growing conditions, both in wild type and transfer-gen plant content without significant difference, After alkaline stress is handled, soluble sugar content and proline content have increased separately 76.54% He in transfer-gen plant 50.91%, which is significantly higher than WT lines under same Stress treatment (being respectively 62.38% and 43%).In addition, After NaCl processing, the increasing degree of proline is significantly higher than with the wild type under processing GmMYB68 transfer-gen plants.To sum up tie Fruit illustrates that under the conditions of saline and alkaline, overexpressions of the GmMYB68 in soybean is conducive to synthesis and the product of soluble sugar and proline It is tired.

4 salt of table, influence of the alkaline stress to GmMYB68 rotaring gene plant blade osmotic adjustments：

Embodiment 8：The investigation of GmMYB68 transfer-gen plant economical characters

By NaCl and Na₂CO₃After processing, the development of plant is significantly affected, is embodied in lateral root reduction, root system Elongation is limited, and lignifying accelerates.Economical character investigation is carried out to transfer-gen plant, leading indicator includes plant height, branch amount, single plant Five pod number, single-strain grain number, 100-grain weight aspects, data result are for statistical analysis using LSD methods.As a result it is as shown in the table, respectively refers to Decline though being marked on and existing after supersalt, alkaline stress processing, significant difference, table are had no between transfer-gen plant and WT lines The economical character that the kind is not influenced while being inserted in enhancing soybean varieties saline and alkaline patience of bright GmMYB68.

Economical character investigation in table 5GmMYB68 transfer-gen plants and WT lines：

Embodiment 9：The expression of anti contravariance related gene in GmMYB68 transfer-gen plants

Using fluorescence real-time quantitative PCR to the expression of anti contravariance related gene in transfer-gen plant and WT lines into Row detection, relative expression quantity are obtained by comparing the WT lines under transfer-gen plant and same treatment conditions.Wherein： GmABI5 is ABA signal pathway key regulators；DREB2 is that with high salt, arid and low temperature stress molecule reaction is adjusted to plant Control gene；GmNHX1 is Na⁺/ H transporter genes；PAL is the key enzyme phenylalanine lyase of phenylpropyl alcohol alkane metabolic pathway； GmNPR1-1 and GmNPR1-2 reacts relevant gene with the plant course of disease, and document is confirmed with plant inverse above-mentioned 5 genes Stress reaction under the stress of border has important relationship.

The primer that table 6 uses in testing：

All anti contravariance related genes have different degrees of response to salt and alkaline stress, in GmMYB68 transfer-gen plants In, anti contravariance related gene has accumulated higher expression quantity, illustrates the transcriptional level of these genes by GmMYB68 overexpressions It influences, and participates in the Saline Alkali Stress response mechanism regulated and controled by GmMYB68.Wherein, compared with wild type, GmABI5 and DREB2 amounts of increase in transfer-gen plant are apparent.In addition, it has been found that two play a role in broad-spectrum disease resistance function turn The factor is recorded, GmNPR1-1 and GmNPR1-2 also have significant high transcriptional level (P ＜ 0.01) in transfer-gen plant, show GmMYB68 also functions to certain effect in terms of biotic.

Sequence table

<110>Jilin University

<120>A kind of soybean salt-tolerance correlation myb transcription factor and its encoding gene and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 780

<212> DNA

<213>Artificial synthesized (artificial sequence)

<400> 1

atggatcgga taaaagggcc atggagtcct gaagaggacg aagcgttacg gaggttggtt 60

cagacttacg gccctaggaa ctggtccgtt ataagcaaat ccattccggg tcggtctggg 120

aaatcgtgcc gcttgcggtg gtgcaaccag ctgtctccgg aggtggagcg ccggcctttc 180

acggcggagg aagacgaggc gatcctgaag gctcacgcca ggttcgggaa caagtgggcc 240

accatcgcgc gcttcctcaa tggccgcacc gacaacgcca tcaagaacca ttggaattcc 300

accctcaaga ggaagtgctc cgagcctctc tccgagcctc ggcctctgaa gagatccgcc 360

accgtttcgg gtagtcaatc cggatccgat ttgagcgatt cgggtttacc cattattctt 420

gcaagaagcg tgagcgtgac ggtagctccc tctaaccacc ttgcggaaac cgcgtcttct 480

tcagtaactg atcctgccac gttattgagt ttgtctctac cgggattcga ttcgtgcgat 540

ggggctaata atgggcctgg gccgaatcag gggcccagtt gcggcccgtt ccaggagata 600

ccgatgcttg gttcccaaaa gcagttgttc agccaagagt ttatgaaggt gatgcaagag 660

atgatacgag tggaagtgag aaattacatg tctgtactgg aacgtaatgg tgtgtgtatg 720

caaaccgatg ccattaggaa ctcggtgttg gagaggatgg gaattggtag ggttgaataa 780

<210> 2

<211> 259

<212> PRT

<213>Artificial synthesized (artificial sequence)

<400> 2

Met Asp Arg Ile Lys Gly Pro Trp Ser Pro Glu Glu Asp Glu Ala Leu

1 5 10 15

Arg Arg Leu Val Gln Thr Tyr Gly Pro Arg Asn Trp Ser Val Ile Ser

20 25 30

Lys Ser Ile Pro Gly Arg Ser Gly Lys Ser Cys Arg Leu Arg Trp Cys

35 40 45

Asn Gln Leu Ser Pro Glu Val Glu Arg Arg Pro Phe Thr Ala Glu Glu

50 55 60

Asp Glu Ala Ile Leu Lys Ala His Ala Arg Phe Gly Asn Lys Trp Ala

65 70 75 80

Thr Ile Ala Arg Phe Leu Asn Gly Arg Thr Asp Asn Ala Ile Lys Asn

85 90 95

His Trp Asn Ser Thr Leu Lys Arg Lys Cys Ser Glu Pro Leu Ser Glu

100 105 110

Pro Arg Pro Leu Lys Arg Ser Ala Thr Val Ser Gly Ser Gln Ser Gly

115 120 125

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

130 135 140

Ser Val Thr Val Ala Pro Ser Asn His Leu Ala Glu Thr Ala Ser Ser

145 150 155 160

Ser Val Thr Asp Pro Ala Thr Leu Leu Ser Leu Ser Leu Pro Gly Phe

165 170 175

Asp Ser Cys Asp Gly Ala Asn Asn Gly Pro Gly Pro Asn Gln Gly Pro

180 185 190

Ser Cys Gly Pro Phe Gln Glu Ile Pro Met Leu Gly Ser Gln Lys Gln

195 200 205

Leu Phe Ser Gln Glu Phe Met Lys Val Met Gln Glu Met Ile Arg Val

210 215 220

Glu Val Arg Asn Tyr Met Ser Val Leu Glu Arg Asn Gly Val Cys Met

225 230 235 240

Gln Thr Asp Ala Ile Arg Asn Ser Val Leu Glu Arg Met Gly Ile Gly

245 250 255

Arg Val Glu

Claims

1. a kind of soybean salt-tolerance correlation myb transcription factor, it is characterised in that：Its amino acid sequence is SEQ ID N0.2.

2. soybean salt-tolerance correlation myb transcription factor as described in claim 1, it is characterised in that：Its nucleotides sequence is classified as SEQ ID N0.1。

3. application of the soybean salt-tolerance correlation myb transcription factor as described in claim 1 in cultivating incultured soybean kind.