CN109207452A - Sorghum resistance related gene SbERECTA and its coding albumen and application - Google Patents

Abstract

The present invention provides a kind of sorghum SbERECTA albumen, its are as follows: 1) protein or 2) be substituted, lack or add one or several amino acid in the amino acid sequence shown in SEQ ID NO.1 and there is the same active protein as derived from 1) that by SEQ ID NO.1 shown in amino acid sequence form.Invention also provides the gene SbERECTA for encoding above-mentioned albumen, and the resistance of plant, especially heat-resisting quantity can be significantly improved by being overexpressed the gene.

Description

Sorghum resistance related gene SbERECTA and its coding albumen and application

Technical field

The present invention relates to genetic engineering fields, more particularly to a sorghum resistance related gene SbERECTA, its volume Code albumen and its application in plant stress-resistance.

Background technique

Sorghum (Sorghum biocolor L.Moench) is a kind of good forage grass source of animal husbandry, can both do herbage It herds, and fine manipulation of green tea leaves feeding, ensiling and hay can be cradled.In recent years, due to overgrazing, soil drifting, water erosion, salinization of soil are aggravated, Pasture animal husbandry based on natural meadow can be sayed without advantage.Develop forage sorghum, is made less to alleviating due to raiseeing more grass At ecology of grassland unbalance be of great significance.The main cultivation sorghum variety genetic resources in China is abundant, major traits (biological production Amount, grain yield etc.) variation amplitude is big, excavates and identification sorghum key function gene, and to cultivating, biological yield is high, resistance is strong Forage sorghum new varieties, solving the agricultural development of land resource desert, livestock breed aquatics, biological new energy development etc. has Significance.

The degeneration-resistant mechanism of crop is the composite defense reaction that polygenes participates in, including the perception of extraneous adverse circumstance signal and transmitting machine System, molecules in response mechanism of environment stress etc..Adverse circumstance will affect the various physiological and biochemical procedures of crop, such as inhibition photosynthesis, Change cell membrane stability, change hormone and secondary metabolites synthesis, even results in plant death.Therefore, research sorghum is degeneration-resistant Functional gene, for increasing the photosynthetic efficiency of sorghum, the biological yield for improving sorghum is of great significance.

ERECTA gene can regulate and control the stomata development of arabidopsis, influence the transpiration efficiency and photosynthetic efficiency of plant, increase The heat resistance of plant is an important functional gene.ERECTA albumen is a receptoroid kinases, is primarily involved in regulation plant Cell division and tissue development.In arabidopsis after overexpression ERECTA gene, Stoma of Leaves density is reduced, and leaf area increases Greatly, water use efficiency (WUE) improves, and plant height increases, and biomass dramatically increases, further study show that ERECTA gene participates in Plant organ is built up, cell expands and iuntercellular correlative connection.In addition, ERECTA gene can influence diphosphoribulose carboxylase (Rubisco) carbonation efficiency and photoelectron transfer ability, adjust the photosynthesis of plant, the PdERECTA cloned from poplar Gene is transferred to arabidopsis, and arabidopsis stomatal frequency reduces, and transpiration efficiency increases, and photosynthetic rate improves, and biomass increases.ERECTA Gene also takes part in the disease resistance of regulation plant, enhances plant heat resistance, the resistance of non-host's property is such as generated to rice blast, excessive Expression significantly improves the heat resistance and biomass of tomato and rice.Therefore, ERECTA gene is not only involved in regulation stomatal frequency, shadow The photosynthesis and biomass of plant are rung, and participates in the disease resistance and heat resistance of regulation plant, is a multi-functional gene.So And so far, there is not yet forage crop, such as the report of sorghum, Sorghum sudanense in relation to the gene.For deep anatomy sorghum SbERECTA (SbER) gene function, this research carry out separation and preliminary functional verification to SbER gene.

Summary of the invention

The present invention provides a kind of sorghum resistance correlation SbERECTA albumen.

Its are as follows: 1) protein (being made of 982 aa) that by SEQ ID No.1 shown in amino acid sequence form；Or 2) One or several amino acid are substituted, lack or added in the amino acid sequence shown in SEQ ID No.1 and are had same living The protein as derived from 1) of property.

It is a further object of the present invention to provide the SbERECTA bases for encoding above-mentioned sorghum resistance correlation SbERECTA albumen Cause.

SbERECTA gene has nucleotide sequence (being made of 3210 nucleotide) shown in SEQ ID No.2.This hair Bright sorghum resistance correlation SbERECTA gene is a gene being cloned into from sorghum.

It should be appreciated that those skilled in the art can disclosed amino acid sequence according to the present invention, not influencing, its is active Under the premise of, replace, lack and/or increase one or several amino acid, obtains the mutant nucleotide sequence of the albumen.

Therefore, sorghum resistance correlation SbERECTA albumen of the invention further includes amino acid sequence shown in SEQ ID No.1 Column are substituted, replace and/or increase one or several amino acid, have the same isoreactivity of sorghum resistance correlation SbERECTA albumen By the protein derived obtained protein of sorghum resistance correlation SbERECTA.Gene of the present invention includes the core of encoding said proteins Acid sequence.Furthermore, it is to be understood that in view of the degeneracy of codon and the preferences of different plant species codon, art technology Personnel can according to need using the codon for being suitble to particular species expression.

It is a further object to provide the plant expression vector for carrying SbERECTA gene, the plant expression Carrier is pRRes14-gSbER.

SbERECTA gene of the invention, the protein sequence and corn, rice, sorghum, wheat, barley, two fringes that it is encoded Similitude similarity between the amino acid sequence of false bromegrass, arabidopsis etc. is protected very much in 80% or more, LRR region amino acid sequence It keeps；The amino acid sites that middle part is about 570 to 600 are the trans-membrane regions of the albumen, poor with the amino acid sequence of other species It is different larger, thus it is speculated that the transmembrane region function difference of different plant species RLKs is larger；The amino acid of 3 ' ends about 268 is leucine/Soviet Union The kinase region of propylhomoserin, sequence is very conservative, including typical glycine (G), lysine (K) and aspartic acid (D), these knots Structure domain and cell division and increment, Apoptosis and differentiation have important relationship.These results imply SbERECTA for regulation Sorghum growth and development has key effect.In sorghum different tissues organ, the expression difference of SbERECTA gene is very big: In bennet tissue, SbERECTA gene expression amount is very high；In young fringe, SbERECTA gene expression amount is higher, but young stem, In leaf sheath and ovary, SbERECTA gene expression amount is substantially reduced, and in spire, anther and seed, destination gene expression amount It is lower, the expression of SbERECTA gene is nearly no detectable in root.These results illustrate SbERECTA gene immature There is biggish transcription amount, and on the ground in the tender tissue of quick differentiation and development in histoorgan, transcription amount is extremely abundant, secretly Show SbERECTA sorghum seedling development early stage just take part in plant development and Morphogenesis at.In ABA, BRs, GA₃With Under IAA processing, the relative expression quantity of sorghum endogenous gene SbActin is very stable, and variation is unobvious, and target gene SbERECTA Relative expression quantity is larger, and differential expression level reaches extremely significant within the different disposal period, and with the HORMONE TREATMENT time Extend, the presentation of SbERECTA gene expression amount first increases the trend reduced afterwards.

The present invention is gene constructed on expression vector pMD18-T, acquiring pRRes14-gSbER overexpression by SbERECTA Carrier, and expand in Escherichia coli numerous.By agrobacterium mediation converted method, the SbERECTA that pRRes14-gSbER is carried Gene is transferred in model plant arabidopsis, obtains transformation of Arabidopsis thaliana plant.The result shows that SbERECTA early stage just takes part in plant Development and Morphogenesis at, have the function of improve transgenic arabidopsis high-temperature stability.

The present invention also provides the cloning vector containing SbERECTA nucleotide sequence or its segment or all kinds of expression vectors, contain There is the host cell of the carrier, planted containing the conversion plant cell and transgenosis of the nucleotide sequence or its specific fragment Object.

It is a further object to provide SbERECTA genes and its coding Protein S bERECTA to improve Genes For Plant Tolerance Application in inverse property, especially high-temperature stability.

The beneficial effects of the present invention are:

(1) sorghum SbERECTA gene and its coding Protein S bERECTA are provided；

(2) stress resistance of plant, especially high-temperature stability are improved by being overexpressed SbERECTA gene in plant.

Therefore, stress resistance of plant, especially high-temperature stability can be improved in the albumen of SbERECTA gene and its coding.

Detailed description of the invention

Fig. 1 is the pRRes14-gSbER recombinant vector of sorghum resistance related gene SbERECTA in the embodiment of the present invention 1 Structural schematic diagram.

Fig. 2 is SbERECTA gene cDNA of the present invention and amino acid sequence analysis schematic diagram.

Fig. 3 is the structural domain schematic diagram of the conserved region sorghum resistance related gene SbERECTA.Wherein, LRRNT is bright ammonia The N-terminal of sour enrichment region；LRR Domain is leucine series connection area；Transmembrane Domain is transmembrane region；S_TKc is silk Propylhomoserin/Serineprotein kinase area, catalysis region；NP_BIND is ATP-binding site (being rich in lysine residue)；ACT_SITE For proton acceptor sites (being rich in asparagicacid residue).

Fig. 4 is that cDNA the and gDNA sequence of SbERECTA gene analyzes schematic diagram.

Fig. 5 is SbERECTA amino acid alignment.Wherein, black line connects SbERECTA leucine area and silk ammonia Acid/threonine kinase differentiation is isolated.Target cross indicates SbERECTA and the highly conserved amino of other species RLKs family Acid sequence；Arrow indicates the typical amino acid in SbERECTA kinases area.It is as follows that each compares species abbreviation number: TaER=is small Wheat；Hv=barley；Bd=two fringe false bromegrass；SbER=sorghum；Zm=corn；Os=rice；Rc=castor-oil plant；Vv=grape；Gm =soybean；At=arabidopsis；Mt=clover；Cs=cucumber；Pt=poplar；Sm=Selaginella tamariscina；Pp=moss.

Fig. 6 is sorghum resistance related gene SbERECTA tissue specific expression histogram.

Fig. 7 is expression pattern lines figure of the sorghum resistance related gene SbERECTA under exogenous hormone.

Fig. 8 is the plant photo of 4 periods after the Arabidopsis thaliana Seedlings high-temperature process for turning SbERECTA gene.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..

Embodiment 1: the extraction of sorghum resistance related gene SbERECTA

1, experimental material

In April, 2016, sowing sorghum variety Tx623B in Anhui Science and Technology College plantation, (known sorghum variety, can be from educating Kind unit purchase).Young root, young stem, spire and leaf sheath sample are taken in seedling stage, take young fringe, bennet, anther and ovary coming into bloom Sample takes seed sample in the maturity period, quick-frozen in liquid nitrogen, saves backup in -80 DEG C of refrigerators, to analyze SbERECTA base Because of the expression characterization in sorghum different tissues, and separate SbERECTA gene.

Meanwhile in sorghum Tx623B seed sowing soil (Nutrition Soil: vermiculite=2:1), in illumination box culture.Culture 26 DEG C of case environment temperature, humidity 50%-70%, daylight 16h, night illumination 8h, 525 μm of ol.s of light intensity^-1.m^-2.10d with Afterwards, by seedling, gently band soil is taken out from soil, and clear water is rinsed well, not destroy root system, after clearing up moisture with filter paper, at once It is respectively placed in ABA (100 μM/L), BRs (0.75 μM/L), GA₃It is cultivated in (30mM/L) and IAA (10 μM/L) solution, control group It is handled using clear water.The processing period is 0h, 1h, 2h, 4h, 6h, 12h, for 24 hours, after 48h and 60h, control group and processing group difference Sampling mixes the root, stem and leaf of plant when sampling, and each sample takes three plants, repeats as biology, liquid nitrogen flash freezer, -80 DEG C It saves backup, analyzes expression pattern of the SbERECTA gene under four kinds of exogenous hormones.

2, experimental method

The extraction of 2.1 sorghum RNA and the synthesis of cDNA

(1) RNA extracts (phenol-chloroform-isoamyl alcohol method)

1. liquid nitrogen grinding plant tissue, is added RNA extracting solution, oscillator is fullyd shake；

2. chloroform/isoamyl alcohol (24:1) of 0.5ml is added, continue vortex 30sec, 4 DEG C of centrifugation 5min；

3. upper strata aqueous phase is transferred in the centrifuge tube of another new 2.0ml, be added equal volume (about 1ml) chloroform/ Isoamyl alcohol (24:1), vortex 30s, 4 DEG C of centrifugation 5min；

4. upper strata aqueous phase is transferred in the centrifuge tube of a new 2.0ml, micro- LiCl (4M) that equal volume is added is mixed After even in refrigerator 4 DEG C be incubated overnight；

5. 4 DEG C of centrifugation 20min discard supernatant, the alcohol of 1ml 70% is added, rinsing precipitating is sufficiently dried in draught cupboard Afterwards, DNA impurity in sample is removed；

It is mixed 6. phenol/chloroform/isoamyl alcohol (25:24:1) that the same volume is added is vortexed, is centrifuged 5min；Supernatant is transferred to In new centrifuge tube；

7. chloroform/isoamyl alcohol (24:1) of the same volume is added, it is vortexed and mixes, is centrifuged 5min；Supernatant be transferred to it is new from In heart pipe；

8. the dehydrated alcohol of 2.5 volumes and the NaOAc solution (3M pH=5.2) of 1/10 volume is added, 1 is small at -80 DEG C When；

9. 4 DEG C of centrifugation 20min, discard supernatant, the alcohol of 1ml 70% rinses precipitating, abandons supernatant, sufficiently dry in the air in draught cupboard It is dry, 20 μ l H are added₂ODepc dissolution；

10. Nanodrop measures RNA concentration, 1 μ g RNA sample is taken, agarose electrophoresis detection saves RNA sample at -80 DEG C It is spare in refrigerator.

(2) synthesis and detection of cDNA

The total serum IgE of 5 μ g is taken, is utilized (Oligo dT)_12-18Primer, as former state, synthesis step is referring to TaKaRa by the cDNA of synthesis The PrimeScript of company^TMII 1st Strand cDNA Synthesis Kit kit (6120A) specification.Synthesis CDNA sample dispenses after diluting 5 times with DEPC water, and -20 DEG C of refrigerators save.

2.2SbERECTA separation and sequence analysis

(1) SbERECTA sequence is analyzed

The TaERECTA sequence (Gene Bank accession JQ599261.2) for the wheat that reference NCBI is announced, Analysis is compared in Phytozome (https: //phytozome.jgi.doe.gov/pz/portal.html#) database, is obtained The cDNA sequence and gDNA sequence of the SbERECTA gene of sorghum.SbERECTA family amino acid translation site uses Geneious 6.0.5 software speculates, the functional structure domain analysis of SbERECTA family using Conserved Domain Search (http: // ) and SMART (http://smart.embl- www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi Heidelberg.de) database etc. carries out.

(2) expression pattern under SbERECTA Tissue-specific expression and exogenous hormone

Using using under implementation quantitative (qRT-PCR) detection SbERECTA Tissue-specific expression and exogenous hormone Expression pattern, the primer of target gene SbERECTA are SbERECTAQ-F:GCCAGCCGTGCCAAGCTACATC (SEQ ID ) and SbERECTAQ-R:CTCCCTGCATCCTTCAACTCCC (SEQ ID No.4) No.3.The primer of reference gene SbActin For SbActin-F:ACGGCCTGGATGGCGACGTACATG (SEQ ID No.5) and SbActin-R: GCAGAAGGACGCCTACGTTGGTGAC(SEQ ID No.6).Using ABI7300 (Applied Biosystems, USA) into Row qRT-PCR analysis, technology repeats each sample three times, and reaction system is 20 μ l, including 10 μ 2 × SYBR of l MIX；0.3μl Upstream primer (10 μM)；Swim primer (10 μM) in 0.3 downstream μ l；The 2 diluted cDNA templates of μ l (50ng), ddH2O supply 20 μ l.Instead Answer program are as follows: 95 DEG C of 20s；95 DEG C of 5s, 61 DEG C of 30s, 40 circulations.QRT-PCR data are analyzed referring to following formula (1):

E in formula_XIt is the amplification efficiency of target gene primer, E_RThe amplification efficiency of house-keeping gene primer, C_t, X and C_t, R points It Wei not the C that is collected in PCR reaction process of purpose gene and house-keeping gene_tValue.If there is multiple house-keeping genes, then formula denominator For geometric mean (the Ramakers et al.2003 of multiple house-keeping gene evaluations；Olsen et al.2012；Rieu and Powers 2009).The significance difference analysis of SbERECTA gene expression amount utilizes SPSS19.0 software (IBM SPSS Statistics,USA)。

(3) separation of SbERECTA full length gene cDNA and gDNA sequence

1) gene magnification

Referring to the primers of SbERECTA in Phytozome database, cDNA sequence separation primer is respectively SbERcDNA-F:GCGAGAACCCACGAAACCAACC (SEQ ID No.7) and SbERcDNA-RF: CCCATGATTCCAACACGGCAAA(SEQ ID No.8)；

GDNA sequence is larger, is expanded using segmentation, and centre is equipped with the region of one section of overlapping, and then digestion links together, Primer is respectively SbERgDNA-1F:ATGCCTGTCCGCAGCTCAGTGGCC (SEQ ID No.9) and SbERgDNA-1R: GGTGGTATCGACCCAGTTAACTTATTG(SEQ ID No.10)；SbERgDNA-2F: CAATAAGTTAACTGGGTCGATACCACC (SEQ ID No.11) and SbERgDNA-2R: CCCCAGACCTCTCTCTCTTCCCTAC(SEQ ID No.12)；Using high fidelity enzyme (KOD FX-101, TOYOBO), with height The bennet tissue cDNA and DNA of fine strain of millet Tx623B is template, carries out PCR amplification, and cDNA sequence length is 3376bp, and gDNA sequence is long Spending is 7276bp, response procedures: 94 DEG C of 2min, 35cycles (98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 1mim/kb), 72 DEG C 10min, 4 DEG C of preservations.The purifying of target fragment PCR product illustrates to carry out according to hundred Tyke kits (DP1711).

2) connection conversion

1. needing to facilitate subsequent in product end plus ployA tail if PCR product after purification is flat end TA clone's connection, 10 μ l:PCR product of reaction system, 6 μ l, Taq buffer, 2 μ l, Taq polymerase 1 μ l, dATP After 0.8 μ l of (10mM) 0.2 μ l, ddH2O, room temperature or 37 DEG C of reaction 30min, 70 DEG C of continuation 30min terminate reaction；

2. above-mentioned PCR product is connected on cloning vector pMD18-T (6011, TaKaRa), reaction system is 12 μ l, 1 μ l, solution I solution of PCR product 6 μ l (appropriate adjustment), pMD18-T Cloning Vector, 5 μ l；

3. mixing gently, 16 DEG C of connection 30min, reaction terminates, centrifuge tube is placed on ice；

4. plus connection product is transferred in 50 μ l Top10 competent cells and (slowly thaws on ice in advance)；

5. 42 DEG C of heat shock 40s, are immediately placed on 2min on ice；

6. 150 μ l liquid LB (antibiotic-free), 200rpm are added, 37 DEG C of incubation 1h；

7. the bacterium solution of conversion is uniformly applied and (containing 100 μ g/ml Amp+, and is attached with 40 μ l 20mg/ on LB plate The IPTG of the X-gal of ml and 60 μ l 200mg/ml)；

8. picking white colonies, it is spare to shake bacterium.

3) digestion is identified, sequencing

50 μ l bacterium solutions are drawn respectively, and 12- is cultivated in 5ml LB liquid medium (containing 100 μ g/ml Amp+ of final concentration) 16h extracts specification (9760, TaKaRa) according to plasmid and extracts plasmid, and double digestion identification, clip size is correct, sample presentation sequencing Analysis, sequencing is correct, and SbERECTA full length gene cDNA and gDNA sequence fragment separates successfully.

4) conversion of overexpression vector building and arabidopsis

SbERECTA gene gDNA sequence is inserted into over-express vector (containing Ubi constitutive promoter) by segmentation, is acquired PRRes14-gSbER over-express vector (specific structure is shown in Fig. 1).In conjunction with the multiple cloning sites of carrier, using restriction enzyme NcoI and The plasmid of the first segment of NotI digestion gSbER-1, the plasmid of restriction enzyme NcoI the second segment of digestion gSbER-2, recycling digestion Product.20 μ l:NotI of endonuclease reaction system, 1 μ L, 10 × Buffer H 2 μ L, 0.1%BSA 2 μ L, 0.1%X- 100 2 μ L, Plasmid DNA≤1 μ g, ddH2O polishing to 20 μ L；1 μ L, 10 × Buffer K of NcoI, 22 μ L of μ L, 0.1%BSA, Plasmid DNA≤1 μ g, ddH2O polishing to 20 μ L.After endonuclease reaction 4h, purification and recovery digestion products.Obtain SbERECTA gene GDNA sequence two parts product gSbER-1 and gSbER-2 segment.

Using restriction enzyme NcoI and NotI digestion over-express vector pRRes14, the gSbER-1 segment that then will be recycled above It is inserted into linear carrier pRRes14,15 μ l:5 × Ligase Reaction Buffer of coupled reaction system, 3 μ l, carrier DNA 1.5 3 μ l of μ l, ddH2O of 1.5 μ l, 6 μ l, T4DNA Ligase (1units) of piece segment DNA, 4 DEG C of connections overnight.Sequencing analysis obtains Obtain pRRes14-gSbER-1 recombinant vector.

Using restriction enzyme NcoI digestion pRRes14-gSbER-1 recombinant vector, the gSbER-2 segment that then will be recycled above It is inserted into linear carrier pRRes14.101-gSbER-1, linked system is as above.Sequencing analysis, NcoI and NotI double digestion identification, is obtained It obtains pRRes14-gSbER and is overexpressed recombinant vector.

PRRes14-gSbER plasmid is extracted, Agrobacterium is converted, then infects arabidopsis (Colombia 0 using Agrobacterium Type) titbit, obtain SbERECTA be overexpressed transgenic arabidopsis seed, step sizing plant three generations, obtain T3 band transgenosis Arabidopsis seed, it is spare.

5) transgenic arabidopsis high-temperature process

The transgenic arabidopsis in T3 generation and wildtype Arabidopsis thaliana seed are sowed respectively on empty MS culture medium, tissue culture room is raw After long 10d, chooses root long isometric transgenic arabidopsis and wildtype Arabidopsis thaliana seedling and be transplanted to soil (Nutrition Soil: leech respectively Stone=2:1) in, in illumination box culture, 26 DEG C of incubator environment temperature, humidity 50%-70%, daylight 16h, night Illumination 8h, 525 μm of ol.s-1.m-2 of light intensity.After 5 days, 42 DEG C of high-temperature process different period, rear incubator of placing continues to train Support 10d, the resurrection situation of observation statistics seedling.

Embodiment 2: the cDNA sequence analysis of sorghum resistance related gene SbERECTA and its amino acid of coding albumen Sequence analysis

1, cDNA sequence and the analysis of amino propylhomoserin

SbERECTA gene cDNA and amino acid sequence analysis schematic diagram of the present invention are shown in Fig. 2, SbERECTA gene cDNA sequence The reading frame open containing 2949bp is arranged, 983 amino acid are encoded, predicts molecular weight of albumen 106KDa, isoelectric point (pI) is 6.21. The albumen discovery of its coding is further analyzed, SbERECTA belongs to typical receptoroid kinase families (RLKs), and N-terminal is leucine Concatenated rich region receives extracellular signal transduction, and it is transmembrane region that centre, which is hydrophobic structure, and C-terminal is serine/threonine egg White kinases area.The structural domain schematic diagram of the conserved region sorghum resistance related gene SbERECTA is shown in Fig. 3, wherein LRRNT is bright ammonia The N-terminal of sour enrichment region；LRR Domain is leucine series connection area；Transmembrane Domain is transmembrane region；S_TKc is silk Propylhomoserin/Serineprotein kinase area, catalysis region；NP_BIND is ATP-binding site (being rich in lysine residue)；ACT_SITE (asparagicacid residue is rich in for proton acceptor sites.SbERECTA gene gDNA sequence contains 7076bp.

2, it is analyzed the characteristics of alternative splicing

CDNA the and gDNA sequence analysis schematic diagram of SbERECTA gene is shown in Fig. 4, compares SbERECTA gene cDNA sequence After gDNA sequence, it is found that the gDNA sequence of SbERECTA has 27 exons, 26 intrones.However, SbERECTA CDNA sequence there are different intrones and exon spliced body form.In-depth analysis shows SbERECTA in gDNA sequence There are the spliced body forms of three kinds of cDNA sequences in the section 4127-4497bp: 1. Sb-cDNA1, aobvious outside length 3110,27 Son, containing there are two intrones；2. Sb-cDNA2, length 2880bp, 26 exons, intronless；3. Sb-cDNA3, length 2949,27 exons, intronless.

3, SbERECTA family amino acid sequence structure is analyzed

It in Phytozome v9.0 database, is compared, downloads using sorghum SbERECTA amino acid sequence as probe RLKs family amino acid of the similarities such as corn, rice, sorghum, wheat, barley, two fringe false bromegrass, arabidopsis 80% or more, Specific amino acid alignment figure is shown in Fig. 5, wherein black line connects SbERECTA leucine area and serine/threonine Kinases differentiation is isolated.Target cross indicates SbERECTA and the highly conserved amino acid sequence of other species RLKs family；Arrow Head indicates the typical amino acid in SbERECTA kinases area.It is as follows that each compares species abbreviation number: TaER=wheat；Hv=is big Wheat；Bd=two fringe false bromegrass；SbER=sorghum；Zm=corn；Os=rice；Rc=castor-oil plant；Vv=grape；Gm=soybean；At =arabidopsis；Mt=clover；Cs=cucumber；Pt=poplar；Sm=Selaginella tamariscina；Pp=moss.Multiple Sequence Alignment prediction: SbERECTA As other species, LRR region amino acid sequence is very conservative；The amino acid sites that middle part is about 570 to 600 are the albumen Trans-membrane region, it is larger with the amino acid sequence differences of other species, thus it is speculated that the transmembrane region function difference of different plant species RLKs It is larger；The amino acid of 3 ' ends about 268 is leucine/threonine kinase region, and sequence is very conservative, including typical sweet ammonia Sour (G), lysine (K) and aspartic acid (D), these structural domains and cell division and increment, Apoptosis and differentiation have weight It is related to.These results imply that SbERECTA has key effect for regulation sorghum growth and development.

Embodiment 3:SbERECTA Tissue-specific expression

In each developmental stage, sorghum different tissues organ material is collected, using SbActin reference gene as reference, It studies SbERECTA gene relative expression's situation, specific expressed histogram in different sorghums tissue and sees Fig. 6.As a result, it has been found that In sorghum different tissues organ, the expression difference of SbERECTA gene is very big: in bennet tissue, SbERECTA gene table It is very high up to measuring；In young fringe, SbERECTA gene expression amount is higher, but in young stem, leaf sheath and ovary, SbERECTA gene Expression quantity is substantially reduced, and in spire, anther and seed, destination gene expression amount is lower, is nearly no detectable in root The expression of SbERECTA gene.These results illustrate that SbERECTA gene has biggish transcription in immature histoorgan Amount, and on the ground in the tender tissue of quick differentiation and development, transcription amount is extremely abundant, implies that SbERECTA is developed in sorghum seedling Early stage just take part in plant development and Morphogenesis at.

Expression pattern of the embodiment 4:SbERECTA under exogenous hormone

In ABA, BRs, GA₃Under IAA processing, the relative expression quantity of sorghum endogenous gene SbActin is very stable, and variation is not Obviously, and target gene SbERECTA relative expression quantity is larger, and differential expression level reaches extremely aobvious within the different disposal period It writes, and with the extension of HORMONE TREATMENT time, expression pattern of the sorghum resistance related gene SbERECTA under exogenous hormone Lines figure is shown in Fig. 7, and the presentation of SbERECTA gene expression amount first increases the trend reduced afterwards.

Embodiment 5:SbERECTA overexpresses the identification of arabidopsis strain high-temperature stability

The Arabidopsis thaliana Seedlings for turning SbERECTA gene handle tetra- times of 4h, 8h, 12h and 15h under 42 DEG C of high temperature respectively Section.(Fig. 8) as the result is shown, after high temperature 4h, transgenic line and WT strain survival rate are all 60%；In high temperature 8h and After 12h, the survival rate of transgenic line and WT strain is all 40% and 20% respectively；After high temperature 15h, transgenic line Also it is respectively 40% and 20% with WT strain survival rate, illustrates under high temperature stress, overexpression SbERECTA gene is intended Southern mustard seedling has very strong vitality, can resist the injury of high temperature bring to a certain extent, and wildtype Arabidopsis thaliana is not With this ability, the high-temperature stability of transgenic arabidopsis is can be improved in the overexpression of preliminary proof SbERECTA gene.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Anhui Science and Technology College

<120>sorghum resistance related gene SbERECTA and its coding albumen and application

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<170> SIPOSequenceListing 1.0

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<211> 982

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<213>sorghum (Sorghum bicolor)

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Met Pro Val Arg Ser Ser Val Ala Met Thr Thr Thr Ala Ala Arg Ala

1 5 10 15

Leu Val Ala Leu Leu Leu Val Ala Val Ala Val Ala Asp Asp Gly Ala

20 25 30

Thr Leu Val Glu Ile Lys Lys Ser Phe Arg Asn Val Gly Asn Val Leu

35 40 45

Tyr Asp Trp Ala Gly Asp Asp Tyr Cys Ser Trp Arg Gly Val Leu Cys

50 55 60

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

65 70 75 80

Leu Glu Gly Glu Ile Ser Pro Ala Val Gly Ser Leu Lys Ser Leu Val

85 90 95

Ser Ile Asp Leu Lys Ser Asn Gly Leu Ser Gly Gln Ile Pro Asp Glu

100 105 110

Ile Gly Asp Cys Ser Ser Leu Arg Thr Leu Asp Phe Ser Phe Asn Asn

115 120 125

Leu Asp Gly Asp Ile Pro Phe Ser Ile Ser Lys Leu Lys His Leu Glu

130 135 140

Asn Leu Ile Leu Lys Asn Asn Gln Leu Ile Gly Ala Ile Pro Ser Thr

145 150 155 160

Leu Ser Gln Leu Pro Asn Leu Lys Ile Leu Asp Leu Ala Gln Asn Lys

165 170 175

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

180 185 190

Tyr Leu Gly Leu Arg Gly Asn His Leu Glu Gly Ser Leu Ser Pro Asp

195 200 205

Met Cys Gln Leu Thr Gly Leu Trp Tyr Phe Asp Val Lys Asn Asn Ser

210 215 220

Leu Thr Gly Val Ile Pro Asp Thr Ile Gly Asn Cys Thr Ser Phe Gln

225 230 235 240

Val Leu Asp Leu Ser Tyr Asn Arg Phe Thr Gly Pro Ile Pro Phe Asn

245 250 255

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

260 265 270

Thr Gly Pro Ile Pro Ser Val Ile Gly Leu Met Gln Ala Leu Ala Val

275 280 285

Leu Asp Leu Ser Tyr Asn Gln Leu Ser Gly Pro Ile Pro Ser Ile Leu

290 295 300

Gly Asn Leu Thr Tyr Thr Glu Lys Leu Tyr Ile Gln Gly Asn Lys Leu

305 310 315 320

Thr Gly Ser Ile Pro Pro Glu Leu Gly Asn Met Ser Thr Leu His Tyr

325 330 335

Leu Glu Leu Asn Asp Asn Gln Leu Thr Gly Ser Ile Pro Pro Glu Leu

340 345 350

Gly Arg Leu Thr Gly Leu Phe Asp Leu Asn Leu Ala Asn Asn His Leu

355 360 365

Glu Gly Pro Ile Pro Asp Asn Leu Ser Ser Cys Val Asn Leu Asn Ser

370 375 380

Phe Asn Ala Tyr Gly Asn Lys Leu Asn Gly Thr Ile Pro Arg Ser Leu

385 390 395 400

Arg Lys Leu Glu Ser Met Thr Tyr Leu Asn Leu Ser Ser Asn Phe Ile

405 410 415

Ser Gly Ser Ile Pro Ile Glu Leu Ser Arg Ile Asn Asn Leu Asp Thr

420 425 430

Leu Asp Leu Ser Cys Asn Met Met Thr Gly Pro Ile Pro Ser Ser Ile

435 440 445

Gly Ser Leu Glu His Leu Leu Arg Leu Asn Leu Ser Lys Asn Gly Leu

450 455 460

Val Gly Phe Ile Pro Ala Glu Phe Gly Asn Leu Arg Ser Val Met Glu

465 470 475 480

Ile Asp Leu Ser Tyr Asn His Leu Gly Gly Leu Ile Pro Gln Glu Leu

485 490 495

Glu Met Leu Gln Asn Leu Met Leu Leu Lys Leu Glu Asn Asn Asn Ile

500 505 510

Thr Gly Asp Leu Ser Ser Leu Met Asn Cys Phe Ser Leu Asn Ile Leu

515 520 525

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

530 535 540

Phe Thr Arg Phe Ser Pro Asp Ser Phe Leu Gly Asn Pro Gly Leu Cys

545 550 555 560

Gly Tyr Trp Leu Gly Ser Ser Cys Arg Ser Thr Gly His His Glu Lys

565 570 575

Pro Pro Ile Ser Lys Ala Ala Ile Ile Gly Val Ala Val Gly Gly Leu

580 585 590

Val Ile Leu Leu Met Ile Leu Val Ala Val Cys Arg Pro His Arg Pro

595 600 605

Pro Ala Phe Lys Asp Val Thr Val Ser Lys Pro Val Arg Asn Ala Pro

610 615 620

Pro Lys Leu Val Ile Leu His Met Asn Met Ala Leu His Val Tyr Asp

625 630 635 640

Asp Ile Met Arg Met Thr Glu Asn Leu Ser Glu Lys Tyr Ile Ile Gly

645 650 655

Tyr Gly Ala Ser Ser Thr Val Tyr Lys Cys Val Leu Lys Asn Cys Lys

660 665 670

Pro Val Ala Ile Lys Lys Leu Tyr Ala His Tyr Pro Gln Ser Leu Lys

675 680 685

Glu Phe Glu Thr Glu Leu Glu Thr Val Gly Ser Ile Lys His Arg Asn

690 695 700

Leu Val Ser Leu Gln Gly Tyr Ser Leu Ser Pro Val Gly Asn Leu Leu

705 710 715 720

Phe Tyr Asp Tyr Met Glu Cys Gly Ser Leu Trp Asp Val Leu His Glu

725 730 735

Gly Ser Ser Lys Lys Lys Lys Leu Asp Trp Glu Thr Arg Leu Arg Ile

740 745 750

Ala Leu Gly Ala Ala Gln Gly Leu Ala Tyr Leu His His Asp Cys Ser

755 760 765

Pro Arg Ile Ile His Arg Asp Val Lys Ser Lys Asn Ile Leu Leu Asp

770 775 780

Lys Asp Tyr Glu Ala His Leu Thr Asp Phe Gly Ile Ala Lys Ser Leu

785 790 795 800

Cys Val Ser Lys Thr His Thr Ser Thr Tyr Val Met Gly Thr Ile Gly

805 810 815

Tyr Ile Asp Pro Glu Tyr Ala Arg Thr Ser Arg Leu Asn Glu Lys Ser

820 825 830

Asp Val Tyr Ser Tyr Gly Ile Val Leu Leu Glu Leu Leu Thr Gly Lys

835 840 845

Lys Pro Val Asp Asn Glu Cys Asn Leu His His Leu Ile Leu Ser Lys

850 855 860

Thr Ala Ser Asn Glu Val Met Asp Thr Val Asp Pro Asp Ile Gly Asp

865 870 875 880

Thr Cys Lys Asp Leu Gly Glu Val Lys Lys Leu Phe Gln Leu Ala Leu

885 890 895

Leu Cys Thr Lys Arg Gln Pro Ser Asp Arg Pro Thr Met His Glu Val

900 905 910

Val Arg Val Leu Asp Cys Leu Val Asn Pro Asp Pro Pro Pro Lys Pro

915 920 925

Ser Ala His Gln Leu Pro Gln Pro Ser Pro Ala Val Pro Ser Tyr Ile

930 935 940

Asn Glu Tyr Val Ser Leu Arg Gly Thr Gly Ala Leu Ser Cys Ala Asn

945 950 955 960

Ser Thr Ser Thr Ser Asp Ala Glu Leu Phe Leu Lys Phe Gly Glu Ala

965 970 975

Ile Ser Gln Asn Met Glu

980

<210> 2

<211> 3210

<212> DNA

<213>sorghum (Sorghum bicolor)

<400> 2

gctctgcccc cattaaggca gggcaggcag aggaggcagg ctcgctgcag cacgcttcgc 60

gctccatcgt cgctgtcctc ttctccctgt aatgtcactc ccccgatgcc tgtccgcagc 120

tcagtggcca tgacgacgac ggccgcccgt gctctcgtcg ccctcctcct cgtcgccgtc 180

gccgtcgccg acgatggggc gacgctggtg gagatcaaga agtccttccg caacgtcggc 240

aacgtactgt acgattgggc cggcgacgac tactgctcct ggcgcggcgt cctgtgcgac 300

aacgtcacat tcgccgtcgc tgcgctcaac ctctctggcc tcaaccttga gggcgagatc 360

tctccagccg tcggcagcct caagagcctc gtctccatcg atctgaagtc aaatgggcta 420

tccgggcaga tccctgatga gattggtgat tgttcatcac ttaggacgct ggacttttct 480

ttcaacaact tggatggcga cataccattt tctatatcaa agctgaagca cctggagaac 540

ttgatattga agaacaacca gctgattggt gcgatcccat caacattgtc acagctccca 600

aatttgaaga ttttggattt ggcacaaaac aaactgactg gggagatacc aaggcttatc 660

tactggaatg aggttcttca atatctgggc ttacggggca atcatttaga aggaagcctc 720

tctcctgata tgtgccagct gactggcctt tggtactttg atgtgaagaa caatagcttg 780

accggggtga taccagacac cattgggaac tgtacaagtt ttcaagtctt ggatttgtct 840

tacaaccgct ttactggacc aatcccattc aacattggtt tcctacaagt ggctacacta 900

tccttgcaag ggaacaagtt caccggtcca attccttcag taattggtct tatgcaggct 960

ctcgctgttc tagatctgag ttacaaccaa ttatctggtc ctataccatc aatactaggc 1020

aacttgacat acactgagaa gctgtacatc caaggcaata agttaactgg gtcgatacca 1080

ccagagttag gaaatatgtc aacacttcat tacctagaac tgaacgataa tcaacttact 1140

gggtcaattc caccagagct tggaaggcta acaggcttgt ttgacctgaa ccttgcgaat 1200

aaccacctgg aaggaccaat tcctgacaac ctaagttcat gtgtgaatct caatagcttc 1260

aatgcttatg gcaacaagtt aaatgggacc attcctcgtt cgttgcggaa acttgaaagc 1320

atgacctatt taaatctgtc atcaaacttc ataagtggct ctattcctat tgagttatca 1380

aggatcaaca atttggacac gctggattta tcctgtaaca tgatgactgg tccaattcca 1440

tcatcaattg gcagcctaga gcatctattg agacttaact tgagcaagaa tggtctagtt 1500

ggattcatcc ccgcggagtt tggtaatttg aggagtgtca tggagattga tttatcctat 1560

aatcaccttg gtggcctgat tcctcaagaa cttgaaatgc tgcaaaacct gatgttgcta 1620

aaactggaaa ataacaatat aactggtgat ctgtcgtctc tgatgaactg cttcagcctc 1680

aatatcttaa atgtgtcgta caataatttg gctggtgttg tccctgctga caacaacttc 1740

acacggtttt cacctgacag ctttttaggt aatcctggac tctgtggata ctggcttggt 1800

tcgtcgtgtc gttccactgg ccaccacgag aaaccgccta tctcaaaggc tgccataatt 1860

ggtgttgctg tgggtggact tgttatcctc ttgatgatct tagtagctgt ttgcaggcca 1920

catcgtccac ctgcttttaa agatgtcact gtaagcaagc cagtgagaaa tgctcccccc 1980

aagctggtga tccttcatat gaacatggcc cttcatgtat acgatgacat aatgaggatg 2040

actgagaact tgagtgagaa atacatcatt ggatacgggg cgtcaagtac agtttataaa 2100

tgtgtcctaa agaattgcaa accggtggca ataaaaaagc tgtatgccca ctacccacag 2160

agccttaagg aatttgaaac tgagcttgag actgttggta gcatcaagca ccggaatcta 2220

gtcagccttc aagggtactc attatcacct gttgggaacc tcctctttta tgattatatg 2280

gaatgtggca gcttatggga tgttttacat gaaggttcat ccaagaagaa aaaacttgac 2340

tgggagactc gcctacggat tgctcttggt gcagctcaag gccttgctta ccttcaccat 2400

gactgcagtc cacggataat tcatcgggat gtaaaatcaa agaatatact ccttgacaaa 2460

gattatgagg cccatcttac agactttgga attgctaaga gcttatgtgt ctcaaaaact 2520

cacacatcaa cctatgtcat gggaactatt ggctacattg atcctgagta cgcccgcact 2580

tcccgtctca acgaaaagtc tgatgtctac agctatggca ttgttctgct ggagctgctg 2640

actggcaaga agccagtgga caacgagtgc aatctccatc acttgatcct atcgaagacg 2700

gcaagcaacg aggtcatgga taccgtggac cctgacatcg gggacacctg caaggacctc 2760

ggcgaggtga agaagctgtt ccagctggcg ctcctttgca ccaagcggca accctcggac 2820

cgaccgacga tgcacgaggt ggtgcgcgtc ctggactgcc tggtgaaccc ggacccgccg 2880

ccaaagccgt cggcgcacca gctgccgcag ccgtcgccag ccgtgccaag ctacatcaac 2940

gagtacgtca gcctgcgggg caccggcgct ctctcctgcg ccaactcgac cagcacctcg 3000

gacgccgagc tgttcctcaa gttcggcgag gccatctcgc agaacatgga gtagggaaga 3060

gagagaggtc tggggagttg aaggatgcag ggagtagtgg gagtagctga ctgacatttt 3120

gcgggatgca ggaggagatt aacatgggga actcagtagg gtgttggtta actgtaaaaa 3180

aagtcatgtg cctctagagc agcagtagag 3210

<210> 3

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gccagccgtg ccaagctaca tc 22

<210> 4

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ctccctgcat ccttcaactc cc 22

<210> 5

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

acggcctgga tggcgacgta catg 24

<210> 6

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gcagaaggac gcctacgttg gtgac 25

<210> 7

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gcgagaaccc acgaaaccaa cc 22

<210> 8

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

cccatgattc caacacggca aa 22

<210> 9

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

atgcctgtcc gcagctcagt ggcc 24

<210> 10

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

ggtggtatcg acccagttaa cttattg 27

<210> 11

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

caataagtta actgggtcga taccacc 27

<210> 12

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

ccccagacct ctctctcttc cctac 25

Claims (10)

1. a kind of sorghum SbERECTA albumen, are as follows:

1) protein that the amino acid sequence shown in SEQ ID NO.1 forms, or

2) one or several amino acid are substituted, lack or added in the amino acid sequence shown in SEQ ID NO.1 and have The same active protein as derived from 1).

2. encoding the sorghum SbERECTA gene of albumen described in claim 1.

3. gene according to claim 2, which is characterized in that nucleotide sequence is as shown in SEQ ID NO.2.

4. the carrier containing gene described in Claims 2 or 3.

5. the host containing carrier described in claim 4.

6. conversion plant cell or genetically modified plants containing gene described in Claims 2 or 3 or its specific fragment.

7. gene described in claim 2 or 3 is adjusting the application in stress resistance of plant.

8. application according to claim 7, which is characterized in that the application is the overexpression sorghum in plant SbERECTA gene improves stress resistance of plant.

9. gene described in claim 2 or 3 is adjusting the application in plant heat-resisting quantity.

10. application according to claim 9, which is characterized in that the application is the overexpression sorghum in plant SbERECTA gene improves plant heat-resisting quantity.