CN107325162A

CN107325162A - SPL genes and its application in enhancing Heat Resistance of Plant performance

Info

Publication number: CN107325162A
Application number: CN201610285625.6A
Authority: CN
Inventors: 陈晓亚; 朝鲁门; 刘尧倩; 曹俊峰; 毛颖波
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2016-04-29
Filing date: 2016-04-29
Publication date: 2017-11-07
Anticipated expiration: 2036-04-29
Also published as: CN107325162B; WO2017185854A1

Abstract

Application the invention provides SPL genes and its in enhancing Heat Resistance of Plant performance.Specifically, the invention provides a kind of function and purposes of SPL genes, SPL genes (the SPL1 or SPL12) key factor necessary to maintaining the reaching maturity and sprouts of seed under the adaptation and tolerance of high temperature, hot conditions that is plant, overexpression SPL1 or SPL12 transgenic arabidopsis can strengthen tolerance of the floral organ to extreme and gentle high temperature.The SPL genes of the present invention are with a wide range of applications, and the crops coerced to cultivate high temperature resistance provide a new way.

Description

SPL genes and its application in enhancing Heat Resistance of Plant performance

Technical field

The present invention relates to botany field, relate more specifically to the application in SPL genes and its enhancing Heat Resistance of Plant performance.

Background technology

Temperature is that one of key physical factor of vital movement is influenceed on the earth.The whole food chain of ambient temperature effect and life State system.High temperature stress has a negative impact to nearly all aspects such as plant growth, development, breeding and yield, including seed Germination is obstructed, blade and the shrivelled of tender shoots, the sunburn of branch and stem, the aging of blade and come off, the growth inhibition of root and stem, fruit Real discoloration and the reduction of yield, or even cause the death of whole plant.All plant tissues are all easily hindered by heat stress Evil, wherein the sensitiveness highest of reproductive organs, just can cause crop yield drastically to decline, very in the flowering period temperature rise several years To the total crop failure of whole agricultural production.Compared to vegetative growth phase, plant reproductive growth period is more sensitive to high temperature, but most of agricultures The florescence of crop is just in summer high temperature period, therefore the research of plant reproductive growth period heat shock response mechanism is extremely urgent.With Global climate warms, and researching high-temperature becomes ever more important to the mechanism that growth and development of plants and crop yield influence.

Plant all forms the signal path of sense ambient temperature change during evolution, and adjusts the metabolism of itself and thin Born of the same parents' function, prevents the body that environment-stress is caused from damaging.These different signal pathways have tissue specificity and species special Property, especially there is very big difference in plant reproductive growth period and vegetative growth phase heat shock response signal.At present to plant reproductive The research of growth period heat resistance remains in form, physiology and biochemistry level, and still few to its Study on Molecular Mechanism, more can not The regulated and control network of reference.

Therefore, this area is in the urgent need to the gene of exploitation regulation and control plant heat resistance property, and its application of function is carried out corresponding Research.

The content of the invention

It is an object of the invention to provide a kind of SPL genes of regulation and control Heat Resistance of Plant performance and its in enhancing plant heat resistance property Application in energy.

In the first aspect of the present invention, there is provided the purposes of a kind of SPL genes or its encoding proteins, the SPL genes choosing From SPL1 genes, SPL12 genes or its combination, and described SPL genes or its encoding proteins are used for the use that is selected from the group On the way：

(a) it is used for the reagent or composition for preparing enhancing Heat Resistance of Plant performance；

(b) it is used to strengthen the heat resistance of plant.

In another preference, described plant is selected from the group：Grass and crucifer.

In another preference, described plant is selected from the group：Arabidopsis, tobacco, paddy rice and wheat.

In another preference, described plant is arabidopsis.

In another preference, described SPL genes are SPL1 genes and SPL12 genes.

In another preference, described " enhancing Heat Resistance of Plant performance " includes the one or more performances being selected from the group：

(i) heat resistance of inflorescence is strengthened；

The germination rate of solid seed under (i i) enhancing hot environment；

(i i i) is used to strengthen tolerance of the plant to hot environment；

(iv) heat shock of plant is responded under enhancing hot environment；

(v) antioxygenic property of plant under hot environment is strengthened；

(vi) heat resistance of enhancing plant roots, stem, and/or leaf；

(vii) setting percentage of plant under hot environment is strengthened.

In another preference, described hot environment refers to the environment that temperature is 30-50 DEG C, preferably 32-45 DEG C Environment, the more preferably environment for 35-42 DEG C, such as 30 DEG C, 35 DEG C, 37 DEG C, 42 DEG C.

In another preference, the heat resistance of described enhancing inflorescence includes：Strengthen the survival of inflorescence under hot environment Rate, and/or flowering rate.

In another preference, described enhancing plant includes to the tolerance of hot environment：Plant is improved in high temperature ring Survival rate under border.

In another preference, described enhancing heat shock response includes the gene expression that up-regulation is selected from the group：

WRKY15、WRKY25、WRKY33、WRKY39、ERF020、ERF1、ERF2、RAP2.1、ERF054、CRJ1、 ABRE1, RAP2.6 or its combination.

In another preference, the antioxygenic property of described plant refers to the ability that plant removes internal ROS.

In another preference, the antioxygenic property of described enhancing plant refers to the SOD expression for improving plant and/or living Property.

In another preference, described " enhancing Heat Resistance of Plant performance " includes the resistance to of enhancing vegetation period and reproduction period Hot property.

In another preference, described SPL genes include wild type SPL genes and saltant type SPL genes.

In another preference, described saltant type includes the mutant form that the function of encoding proteins after mutation does not change Formula (i.e. function is identical or essentially identical with wild type encoding proteins).

In another preference, the polypeptides of described saltant type SPL gene codes is more with wild type SPL coded by said gene Peptide is identical or essentially identical.

In another preference, described saltant type SPL genes are included compared with wild type SPL genes, and homology >= 80% (preferably >=90%, more preferably >=polynucleotides 95%).

In another preference, described saltant type SPL genes are included in the 5 ＇ ends and/or 3 ＇ ends of wild type SPL genes Truncate or add the polynucleotides of 1-60 (preferably 1-30, more preferably 1-10) nucleotides.

In another preference, described gene includes genomic DNA, cDNA, and/or mRNA.

In another preference, the CDS sequences such as SEQ ID NO. of described SPL1 genes:Shown in 1.

In another preference, the encoding proteins such as SEQ ID NO. of described SPL1 genes:Shown in 2.

In another preference, the genome sequence such as SEQ ID NO. of described SPL1 genes:Shown in 3.

In another preference, the CDS sequences such as SEQ ID NO. of described SPL12 genes:Shown in 4.

In another preference, the encoding proteins such as SEQ ID NO. of described SPL12 genes:Shown in 5.

In another preference, the genome sequence such as SEQ ID NO. of described SPL12 genes:Shown in 6.

In another preference, described SPL gene sources are in plant, preferably from grass and cross Flower section plant, more preferably derives from：Arabidopsis, tobacco, paddy rice and wheat.

In the second aspect of the present invention, there is provided a kind of method for changing Heat Resistance of Plant performance, including step：

(a) construction of external source is imported into plant cell, wherein described construction contain external source SPL gene orders, Promote the exogenous nucleotide sequence of SPL gene expressions or suppress the exogenous nucleotide sequence of SPL gene expressions, so as to be led Enter the plant cell of external source construction；

(b) plant cell for the importing external source construction for obtaining previous step, regenerates plant：With

(c) optionally the plant of the regeneration is identified, so as to obtain the plant of heat resistance change；

Wherein, the SPL genes are selected from SPL1 genes, SPL12 genes or its combination.

In another preference, the plant that described heat resistance changes refers to that compared with mother plant, heat resistance changes Become.

In another preference, the SPL gene orders of the external source are also comprising the promoter being connected with ORF series of operations And/or terminator.

In another preference, described promoter is selected from the group：Constitutive promoter, tissue-specific promoter, lure Conductivity type promoter and strong promoter.

In another preference, described composition promoter includes 35S promoter.

In another preference, described exogenous nucleotide sequence includes the nucleotides sequence for disturbing the SPL gene expressions Row.

In another preference, described exogenous nucleotide sequence includes RNA interference sequences.

Strengthen the method for Heat Resistance of Plant performance there is provided a kind of in the third aspect of the present invention, methods described includes following Step：In the plant, promote the expression of SPL genes or promote the activity of SPL albumen, wherein, the SPL genes are selected from SPL1 genes, SPL12 genes or its combination.

In another preference, methods described includes the accelerator for giving plant SPL genes or the polypeptide of its coding.

In another preference, methods described includes importing the SPL genes of external source into plant.

In another preference, methods described includes step：

(i) plant or plant cell are provided；With

SPL gene orders are imported the plant or plant cell by (i i), so that the plant or plant that obtain transgenosis are thin Born of the same parents.

In another preference, methods described includes step：

(a) Agrobacterium for the expression vector for carrying SPL gene orders is provided；

(b) plant cell or tissue or organ are contacted with the Agrobacterium in step (a), so that SPL gene order Plant cell is transferred to, and is incorporated on the chromosome of plant cell；

(c) selection has been transferred to the plant cell or tissue or organ of SPL gene orders；With

(d) it is plant by the plant cell or tissue in step (c) or neomorph.

In the fourth aspect of the present invention there is provided the purposes of a kind of SPL genes or the adjusting control agent of its encoding proteins, for adjusting The heat resistance of plant is controlled, or for preparing the reagent or composition of regulation and control Heat Resistance of Plant performance, wherein, the SPL genes choosing From SPL1 genes, SPL12 genes or its combination.

In another preference, described composition includes agriculturally useful compositions.

In another preference, described adjusting control agent includes accelerator, inhibitor.

In another preference, described adjusting control agent is accelerator, and described regulation and control refer to enhancing plant heat resistance property Energy.

In another preference, described adjusting control agent is inhibitor, and described regulation and control refer to decrease plant heat resistance property Energy.

In another preference, described adjusting control agent includes micromolecular compound or nucleic acid material.

In another preference, described nucleic acid material is selected from the group：MiRNA, shRNA, siRNA or its combination.

In the fifth aspect of the present invention there is provided a kind of transfer-gen plant, SPL genes are imported in the transfer-gen plant, The SPL genes are selected from SPL1 genes, SPL12 genes or its combination.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.

Brief description of the drawings

Fig. 1 shows the tissue expression feature of SPL1 and SPL12 genes.

Figure 1A shows the gus gene expression pattern of SPL1 promoters driving.pSPL1:GUS transfer-gen plants GUS is dyed： Upper strata is from left to right followed successively by seedling, root maturation zone, the tip of a root and the stem that 7d is grown on 1/2MS culture mediums；Lower floor is from left to right To grow 20d seedling, inflorescence and Fruit pod.

Figure 1B shows the gus gene expression pattern of SPL12 promoters driving.pSPL12:GUS transfer-gen plants GUS contaminates Color, the same Figure 1A of order.

Fig. 1 C-1F respectively illustrate pSPL1:The dyeing of GUS transfer-gen plant flower development phase different phases (stage1-16) As a result.Wherein, Fig. 1 C show period 1-12；Fig. 1 D and Fig. 1 E show period 13-14, and red arrow indicates pollen；Fig. 1 F Show period 15-16.

Fig. 1 G show the result of expressions of the Real-Time PCR detections SPL1 and SPL12 in spending, including petal (stage12 and before) and flower of having bloomed (stage13 and afterwards).

Fig. 2 shows SPL1, and influence flower development and seed production are lowered in SPL12 expression.

Fig. 2A shows col-0, spl1, spl12 and spl1spl12 reproductive stage phenotype.Wherein, spl1spl12 is spent Dysplasia, part abortion；

Fig. 2 B show that the col-0 same day blooms (stage13) phenotype.

Fig. 2 C show that the spl1spl12 same day spends (stage13) phenotype, and flower can not normally deploy.

Fig. 2 D show to choose out the external morphology structure in D after calyx with sharp tweezer；Spl1spl12 calyx adhesions.

Fig. 2 E show that col-0 flower maturities come off process.

Fig. 2 F show that spl1sgpl12 flower maturities come off process.

Fig. 2 G show genetically modified plants 35S:Myc-gSPL1, RNAi-1 and RNAi-2 phenotype.

Fig. 2 H show Realtime-PCR detection genetically modified plants 35S:GSPL1 (line1 and line4), 35S:myc- GSPL1 (line4 and line12), pSPL1:SPL1 in SPL1 (line3 and line4) and mutant spl1 and spl1spl12 Expression.

Fig. 2 I show Realtime-PCR detection genetically modified plants 35S:GSPL12 (line2 and line4), RNAi-1, SPL12 expression in RNAi-2 and mutant spl12 and spl1spl12.(RNAi-1 and RNAi-2 are under spl1 backgrounds Silence SPL12 two transgenosis systems)

Fig. 2 J are shown in Col-0, spl1-1spl12-1, ox-MS1d-1, ox-MS1d-4, ox-MS1d-7 and ox-MS1c-1 SPL12 tables in SPL1 expression quantity and Col-0, spl1-1spl12-1, ox-S12c-3 and ox-S12c-4, RNA1-1, RNAi-2 Up to amount.

Fig. 3 shows that stem terminal inflorescence averagely spends openness situation analysis daily.

Col-0 under the conditions of Fig. 3 A show 22 DEG C and 30 DEG C, spl1-1spl12-1 and ox-SPL1 stem terminal inflorescences are daily Flower openness (NOF) average value.Wherein, n=20-30, ± SD；* represents p-value≤0.01, * and represents p-value≤0.05

Fig. 3 B show NOF average values relative changing value in Fig. 3 A.22 DEG C of absolute change value divided by corresponding plant are averaged NOF is obtained.

Fig. 4 shows that stem terminal inflorescence spends openness statistical analysis and morphological feature daily.

Col-0 under the conditions of Fig. 4 A show 22 DEG C and 30 DEG C, spl1-1spl12-1 and ox-SPL1 stem terminal inflorescences are daily Flower openness statistics.One of 0,1 and 4d statistic, n=20-30, three reproducible results of displaying are shown in figure.

Col-0 under the conditions of Fig. 4 B show 22 DEG C and 30 DEG C, spl1-1spl12-1 and ox-SPL1 stem terminal inflorescence form Feature.

Fig. 5 shows tolerated of the thaliana flower organ to thermal extremes.

Fig. 5 A show 37 DEG C of processing 1d to ox-SPL1, the shadow of col-0 and spl1-1spl12-1 flower organ morphology features Ring.

Fig. 5 B show that Real-time PCR detect SPL1 in ox-SPL1, col-0 and spl1-1spl12-1 plant inflorescences Expression.

Fig. 5 C show 37 DEG C of processing 1d to ox-SPL1, the influence of col-0 and spl1-1spl12-1 floral organ survival rates. Wherein, 22 DEG C of growth 1d will be restored to after 37 DEG C of processing 1d, observe and count completely shrivelled and can not normally bloom Inflorescence number, using the plant that does not process as control, its survival rate is 100%；(n=20-30, ± SD；* represents p- Value≤0.01, * represents p-value≤0.05)

Fig. 6 shows influence of the high-temperature process to arabidopsis floral SOD activity, seed yield and germination rate.

Fig. 6 A show influence of the high-temperature process to col-0 and spl1-1spl12-1 inflorescences SOD activity.Wherein, CK generations 22 DEG C of table, HS represents 37 DEG C of processing 4h, and the arabidopsis of growth 5 weeks is material.(3times, ± SD；* represent p-value≤ 0.01)；

Fig. 6 B show influence of the high-temperature process to col-0 and spl1-1spl12-1 seed yields.Wherein, CK represents 22 DEG C, HS recovers 22 DEG C of growths after representing 42 DEG C of processing 1h.(n=18, ± SD；* represents p-value≤0.01, * and represents p- value≤0.05)；

Fig. 6 C show influence of the high-temperature process to col-0 and spl1-1spl12-1 seed germination rates.Wherein, CK is represented 22 DEG C, HS represent 30 DEG C growth 7 days after count seed germination rate.(n=60-70,3times, ± SD；* represent p-value≤ 0.01, * represents p-value≤0.05)

Fig. 7 shows the heat shock response expression of the Real-time PCR detection candidate transcription factors.

Fig. 7 A show the heat shock response expression of WRKY transcription factors.

Fig. 7 B and Fig. 7 C show the heat shock response expression of ERF class transcription factors.Wherein, total serum IgE is taken respectively from 37 DEG C 1 and 4h inflorescence is managed, using the inflorescence under the conditions of untreated 22 DEG C as control (0h).

Fig. 8 displays are overexpressed SPL1 or SPL12 and improve plant heat resistance.

Fig. 8 A show wild type of 42 DEG C of processing to growth 14 days, spl1-1spl12-1 and genetically modified plants ox- The influence of MS1c-1, ox-MS1d-1 and ox-S12c-3 growth.2d 42 DEG C of high-temperature process are carried out to the plant of growth 14 days, it is extensive Taken pictures after multiple 5 days.

Fig. 8 B show wild type of 42 DEG C of processing to growth 14 days, spl1-1spl12-1 and genetically modified plants ox- The influence of MS1c-1, ox-MS1d-1 and ox-S12c-3 survival rate.2d 42 DEG C of high-temperature process are carried out to the plant of growth 14 days, Survival rate is counted after recovering 5 days.

Fig. 8 C show wild type of 42 DEG C of processing to growth 5 weeks, spl1-1spl12-1 and genetically modified plants ox-MS1d- The influence of 1, ox-MS1d-4 and ox-MS1d-7 inflorescence growths.Plant after 42 DEG C of high-temperature process 5h is carried out to the plant of growth 5 weeks Thing state.

Fig. 8 D show wild type of 42 DEG C of processing to growth 5 weeks, spl1-1spl12-1 and genetically modified plants ox-MS1d- The influence of 1, ox-MS1d-4 and ox-MS1d-7 inflorescence growths.The plant of growth 5 weeks is carried out after 42 DEG C of high-temperature process 5h to flower Sequence compromise state is counted.

Experiment in Fig. 8 A- Fig. 8 D shows representational once result at least in triplicate, in figure.

Fig. 8 E show that genetically modified plants ox-MS1c-1, ox-MS1d-1, ox-S12c-3 and ox-S12c-3 are coerced in high temperature Compel lower with higher seed production.It is left：Always in 22 DEG C of growths；In：Plant recovery after 42 DEG C of processing 5h of 5 weeks will be grown To 22 DEG C of growths；It is right：The plant for growing 32 days is moved into 30 DEG C of incubators and grows to maturation.

Fig. 9 A show SPL1, SPL12, SPL2, SPL9 and SPL11 sequence analysis result.Wherein, underlined in red generation The conservative SBP-box domains of table.

Fig. 9 B show SPL1, SPL12, SPL2, SPL9 and SPL11 evolution tree graph.

Figure 10 shows the homologous bases of SPL1 and SPL12 in arabidopsis (At), tobacco (Nt), paddy rice (Os) and wheat (Ta) The protein sequence comparison result of cause.Wherein, underlined in red represents conservative SBP-box domains, and green represents Ankrin Repeat (ANK) domain, blueness represents cross-film (TM) domain.

Embodiment

The present inventor is by extensively and in depth studying, and be found that a kind of can regulate and control the SPL of Heat Resistance of Plant performance first Gene.Experiment shows, SPL1 and SPL12 are thaliana flower organs to maintaining seed under the adaptation and tolerance of high temperature, hot conditions Reach maturity and key factor necessary to sprouting, floral organ can be strengthened by being overexpressed SPL1 or SPL12 transgenic arabidopsis Official further demonstrates SPL1 and SPL12 and intends south by the regulation and control of a plurality of signal pathway to the tolerance of extreme and gentle high temperature The resistance to thermal process of mustard.This result of study is to understand how plant is protected under heat stress conditions and maintain its normal reproductive growth Development, and provided for further further investigation plant reproductive growth period heat shock response mechanism with the crops for cultivating high temperature resistance stress One New Century Planned Textbook.The present invention is completed on this basis.

Specifically, the present invention identify the SBP-box transcription factor genes SPL1 that participates in regulation and control arabidopsis heat shock response and SPL12.SPL1 and SPL12 protein sequence homologies are up to 72%, the wide expression in arabidopsis tissue, and flower development late period high table Reach.Both participate in flower development late period maturation jointly is shown to the phenotypic analysis of SPL1 and SPL12 afunction mutant, Spl1-1spl12-1 double-mutants floral organ shows the phenotype of sensitivity to extreme and gentle high temperature, causes its high temperature stress Under seed production significantly reduced compared to wild type, it is that thaliana flower organ tolerance's high temperature stress must to illustrate SPL1 and SPL12 The key gene needed, and functional redundancy.

In order to explore response mechanism of the arabidopsis reproductive stage to high temperature, and find the heat shock of SPL1 and SPL12 regulation and control The gene of response approach and correlation, we are warm in control with the wild type and spl1-1spl12-1 double-mutants plant for growing 5 weeks The inflorescence material progress full-length genome transcription spectrum sequencing spent under the conditions of (22 DEG C) and heat treatment (42 DEG C, 1h).By to wild type With the analysis of heat shock response difference expressing gene in spl1-1spl12-1 double-mutants, it was demonstrated that SPL1 and SPL12 missing is tight Transcriptional control of the arabidopsis to high temperature stress is have impact on again.

Importantly, present invention obtains the transgenosis that some SPL1 with high heat resistance and SPL12 are overexpressed Arabidopsis, compared to wild type, they in reproductive stage and vegetative growth phase show heat-resisting phenotype, under high temperature stress Obtain higher yield.

Term

As used herein, term " functional redundancy " refers to that two or more (n) gene has an identical function, missing or Reduce the expression quantity of wherein one or more (n-1), the phenotype that individual is still acted normally.

As used herein, term " specific expressed " refers to target gene specific time and/or specific in plant The expression of tissue.

As used herein, " external source " or " heterologous " refers to the two or more pieces nucleic acid or protein sequence of separate sources Between relation.For example, if the combination of promoter and objective gene sequence is not usually naturally occurring, promoter for It is external source for the target gene.Particular sequence for its cell inserted or is " external source " for organism.

SPL genes

As used herein, term " SPL genes ", " heat resistance related gene ", " gene of the present invention " can with used interchangeably, All referring to the gene with regulation and control Heat Resistance of Plant performance of the present invention.

In a preference, gene of the present invention refers to SPL1 genes and/or SPL12 genes.More preferably, SPL genes of the present invention From arabidopsis.

SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) one class plant of gene code is distinctive to be included The albumen for the highly conserved SBP-box being made up of 76 amino acid.Arabidopsis contains 17 SPL genes, wherein, SPL1 and SPL12 does not contain miR156 regulating and controlling sequences, there is no function to report so far.

Present invention firstly discovers that SPL1 and SPL12 are thaliana flower organs under the adaptation and tolerance of high temperature, hot conditions Maintain reaching maturity and key factor necessary to sprouting for seed, overexpression SPL1 or SPL12 transgenic arabidopsis can be with Strengthen tolerance of the floral organ to extreme and gentle high temperature, and further demonstrate SPL1 and SPL12 by a plurality of signal pathway Regulate and control the resistance to thermal process of arabidopsis.This result of study is to understand how plant is protected under heat stress conditions and maintain its normal Reproductive growth is developed, and is further further investigation plant reproductive growth period heat shock response mechanism and the agriculture for cultivating high temperature resistance stress Crop provides a New Century Planned Textbook.

The SPL1 genes and/or SPL12 genes of the present invention can be DNA form or rna form.DNA form include cDNA, Genomic DNA or artificial synthesized DNA.Genomic DNA can be and SEQ ID NO.:3rd, the sequence shown in 6 is identical either The variant of degeneracy.The DNA of the present invention can be single-stranded or double-strand, and DNA can be coding strand or noncoding strand.Coding The coding region sequence of mature polypeptide can be with SEQ ID NO.:1st, the coding region sequence shown in 4 is identical or variation of degeneracy Body.

As used herein, " variant of degeneracy " refers to that coding has SEQ ID NO. in the present invention:2nd, 5 albumen Matter, but with SEQ ID NO.:1st, shown in 4 coding region sequence or SEQ ID NO.:3rd, the genome sequence shown in 6 is differentiated Nucleotide sequence.

Encode SEQ ID NO.:2nd, the polynucleotides of 5 mature polypeptide include：The coded sequence of encoding mature polypeptide； The coded sequence of mature polypeptide and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide And non-coding sequence.

Term " polynucleotides of coded polypeptide " can be included encoding the polynucleotides of this polypeptide or also include The polynucleotides of additional code and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotides, it is encoded has many of identical amino acid sequence with the present invention The fragment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant that naturally occurs or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this Known to field, allelic variant is the alternative forms of a polynucleotides, it be probably one or more nucleotides substitution, Missing or insert, but not from substantially change its coding polypeptide function.

The invention further relates to have at least 50% between above-mentioned sequence hybridization and two sequences, preferably at least 70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention many The interfertile polynucleotides of nucleotides.In the present invention, " stringent condition " refers to:(1) compared with low ionic strength and higher temperature Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant during (2) hybridization, such as 50% (v/v) first Acid amides, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or the phase same sex of (3) only between two sequences is at least 90% More than, just hybridize when more preferably more than 95%.Further, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.:2 Shown mature polypeptide has identical biological function and activity.

The invention further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " is extremely Less containing 15 nucleotides, preferably at least 30 nucleotides, preferably more preferably at least 50 nucleotides, at least 100 nucleosides It is more than acid.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid to determine and/or separate coding heat resistance related polypeptide Polynucleotide.

The polypeptide of SPL gene codes

As used herein, term " heat resistance related polypeptide ", " polypeptide of the present invention ", " polypeptide of SPL gene codes ", " albumen of SPL gene codes ", " SPL polypeptides " can have regulation and control Heat Resistance of Plant performance with used interchangeably all referring to the present invention Polypeptide.

In a preference, polypeptide of the present invention refers to SPL1 and/or SPL12.More preferably, polypeptide of the present invention is southern from intending Mustard.

The polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.The present invention's is more Peptide can be native purified product, or chemical synthesis product, or using recombinant technique from protokaryon or eucaryon host (example Such as, bacterium, yeast, higher plant, insect and mammalian cell) middle generation.Host according to used in recombinant production scheme, this The polypeptide of invention can be glycosylated, or can be nonglycosylated.The polypeptide of the present invention may also include or do not include starting Methionine residues.

Present invention additionally comprises the fragment of SPL polypeptides, derivative and analog.As used herein, term " fragment ", " derivative Thing " and " analog " refer to the polypeptide for the natural SPL polypeptides identical biological function or activity for being kept substantially the present invention.This The polypeptide fragment of invention, derivative or the like can be that (i) has one or more conservative or non-conservative amino acid residues (excellent Select conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be may not be by losing Cipher coding is passed, or (ii) has the polypeptide of substituted radical, or (iii) mature polypeptide in one or more amino acid residues Formed polypeptide is merged with another compound (such as extending the compound of polypeptide half-life period, such as polyethylene glycol), or (iv) additional amino acid sequence is fused to polypeptide formed by this peptide sequence (such as targeting sequencing or secretion sequence or for pure Change the sequence or proprotein sequence of this polypeptide, or fusion protein).According to teaching herein, these fragments, derivative and analog Belong to scope known to those skilled in the art.

In preference, polypeptide of the present invention refers to the SEQ ID NO. with heat resistance:The polypeptide of 2 sequences.Also include tool There are with SPL polypeptide identical functions, SEQ ID NO.:The variant form of 2 sequences.These variant forms include (but not limiting In)：One or more (being usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid lack Lose, insert and/or replace, and it (is usually within 20, preferably to add one or several in C-terminal and/or N-terminal Within 10, more preferably within 5) amino acid.For example, in the art, being carried out with similar nature or similar amino acid During substitution, it will not generally change the function of protein.Again such as, one or several amino acid are added in C-terminal and/or N-terminal The function of protein will not generally also be changed.The term also includes the active fragment and reactive derivative of SPL polypeptides.

The variant form of the polypeptide includes：Homologous sequence, conservative variant, allelic variant, natural mutation, induction Mutant, can be with the albumen coded by the DNA of the DNA hybridization of SPL polypeptides and using anti-under the conditions of high or low stringency Many peptide or proteins that the antiserum of SPL polypeptides is obtained.Present invention also offers other polypeptides, such as comprising SPL polypeptides or its fragment Fusion protein.In addition to the almost polypeptide of total length, present invention includes the soluble fragments of SPL polypeptides.Generally, the fragment At least about 10 continuous amino acids with SPL peptide sequences, typically at least about 30 continuous amino acids, preferably at least about 50 Individual continuous amino acid, more preferably at least about 80 continuous amino acids, most preferably at least about 100 continuous amino acids.

The present invention also provides SPL polypeptides or its analog.The difference of these analogs and natural SPL polypeptides can be amino Difference on acid sequence or the difference on the modified forms of sequence is not influenceed, or had both at the same time.These polypeptides include Natural or induction genetic variant.Induction variant can be obtained by various technologies, such as by radiation or exposed to mutagenesis Agent and produce random mutagenesis, can also be by the technology of site-directed mutagenesis or other known molecular biology.Analog also includes tool There is the analog of the residue (such as D- amino acid) different from natural L-amino acids, and with non-naturally occurring or synthesis ammonia The analog of base acid (such as β, gamma-amino acid).It should be understood that the present invention polypeptide be not limited to it is above-mentioned enumerate it is representational many Peptide.

Modification (not changing primary structure generally) form includes：The chemically derived form such as acetyl of inner or in vitro polypeptide Change or carboxylated.Modification also includes glycosylation.Modified forms also include have phosphorylated amino acid residue (such as phosphotyrosine, Phosphoserine, phosphothreonine) sequence.Also include being modified improving its anti-proteolysis performance or optimizing molten Solve the polypeptide of performance.

In the present invention, " SPL polypeptide conservative variations polypeptide " refers to and SEQ ID NO.:2 amino acid sequence is compared, and is had At most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are by the similar or close ammonia of property Base acid is replaced and forms polypeptide.In the albumen, when being replaced with similar nature or similar amino acid, generally will not Change the function of protein, C-terminal and/or end add one or several amino acid and will not generally also change protein Function.These conservative variation's polypeptides carry out amino acid substitution preferably based on following table and produced.

The homology of SPL albumen

Arabidopsis altogether containing 17 SPL genes, all SPL albumen all include by 76 amino acid constitute it is highly conserved SBP-box albumen.Arabidopsis SPL1 is 69%, SPL1 and SPL12 and other arabidopsis SPL with SPL12 albumen homologies Albumen, such as SPL2, SPL9 and SPL11 homology are respectively less than 20%, and sequence analysis result is as shown in following table and Fig. 9.

	SPL1	SPL12	SPL2	SPL9	SPL11
						SPL1	100	69	10	19	10
SPL12		100	8	15	10
						SPL2			100	59	69
SPL9				100	58
						SPL11					100

Arabidopsis, tobacco, the tetraploid rice of paddy rice and wheat

Arabidopsis, tobacco, the tetraploid rice result of paddy rice and wheat

The albumen sequence of SPL1 and SPL12 homologous genes in arabidopsis (At), tobacco (Nt), paddy rice (Os) and wheat (Ta) Row comparison result shows that SPL1 albumen homologies are between 50-62% between each species, and the homology between each species of SPL12 is in 55- 61% or so.

Recombinant technique and plant improvement

The full length sequence or its fragment of heat resistance related gene of the present invention generally can with PCR TRAPs, recombination method or Artificial synthesized method is obtained.For PCR TRAPs, can especially it be opened according to relevant nucleotide sequence disclosed in this invention Reading frame sequence is put to design primer, and with commercially available cDNA storehouses or as prepared by conventional method well known by persons skilled in the art CDNA storehouses as template, amplification and relevant sequence.When sequence is longer, it is often necessary to carry out twice or multiple PCR is expanded, Then the fragment again amplified each time is stitched together by proper order.

Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then by conventional method from the host cell after propagation isolated relevant sequence.

In addition, relevant sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.Generally, lead to After first synthesizing multiple small fragments, sequence very long fragment can be obtained by being then attached again.

At present, it is already possible to obtain encoding albumen of the present invention (or its fragment, or its derivative by chemical synthesis completely Thing) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.It is introduced into addition, be able to will be also mutated by chemical synthesis in protein sequence of the present invention.

Compiled the present invention also relates to the carrier of the polynucleotides comprising the present invention, and with the carrier or SPL polypeptides of the present invention The host cell that code sequence is produced through genetic engineering, and the method through recombinant technique generation polypeptide of the present invention.

Pass through conventional recombinant DNA technology (Science, 1984；224：1431), using the polynucleotide of the present invention Sequence can be used to express or produce the SPL polypeptides of restructuring.In general there are following steps：

(1) polynucleotides (or variant) of the present invention are used, or are converted with the recombinant expression carrier containing the polynucleotides Or suitable host cell of transduceing；

(2) host cell cultivated in suitable culture medium；

(3) separation, protein purification from culture medium or cell.

The polynucleotide sequence of the present invention can be plugged into recombinant expression carrier.Term " recombinant expression carrier " refers to this area Well known bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other carriers.In a word, As long as can be replicated in host and stably, any plasmid and carrier can be used.One key character of expression vector is logical Often contain replication orgin, promoter, marker gene and translation control element.

Method well-known to those having ordinary skill in the art, which can be used to build, contains polynucleotides of the present invention and suitable transcription/translation The expression vector of control signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..Institute The DNA sequence dna stated can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Expression vector also includes The ribosome bind site and transcription terminator of translation initiation.

In addition, expression vector preferably includes one or more selected markers, it is used to select conversion to provide Dihyrofolate reductase, neomycin resistance and the green fluorescence egg of the phenotypic character of host cell, such as eukaryotic culture In vain (GFP), or tetracycline or amicillin resistance for Escherichia coli.

The carrier of above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence is included, can be used for conversion suitable When host cell, allow it to marking protein.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；Or it is high Deng eukaryotic, such as plant cell (cells of such as crops and forestry plant).Representative example has：Escherichia coli, streptomycete Category, Agrobacterium；Fungal cell's such as yeast；Plant cell etc..

When the polynucleotides of the present invention are expressed in higher eucaryotic cells, if will when inserting enhancer sequence in the carrier Transcription can be strengthened.Enhancer is DNA cis-acting factors, generally about has 10 to 300 base-pairs, acts on and open Mover is to strengthen the transcription of gene.

Persons skilled in the art are aware that how to select appropriate carrier, promoter, enhancer and host cell.

It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art.When host is original When core biology is such as Escherichia coli, can absorb DNA competent cell can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method is carried out.When host is eucaryote, following DNA transfection methods are can select：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The methods such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as leaf disk method can also be used in conversion plant.For the plant of conversion Thing cell, tissue or organ can regenerate plant with conventional method, so as to obtain the plant of heat resistance change.

The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, culture medium used may be selected from various conventional mediums in culture.Under conditions of suitable for host cell growth Cultivated.After host cell growth is to appropriate cell density, with suitable method (such as temperature transition or chemical induction) The promoter of selection is induced, cell is further cultured for a period of time.

Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in the cell or on cell membrane.Such as Fruit needs, can be separated using its physics, chemistry and other characteristics by various separation methods and purification of Recombinant albumen.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：Renaturation process, the use of routine Protein precipitant processing (salting-out method), centrifugation, the broken bacterium of infiltration, hyperfiltration treatment, ultracentrifugation, sieve chromatography (gel filtration), Adsorption chromatography, ion-exchange chromatography, the knot of high performance liquid chroma- tography (HPLC) and other various liquid chromatography technologies and these methods Close.

The SPL polypeptides of restructuring are of use in many ways.For example for screening the compound with regulation and control heat resistance, polypeptide Or other parts.It can be used for finding valuable can suppress or promotion plant with the screening peptide library of the restructuring SPL polypeptides of expression The peptide molecule of heat resistance.

On the other hand, present invention additionally comprises there is specific polyclonal antibody and monoclonal antibody to SPL polypeptides, especially It is monoclonal antibody.The present invention not only includes complete monoclonal or polyclonal antibody, but also including with immunocompetent Antibody fragment or chimeric antibody.

The antibody of the present invention can be prepared by various technologies known to a person skilled in the art.For example, purifying SPL polypeptide gene products or its there is antigenic fragment, animal can be applied to induce the generation of polyclonal antibody. Each antibody-like of the present invention can utilize fragment or the functional areas of heat resistance relevant gene product, be obtained by common immunological techniques .These fragments or functional areas can prepare or utilize Peptide synthesizer synthesis using recombination method.With heat resistance dependency basis Because the antibody that the unmodified form of product is combined can be immunized with the gene outcome of production in prokaryotic (such as E.Coli) Animal and produce；The antibody (such as the albumen or polypeptide of glycosylation or phosphorylation) combined with posttranslational modification form, can be with very The gene outcome produced in nucleus (such as yeast or insect cell) is obtained animal is immunized.The antibody of anti-SPL polypeptides can For detecting the heat resistance related polypeptide in sample.

The invention further relates to the method for testing of quantitative and detection and localization heat resistance related polypeptide level.These experiments are these Known to field.The heat resistance related polypeptide level detected in experiment, is adjusted available for explanation heat resistance related polypeptide Control the function of heat resistance.

A kind of method in detection sample with the presence or absence of heat resistance related polypeptide is the specific antibody using SPL polypeptides Detected, it includes：Sample is contacted with SPL polypeptide specific antibodies；See whether to form antibody complex, form anti- Nanocrystal composition, which is meant that in sample, has heat resistance related polypeptide.

Part or all of the polynucleotides of the present invention can be fixed on microarray (microarray) or DNA as probe On chip (also known as " genetic chip "), the Differential expression analysis for analyzing gene in tissue.With the primer of SPL polypeptides Carry out the transcription product that RNA- polymerase chain reactions (RT-PCR) amplification in vitro also can detect SPL polypeptides.

Main advantages of the present invention include：

(a) Heat Resistance of Plant performance related gene and its coded polypeptide are provided；

(b) SPL genes of the invention can strengthen the heat resistance of plant；

(c) a kind of method of the heat resistance of targetedly enhancing plant is provided；

(d) the SPL genes and method that provide are applied to the genetic improvement of various plants.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are calculated by weight.

Universal method

A. arabidopsis thaliana genomic dna is extracted

Plant tissue 0.1g, plus lysis buffer 0.3mL are taken, is homogenized.Plus 0.3mL phenol/chloroform (1:1), mix.12, 000rpm centrifuges 5min, and supernatant is transferred to another centrifuge tube, adds 30 μ L 3M NaAc (pH 5.2) and 500 μ L absolute ethyl alcohols.It is mixed It is even.10min is centrifuged, precipitation is dissolved in 50 μ L TE (pH 8.0) through 70% ethanol wash, vacuum drying.

B. the extraction of arabidopsis total serum IgE

Draw materials (about 100mg) be fully ground in liquid nitrogen.It is transferred in 1.5mL centrifuge tubes, adds 1mL Trizol (Invitrogen, Cat.15596-018), is mixed, and room temperature places 5min.12,000rpm centrifugation 10min, abandon or adopt precipitation.Supernatant 200 μ L chloroforms of middle addition, are mixed, 12,000rpm centrifugation 10min.Supernatant is taken, 500 μ L isopropanol precipitatings RNA are added.12, 000rpm centrifuges 10min, and precipitation is washed with 70% ethanol, is dried in vacuo, is dissolved in 20-50 μ L H₂O(RNase free).Treat RNA All after dissolving, RNase free DNase (0.5-1 μ L), 37 DEG C of placement half an hour, 65 DEG C of inactivation 20min are added.With NANODROP 2000c (Thermo) determine RNA concentration.

C.cDNA reverse transcriptions

The reverse transcription of the first chains of PolyA mRNA using M-MLV Reverse Transcriptase systems (Invitrogen, Cat.C28025-021), reaction system is as follows：

System is sufficiently mixed, 65 DEG C of reaction 5min, is immediately placed on ice, is placed 5min.Then added into system：

System is sufficiently mixed, 37 DEG C of reaction 2min, is eventually adding 1 μ L M-MLV Reverse Transcriptase, is mixed Even, 37 DEG C of reactions 50min, 70 DEG C of reaction 15min inactivate reverse transcriptase, and 95 DEG C of heating 5min are placed on ice.Reverse transcription product QRT-PCR detections are can be directly used for after 3-10 times of dilution.

D.PCR reacts

Ex-Taq PCR reaction systems (20 μ L) are as follows：

The renaturation temperature and extension of time of PCR reactions are determined by primer and expanding fragment length.General reactions condition is：94 DEG C denaturation 5min；94 DEG C of denaturation 30s, 57 DEG C of renaturation 30s, 72 DEG C of extension 30s, expand 30 to 35 circulations；72 DEG C of insulations 10min.4 DEG C of insulations.

PCR primer sequence such as SEQ ID NO.:Shown in 7-16.

PrimeSTAR HS reaction systems (50 μ L) are as follows：

General reactions condition is：98 DEG C of denaturation 10sec；55 DEG C of renaturation 5 or 15s, 72 DEG C extension 1min/kb, amplification 30 to 35 circulations；4 DEG C of insulations.

E.Real-time-PCR is analyzed

Real-time RT-PCR detection use chimeric fluorescents method (Premix Ex Taq^TMII, TaKaRa, DRR041A).PCR primer sequence such as SEQ ID NO.:Shown in 11-16.Reaction system is as follows：

Referred to using arabidopsis S18 (AT1G07210) gene as internal standard.Data analysis uses Realplex v2.0 (Eppendorf, Hamburg, Germany).Experiment in triplicate, takes the average value and variance of each group of data, drawn a diagram.

F. vector construction

DNA agarose gel electrophoresis, the digestion of fragment, purifying and connection reference《Molecular cloning》(Sambrook and Russell, 2001) and related reagent and enzyme manufacturer operating instruction.The promoter and gene coded sequence of carrier construction Obtained using High fidelity PCR enzymatic amplification, TA clones and be sequenced to ensure that sequence is correct.

Embodiment 1

SPL1, SPL12 gene cloning and vector construction

By pBI121 35S promoter and NOS terminator by high-fidelity enzymatic amplification, respectively through EcoRI/SacI and PstI/HindIII is connected into pCAMBIA1300 multiple cloning sites, then by the 35S promoter of resistant gene NOS promoters (coming from pBI121) substitutes, and obtains pCAMBIA1300S.With pBSK-tag templates, high-fidelity enzymatic amplification 6 × myc fragments, product Through KpnI/SmaI enzyme cutting clones into pCAMBIA1300S carriers, pCAMBIA1300S-myc is obtained.Use PrimeSTAR HS Archaeal dna polymerase expands SPL1 genome sequences, and product is obtained through BamH1 enzyme cutting clones to pCAMBIA1300S-myc carriers 35S:myc-gSPL1.SPL12 genome sequence is expanded with PrimeSTAR HS archaeal dna polymerases, SPL12 products are cloned through TA JW819 (pCAMBIA3300 is obtained by transformation) carrier is connected to, p35S is obtained:gSPL12.

Embodiment 2

The tissue specificity analysis of SPL1 and SPL12 gene expressions

SPL1 the and SPL12 genes of separation are separately encoded the albumen containing 881 and 921 amino acid, and sequence homology is 72%.The nucleotide sequence of SPL1 and SPL12 genes such as SEQ ID NO.:1 and SEQ ID NO.:Shown in 4, the ammonia coded by it The sequence such as SEQ ID NO. of base acid:2 and SEQ ID NO.:Shown in 5.

For research SPL1, tissue expression specificities of the SPL12 in Arabidopsis plant, by SPL1, SPL12 promoter The carrier pSPL1 of (ATG upstreams about 3.1kb) driving gus gene expression:GUS and pSPL12:GUS is transformed into wildtype Arabidopsis thaliana In.Transformation of Arabidopsis thaliana uses infusion method, and it is 3 that resistance ratio is selected in T2 is for plant:1 list inserts independent strain, T3 generation screenings The strain of homozygosis is used for subsequent analysis.Plant is cultivated in 22 DEG C, phjytotron dark 16h illumination/8h.

Multiple independent transgenic lines (each at least eight) are carried out with GUS dyeing observations.The Coloration experiment of reporter gene shows Show pSPL1:GUS and pSPL12:GUS wide expression (Figure 1A, Figure 1B), including 1) cotyledon, lower embryo in arabidopsis each tissue Axle vascular tissue；2) taproot and lateral root (removing root cap)；3) expression quantity in lotus throne leaf (containing epidermal hair), its old leaf is higher than tender leaf； 4) stem epidermis (containing epidermal hair)；5) inflorescence and common peduncle；6) Fruit pod top, Fruit pod and carpopodium junction and ripe Fruit pod, but Do not expressed in seed.pSPL12:The dye levels of reporter gene are slightly weaker than pSPL1 in GUS transgenic lines:GUS, except not existing Expressed in hypocotyl and stem epidermal hair, expression pattern and pSPL1 in remaining tissue:GUS is similar, implys that SPL1 and SPL12 Possible functional redundancy, and there is portion of tissue specificity.

SPL1 and SPL12 starts expression on petal top in flower development late period (period 10-16), is extended to entirely to the later stage Calyx, petal, column cap, stamen and pollen grain (Fig. 1 C- Fig. 1 F).Real-Time PCR experiment results display that, SPL1 and Expression quantity is apparently higher than the petal (Fig. 1 G) do not opened in the flower (stage13 and afterwards) opened by SPL12, and hint may be in floral organ Play the role of in official's late period growth course important.

Embodiment 3

SPL1, SPL12 expression lower influence flower development and solid

In order to disclose SPL1 and SPL12 biological function, its T-DNA insertion mutation body, respectively spl1-1 is analyzed And spl12-1 (SALK_142295) (Fig. 2A) (SALK_134584).RT-PCR detections show SPL1 in spl1-1 mutant Expression and spl12-1 mutant in SPL12 expression it is almost nil (Fig. 2 B), it is function to illustrate spl1-1 and spl12-1 Complete depletion mutant.Spl1-1spl12-1 double-mutants are obtained by hybridizing spl1-1 and spl12-1 single mutants, i.e., The complete depletion mutant of SPL1 and SPL12 functions (Fig. 2 B).

Under normal illumination and temperature conditionss, spl1-1 and spl12-1 single mutants do not have any visible abnormal phenotype, and Spl1-1spl12-1 double-mutant plant arrived reproductive stage show part flower can not normally deploy (Fig. 2 C, Fig. 2 E, figure 2F), calyx edge adhesion (Fig. 2 G), due to blooming, same day floral organ can not be sufficiently spread out, and spatially prevent fertilization process, from And cause setting percentage to reduce (Fig. 2 C, Fig. 2 D).Wild type flower comes off naturally quickly in Post flowering, and double-mutant flower maturity and de- Fall process slower, calyx can not normally come off (Fig. 2 H, Fig. 2 I).

Use 35S:Myc-gSPL1 and 35:The transgenosis that gSPL12- carriers conversion spl1-1spl12-1 double-mutants are obtained Plant can return to wild type phenotype (Fig. 2 D).In addition, RNAi (under spl1-1 backgrounds, silence SPL12) transgenic line RNAi-1 plants (Fig. 2 J) also show the phenotype (Fig. 2 D) of similar spl1-1spl12-1 double-mutants.

These results suggest that spl1-1spl12-1 double-mutant flower developments abnormal phenotype is strictly by SPL1 and SPL12 bases Because missing caused by, and there is functional redundancy (Fig. 2) between SPL1 and SPL12.

Embodiment 4

SPL1, SPL12 expression influence response of the thaliana flower organ to gentle high temperature

To wild type Col-0, spl1-1spl12-1 double-mutant and genetically modified plants ox-MS1c-1 (35S:myc- GSPL1, Col-0 background) the flower openness after the processing of (30 DEG C) of gentle high temperature carried out the tracking statistics of continuous 4 days.By stem Its calyx of the flower that the same day opens on terminal inflorescence is fully deployed, and the situation that petal stretches naturally is considered as open to the outside world, calyx or petal Situation about can not deploy naturally is considered as " losing out ", counts the number that the same day blooms on each stem terminal inflorescence, referred to as daily flower Openness (Number of open flowers per inflorescence per day, abbreviation NOF).

The arabidopsis of growth five weeks, the average NOF situations of stem terminal inflorescence are as shown in Figure 3 in the continuous four day time：22 DEG C of feelings Under condition, wildtype Arabidopsis thaliana NOF=2.5, and spl1-1spl12-1 double-mutants NOF=1.4.And 30 DEG C of continuous processings four days, Wildtype Arabidopsis thaliana NOF=1.7, spl1-1spl12-1 double-mutant NOF=0.4.Without notable between ox-SPL1 and wild type Difference, its 22 DEG C and 30 DEG C of NOF is respectively 2.9 and 1.9.Illustrating the gentle high temperature influence arabidopsis of accumulation, daily the flowers are in blossom puts Degree, spl1-1spl12-1 double-mutants are more sensitive to high temperature.

Fig. 4 shows arabidopsis stem terminal inflorescence morphological feature and daily flower openness statistical result at 22 DEG C and 30 DEG C, Respectively illustrate 0d, 1d and 4d situation.It can be seen that, (0d-4d) wildtype Arabidopsis thaliana stem top under normal 22 DEG C of growth conditions It is 2-3 that open daily spending, which is counted, on the inflorescence of end, and proportion is 80-90%.And it is open daily spend number for 0,1,4,5 accountings Example is added up as 10-20%.And the ratio that it is 2-3 that open daily the spending of spl1-1spl12-1 double-mutant stems terminal inflorescence, which is counted, Only~45%, remaining accounts for~55%, wherein predominantly 0 and 1, and 4 and 5 proportions are only~1%.Illustrate SPL1 and SPL12 Missing reduce daily flower openness (Fig. 4 A, Fig. 4 B) under arabidopsis normal temperature.

After 30 DEG C of processing first day (1d), wildtype Arabidopsis thaliana spends openness that obvious change does not occur daily, and Spl1-1spl12-1 double-mutants spend openness to significantly reduce daily, open to spend number to be dropped to for 2 and 3 proportion from 45% 12%, and 0 and 1 proportion rises to 88% from 51%.The 4th day after to processing, wildtype Arabidopsis thaliana flower openness also has Reduce to a certain degree, the flowers are in blossom puts number and drop to 29%, 0 and 1 ratio for 2 and 3 ratios from 88% and be raised to 70% from 9%.spl1- 1spl12-1 double-mutants spend that openness amplitude of variation is bigger daily, and it 95%, 1 is 5% that the flowers are in blossom, which puts the ratio that number is 0 to account for, it Remaining is 0%.Ox-SPL1 phenotype does not have obvious difference (Fig. 4 A, Fig. 4 B) with wild type.The temperature of accumulation is these results suggest that Change the growth and morphological development of arabidopsis floral to a certain extent with high temperature, and SPL1 and SPL12 missing have impact on Response of the thaliana flower to gentle high temperature.

Analyze based on the above results and to the Morphological And Anatomical that double-mutant is spent, thus it is speculated that SPL1 and SPL12 are not only involved in regulation and control Thaliana flower opening and reaching maturity, while played a significant role in response process of the plant to gentle high temperature.

Embodiment 5

Tolerance of SPL1, SPL12 expression the influence thaliana flower organ to thermal extremes

Further analysis thaliana flower organ will grow the plant of five weeks at 22 DEG C with 37 DEG C to the tolerance of thermal extremes Processing just can see the young tender stem in wild-type plant top for 1 minute and floral organ is quickly wilted, but after half an hour and gradually Recover normal, ox-MS1c-1 is similar with wild type, but wilting degree is not so good as wild type.And spl1-1spl12-1 double-mutants Wilting degree is more serious, and part inflorescence can not recover normal condition after half an hour, or even some are quick shrivelled.By 37 DEG C of processing Inflorescence (Fig. 5 A) is restored to 22 DEG C and grown 1 day after 1 day, observes and counts inflorescence that is completely shrivelled and can not normally blooming Number, it is ox-MS1c-1 64.5%, wild type 43.2%, the double mutation of spl1-1spl12-1 respectively as a result to show flower survival rate Body 16.7% (Fig. 5 C), using the plant that does not process as control, its survival rate is 100%.Illustrate lacking for SPL1 and SPL12 Mistake reduces tolerance of the thaliana flower organ to thermal extremes, and SPL1 overexpression strengthens its tolerance, its expression quantity With high temperature resistance positive correlation (Fig. 5 B, Fig. 5 C).

Superoxide dismutase (Super Oxide Dismutase, SOD) is the primary thing that free radical is removed in plant Matter.The level height of SOD in vivo is aging and dead index directly perceived.The height indirect reaction of SOD vigor body Remove ROS ability.High temperature stress can cause the accumulation of ROS in plant, and the active oxygen largely accumulated causes wound to cell Evil, can cause the death of cell when serious.It has detected 22 DEG C of wild types for growing 5 weeks and spl1-1spl12-1 double-mutants exist The SOD activity of inflorescence after 22 DEG C and 37 DEG C processing 4h.Wildtype Arabidopsis thaliana inflorescence SOD activity reduction after high temperature stress, in spl1- Even more serious (Fig. 6 A) is reduced in 1spl12-1, illustrates that SPL1 and SPL12 remain certain to arabidopsis floral under high temperature stress Oxidation resistance plays an important role.It is that arabidopsis reproductive organs maintains high temperature tolerance that result above, which implies SPL1 and SPL12, Key gene necessary to property.

Embodiment 6

SPL1, SPL12 expression decline the influence to seed production and germination rate under arabidopsis high temperature

22 DEG C of cultures are restored to after the wild type and double-mutant plant that grow 6 weeks are handled 1 day at 42 DEG C, finally The seed gross dry weight of every plant is counted, the wild type seeds yield using 22 DEG C of cultures counts mutant and do not existed together as 100% Manage the seed yield of plant.As a result show, spl1-1spl12-1 double-mutants seed production is wild type under normal condition 69%.High-temperature process causes wild type seeds yield to be reduced to 85%, and double-mutant seed yield is reduced to 25%, explanation SPL1, SPL12 missing not only have impact on seed production, and this influence even more serious at high temperature (Fig. 6 B).

Seed germination rate under high temperature stress is have detected, the seed germination rate of wild type and double-mutant under the conditions of 22 DEG C is found It is more than 90%, the wild type germination rate after 30 DEG C of growths 7 days is for 80%, spl1-1spl12-1 double-mutant germination rates 47% (Fig. 6 C), illustrates that influence of 30 DEG C of high temperature to wild type seeds germination rate is weaker, and spl1-1spl12-1 double-mutant kinds Sub- germination rate is to 30 DEG C of sensitives.And thermal extremes wild type and spl1-1spl12-1 double-mutant kinds such as in the case of 37 DEG C Sub- germination rate, which is 0,37 DEG C, can completely inhibit Seed Germination of Arabidopsis Pumila.Result above shows SPL1 and SPL12 for intending south Mustard maintains to play an important roll in terms of seed production and germination rate at high temperature.

Embodiment 7

QRT-PCR verifies SPL1, the heat shock response transcription factor of SPL12 regulation and control

Transcription factor is in growth and development of plants and with playing highly important role in environment Interaction.Pass through transcript profile With the analysis of differential transcription factor expression gene, more than ten participation drought stresses being reported, ABA signals-modulatings way have been screened The related transcription factor gene of footpath, heat resistanceheat resistant approach, analyzes their heat shock response expressions in inflorescence.Real-time PCR experiment testing result shows that these genes are expressed in wild type by high temperature induction, and in spl1-1spl12-1 double-mutants Middle induction is obstructed, (Fig. 7 A, Fig. 7 B, Fig. 7 C) consistent with RNA-seq results.Illustrate the heat shock response derived need of these genes SPL1, SPL12 participation.It is probably the heat shock response transcription factor of SPL1, SPL12 regulation and control further to confirm these genes, Arabidopsis floral maintains to play an important role during heat resistance.

Embodiment 8

It is overexpressed SPL1 or SPL12 and improves plant heat resistance

In order to further explore the effect of SPL1 and SPL12 in arabidopsis heat resistance, divide in wild type and double-mutant Not Zhuan Hua 35S promoter driving 6 × myc-SPL1 (ox-MS1) or SPL12 (ox-S12) carrier.It has selected target gene table Up to amount 6 transgenic lines of highest, wherein ox-MS1c-1, ox-S12c-3, and ox-S12c-4 is wild type background, ox- MS1d-1, ox-MS1d-4 and ox-MS1d-7 are spl1-1spl12-1 double-mutants background (Fig. 2 J).To 14 days wild of growth Type, spl1-1spl12-1 and genetically modified plants ox-MS1c-1, ox-MS1d-1 and ox-S12c-3 are carried out at 2d 42 DEG C of high temperature Reason, counts survival rate after recovering 5 days, as a result shows that ox-MS1c-1, ox-MS1d-1 and ox-S12c-3 survival rate are significantly high In wild type and double-mutant (Fig. 8 A, Fig. 8 B).Ox-MS1d-1, ox-MS1d-4 and ox-MS1d-7 inflorescence are at 42 DEG C of high temperature Higher survival rate (Fig. 8 C, Fig. 8 D) is shown after reason.In addition, at 42 DEG C, ox-MS1c-1, ox-MS1d-1, ox-S12c-3 is obtained Get Geng Gao seed production (Fig. 8 E), at 30 DEG C, ox-MS1c-1, ox-MS1d-1, ox-S12c-4 obtains higher seed production Amount.It these results suggest that overexpression SPL1 or SPL12 can improve the heat resistance of arabidopsis vegetative growth phase and reproductive stage.

All documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Claims

1. the purposes of a kind of SPL genes or its encoding proteins, it is characterised in that the SPL genes are selected from SPL1 genes, SPL12 Gene or its combination, and described SPL genes or its encoding proteins are used for the purposes that is selected from the group：

(b) it is used to strengthen the heat resistance of plant.

2. purposes as claimed in claim 1, it is characterised in that described plant is arabidopsis.

3. purposes as claimed in claim 1, it is characterised in that described SPL genes are SPL1 genes and SPL12 genes.

4. purposes as claimed in claim 1, it is characterised in that described " enhancing Heat Resistance of Plant performance " includes what is be selected from the group One or more performances：

(i) heat resistance of inflorescence is strengthened；

(ii) germination rate of seed solid under enhancing hot environment；

(iii) it is used to strengthen tolerance of the plant to hot environment；

(iv) heat shock of plant is responded under enhancing hot environment；

(v) antioxygenic property of plant under hot environment is strengthened；

(vi) heat resistance of enhancing plant roots, stem, and/or leaf；

(vii) setting percentage of plant under hot environment is strengthened.

5. purposes as claimed in claim 1, it is characterised in that described " enhancing Heat Resistance of Plant performance " includes enhancing plant life The heat resistance in long-term and reproduction period.

6. purposes as claimed in claim 1, it is characterised in that described SPL genes include wild type SPL genes and saltant type SPL genes.

7. a kind of method for changing Heat Resistance of Plant performance, it is characterised in that including step：

(a) construction of external source is imported into plant cell, wherein described construction contains the SPL gene orders of external source, promotion The exogenous nucleotide sequence of SPL gene expressions or the exogenous nucleotide sequence for suppressing SPL gene expressions, so that it is outer to obtain importing The plant cell of source construction；

8. method as claimed in claim 7, it is characterised in that the SPL gene orders of the external source are also included and grasped with ORF sequences The promoter and/or terminator of the property made connection.

9. a kind of strengthen the method for Heat Resistance of Plant performance, it is characterised in that the described method comprises the following steps：In the plant In, promote the expression of SPL genes or promote the activity of SPL albumen, wherein, the SPL genes are selected from SPL1 genes, SPL12 bases Cause or its combination.

10. a kind of purposes of the adjusting control agent of SPL genes or its encoding proteins, it is characterised in that the heat resistance for regulating and controlling plant Can, or for prepare regulation and control Heat Resistance of Plant performance reagent or composition, wherein, the SPL genes be selected from SPL1 genes, SPL12 genes or its combination.