CN102260341B - Molecular switch for controlling pollen male sterility calcium fluctuation and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to a molecular switch for controlling pollen male sterility calcium fluctuation and an application thereof. A calcium signal switch molecule according to the invention contains a Ca2+/Mg2+ channel molecule CAC-1, an inhibitor CAC-2 of the Ca2+/Mg2+ channel molecule CAC-1 and a calcium binding protein CMA, wherein the calcium channel activity of the Ca2+/Mg2+ channel molecule CAC-1 is inhibited by the CAC-2; and the CMA simultaneously interacts with the CAC-2 and the CAC-1 so that the inhibition of the CAC-2 to the calcium ion transport activity of the CAC-1 is released.

Description

Control molecular switch and the application thereof of the fluctuation of pollen male sterile calcium

Technical field

The present invention relates to genetically engineered field, relate to the molecular switch and application thereof that control the fluctuation of pollen male sterile calcium particularly.

Background technology

The normal germination and growth of pollen is that spermatid arrives blastular smoothly and realizes amphigameous prerequisite, and plays vital effect to higher plant syngenesis.The growth of pollen tube relates to the process of series of complex, and various mineral element wherein plays an important role at this, and large quantity research shows that the germination in vitro of pollen and pollen tube growth need suitable calcium ion concn.Ca ²⁺affect the growth of pollen germination and pollen tube by number of ways, wherein relate to the assembling of cytoskeleton, the transport of secretory vesicle, all respects such as fusion pollen tube top pulsed growth and the regulation and control of pollen tube growth direction etc.So there is which type of molecular mechanism in the behind of these phenomenons to accomplish the accuracy controlling of plant polar growth actually.

Ca ²⁺as second messenger most important in vegetable cell, the polar growth growth of involved in plant and the transduction to adverse circumstance signal.Under abiotic stress conditions, the calcium ion in plant cytoplasm there will be specific variations in time, space and concentration, namely brings out generation Ca2+ oscillations.Ca2+ oscillations is undertaken experiencing and transduceing by the calcium binding protein in its downstream again, and then in cell, cause a series of biochemical reaction come coordinate plant growth or adaptation or resist various environment stress.Ca is found at present in vegetable cell ²⁺/ CDPK, Ca ²⁺/ CaM and Ca ²⁺/ CBL 3 class Ca2+ oscillations system.Wherein CaM is a most important class Ca2+ oscillations sensing member in vegetable cell, and containing the amino-acid residue of about 150, and between different calmodulin, structure is very conservative.Be the EF hand-type structure of 12 amino-acid residues containing 4 length in each calmodulin, their each and calcium bindings, cause calmodulin conformation to change thus activate calmodulin and exercise signal transduction functionality.Calmodulin as the important signal transmission courier of a class mainly by combining to regulate more intracellular biochemical reactions with CaM associated proteins (CaMBP).We are by research Ca ²⁺, calmodulin, channel protein illustrate to introduce a kind ofly have the Ca2+ oscillations undulated control of whole biological meaning and the molecular switch of decoding.

Summary of the invention

The object of this invention is to provide a kind of Ca ²⁺/ Mg ²⁺passage molecule CAC-1.

Another object of the present invention is to provide above-mentioned Ca ²⁺/ Mg ²⁺the inhibitor C AC-2 of passage molecule CAC-1.

Another object of the present invention is to provide a kind of and above-mentioned Ca ²⁺/ Mg ²⁺the interactional calcium binding protein CMA of passage molecule CAC-1 and inhibitor C AC-2.

Another object of the present invention is to provide Ca2+ oscillations switch molecule.

According to Ca of the present invention ²⁺/ Mg ²⁺passage molecule CAC-1, its aminoacid sequence is as shown in SEQ ID No.1; Its nucleotide sequence is as shown in SEQ ID No.2.

Ca according to the present invention ²⁺/ Mg ²⁺the inhibitor C AC-2 of passage molecule CAC-1, its aminoacid sequence is as shown in SEQ ID No.3; Its nucleotide sequence is as shown in SEQ ID No.4.

According to calcium binding protein CMA of the present invention, its aminoacid sequence is as shown in SEQ ID No.5; Its nucleotide sequence is as shown in SEQ ID No.6.

According to Ca2+ oscillations switch molecule of the present invention, comprise above-mentioned Ca ²⁺/ Mg ²⁺passage molecule CAC-1, Ca ²⁺/ Mg ²⁺the inhibitor C AC-2 of passage molecule CAC-1 and calcium binding protein CMA, wherein, CAC-2 suppresses Ca ²⁺/ Mg ²⁺the activity of calcium channels of passage molecule CAC-1, CMA and CAC-2 and CAC-1 interacts simultaneously, relieves the suppression of CAC-2 to CAC-1 calcium ion transport activity.

Present invention also offers the application of calcium current fluctuation in above-mentioned Ca2+ oscillations switch molecule regulating plant body.

Present invention also offers above-mentioned Ca2+ oscillations switch molecule for the male sterile application of regulating plant.

According to a particular embodiment of the invention, for the molecular switch of the polar growth of regulating plant pollen tube, be described in detail, the albumen of two homologous genes encodings regulates and controls mutually the Ca2+ oscillations switch molecule forming exquisiteness with the 3rd calcium binding protein, the common activity of adjustment pollen tube calcium channel and the fluctuation of Calcium Signal and decoding, thus accurately control extension or the shortening of Pollen Tubes, with the male sterile of regulating plant, this not only has great using value, more has general biological significance.

The contriver of invention finds that gene C AC-1 is the calcium channel in Pollen Tubes after deliberation, and autoploid CAC-2 can suppress the activity of CAC-1, but deposits at CMA and can remove this suppression in case, particularly:

1, CAC-1 has calcium ion and the magnesium ion channel characteristic of inward rectification;

2, autoploid CAC-2 can interact with CAC-1 and suppress the activity of CAC-1 calcium channel;

3, there are two kinds of mode: CAC-2 and CAC-1 to hold direct effect by C-according to the interaction of each element of Ca2+ oscillations switch molecule of the present invention, thus suppress the activity of CAC-1 calcium channel; CAC-2 is acted on by CMA and CAC-1;

4, CMA and CAC-2 and CAC-1 interacts simultaneously, relieves the suppression of CAC-1 to CAC-1 calcium ion transport activity;

5, flow of calcium ions can be combined with CMA, the affinity of feedback regulation CAC-1 and CAC-2, and causes the fluctuation of flow of calcium ions;

6, PROTEIN C AC-1, the CAC-2 of two homologous genes encodings regulate and control mutually to form exquisite switch molecule with the 3rd calcium binding protein CMA, the pollen tube controlled thus extension or shortening, accuracy controlling plants male sterility;

7, above-mentioned Ca2+ oscillations fluctuation mechanism and decoding, be present in the extension of plant root hair, the growth of guard cell, the reaction of pathology or other physiological process equally, have general biological significance and application thereof.

Accompanying drawing explanation

Fig. 1 shows the ion transport characteristic of CAC-1 and CAC-2;

Fig. 2 shows CAC-2 and inhibits the calcium ion transport of CAC-1 active, and CMA relieves this restraining effect;

Fig. 3 shows the interaction between CAC-1, CAC-2 and CMA and acts on specific site.

Fig. 4 shows the prolongation that CAC-1 and CAC-2 interaction maintains pollen tube, and CMA removes the effect that CAC-2 suppresses CAC-1, suppresses pollen tube prolongation;

Embodiment

The clone of embodiment 1, CAC-1, CAC-2, CMA gene

1, the cultivation of Arabidopis thaliana

When cultivating Arabidopis thaliana in soil, be seeded in equably on the Nutrition Soil according to 1/3 ratio mixing vermiculite by saturated for water suction and after processing 2-3 days through 4 DEG C seed, dark lucifuge sprout growth, cultivates under moving to normal illumination after growing true leaf.Growing period waters maintenance ground moistening.

In culture dish during sterile culture Arabidopis thaliana, first to carry out surface sterilization to seed.Appropriate dry Arabidopis thaliana seed is placed in the little centrifuge tube of 1.5mL, first with appropriate dehydrated alcohol sterilization 30sec, 5-10min is soaked again with the chlorine bleach liquor of 1mL 10-15%, then rinsed with sterile water 3-5 time is used, be placed in 4 DEG C of synchronization process after 2 ~ 3 days, dibbling is on 1/2MS substratum equably.

Culture condition:

Temperature 20-23 DEG C, intensity of illumination 80-200 μm of ol m ^-2sec ^-1, the photoperiod is that 16h illumination/8h dark replaces or continuous illumination, and humidity is about 70%.

2, the extraction of total serum IgE

3, total serum IgE reverse transcription

1) undertaken by Invitrogen Super ScripIII process specifications, the first step reaction system is:

Be heated to 65 DEG C, 5min.

2) at least 1min is placed on ice

3) second step reaction system is:

Reaction conditions is: 50 DEG C of 90min;

70℃15min。

4, the acquisition of CAC-1 full length gene

Design primer Primer1:5-CGGGATCCATGAATAAAATCCGGTCT-3, Primer2:5-GGAATTCTTAAACGTCTTCTTTATC-3, dilutes 50 times for touching plate by above-mentioned reverse transcription product, reaction system and condition as follows:

Reaction parameter:

5, the recovery of PCR primer

By PCR primer electrophoresis on 1% agarose, cut object band under ultraviolet lamp and after shredding blob of viscose, operate (being produced by OMEGA) by OMEGA Gel Extraction Kit.

6, the connection of PCR primer and conversion

Get each 1 μ l of the PCR primer after purifying, with Promega company -T easy vector connects (operating according to its product description).

Connection product thermal shock is transformed into E.coli DH5 α competent cell, selects positive colony order-checking.

The sequencing result display of CAC-1 gene, this full length gene 2807bp, has the open reading frame of 2121bp, 707 amino acid whose protein of encoding.

CAC-1 gene has following open reading frame nucleotide sequence:

ATGAATAAAATCCGGTCTCTCCGCTGCCTCCTCCCGGAGACCATAACCTCCGCC

TCCACCGCCGCCTCAAATCGAGGATCTGACGGTAGCCAATTCAGCGTCCTATGG

CGGCATCAGATCCTGGACCCGGACAGCAACATCGTCACCTATTGGAACCACGT

CTTTCTCATTACCTCAATCCTCGCTCTCTTCCTCGATCCTTTCTATTTCTACGTCC

CTTACGTCGGTGGTCCGGCGTGTCTCTCCATCGACATCAGCCTCGCCGCTACTG

TCACTTTCTTCCGTACAGTCGCCGATATCTTCCACCTCTTGCACATTTTCATGAA

ATTCAGAACTGCGTTCGTCGCGAGGAGTTCTCGTGTTTTTGGTCGCGGTGAGCT

TGTTATGGATTCTAGAGAGATTGCTATGAGATACTTGAAGACAGATTTCCTCATT

GATGTCGCCGCCATGCTTCCCCTTCCTCAGCTTGTTATTTGGCTAGTAATTCCAG

CGGCCACAAATGGAACTGCTAATCATGCCAATAGCACTCTCGCACTTATCGTTC

TTGTTCAATACATTCCAAGATCATTCATCATATTCCCACTCAACCAAAGGATTATC

AAAACAACCGGGTTTATCGCCAAGACTGCTTGGGCAGGAGCTGCATACAACCT

CCTATTGTACATCCTTGCTAGTCACGTGTTAGGAGCAATGTGGTATCTGTCATCA

ATCGGGCGGCAGTTTTCGTGCTGGTCCAACGTATGCAAGAAAGACAACGCTTT

GAGAGTTTTGGATTGTCTTCCTAGCTTCTTGGATTGCAAGAGTTTAGAACAACC

AGAGCGCCAGTATTGGCAGAACGTTACTCAGGTCCTTTCTCATTGTGATGCTAC

AAGCAGCACTACCAATTTCAAATTCGGAATGTTTGCTGAAGCCTTTACCACTCA

AGTTGCTACAACAGATTTTGTGTCCAAGTACTTGTATTGTCTTTGGTGGGGTTTA

AGAAATCTAAGTTCTTATGGACAGAATATAACCACAAGTGTATACCTTGGCGAG

ACCTTGTTCTGTATAACAATTTGTATTTTTGGGTTGATTCTCTTCACACTCCTGAT

TGGTAACATGCAATCTTCTCTGCAATCGATGTCAGTAAGAGTTGAGGAATGGAG

AGTTAAGAGAAGAGACACTGAAGAATGGATGAGACATCGTCAACTACCTCCAG

AGCTTCAAGAACGTGTCCGAAGATTTGTCCAATACAAATGGCTTGCAACCAGA

GGTGTCGATGAAGAATCAATCCTCCATTCATTACCTACAGATTTACGCCGTGAAA

TCCAACGTCATCTTTGTCTCTCCCTTGTTCGCCGGGTCCCGTTCTTCTCTCAAAT

GGATGATCAACTTCTTGATGCTATATGCGGATGCCTTGTCTCATCTTTAAGCACG

GCCGGGACATACATATTCCGTGAAGGTGATCCGGTGAATGAGATGCTATTTGTG

ATCCGAGGACAAATAGAGAGCTCAACAACAAATGGAGGAAGATCTGGTTTCTT

CAACTCCACTACTCTACGACCTGGCGATTTCTGTGGGGAAGAACTTCTTACATG

GGCCTTAATGCCAAACTCTACACTCAATCTTCCGTCTTCAACACGAAGCGTCCG

TGCCCTCTCTGAAGTAGAAGCCTTTGCATTAAGCGCAGAGGATCTTAAGTTTGT

TGCTCATCAGTTCAAACGTCTTCAAAGCAAAAAGCTTCAACACGCTTTCAGGT

ACTACTCACATCAATGGAGAGCATGGGGAGCATGTTTTGTCCAATCTGCATGGA

GAAGATACAAGCGAAGAAAGCTTGCGAAAGAGCTCAGCCTTCACGAGAGTAG

CGGATACTATTACCCGGATGAAACAGGTTACAATGAAGAAGACGAAGAAACTA

GAGAGTATTACTATGGAAGTGACGAGGAAGGCGGATCAATGGACAACACAAAT

CTGGGAGCAACGATCTTAGCATCTAAATTTGCGGCGAACACGAGAAGAGGAAC

AAATCAAAAGGCTTCAAGTAGTAGTACTGGTAAGAAAGATGGATCTTCCACCA

GTTTGAAGATGCCTCAACTATTCAAACCAGATGAGCCTGATTTCTCTATAGATAA

AGAAGACGTTTAA 2121

The aminoacid sequence of the protein of its coding is:

MNKIRSLRCLLPETITSASTAASNRGSDGSQFSVLWRHQILDPDSNIVTYWNHVFLITSILALFLDPFYFYVPYVGGPACLSIDISLAATVTFFRTVADIFHLLHIFMKFRTAFVARSSRVFGRGELVMDSREIAMRYLKTDFLIDVAAMLPLPQLVIWLVIPAATNGTANHANSTLALIVLVQYIPRSFIIFPLNQRllKTTGFIAKTAWAGAAYNLLLYILASHVLGAMWYLSSIGRQFSCWSNVCKKDNALRVLDCLPSFLDCKSLEQPERQYWQNVTQVLSHCDATSSTTNFKFGMFAEAFTTQVATTDFVSKYLYCLWWGLRNLSSYGQNITTSVYLGETLFCITICIFGLILFTLLIGNMQSSLQSMSVRVEEWRVKRRDTEEWMRHRQLPPELQERVRRFVQYKWLATRGVDEESILHSLPTDLRREIQRHLCLSLVRRVPFFSQMDDQLLDAICGCLVSSLSTAGTYIFREGDPVNEMLFVIRGQIESSTTNGGRSGFFNSTTLRPGDFCGEELLTWALMPNSTLNLPSSTRSVRALSEVEAFALSAEDLKFVAHQFKRLQSKKLQHAFRYYSHQWRAWGACFVQSAWRRYKRRKLAKELSLHESSGYYYPDETGYNEEDEETREYYYGSDEEGGSMDNTNLGATILASKFAANTRRGTNQKASSSSTGKKDGSSTSLKMPQLFKPDEPDFSIDKEDV

7, the acquisition of CAC-2 full length gene

Design 5 ' end primer Primer1:5-CGGGATCCATGTCATCTAATGCTACG-3,3 ' end primer Primer2:5-GCTCTAGATTAGTTCAAGTCATCTGT-3, dilutes 50 times for touching plate by above-mentioned reverse transcription product, reaction system and condition as follows:

Reaction parameter:

The sequencing result display of CAC-2 gene, this full length gene 2694bp, has the open reading frame of 2262bp, 753 amino acid whose protein of encoding.

CAC-2 gene has following open reading frame nucleotide sequence:

ATGTACAAATCTCAATATATAAGTGGCCATAGAGAAAAATTCGTAAGATTAGATG

ATACAGATTCGAGGGTTTCAATGTCATCTAATGCTACGGGAATGAAGAAGCGAT

CATGTTTTGGATTGTTTAACGTAACTAGTCGTGGAGGAGGAAAGACAAAGAAC

ACATCAAAATCATTTCGAGAAGGTGTTAAAATCGGATCAGAGGGTCTAAAAAC

GATCGGAAAATCATTCACATCAGGTGTAACAAGAGCTGTTTTCCCTGAAGATCT

AAGAGTCTCCGAGAAGAAAATCTTTGATCCTCAAGACAAAACACTTTTATTATG

GAACAGAATGTTTGTTATCTCTTGCATTCTTGCTGTCTCTGTTGATCCTCTGTTCT

TCTATCTCCCTATTGTCGATAACTCAAAGAACTGCATTGGGATTGACTCTAAATT

AGCTGTGACAACTACTACTCTAAGAACGATCATCGATGTTTTTTATCTTACACGA

ATGGCTCTTCAGTTCCGTACCGCTTACATTGCTCCTTCTTCTCGTGTGTTTGGAA

GAGGTGAGCTTGTGATCGACCCTGCAAAGATCGCTGAACGGTATTTGACTCGTT

ATTTCATTGTCGATTTCCTCGCGGTTCTTCCTTTACCTCAGATTGCTGTGTGGAA

GTTTCTTCACGGATCTAAAGGAACAGATGTATTACCAACAAAACAGGCGCTATT

ACACATTGTCATCACACAGTACATACCAAGATTCGTTAGGTTTATTCCATTAACT

TCAGAGCTCAAGAAAACAGCAGGAGCTTTCGCTGAAGGTGCTTGGGCTGGTG

CTGCTTATTACCTCCTCTGGTATATGCTTGCAAGTCACATAACTGGAGCGTTCTG

GTACATGTTGTCAGTGGAACGTAACGATACATGCTTGAGATCCGCTTGTAAAGT

CCAGCCTGATCCCAAGGTCTGTGTTCAGATTCTTTATTGTGGCAGCAAATTAATG

AGCAGTCGCGAAACCGACTGGATCAAATCAGTCCCTGATCTTTTTAAGAACAAT

TGTTCTGCTAAAAGCGATGAGTCTAAATTCAACTACGGGATCTATAGCCAGGCT

GTGTCTTCCGGTATTGTATCTTCGACAACGTTTTTCTCCAAGTTTTGTTATTGTCT

ATGGTGGGGTCTCCAAAATCTCAGCACGTTAGGTCAAGGGCTGCAAACAAGTA

CATATCCAGGAGAAGTTTTGTTTTCAATTGCAATTGCTGTAGCTGGACTTCTTTT

GTTTGCTCTTCTCATTGGGAATATGCAAACTTATCTTCAGTCACTTACGGTTCGC

TTAGAGGAAATGAGGATTAAAAGACGTGATTCCGAACAGTGGATGCATCACAG

GTCACTTCCACAAAACCTGAGGGAACGAGTCAGACGTTATGATCAATACAAAT

GGTTGGAAACAAGAGGAGTCGACGAAGAAAACATAGTCCAGAGTTTACCAAA

AGATCTTAGAAGAGACATCAAACGCCATCTCTGTCTAAATTTAGTTCGCAGGGT

TCCTCTGTTTGCTAATATGGATGAGAGATTACTAGACGCAATATGTGAGAGACTA

AAGCCAAGTCTATACACAGAGAGCACTTACATAGTGCGAGAAGGAGACCCGGT

TAACGAAATGCTCTTCATAATCCGAGGCCGGTTAGAGAGTGTAACAACAGATGG

TGGAAGAAGCGGTTTCTTTAACAGAGGTTTATTGAAAGAAGGTGACTTTTGTG

GTGAAGAGCTTCTGACTTGGGCACTTGACCCTAAAGCAGGCTCAAACTTACCT

TCTTCCACACGAACAGTGAAGGCTCTAACTGAAGTAGAGGCTTTCGCTTTAGA

AGCGGAAGAGCTCAAGTTTGTGGCTAGTCAGTTCAGGCGTCTTCACAGTCGTC

AGGTTCAACAAACTTTCAGGTTTTACTCTCAACAATGGAGGACTTGGGCTGCTT

GTTTCATCCAAGCCGCTTGGCGTAGACATTTAAGAAGGAAAATCGCAGAGCTTA

GACGTAAAGAAGAAGAAGAAGAAGAAATGGATTATGAAGATGATGAATATTAT

GATGATAATATGGGTGGTATGGTTACAAGGAGTGATAGTTCTGTTGGATCAAGTT

CTACATTACGTTCCACGGTATTTGCATCAAGATTTGCTGCTAACGCGCTTAAGGG

TCATAAACTAAGGGTCACCGAGAGTTCAAAGAGTTTAATGAATTTAACAAAGC

CATCAGAACCTGACTTTGAGGCTCTTGATACAGATGACTTGAACTAA

The aminoacid sequence of the protein of its coding is:

MYKSQYISGHREKFVRLDDTDSRVSMSSNATGMKKRSCFGLFNVTSRGGGKTKNTSKSFREGVKIGSEGLKTIGKSFTSGVTRAVFPEDLRVSEKKIFDPQDKTLLLWNRMFVISCILAVSVDPLFFYLPIVDNSKNCIGIDSKLAVTTTTLRTIIDVFYLTRMALQFRTAYIAP S SRVFGRGELVIDPAKIAERYLTRYFIVDFLAVLPLPQIAVWKFLHGSKGTDVLPTKQALLHIVITQYIPRFVRFIPLTSELKKTAGAFAEGAWAGAAYYLLWYMLASHITGAFWYMLSVERNDTCLRSACKVQPDPKVCVQILYCGSKLMSSRETDWIKSVPDLFKNNCSAKSDESKFNYGIYSQAVSSGIVSSTTFFSKFCYCLWWGLQNLSTLGQGLQTSTYPGEVLFSIAIAVAGLLLFALLIGNMQTYLQSLTVRLEEMRIKRRDSEQWMHHRSLPQNLRERVRRYDQYKWLETRGVDEENIVQ SLPKDLRRDIKRHLCLNLVRRVPLFANMDERLLDAICERLKPSLYTESTYIVREGDPVNEMLFIIRGRLESVTTDGGRSGFFNRGLLKEGDFCGEELLTWALDPKAGSNLPSSTRTVKALTEVEAFALEAEELKFVASQFRRLHSRQVQQTFRFYSQQWRTWAACFIQAAWRRHLRRKIAELRRKEEEEEEMDYEDDEYYDDNMGGMVTRSDSSVGSSSTLRSTVFASRFAANALKGHKLRVTESSKSLMNLTKPSEPDFEALDTDDLN

8, the acquisition of CMA full length gene

Design 5 ' end primer Primer1:5-ATGTCAGAAA CATCAAAGTC AGAGTC-3,3 ' end primer Primer2:5-CTATGAGTGGCTATCTTGTCCTGAACCTTG-3, dilutes 50 times for touching plate by above-mentioned reverse transcription product, reaction system and condition as follows:

Reaction parameter:

The sequencing result display of CMA gene, this full length gene 1165bp, has the open reading frame of 486bp, 162 amino acid whose protein of encoding.

CMA gene has following open reading frame nucleotide sequence:

ATGGCGGATCAGCTCACAGACGATCAGATCTCAGAATTCAAGGAAGCCTTCAG

CTTATTCGACAAGGATGGTGATGGTATGCTTCATCCTCCCTTTCCCTCTATCATCG

TAGGTTGCATTACCACAAAGGAGCTTGGTACCGTGATGCGTTCCCTCGGTCAAA

ACCCAACCGAAGCTGAGCTTCAGGACATGATCAACGAAGTTGATGCGGATGGT

AACGGAACCATTGATTTCCCGGAGTTCTTGAACCTAATGGCTAGGAAAATGAA

GGACACTGACTCTGAGGAAGAACTCAAGGAAGCTTTCAGAGTTTTCGACAAA

GACCAGAACGGTTTCATCTCAGCTGCTGAATTGAGACATGTGATGACTAACCTC

GGCGAGAAGCTTACTGATGAAGAAGTTGATGAGATGATTAAGGAAGCTGATGT

TGATGGTGATGGTCAGATCAACTACGAAGAGTTTGTGAAGGTTATGATGGCTAA

GTGA

The aminoacid sequence of the protein of its coding is:

MADQLTDDQISEFKEAFSLFDKDGDGMLHPPFPSIIVGCITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMARKMKDTD SEEELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYEEFVKVMMAK

Embodiment 2, CAC-2, CMA regulate the checking of CAC-1 calcium ion transport activity

1, the preparation of pGEMHE vector construction and Capping RNA

PGEMHE plasmid there is T7 promotor, by gene constructed to CAC-1, CAC-2, CMA in this plasmid, in-vitro transcription test kit mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit can be utilized, obtain the mRNA of CAC-1, CAC-2, CMA full length gene.Concrete grammar is by CAC-1 and CAC-2 gene respectivelyinsert in pGEMHE plasmid, obtain pGEMHE-CAC-1, CAC-2, CMA.And be template as PCR, by primer M13/M13R amplification object segment, make the DNA after purifying about 1 ~ 2 μ g carry out in-vitro transcription, the reagent provided according to in-vitro transcription test kit mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit and step are carried out.Once transcribe and can obtain 20 μ l about 15 ~ 20 μ gmRNA, get 1 μ l biophotometer and measure its concentration, then by the concentration (0.5 μ g/ μ l) being diluted to needs through DEPC process and through the distilled water of High Temperature High Pressure, be dispensed in 1.5mL centrifuge tube, often pipe 2-3 μ L ,-80 DEG C save backup.

2, the cultivation of Africa xenopus and the preparation of frog's egg

The source of Africa xenopus is Nasco company of the U.S.

1), after ice cube anaesthetizes African toad, surgical operation takes out appropriate pieces of an egg;

2) at ambient temperature with the pieces of an egg 1.5h that I-type collagen enzymic digestion is taken out, be placed on low speed shaking table in enzymolysis process, to promote dissociating of pieces of an egg;

3) with without after the flushing of calcium ND96 nutrient solution, move into and fill in the culture dish of ND96.Be placed in 18 DEG C of overnight incubation.

3, the formula of voltage clamp body lotion

1) frog's egg culture solution formula: ND96

NaOH regulates PH to 7.4

2), Bath Solution fills a prescription:

CaCl 30mmol

MgCl 30mmol

MES 10mmol

4, the embedding voltage clamp record of two-electrode voltage and analysis

First in the frog's egg taken out, inject the mRNA of CAC-1 and CAC-2 gene, each ovocyte injection 46nl cRNA, control group injects isopyknic deionized water.After injection, ovocyte is put back to 18 DEG C of incubators and is cultivated 48 ~ 72h, then records electrical signal.Injection have the ovocyte of CAC-1 gene mRNA can be recorded in 30mM calcium chloride solution in one to electric current, and the electric current of 1000mA can be inspired under the voltage of-140mV stimulates.This electric current causes to transport features by the calcium ion of CAC-1 gene, has the dependency of voltage and time.And compared with control cells only has the about 300mA of ovocyte background current.Inject the size of current having the ovocyte of CAC-2 gene mRNA to be recorded in 30mM calcium chloride solution consistent with compared with control cells.So CAC-2 gene does not have calcium ion transport characteristic.CAC-1 is recorded in 30mM calcium chloride solution, the ovocyte that CAC-2 gene mRNA is expressed simultaneously, we find that the Calcium Current originally produced by CAC-1 disappears, and show the rectification characteristic same with compared with control cells, can find out that CAC-2 can suppress the Calcium Current of CAC-1.CAC-1 and CAC-2 has CMA binding site, so whether there is there what relation between they three? this three expresses in ovocyte by we simultaneously, by voltage-clamp recording, we find to be resumed due to the calcium ion transport characteristic of the expression CAC-1 of CMA, and that is CMA relieves the suppression of CAC-2 for CAC-1.

Embodiment 3, the interactional checking of CAC-1, CAC-2, CMA

Yeast two-hybrid assay

1, bait gene transforms sieve storehouse Host Strains AH109

1) 3 1mm clones are inoculated in 3mlYPD liquid nutrient medium 30 DEG C of shaking culture 20 hours (spending the night), and OD600 reaches about 1.2;

2) cultivating 4 hours by this 3ml bacterium liquid access large volume (volume is fixed depending on transforming number) YPD, making nutrient solution OD600 reach 0.6;

3) 50ml centrifuge tube collects bacterium liquid (Falcon, BD), Eppendorf Centrifuge 5810R, 700g (1865rpm), and 5 minutes collected by centrifugation thalline, abandon supernatant;

4) the resuspended thalline of 20ml ddH2O, Eppendorf Centrifuge 5810R, 700g, 5 minutes collected by centrifugation thalline, abandon supernatant;

5) the resuspended thalline of 20ml 1XTE/LiAc, Eppendorf Centrifuge 5810R, 700g5 minute collected by centrifugation thalline, abandons supernatant;

6) add the resuspended thalline of 1XTE/LiAc according to the amount of each conversion needs 50 μ l bacterium liquid, be dispensed in Eppendorf pipe, often pipe 50 μ l bacterium liquid;

7) every Guan Jiayue 100ng Bait gene plasmid, the Carrier DNA of 5 μ l denaturations, 500 μ l 1XTE/LiAc/PEG;

8) be positioned in 30 DEG C of incubators or water-bath and hatch 30 minutes, when 15 minutes, mixing once;

9), after 30 minutes, often pipe adds 15 μ l DMSO (DIMETHYL SULFOXIDE), mixing of turning upside down gently;

10) 42 DEG C of water-bath heat shocks 20 minutes, turn upside down mixing for every 10 minutes once;

11) ice bath 5 minutes;

12) 700g, 5 minutes collected by centrifugation thalline, abandon supernatant;

13) ddH ₂o washs a thalline;

14) utilize the resuspended thalline of residual liquid, coat on SD/-Trp flat board; 15.30 DEG C incubator is cultivated.Within 2nd day, namely can be observed clone and grow, within the 3rd day, namely clone grows to required size; Carrier DNA:Herring Testes Carrier DNA, Denatured.Clontech。10mg/ml。

Original Carrier DNA first 100 DEG C of sex change 10 minutes, are placed in ice bath 2 minutes immediately, and then 100 DEG C of sex change 10 minutes, be placed on ice bath for subsequent use.Used Carrier DNA only needs 100 DEG C of sex change once.1XTE/LiAc:V10XTE: V10XLiAc: VH2O=1: 1: 81XTE/LiAc/PEG:V10XTE: V10XLiAc: V50%PEG=1: 1: 850%PEG:50% is mass volume ratio.PEG3350 is dissolved in ddH20 generally needs heating for dissolving 10XTE:0.1M Tris-HCl, 10mM EDTA, pH7.510XLiAc:1M LiAc, pH7.5

2, after growing clone containing the AH109 conservation Bait gene of Bait gene plasmid, general with bait gene Auele Specific Primer directly in yeast colony PCR identify the plasmid whether proceeding to correct Bait gene, then through identifying that correct clone carries out conservation.

3, yeast colony PCR

1) with containing the plasmid of Bait gene as positive control, with Host Strains AH109 as negative control.

2) PCR:ExTaq Premix (Takara) 10 μ l (2X) 5 ' Primer 0.5 μ l (10 μMs) 3 ' Primer 0.5 μ l (10 μMs) ddH208.9 μ l bacterium 0.1 μ l is carried out according to following reaction system

3) PCR is carried out according to following reaction conditions:

95 DEG C 5 minutes

94 DEG C 30 seconds

58 DEG C 30 seconds

72 DEG C 2 minutes

30 circulations

72 DEG C 10 minutes 72 DEG C to extend 2 minutes be Bait gene for being less than 2Kb, corresponding time expand, is wanted for the Bait gene being longer than 2Kb.

4) PCR primer carries out electrophoresis detection.

4, positive colony conservation

1) positive colony is inoculated in 3ml respective liquid substratum, 30 DEG C of shaking culture 2-3 days;

2) 700g, 5 minutes, collected by centrifugation thalline;

3) liquid nutrient medium and the Sterile Glycerol Solution mixing solutions of 1: 1 is added;

4) fully suspend thalline, is put in-80 DEG C of preservations.Sterile Glycerol Solution：65％(V/V)glycerol，0.1M MgSO4，0.5mM Tris-HCl，pH7.4

5, the self activation of Bait gene detects the general method adopting film inspection, detects the expression of LacZ gene.Film detecting method is as follows:

1) by positive colony bacterial colony photographic reprinting on filter paper;

2) be dipped in completely in liquid nitrogen by filter paper, the time is longer than 30 seconds, dries after taking-up.

3) culture dish adds appropriate nitrite ion (Fresh), and pad one deck filter paper, makes it completely moistening, above being layered on by the filter paper after freeze-thaw, make nitrite ion penetrate into top layer.9CMa culture dish adds 2ml nitrite ion, and 15CMa culture dish adds 5ml nitrite ion;

4) cover ware lid, be placed in 37 DEG C of incubators, lucifuge is placed;

5) starting after 20 minutes to observe with or without becoming blue positive findings and record, being generally as the criterion with 3 hours, whether in particular cases can seeing spends the night becomes blue;

6) open ware lid after reacted, filter paper is dried, preserve and record; Nitrite ion: 100ml Z buffer, 0.27ml β-mercaptoethanol, 1.67ml X-gal Z buffer:16.1g/L Na ₂hPO ₄7H ₂o, 5.50g/LNaH ₂pO ₄h ₂o, 0.75g/LKCl, 0.246g/L MgSO47H ₂o, pH7.0.Room temperature can deposit 1 year.X-gal:X-gal (5-bromo-4-chloro-3-indoly-β-D-galactopyranoside) is dissolved in DMF (N, N-dimethylformamide), and final concentration is 20mg/ml.

6, bait gene sieve storehouse

1) 5-10 2mm clone is inoculated in 1ml SD/-Trp liquid nutrient medium, proceeds to 150mlSD/-Trp shaking culture 20 hours (spending the night), to about OD600=1.2 after mixing;

2) 150ml nutrient solution proceeds to mixing in 1000ml YPDA (YPD+Adenine), now OD600=0.2-0.3;

3) about 4-5 is cultivated little of OD600 > 0.6;

4) 500ml centrifuge tube collects bacterium liquid.Beckman Coulter Centrifuge Avanti J-25 (following steps all use this whizzer), 700g, 5 minutes, RT (room temperature).Simultaneously by 2000 μ l Carrier DNA denaturation twice;

5) 500ml ddH2O washs once, 700g, 5 minutes, RT, collects thalline;

6) 30ml 1X TE/LiAC washs thalline (preparing 50ml 1X TE/LiAC in advance), proceeds to 100ml centrifuge tube;

7) 700g, 5 minutes, RT.(preparing 50ml 1XTE/LiAC/PEG in advance);

8) the resuspended thalline of 12ml 1X TE/LiAc is added;

9) add 100-500 μ g library DNA, the Carrier DNA of 2000 μ l denaturations, mixes gently;

10) 50ml 1XTE/LiAc/PEG is added, violent vortex mixing;

11) hatch 45 minutes for 30 DEG C, mixing in every 15 minutes once;

12) add 3.2ml DMSO, mix gently;

13) 42 DEG C of heat shocks 20 minutes, mixing in every 10 minutes is once;

14) ice bath 5 minutes;

15) 700g, 5 minutes, collects thalline;

16) 1000ml YPDA is added, 30 DEG C of shaking culture 60 minutes;

17) 700g, 5 minutes, collects thalline;

18) add 2ml 0.9%NaCl suspension thalline, final volume is about about 5ml.Get 20 μ l bacterium liquid and do titer determination, all the other bacterium liquid 200 μ l/ block, coats on 15CMa SD/-Trp/-Leu/-His/+30mM3-AT (3-amino-1,2,4-triazole) flat board.Within 3rd day, rise and will note observing dull and stereotyped yeast clone growth conditions;

19) titre (stock relocation efficiency) measures: 20 μ l+180 μ l NaCl (0.9%) 1: 10; 20 μ l+180 μ lNaCl (0.9%) 1: 100; 20 μ l+180 μ l NaCl (0.9%) 1: 1000; 20 μ l+180 μ l NaCl (0.9%) 1: 10,000 20 μ l+180 μ l NaCl (0.9%) 1: 1000001: 1000; 1: 10000; 1: 100,000 three kind of concentration is respectively got 100 μ l and is coated with SD/-Trp/-Leu flat board

20) calculating of stock relocation efficiency: to the colony count of growth on SD/-Trp/-Leu flat board, selected clone number extent of dilution between 30-300 calculates stock relocation efficiency

21) sieve storehouse to complete, bacterium at SD/-Trp/-Leu/-His+30mM3-AT grow on plates after 7-14 days, can be cloned number (cfu) X and always to suspend volume

Be coated with plate bulk X extent of dilution X library consumption (μ g)

＝cfu/μg DNA

Have positive colony to grow out, positive colony is generally greater than 3mm.The a small amount of bacterium of picking is cooked film inspection.From film inspection becomes blue yeast, extracting prey plasmid and Bait plasmid do corotation and verify.

7, extracting yeast plasmid

1) the positive colony toothpick that film inspection change is blue is coated on SD/-Trp-Leu-His+30mM3-AT flat board, and about 2CMa2, is then positioned in 30 DEG C of incubators and cultivates 3-4 days;

2) get an Eppendorf pipe, add the 5u/ μ l Lyticase of 30 μ l.Bacterium toothpick is scraped in lyticase, and Vortex fully suspends.Hatch 30 minutes for 37 DEG C;

3) adding 170 μ l Lysis Buffer makes final volume be 200 μ l.Add 200 μ l granulated glass spherees (425-600microns) again, 200 μ l phenol/chloroform/primary isoamyl alcohol (25: 24: 1), vortex thermal agitation 5 minutes;

4) 12,000rpm centrifugal 10 minutes, supernatant is proceeded in a clean Eppfendorf pipe.Note, protein delivery is not gone over;

5) add 8 μ l 10M NH4Ac and 500 μ l dehydrated alcohols, mixing, place 1 hour for-80 DEG C;

6) 12,000rpm centrifugal 10 minutes, supernatant is removed;

7) 500 μ l 70% washing with alcohol precipitations are added, centrifugal 5 minutes of 12,000rpm;

8) abandon supernatant, vacuum drains precipitation;

9) precipitation is resuspended in 10 μ l ddH2O, generally gets 2-3 μ l transformation of E. coli; Lyticase: Lyticase dry powder is dissolved in 1XTE, final concentration is 5u/ μ l; Lysis Buffer:50mM Tris-HCl (pH8.0), 0.1%Triton X-100,0.5%SDS

8, the method that turns of the general electricity consumption of the amplification of yeast plasmid, increases the Prey plasmid of extracting in the positive colony from sieve storehouse gained in intestinal bacteria.Intestinal bacteria electricity turns competence preparation:

1) choose a mono-clonal access 3ml LB substratum, shake training 14-16 little of OD600=1.0-1.2;

2) by shaking the switching of the bacterium liquid after training 1L LB substratum, training 4-5 is shaken little of OD600=0.7-0.8;

3) centrifugal receipts bacterium, 10ml frozen water dissolves;

4) 5000g, within centrifugal 10 minutes, receive bacterium for 4 DEG C, 400ml frozen water is resuspended by bacterium, ice bath 10 minutes;

5) 5000g, within centrifugal 10 minutes, receive bacterium for 4 DEG C, 40ml precooling 10% glycerine is resuspended, ice bath 10 minutes;

6) 5000g, receives bacterium in centrifugal 10 minutes, adds 1ml 10% glycerine resuspended for 4 DEG C;

7) with rifle measure resuspended after bacteria liquid amass, add with bacterium liquid isopyknic 10% glycerine (note: equal-volume 10% glycerine refer to resuspended after bacteria liquid long-pending deduct 1ml.Example: after adding 1ml10% glycerine, resuspended bacteria liquid amasss as 4ml, then adding 10% amounts of glycerol is 3ml);

8) be dispensed in Eppendorf pipe, often pipe 100 μ l, operates on ice; 9.-80 DEG C Refrigerator store; 10. take out two pipes and do feminine gender, positive control; 10% glycerine: V glycerine: V water=1: 9.

9, electroporated

1) competence is taken out from-80 DEG C of refrigerators, to dissolving on ice.Meanwhile, by electric revolving cup to cooled on ice, open electroporation and prepare 30 minutes;

2), after competence is dissolved, the plasmid (or connecting product or other materials that will transform) that will transform adds competence, even with rifle piping and druming.Be necessary when electricity turns to carry out negative control, positive control, to check, whether competence is contaminated and whether competence is normal;

3) mixed competence is added the electric revolving cup of precooling;

4) wall on electric revolving cup top electrode both sides is dried;

5) electric revolving cup is put into electroporation, electric shock;

6), after electric shock, with 1ml SOC, competence is washed out, hatch 45 minutes for 37 DEG C;

7) 6,000rpm, 3 minutes centrifugal receipts bacterium;

8) coated plate, the suitableeest voltage of different electric revolving cup is different, through experiment: 1mm electricity revolving cup the suitableeest voltage be 1900-2200V 4mm electricity revolving cup the suitableeest voltage be 2300-2500V 8mm electricity revolving cup the suitableeest voltage be 2500-2700V SOC:20g/L peptone, 5g/L yeast extract, 0.5g/L NaCl, 2.5mM KCl, pH7.0.121 DEG C, sterilizing in 20 minutes.When being cooled to below 60 DEG C, often liter of 1M glucose solution adding 20ml sterilizing.Before this solution uses, often liter adds the 2M MgCl of 5ml sterilizing ₂.

Bimolecular fluorescence complementary is tested

Molecular fluorescence complementation (bimolecular fluorescence complementation, BiFC) analytical technology, that the one reported at first in 2002 by Hu etc. is directly perceived, judge the location of target protein in viable cell and interactional new technology rapidly. this technology dexterously by two of fluorescent protein molecule complementary fragments respectively with target protein amalgamation and expression, if fluorescin activation recovering, show that two target proteins there occurs interaction. the multicolor fluorescence complementary technology (multicolorBiFC) developed thereafter, the formation of multiple proteins complex body can not only be detected simultaneously, can also compare producing between different proteins interactional power. at present, this technology is for transcription factor, the dimeric forms of G-protein β γ subunit, produce between different proteins and interact in the research work of the aspects such as strong and weak comparison and protein ubiquitination.

Utilize yellow fluorescence protein (yellow fluorescent protein, YFP) as reporter gene, fluorescin is divided into two molecule fragments without fluorescence activity, be connected with target protein respectively again, if two target proteins are close because there being interaction, just make two of fluorescin molecule fragments spatially close to each other, again form active fluorophor and send fluorescence. under fluorescent microscope, just can observe directly two target proteins and whether there is interaction, and under the condition closest to viable cell physiological status, observe it interact the time occurred, position, strong and weak, form the stability of protein complex, and cell signaling molecule is on its interactional impact.

The one of carbon tip of CAC-1 and CAC-2 is structured in pGADT7 and pGBKT7 respectively and is used for doing yeast two-hybrid.At leucine, tryptophane, the yeast that the substratum of Histidine disappearance have expressed CAC-1 and CAC-2 can grow, and illustrates that CAC-1 and CAC-2 can interact.CAC-1 and CAC-2 can interact with CMA simultaneously, but doing mutually of CAC-2 and CMA is stronger.

Can find out equally have CAC-1-YFP turning in the experiment of bimolecular fluorescence complementary ⁿand CAC-2-YFP ^cprotoplastis in viridescent fluorescence and being positioned on cytolemma.At CAC-2-YFP ⁿand CMA-YFP ^cthe protoplastis of expressing also has green fluorescence and is positioned on cytolemma.But at CAC-1-YFP ⁿand CMA-YFP ^cthe protoplastis of expressing does not have obvious green fluorescence.

By these two experiments we can draw can to mutual effect at such conclusion: CAC-1 and CAC-2, the interaction of CMA and CAC-2 is better than CAC-1.

Embodiment 4, CAC-1, CAC-2, CMA regulate and control pollen tube and stretch, affect male sterile checking

The sprouting of pollen and cultivation

1) pollen germination substratum 1mM CaCl is configured ₂, 1mM Ca (NO ₃) ₂, 1mM MgSO ₄, 0.01% (w/v) H ₃bO ₃, and 18% (w/v) sucrose 0.8% (w/v) agar, pH 7.0.

2) collect 20 tobaccos and put into 10mL centrifuge tube, shake 1 minute, centrifugal 2 minutes of 500g.

3) pollen of collection is sprinkling upon on substratum and cultivates 30 minutes.

Biolistic bombardment

1) CAC-1 containing GFP that will build, CAC-2 and CM plasmid bronze bag quilt (bronze of the plasmid use 3mg of 30ug)

2) use the PDS-1000/He biolistic system of Bio-Rad company to enter in cultured pollen tube by the plasmid bombardment being coated with bronze, each sample bombards 2 times.Parameters is the Hg vacuum chamber of 28 feet, 1100-psi safety film, 0.25 foot of gap distance.

3) cultivate after 30 minutes and use confocal laser scanning microscope.

Compared with the control, what the pollen tube of CAC-1 process LAN obviously became has thick short again, may be caused by the accumulation due to calcium ion.But the pollen tube of expressing CAC-1 with CAC-2 has recovered the phenotype the same with contrast simultaneously.If process LAN CAC-1 simultaneously, the short again of CAC-2 and CMA pollen tube change has slightly, consistent with the pollen tube phenotype of process LAN CAC-1.

Claims (2)

1. the Ca of aminoacid sequence as shown in SEQ ID No.1 ²⁺/ Mg ²⁺passage molecule CAC-1, the aminoacid sequence Ca as shown in SEQ ID No.3 ²⁺/ Mg ²⁺the inhibitor C AC-2 of passage molecule CAC-1 and the aminoacid sequence calcium binding protein CMA as shown in SEQ ID No.5 combines the application as calcium current fluctuation in Ca2+ oscillations switch molecule regulating plant body, and it is characterized in that, CAC-2 suppresses Ca ²⁺/ Mg ²⁺the activity of calcium channels of passage molecule CAC-1, CMA and CAC-2 and CAC-1 interacts simultaneously, relieves the suppression of CAC-2 to CAC-1 calcium ion transport activity.

2. the Ca of aminoacid sequence as shown in SEQ ID No.1 ²⁺/ Mg ²⁺passage molecule CAC-1, the aminoacid sequence Ca as shown in SEQ ID No.3 ²⁺/ Mg ²⁺the inhibitor C AC-2 of passage molecule CAC-1 and the aminoacid sequence calcium binding protein CMA as shown in SEQ ID No.5 combines and is used for the male sterile application of regulating plant as Ca2+ oscillations switch molecule, and it is characterized in that, CAC-2 suppresses Ca ²⁺/ Mg ²⁺the activity of calcium channels of passage molecule CAC-1, CMA and CAC-2 and CAC-1 interacts simultaneously, relieves the suppression of CAC-2 to CAC-1 calcium ion transport activity.