CN102260341A - 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

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

Technical field

The present invention relates to the genetically engineered field, relate to the molecular switch and the application thereof of the fluctuation of control 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 higher plant syngenesis is played crucial effects.The growth of pollen tube relates to the process of a series of complexity, and various mineral elements wherein play an important role at this, studies show that in a large number the stripped sprouting of pollen and pollen tube growth need suitable calcium ion concn.Ca ²⁺The growth of pollen germination and pollen tube be can influence by number of ways, the assembling of cytoskeleton, all respects such as transportation, fusion pollen tube top pulsed growth and the regulation and control of pollen tube growth direction of secretory vesicle wherein related to.Exist which type of molecular mechanism to accomplish the accuracy controlling of plant polar growth in the behind of these phenomenons so actually.

Ca ²⁺As most important second messenger in the vegetable cell, the polar growth of involved in plant is grown and to the transduction of adverse circumstance signal.Under the abiotic stress condition, the calcium ion in the plant cytoplasm specific variations can occur on time, space and concentration, promptly brings out to produce the calcium signal.The calcium signal is experienced by the calcium binding protein in its downstream and is transduceed, and then causes that in cell a series of biochemical reaction comes coordinate plant growth or adaptation or resists various environment stresses.In vegetable cell, find Ca at present ²⁺/ CDPK, Ca ²⁺/ CaM and Ca ²⁺/ CBL 3 class calcium signalling systems.Wherein CaM is a most important class calcium signal sensing element in the vegetable cell, contain the amino-acid residue about 150, and structure is very conservative between the different calmodulin.The EF hand-type structure that all to contain 4 length in each calmodulin be 12 amino-acid residues, they each combine with a calcium ion, thereby causing that the calmodulin structure looks like to change activates calmodulin and exercises signal transduction functionality.Calmodulin transmits the courier as the important signal of a class and combines by conjugated protein with CaM (CaMBP) to regulate more intracellular biochemical reactions.We are by research Ca ²⁺, calmodulin, channel protein explanation introduce a kind of molecular switch with the control of calcium signal fluctuation with decoding of general biological significance.

Summary of the invention

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

A further object of the present invention provides above-mentioned Ca ²⁺/ Mg ²⁺The inhibitor C AC-2 of passage molecule CAC-1.

Another object of the present invention provides 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 provides calcium signaling switch molecule.

According to Ca of the present invention ²⁺/ Mg ²⁺Passage molecule CAC-1, its aminoacid sequence is shown in SEQ ID No.1; Its nucleotide sequence is 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 shown in SEQ ID No.3; Its nucleotide sequence is shown in SEQ ID No.4.

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

According to calcium signaling 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 calcium channel activity of passage molecule CAC-1, CMA and CAC-2 and CAC-1 interact simultaneously, have removed CAC-2 to the active inhibition of CAC-1 calcium ion transport.

The present invention also provides the application of calcium current fluctuation in the above-mentioned calcium signaling switch molecular regulation plant materials.

The present invention also provides above-mentioned calcium signaling switch molecule to be used to regulate and control the application of plants male sterility.

According to a particular embodiment of the invention, molecular switch with the polar growth of regulating the plant flowers tube cell is an example, be described in detail, the albumen of two homologous genes encodings regulation and control is mutually formed exquisite calcium signaling switch molecule with the 3rd calcium binding protein, the activity of common adjusting pollen tube calcium channel and the fluctuation and the decoding of calcium ion signal, thereby the accurately extension or the shortening of controlling plant pollen tube, male sterile with the regulation and control plant, this not only has great application value, and general biological significance is more arranged.

The contriver of invention finds that after deliberation gene C AC-1 is the calcium channel in the plant flowers tube cell, and autoploid CAC-2 can suppress the activity of CAC-1, but can remove this inhibition under the situation that CMA exists, particularly:

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

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

3, the interaction according to each element of calcium signaling switch molecule of the present invention has dual mode: CAC-2 and CAC-1 be by the directly effect of C-end, thereby suppress the activity of CAC-1 calcium channel; CAC-2 is by CMA and CAC-1 effect;

4, CMA and CAC-2 and CAC-1 interact simultaneously, have removed CAC-1 to the active inhibition of CAC-1 calcium ion transport;

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

6, the PROTEIN C AC-1 of two homologous genes encodings, CAC-2 regulation and control and the 3rd the exquisite switch molecule of calcium binding protein CMA composition mutually, the pollen tube of control extends or shortens the accuracy controlling plants male sterility thus;

7, above-mentioned calcium signal fluctuation mechanism and decoding are present in the extension of plant root hair, guard cell's growth, the reaction of pathology or other physiological process equally, have general biological significance and application thereof.

Description of drawings

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

Fig. 2 shows that CAC-2 has suppressed the calcium ion transport activity of CAC-1, and CMA has removed this restraining effect;

Fig. 3 has shown the interaction and effect specific site between CAC-1, CAC-2 and the CMA.

Fig. 4 shows that CAC-1 and CAC-2 interact and keeps the prolongation of pollen tube that CMA removes the effect that CAC-2 suppresses CAC-1, suppresses pollen tube and prolongs;

Embodiment

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

1, the cultivation of Arabidopis thaliana

When cultivating Arabidopis thaliana in the soil, suction is saturated and on 4 ℃ of seeds after handling 2-3 days are seeded in nutrition soil according to 1/3 mixed vermiculite equably, and dark lucifuge sprout growth moves to cultivation normal illumination under after waiting to grow true leaf.The growing period maintenance ground moistening that waters.

During the sterile culture Arabidopis thaliana, carry out surface sterilization to seed earlier in the culture dish.An amount of exsiccant Arabidopis thaliana seed is placed the little centrifuge tube of 1.5mL, earlier with an amount of dehydrated alcohol sterilization 30sec, chlorine bleach liquor with 1mL 10-15% soaks 5-10min again, use rinsed with sterile water 3-5 time then, place 4 ℃ of synchronization process after 2～3 days, dibbling is on the 1/2MS substratum equably.

Culture condition:

Temperature 20-23 ℃, intensity of illumination 80-200 μ mol m ^-2Sec ^-1, the photoperiod is the alternately dark or continuous illumination of 16h illumination/8h, humidity is about 70%.

2, the extraction of total RNA

3, total RNA reverse transcription

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

Be heated to 65 ℃, 5min.

2) place 1min at least on ice

3) the second step reaction system is:

Reaction conditions is: 50 ℃ 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 with above-mentioned reverse transcription product, and reaction system and condition are as follows:

Reaction parameter:

5, the recovery of PCR product

With PCR product electrophoresis on 1% agarose, after cutting the purpose band under the ultraviolet lamp and shredding blob of viscose, press OMEGA Gel Extraction Kit operation (producing) by OMEGA.

6, the connection of PCR product and conversion

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

To connect the product thermal shock and be transformed into E.coli DH5 α competent cell, select the positive colony order-checking.

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

The 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 its encoded protein matter 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 with above-mentioned reverse transcription product, and reaction system and condition are as follows:

Reaction parameter:

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

The 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 its encoded protein matter 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 with above-mentioned reverse transcription product, and reaction system and condition are as follows:

Reaction parameter:

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

The CMA gene has following open reading frame nucleotide sequence:

ATGGCGGATCAGCTCACAGACGATCAGATCTCAGAATTCAAGGAAGCCTTCAG

CTTATTCGACAAGGATGGTGATGGTATGCTTCATCCTCCCTTTCCCTCTATCATCG

TAGGTTGCATTACCACAAAGGAGCTTGGTACCGTGATGCGTTCCCTCGGTCAAA

ACCCAACCGAAGCTGAGCTTCAGGACATGATCAACGAAGTTGATGCGGATGGT

AACGGAACCATTGATTTCCCGGAGTTCTTGAACCTAATGGCTAGGAAAATGAA

GGACACTGACTCTGAGGAAGAACTCAAGGAAGCTTTCAGAGTTTTCGACAAA

GACCAGAACGGTTTCATCTCAGCTGCTGAATTGAGACATGTGATGACTAACCTC

GGCGAGAAGCTTACTGATGAAGAAGTTGATGAGATGATTAAGGAAGCTGATGT

TGATGGTGATGGTCAGATCAACTACGAAGAGTTTGTGAAGGTTATGATGGCTAA

GTGA

The aminoacid sequence of its encoded protein matter is:

MADQLTDDQISEFKEAFSLFDKDGDGMLHPPFPSIIVGCITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMARKMKDTD?SEEELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYEEFVKVMMAK

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

1, the preparation of pGEMHE vector construction and Capping RNA

The T7 promotor is arranged on the pGEMHE plasmid, CAC-1, CAC-2, CMA is gene constructed in this plasmid, can utilize in-vitro transcription test kit mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit, obtain the mRNA of CAC-1, CAC-2, CMA full length gene.Concrete grammar is with CAC-1 and CAC-2 gene RespectivelyInsert in the pGEMHE plasmid, obtain pGEMHE-CAC-1, CAC-2, CMA.And as PCR is template, with primer M13/M13R amplification purpose segment, make about 1～2 μ g of DNA behind the purifying carry out in-vitro transcription, carry out according to reagent and step that in-vitro transcription test kit mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit is provided.Once transcribe and to obtain about 15～20 μ gmRNA of 20 μ l, get 1 μ l and measure its concentration, use then through DEPC and handle and be diluted to the concentration (0.5 μ g/ μ l) that needs, divide to install in the 1.5mL centrifuge tube through the distilled water of High Temperature High Pressure with biophotometer, every pipe 2-3 μ L ,-80 ℃ of preservations are standby.

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

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

1) after ice cube was anaesthetized African toad, surgical operation was taken out an amount of pieces of an egg;

2) the pieces of an egg 1.5h that takes out with the type i collagen protease digestion at ambient temperature places in the enzymolysis process on the low speed shaking table, to promote dissociating of pieces of an egg;

3) with after the no calcium ND96 nutrient solution flushing, immigration fills in the culture dish of ND96.Place 18 ℃ of overnight incubation.

3, the prescription of voltage clamp body lotion

1) frog's egg culture solution prescription: ND96

NaOH regulates PH to 7.4

2), Bath Solution prescription:

CaCl 30mmol

MgCl 30mmol

MES 10mmol

4, record of two electrodes voltage embedding voltage clamp and analysis

At first in the frog's egg that takes out, inject the mRNA of CAC-1 and CAC-2 gene, each ovocyte injection 46nl cRNA, control group is injected isopyknic deionized water.After the injection, ovocyte is put back to 18 ℃ of incubators and is cultivated 48～72h, writes down electrical signal then.Injection have the ovocyte of CAC-1 gene mRNA in the 30mM calcium chloride solution, can record in one to electric current, and under the voltage of-140mV stimulates, can inspire the electric current of 1000mA.Cause to have the dependency of voltage and time to transport features in the calcium ion of this electric current by the CAC-1 gene.And control cells has only the about 300mA of ovocyte background current.The size of current that injection has the ovocyte of CAC-2 gene mRNA to record in the 30mM calcium chloride solution is consistent with control cells.So the CAC-2 gene does not have the calcium ion transport characteristic.In the 30mM calcium chloride solution, write down CAC-1, the ovocyte that the CAC-2 gene mRNA is expressed simultaneously, we find original calcium ion current vanishes that is produced by CAC-1, show the rectification characteristic same with control cells, and CAC-2 can suppress the calcium ion electric current of CAC-1 as can be seen.CAC-1 and CAC-2 have the CMA binding site, so whether is there there what relation between they three? we are expressed in this three in the ovocyte simultaneously, writing down us by voltage clamp finds to that is to say that CMA has removed the inhibition of CAC-2 for CAC-1 because the calcium ion transport characteristic of the expression CAC-1 of CMA is resumed.

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

The yeast two-hybrid experiment

1, bait gene transforms sieve storehouse host bacterium AH109

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

2) with cultivating about 4 hours among this 3ml bacterium liquid access large volume (it is fixed that volume is looked the conversion number) YPD, make nutrient solution OD600 reach 0.6;

3) the 50ml centrifuge tube collect bacterium liquid (Falcon, BD), Eppendorf Centrifuge 5810R, 700g (1865rpm), 5 minutes centrifugal collection thalline are abandoned supernatant;

4) the resuspended thalline of 20ml ddH2O, Eppendorf Centrifuge 5810R, 700g, 5 minutes centrifugal collection thalline are abandoned supernatant;

5) the resuspended thalline of 20ml 1XTE/LiAc, Eppendorf Centrifuge 5810R, 700g5 minute centrifugal collection thalline abandoned supernatant;

6) add the resuspended thalline of 1XTE/LiAc according to each amount that transforms needs 50 μ l bacterium liquid, divide to install in the Eppendorf pipe every pipe 50 μ l bacterium liquid;

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

8) be positioned in 30 ℃ of incubators or the water-bath and hatched 30 minutes, mixing once in the time of 15 minutes;

9) after 30 minutes, every pipe adds 15 μ l DMSO (DIMETHYL SULFOXIDE), and mixing gently turns upside down;

10) 42 ℃ of water-bath heat shocks are 20 minutes, turn upside down mixing once in per 10 minutes;

11) ice bath is 5 minutes;

12) 700g, 5 minutes centrifugal collection thalline are abandoned supernatant;

13) ddH ₂O washs a thalline;

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

Original Carrier DNA 100 ℃ of sex change earlier 10 minutes placed ice bath 2 minutes immediately, and then 100 ℃ of sex change 10 minutes, placed on the ice bath standby afterwards.Used Carrier DNA only needs 100 ℃ of sex change once to get final product.1XTE/LiAc:V10XTE: V10XLiAc: VH2O=1: 1: 81XTE/LiAc/PEG:V10XTE: V10XLiAc: V50%PEG=1: 1: 850%PEG:50% is a mass volume ratio.PEG3350 is dissolved in generally needs heating for dissolving 10XTE:0.1M Tris-HCl, 10mM EDTA, pH7.510XLiAc:1M LiAc, pH7.5 among the ddH20

2, after the AH109 that contains the Bait gene plasmid protects kind of a Bait gene and grows the clone, generally with the bait gene Auele Specific Primer directly in yeast colony the PCR evaluation whether change the plasmid of correct Bait gene over to, then through identifying that correct clone protects kind.

3, yeast colony PCR

1) with the plasmid that contains the Bait gene as positive control, with host bacterium AH109 as negative control.

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

3) carry out PCR according to following reaction conditions:

95 ℃ 5 minutes

94 ℃ 30 seconds

58 ℃ 30 seconds

72 ℃ 2 minutes

30 circulations

72 ℃ of 72 ℃ of extensions in 10 minutes were at the Bait gene less than 2Kb in 2 minutes, wanted corresponding time expand for the Bait gene of being longer than 2Kb.

4) the PCR product carries out electrophoresis detection.

4, positive colony is protected and is planted

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

2) 700g, 5 minutes, centrifugal collection thalline;

3) adding 1: 1 liquid nutrient medium and Sterile Glycerol Solution mixing solutions;

4) thalline that fully suspends is put in-80 ℃ 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 that adopts the film inspection, detects LacZ expression of gene situation.The film detecting method is as follows:

1) with the positive colony bacterial colony photographic reprinting on filter paper;

2) filter paper is dipped in the liquid nitrogen fully, the time was longer than 30 seconds, dried after the taking-up.

3) culture dish adds an amount of colour developing liquid (fresh preparation), and pad one deck filter paper makes it moistening fully, will freeze above filter paper after molten is layered on, and makes colour developing liquid be penetrated into the top layer.The 9CMa culture dish adds 2ml colour developing liquid, and the 15CMa culture dish adds 5ml colour developing liquid;

4) cover the ware lid, be placed in 37 ℃ of incubators, lucifuge is placed;

5) begin to observe positive findings and the record that has or not change blue after 20 minutes, generally be as the criterion with 3 hours, whether in particular cases can seeing spends the night becomes blue;

6) the ware lid is opened in reaction later, with the filter paper oven dry, preserves and record; Colour developing liquid: 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 be deposited 1 year.X-gal:X-gal (5-bromo-4-chloro-3-indoly-β-D-galactopyranoside) be dissolved in DMF (N, N-dimethylformamide) in, final concentration is 20mg/ml.

6, bait gene sieve storehouse

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

2) the 150ml nutrient solution changes mixing among the 1000ml YPDA (YPD+Adenine) over to, at this moment OD600=0.2-0.3;

3) cultivate about 4-5 hour to OD600＞0.6;

4) the 500ml centrifuge tube is collected bacterium liquid.Beckman Coulter Centrifuge Avanti J-25 (following steps are all used this whizzer), 700g, 5 minutes, RT (room temperature).Simultaneously with twice of the pre-sex change of 2000 μ l Carrier DNA;

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

6) 30ml 1X TE/LiAC washing thalline (preparing 50ml 1X TE/LiAC in advance) changes the 100ml centrifuge tube over to;

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

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

9) add 100-500 μ g library DNA, the Carrier DNA of the pre-sex change of 2000 μ l, mixing gently;

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

11) hatched 45 minutes for 30 ℃, per 15 minutes mixings once;

12) add 3.2ml DMSO, mixing gently;

13) 42 ℃ of heat shocks are 20 minutes, and per 10 minutes mixings once;

14) ice bath is 5 minutes;

15) 700g 5 minutes, collects thalline;

16) add 1000ml YPDA, 30 ℃ of shaking culture 60 minutes;

17) 700g 5 minutes, collects thalline;

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

19) titre (stock relocation efficient) is measured: 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; Three kinds of concentration were respectively got 100 μ l coating SD/-Trp/-Leu flat board in 1: 100000

20) calculating of stock relocation efficient: to being grown in the clone's counting on the SD/-Trp/-Leu flat board, selected clone number extent of dilution between 30-300 calculates stock relocation efficient

21) finish in the sieve storehouse, and bacterium after 7-14 days, can be cloned number (cfu) the X volume that always suspends in growth on the SD/-Trp/-Leu/-His+30mM3-AT flat board

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 the film inspection.Extracting prey plasmid and Bait plasmid are done the corotation checking from the blue yeast of film inspection change.

7, extracting yeast plasmid

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

2) get Eppendorf pipe, add the 5u/ μ l Lyticase of 30 μ l.Bacterium is scraped among the lyticase with toothpick, and Vortex fully suspends.Hatched 30 minutes for 37 ℃;

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

4) 12, centrifugal 10 minutes of 000rpm changes supernatant in the one clean Eppfendorf pipe over to.Note, albumen is not shifted in the past;

5) add 8 μ l 10M NH4Ac and 500 μ l dehydrated alcohols, mixing was placed 1 hour for-80 ℃;

6) 12, centrifugal 10 minutes of 000rpm removes supernatant;

7) add 500 μ l, 70% washing with alcohol precipitation, 12, centrifugal 5 minutes of 000rpm;

8) abandon supernatant, vacuum is drained precipitation;

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

8, the method for the general electricity consumption commentaries on classics of the amplification of yeast plasmid increases extractive Prey plasmid from the positive colony of sieve storehouse gained in intestinal bacteria.The intestinal bacteria electricity changes the competence preparation:

1) chooses a mono-clonal and insert 3ml LB substratum, shake and train 14-16 hour to OD600=1.0-1.2;

2) will shake bacterium liquid switching 1L LB substratum after the training, shake training 4-5 hour to OD600=0.7-0.8;

3) centrifugal receipts bacterium, the dissolving of 10ml frozen water;

4) 5000g, 4 ℃ of centrifugal 10 minutes receipts bacterium, the 400ml frozen water is resuspended with bacterium, ice bath 10 minutes;

5) 5000g, 4 ℃ of centrifugal 10 minutes receipts bacterium, 40ml precooling 10% glycerine is resuspended, ice bath 10 minutes;

6) 5000g, 4 ℃ of centrifugal 10 minutes receipts bacterium, it is resuspended to add 1ml 10% glycerine;

7) bacteria liquid of measuring after resuspended with rifle is long-pending, adds that (annotate: equal-volume 10% glycerine refers to the long-pending 1ml that deducts of bacteria liquid after resuspended with isopyknic 10% glycerine of bacterium liquid.Example: resuspended bacteria liquid amasss and is 4ml behind the adding 1ml10% glycerine, and then adding 10% amounts of glycerol is 3ml);

8) branch installs in the Eppendorf pipe, every pipe 100 μ l, operation on ice; 9.-80 ℃ refrigerator is preserved; 10. take out two pipes and do feminine gender, positive control; 10% glycerine: V glycerine: V water=1: 9.

9, electric shock transforms

1) takes out competence from-80 ℃ of refrigerators, to dissolving on ice.Simultaneously, electric revolving cup to cooled on ice, is opened electroporation and prepared 30 minutes;

2) after the competence dissolving, the plasmid that will transform (or connecting product or other materials that will transform) adds competence, with rifle piping and druming evenly.Electricity is necessary to carry out negative control, positive control when changeing, so that whether the check competence is contaminated and whether competence is normal;

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

4) dry the wall on electric revolving cup top electrode both sides;

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

6) after the electric shock, competence is washed out, hatched 45 minutes for 37 ℃ with 1ml SOC;

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

8) coated plate, the suitableeest voltage of different electric revolving cups has nothing in common with each other, through experiment: the suitableeest voltage of 1mm electricity revolving cup is that the suitableeest voltage of 1900-2200V 4mm electricity revolving cup is that the suitableeest voltage of 2300-2500V 8mm electricity revolving cup is 2500-2700V SOC:20g/L peptone, the 5g/L yeast extract, 0.5g/L NaCl, 2.5mM KCl, pH7.0.121 ℃, sterilization in 20 minutes.When being cooled to below 60 ℃, every liter of 1M glucose solution that adds the 20ml sterilization.Every liter of 2M MgCl that adds the 5ml sterilization before this solution uses ₂

The experiment of bimolecular fluorescence complementary

Molecular fluorescence complementation (bimolecular fluorescence complementation, BiFC) analytical technology, be by Hu etc. in 2002 report a kind of directly perceived at first, judge location and the interactional new technology of target protein in viable cell apace. this technology dexterously with two complementary fragments of fluorescin molecule respectively with the target protein amalgamation and expression, if fluorescin activation recovering then show that interaction has taken place two target proteins. develop the multicolor fluorescence complementary technology (multicolorBiFC) that thereafter, can not only detect the formation of multiple proteins complex body simultaneously, can also compare producing interactional power between different proteins. at present, this technology has been used for transcription factor, the dimeric forms of G albumen β γ subunit produces between different proteins in the research work of the aspects such as strong and weak comparison and protein ubiquitinization that interact.

Utilize yellow fluorescence protein (yellow fluorescent protein, YFP) as reporter gene, fluorescin is divided into two molecule fragments that do not have fluorescence activity, be connected with target protein respectively again, if two target proteins are close because interaction is arranged, just make that two molecule fragments of fluorescin are spatially close mutually, again form active fluorophor and send fluorescence. under fluorescent microscope, just can observe directly two target proteins and whether have interaction, and observing the time that it interacts and takes place under the condition near the viable cell physiological status, the position, strong and weak, the stability of the protein complex that forms, and the cell signal molecule is to its interactional influence.

The one of carbon tip of CAC-1 and CAC-2 is structured in respectively is used to do yeast two-hybrid among pGADT7 and the pGBKT7.At leucine, tryptophane has been expressed the yeast of CAC-1 and CAC-2 and can have been grown on the substratum of Histidine disappearance, illustrate 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.

Equally in bimolecular fluorescence complementary experiment as can be seen, in commentaries on classics CAC-1-YFP is arranged ^NAnd CAC-2-YFP ^CProtoplastis in viridescent fluorescence and being positioned on the cytolemma.At CAC-2-YFP ^NAnd CMA-YFP ^CThe protoplastis of expressing also has green fluorescence and is positioned on the cytolemma.But at CAC-1-YFP ^NAnd CMA-YFP ^CThe protoplastis of expressing does not have tangible green fluorescence.

By these two experiments we can draw can be 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 regulation and control pollen tube stretch, influence male sterile checking

The sprouting of pollen and cultivation

1) configuration pollen germination substratum 1mM CaCl 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 the 10mL centrifuge tube, shook centrifugal 2 minutes of 500g 1 minute.

3) pollen of collecting is sprinkling upon on the substratum cultivated 30 minutes.

The particle gun bombardment

The CAC-1 that contains GFP that 1) will make up, CAC-2 and CM plasmid with the bronze bag by (plasmid of 30ug uses the bronze with 3mg)

2) the plasmid bombardment of using the PDS-1000/He biolistic system of Bio-Rad company will be coated with bronze enters in the cultured pollen tube each sample bombardment 2 times.Parameter be set be 28 feet Hg vacuum chamber, 1100-psi safety film, 0.25 foot gap distance.

3) cultivation was observed with laser confocal microscope after 30 minutes.

Compared with the control, CAC-1 crosses obvious the having slightly of becoming of pollen tube of expression and lacks again, may be because due to the accumulation of calcium ion.But the pollen tube of expressing CAC-1 and CAC-2 has simultaneously recovered the phenotype the same with contrast.If cross expression CAC-1 simultaneously, the weak point again that CAC-2 and CMA pollen tube become has thick, and the pollen tube phenotype of expressing CAC-1 is consistent with crossing.

Claims (9)

1. Ca ²⁺/ Mg ²⁺Passage molecule CAC-1 is characterized in that, its aminoacid sequence is shown in SEQ ID No.1.

2. Ca according to claim 1 ²⁺/ Mg ²⁺Passage molecule CAC-1 is characterized in that, its nucleotide sequence is shown in SEQ ID No.2.

3. the described Ca of claim 1 ²⁺/ Mg ²⁺The inhibitor C AC-2 of passage molecule CAC-1 is characterized in that, its aminoacid sequence is shown in SEQ ID No.3.

4. Ca according to claim 3 ²⁺/ Mg ²⁺The inhibitor C AC-2 of passage molecule CAC-1 is characterized in that, its nucleotide sequence is shown in SEQ ID No.4.

5. a calcium binding protein CMA is characterized in that, its aminoacid sequence is shown in SEQ ID No.5.

6. calcium binding protein CMA according to claim 5 is characterized in that its nucleotide sequence is shown in SEQID No.6.

7. calcium signaling switch molecule is characterized in that, comprises the described Ca of claim 1 ²⁺/ Mg ²⁺Passage molecule CAC-1, the described Ca of claim 3 ²⁺/ Mg ²⁺The described calcium binding protein CMA of the inhibitor C AC-2 of passage molecule CAC-1 and claim 5, wherein, CAC-2 suppresses Ca ²⁺/ Mg ²⁺The calcium channel activity of passage molecule CAC-1, CMA and CAC-2 and CAC-1 interact simultaneously, have removed CAC-2 to the active inhibition of CAC-1 calcium ion transport.

8. the application of calcium current fluctuation in the described calcium signaling switch of the claim 7 molecular regulation plant materials.

9. the described calcium signaling switch of claim 7 molecule is used to regulate and control the application of plants male sterility.

Images