CN104884619A - Cotton isopentenyl transferase ipt2, gene for encoding same, and application thereof - Google Patents
Cotton isopentenyl transferase ipt2, gene for encoding same, and application thereof Download PDFInfo
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Abstract
Provided are an isopentenyl transferase (IPT2) from cotton, a gene for encoding same, and an application thereof in culturing transgenic plants with improved drought-tolerance.
Description
A kind of cotton isopentene group transferase I PT2 and its encoding gene and application
The present invention relates to vegetable protein and its encoding gene and application, more particularly to one prenyltransferase from cotton for technical field（IPT2) albumen and its encoding gene, and its application in the genetically modified plants that drought resistance is improved are cultivated.Background technology abiotic stress, such as arid, salt marsh, extreme temperature, chemical contamination and oxygen injury can cause serious harm to growing for plant, and extreme loss is caused to crop yield.Wherein influence of the arid to crop yield, first place is accounted in many natural adverse circumstances, and it is endangered equivalent to other disaster sums, be many areas be agricultural development bottleneck.According to statistics, world's arid, semiarid zone account for the 34% of land area;China's arid, semiarid zone account for the 52% of area, year area suffered from drought up to 200-270 ten thousand hectares, the national billion cubic meter of the annual water shortage in irrigation district about 30 receives hundred million kilograms of grain 350-400 because of water shortage less；Particularly China relationship and primary grain producing such as North China, northeast and northwest, is the area of China's water shortage most serious, and spring drought frequently reaches 10 years nine chances.
Because the resistance to coercive of plant belongs to quantitative character mostly, existing available germ plasm resource is deficient, and the difficulty for improveing stress tolerance in plants using traditional breeding method is quite big, cultivates really resistance to stress kind just particularly difficult.In recent years, with to plant stress-resistance molecular mechanism research deepen continuously and Protocols in Molecular Biology fast development, degeneration-resistant research is deep into molecular level from physiological level, promotes the development of plant stress-resistance genetic engineering.Corresponding responsing reaction can be produced when plant is being forced, to reduce or eliminate the harm brought to plant.This responsing reaction of plant is one and is related to polygenes, multi signal approach, the complex process of polygenes product.These genes and its expression product can be divided into 3 classes：（1) gene and product of signal cascade amplification system and transcription control are participated in；（2) gene and its expression product directly worked to protection biomembrane and protein；（3) protein related with transhipment to the intake of water and ion.In recent years, research of the plant to stress-tolerance ability is improved by transgenic technology, and to coercing the crops with tolerance, the research of xerophyte and halophytes all achieves significant achievement, stress-related genes and signal transduction system there has also been with further understanding (the Two transcription factors of Liu Q. 1998., DREB1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought- and low temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10: 1391-1406； KANG JY. 2002. Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell, 14:
343-357； ABE H. 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78. ).
But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry under adverse circumstance and physiological response mechanism.The research that the related signaling pathways of plant stress-resistance and signal are transmitted with network system is provided important basis by the research in terms of the function and expression regulation of degeneration-resistant response gene.Content of the invention the present inventor utilizes SSH (Subtractive hybridizations）With RACE (cDNA ends rapid amplifyings）The method being combined has cloned a prenyltransferase for cotton（Be named as IPT2 herein) encoding gene, and determine its DNA sequence dna.And it was found that being conducted into by transgenosis after plant, the drought resistance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.
First aspect present invention provides a prenyltransferase IPT2 for cotton encoding gene（Gh IPT2 are named as herein), its sequence is SEQ ID N0: 2.
Second aspect of the present invention provides a kind of recombinant expression carrier, and it contains the gene described in first aspect present invention and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector；Preferably, the carrier is the rd29A-Gh IPT2-2300 carriers shown in accompanying drawing 2.
Third aspect present invention provides a kind of recombinant cell, and it contains the recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention；Preferably, the recombinant cell is restructuring agrobatcerium cell.
Fourth aspect present invention provides a kind of method of improvement plant drought resistance, including：Gene described in first aspect present invention or the recombinant expression carrier described in second aspect of the present invention are imported into plant or plant tissue and make the gene expression；Preferably, the plant is tobacco.
Fifth aspect present invention provides a kind of method of prepare transgenosis plant, including：The plant containing gene described in first aspect present invention or the recombinant expression carrier described in second aspect of the present invention or plant tissue are cultivated under conditions of plant is effectively produced；Preferably, the plant is tobacco.
Gene, the recombinant expression carrier described in second aspect of the present invention or the recombinant cell described in third aspect present invention that sixth aspect present invention is provided described in first aspect present invention are used to improve plant drought resistance and the purposes for plant breeding；Preferably, the plant is tobacco.
Seventh aspect present invention provides the protein of the gene code described in first aspect present invention, its amino acid sequence such as SEQ ID N0:Shown in 1.
Brief description of the drawings Fig. 1 is GMPT2 plant expression vector (rd29A-GhIPT2-2300;>Structure flow.
Fig. 2 is GMPT2 plant expression vector Crd29A-GhIPT2-2300) plasmid figure.
Fig. 3 is the drought resistance growing state for compareing tobacco and transgene tobacco（Before Fig. 3 a is arids, after Fig. 3 b is arids）；CK (left side)：Compare tobacco；T1Q4 is (right）：Transgene tobacco strain.
Fig. 4 is the result that drought-resistant T1 is for transgenic tobacco plant and not drought-resistant control tobacco plant is on transcriptional level.M is Marker (DL2000), 1-8 be drought-resistant T1 for transgenic tobacco plant, 9 be plasmid PCR positive control, and 10-13 is not drought-resistant control tobacco plant.Embodiment provides following examples, to facilitate those skilled in the art to more fully understand the present invention.The embodiment is not intended to limit the scope of the present invention merely for exemplary purpose.Cotton SSH library constructions under the drought stress of embodiment 1.：
Specific method is：
Subtracted library is built by Subtractive hybridization method using the method shown in the PCR-selectTM cDNA Subtraction Kit of Clontech companies.The mRNA extracted in experimentation using in the blade of the cotton seedling of Osmotic treatment is used as sample（), tester the mRNA extracted using in the blade of untreated cotton seedling is used as control (driver).Specific steps are summarized as follows：
(1) material to be tested：
African cotton（National Cotton mid-term storehouse, obtains unit Cotton research institute, Unified number：ZM-06838) it is seeded on sterilized vermiculite, in 25 °C, photoperiod 16h illumination/8h dark（The Lx of light intensity 2000-3000) under the conditions of cultivate, 1/2MS culture mediums are poured weekly（Contain 9.39 mM KN03, 0.625 mM KH2P04, 10.3 mM NH4NO3,0.75 mM MgS04, 1.5 mM CaCl2, 50 μM of KI, 100 μM of H3B03, 100 μM of MnS04, 30 μM of ZnS04, 1 μ M Na2Mo04, 0.1 μ M CoCl2, 100 μM of Na2EDTA, 100 μ M FeS04) once.It is used to test when seedling plant height is up to 25-30cm.
(2) material process：
2 groups, every group of 4 basins, per 1 plant of basin will be divided into for examination seedling.First group is control group, in 25 °C, photoperiod 16h illumination/8h dark（The Lx of light intensity 2000-3000) culture is normal to pour.Second group is Osmotic treatment group, 25 °C, photoperiod 16h illumination/8h dark（The Lx of light intensity 2000-3000) under the conditions of cultivate, stop pour, handle 10 days, the blade of two groups of seedling apicals 1/3 of timely clip after being disposed, after liquid nitrogen quick freeze, in-70 °C
Preserved in refrigerator.
(3) Total RNAs extraction：
Control group and the g of the cotton leaf of Osmotic treatment group 0.5 are taken respectively, use plant RNA extraction kit（Purchased from invitrogen) extract cotton leaf total serum IgE.Total serum IgE is determined in 260 nm and 280 nm absorbance, OD with the ultraviolet specrophotometer U-2001 of HITACHI companies26Q/OD28QRatio is 1.8-2.0, shows that total serum IgE purity is higher, detects the integrality of total serum IgE with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.MRNA is separated using the Oligotex mRNA purification kits (purification of poly A+ RNA from total RNA) of Qiagen companies.
(4) Subtractive hybridization：
By the PCR-select of Clontech companiesTMMethod shown in cDNA Subtraction Kit kits carries out Subtractive hybridization.Driver mRNA and Tester mRNA are first distinguished into reverse transcription, double-strand cDNA, then the progress subtractive hybridization using 2 μ g Tester cDNA and 2 μ g Driver cDNA as parent material is obtained.Then the Tester cDNA after digestion are divided into joints different in two equal portions, connection by Tester cDNA and Driver the cDNA h of Rsa I digestions 1.5 respectively under 37 °C of water-baths, and Driver cDNA are not connected to head.Two kinds of Tester cDNA for being connected with different joints are mixed with excessive Driver respectively, carry out positive subtractive hybridization for the first time.The products of two kinds of positive subtractives hybridization for the first time are mixed, then second of positive subtractive is carried out with the Driver cDNA that are newly denatured and are hybridized, the fragment of differential expression is expanded by inhibition PCR twice, it is enriched with.
(5) structure of cDNA subtracted libraries and preliminary screening, clone, identification
According to the program of pGEM-T Easy kits, described second positive subtractive is hybridized to second of inhibition PCR primer of cDNA fragments（Purified using QIAquick PCR Purification Kit, purchased from Qiagen) it is connected with pGEM-T Easy (being purchased from Promega kits) carrier, it is comprised the following steps that：Following ingredients are sequentially added with the PCR pipes of 200 μ 1：The positive subtractive of purifying hybridizes second of PCR primer, 31,2 Χ Τ of μ, 4 ligase buffer solutions 5 μ 1, the μ 1 of pGEM-T Easy carriers 1, the μ 1 of T4 DNA ligases 1 of cDNA fragments, is stayed overnight in 4 °C of connections.10 μ coupled reaction products are taken, the competence e. coli jm109 C of 100 μ 1 are added to purchased from TAKARA) in, the min of ice bath 30, heat shock 60 seconds, the min of ice bath 2, the another LB nutrient solutions of 250 μ of power B 1（Containing 1% Tryptone (being purchased from OXOID), 0.5% Yeast Extract (are purchased from OXOID), and 1% NaCl (is purchased from traditional Chinese medicines））Put in 37 °C of water-baths, with the min of 225 rpm shaken cultivations 30, take the bacterium solutions of 200 μ 1 to be inoculated in the LB containing 50 μ g/ml ampicillins (ibid）On/X-gal/IPTG (X-gal/IPTG is purchased from TAKARA) culture plate, 37 °C of 18 h of cultivation.Count diameter in culture plate>1 mm clear white and blue colonies number, random 540 white colonies of picking (numbering：Gh-D2-001 to Gh-D2-540).In 96 porocyte culture plates (CORNING) that all white colonies are inoculated in the LB fluid nutrient mediums containing 50 μ g/ml ampicillins, glycerol adding is saved backup to final concentration 20% in -80 °C after 37 °C of overnight incubations.With nest-type PRC primer Primer 1 and the Primer 2R (PCR-select of Clontech companiesTM
CDNA Subtraction Kit kits are carried）Bacterium solution PCR amplifications are carried out, 452 positive colonies is obtained, then send Invitrogen by all positive colonies（Shanghai）Trade Co., Ltd is sequenced.
(6) the cDNA sequencing analysis of differential cloning：
After the cDNA that DNA sequencing result is removed to carrier and indefinite sequence and redundancy, 405 effective EST (the cotton isopentenyl transferase genes GMPT2 of unigene embodiments 2 clones are obtained
Clone Gh-D2-141 removes after redundant DNA, and sequence is SEQIDNo:3, sequence analysis shows that the albumen of the sequential coding belongs to prenyltransferase.Herein by SEQ IDNo:The corresponding total length encoding gene of 3 sequences is named as GhIPT2, and its corresponding albumen is named as IPT2.
SEQ ID No: 3：
1 A'CT C GASC CGAACCG TTS ACSAGA 3¾C.aACGTGC TAA¾C SC G CAAGAAGCAA
61 TCCAAGAaAT CAAAAGAAAC ACTTGCAGAT TA¾C TGTCG TCAATTGGAG AAGATTCAGC
121 GGC GA'SGAA CAAGAASAAT GGACGA AC ACA.GGC GGA CGCCACCi'SAA GT C CA
131 GGCG GGAAA AGGAGCT'GAT GAS>5CTT&GG AAGAGC TGT AGCTGATCCA A¾~
The clone of GMPT2 total length encoding genes
According to the SEQIDNo obtained:3 sequences, design following two specific primers, are used as 3'RACE 5' end primers.
GMPT2 GSP1: SEQ ID No: 4:
TTAGACAAGA GACAACGTGC TA GMPT2 GSP 1： SEQ ID No : 5:
TAAACTGCTG CAAGAAGCAA TC
Experimental procedure is operated by kit specification（3 ' RACE System for Rapid Amplification of cDNA Ends kits are purchased from invitrogen companies）.
With SEQ ID NO:4 (kit is carried with general bow I things AUAP）, the amplification of first round PCR is carried out by template of the cDNA of mRNA reverse transcriptions.Comprise the following steps that：
The PCR reaction systems of 50 μ 1：The mM of 5 μ, 13 μ of Ι Ο Χ Ε χ Buffer 12.5 dNTP, the cDNA of the mRNA reverse transcriptions of 2.0 μ 1, the Ex Taq of 1.0 μ 1 (being purchased from TAKARA), 10 μM of primer SEQ ID NO:Each distilled waters of 2.0 μ, 1 and 35 μ 1 of 4 and AUAP.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension lmin), 72 °C of 10 min of extension.
The PCR primer of gained takes 2.0 μ 1 as template after diluting 50 times with distilled water, with SEQ ID NO:5 carry out second with universal primer AUAP takes turns PCR amplifications, comprises the following steps that：
50 ylPCR reaction systems：The mM of 5 μ, 1 lOXEx Buffer, 3 μ 12.5 dNTP, 2.0 μ 1 dilute
First round PCR primer, the μ Μ of 1.0 μ Ι Ε χ Taq 10 primer SEQ ID NO:Each μ of 2.0 μ 1 and 35 of 5 and AUAP distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension lmin), 72 °C of 10 min of extension.Reclaim the band that fragment in second of PCR primer is about 400 bp（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy Vector are coupled with, then it is transformed into e. coli jm109 (specific method is ibid).Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/ml ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup.Use SEQIDNO:5 carry out bacterium solution PCR amplifications with universal primer AUAP, obtain 4 positive colonies, 4 positive colonies are delivered into Invitrogen（Shanghai）Trade Co., Ltd's sequencing sequencing, obtains the cDNA of gene 3' ends.
According to the GMPT2 genetic fragments obtained, following three specific primers are designed, 5'RACE 3' end primers are used as.
GMPT2 GSP3: SEQ ID No: 6:
ACAAGCTCTT CCCAAGCCTC AT
GMPT2 GSP4: SEQ ID No: 7:
TGAGGAACAC TTCGGTGGCG TC
GMPT2 GSP5: SEQ ID No: 8:
ATTCTTCTTG TTCCTCAGCC
Experimental procedure is operated by kit specification（5 ' RACE System for Rapid Amplification of cDNA Ends kits are purchased from invitrogen companies）.
Use SEQIDNO:7 (kit is carried with universal primer AAP）, with cDNA (the reverse transcription primer SEQIDNO of mRNA reverse transcriptions:6) amplification of first round PCR is carried out for template, comprised the following steps that：
The PCR reaction systems of 50 μ 1：The mM of 5 μ, 13 μ of Ι Ο Χ Ε χ Buffer 12.5 dNTP, the cDNA of the mRNA reverse transcriptions of 2.0 μ 1, the Ex Taq of 1.0 μ 1 (being purchased from TAKARA), 10 μM of primer SEQ ID NO:Each μ of 2.0 μ 1 and 35 of 7 and AAP distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 55 °C of annealing 30 s, 72 °C of extension lmin), 72 °C of 10 min of extension.
The PCR primer of gained takes 2.0 μ 1 as template after diluting 50 times with distilled water, with SEQ ID NO:8 carry out second with primer AUAP takes turns PCR amplifications, comprises the following steps that：
50 ylPCR reaction systems：The first round PCR primer of the mM of 5 μ, 1 lOXEx Buffer, 3 μ 12.5 dNTP, 2.0 μ 1 dilution, the μ Μ of 1.0 μ Ι Ε χ Taq 10 primer SEQIDNO:Each μ of 2.0 μ 1 and 35 of 8 and AUAP distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 1 min of extension), 72 °C of 10 min of extension.Reclaim the band that fragment is about 800bp in second of PCR primer（Gel Extraction Kit are purchased from OMEGA), and it is coupled with pGEM-T Easy
Vector, being then transformed into JM109, (specific method is ibid）.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 μ g/ml ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup.With SEQ ID NO:8 carry out bacterium solution PCR amplifications with primer AUAP（Reaction system and reaction condition are ibid）, 5 positive colonies are obtained, wherein 4 clones is chosen and delivers to Invitrogen（Shanghai）Trade Co., Ltd's sequencing sequencing, obtains the cDNA of the gene 5 ' ends.
After 5 ' the RACE product clonings sequencing of gained, by itself and 3 ' RACE products sequencing results and SEQ ID No:3 sequences are spliced.Obtain GMPT2 full length cDNA sequence SEQ ID No: 9：
SEQ ID No: 9:
1 CTTTGCTTGA TCATTCTTTG GACTAGAAAG GTTAATGATG ATGACAGTTT CAATGTCAAT
61 GTGCAAAGAA ATGGTGTCTT TACTGGACAT AAGTTCTGGG GTGCTGAAAT CGGACTTCTT
121 TAGTCCTAGG AGACATAAAG AAAAGGTTGT GATCCTAATG GGAGCAACCG GAACAGGAAA
181 ATCGAGGCTC TCCATTGATC TCGCAACCCG ATTCCCAGCA GAAATTATAA ATTCGGATAA
241 GA ACAAGTC CACGAAGGGC TTGACATAGT ACCAACAAG ATCAGTGAGG AAGAACGGTG
301 TGGGATTCCG CACCATTTGT TAGGTGTAAT AAGTCCCAAC ATAGATTTTA CAGTGACGAA
361 TTTCGTGGAC ATGGCTTCGC GTGCCACAGC TTCGATCGTG TCCCGCAGCC AGCTTCCGAT
421 CATAGCCGGC GGCTCGAATT CATACATTGA GGCTTTGGTC GATGACGAAA GGTCTAGATT
481 CCGGTCGAAA TATGAATGTT GTTTCCTTTG GGTAGATGTG GCAATGCCAG TGTTACATCA
541 GTATGTATCA GAGAGAGTAG ATAAGATGGT TGAAAATGGG ATGGTTGATG AGGCAAGAAG
601 CTTCTTTGAT TACAATGCAA ATTATTCAAA GGGGATTAGG AAAGCTATCG GAGTCCCCGA
661 ATTTGACCGG TACTTCCGAG CCGAACCGTT TTTAGACAAG AGACAACGTG CTAAACTGCT
721 GCAAGAAGCA A CCAAGAAA TCAAAAGAAA CACTTGCAGA TTAGCTTGTC GTCAATTGGA
781 GAAGATTCAG CGGCTGAGGA ACAAGAAGAA TTGGACGATA CACAGGCTCG ACGCCACCGA
841 AGTGTTCCTC AGGCGTGGAA AAGGAGCTGA TGAGGCTTGG GAAGAGCTTG TAGCTGATCC
901 AAGTACAGAA ATAGTAGCAC AATTTCTATG TGATATCAGC TCAGGAGCAC TGCTCAGTAC
961 TAGTCCTGCC ATTGAGTTCC TTGTGGTATA ATGCTGTAAA ACCATATCTG ACCCAGGAAT
1021 AACTAGTAAA GCCATAGATC TCAATGTTAA TTAACTTAAA ATAATGAAGA TATGCTACTT
1081 AATGGAAGTC ACTTTTGGTG AAAAAGAAAT AGATACTTAG ATTTCATGGC CAAAAAAAAA
1141 AAAAAAAAAA A are according to SEQ ID NO:9 sequences Design pair of primers are as follows：
SEQ ID No: 10:
ATGACAGTTT CAATGTCAAT GT SEQ ID No: 11 :
TTATACCACA AGGAACTCAA TGG pass through SEQ ID NO:10 and SEQ ID NO:11 clone GMPT2 total length encoding genes.
Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of cotton.The PCR reaction systems of 50 μ 1：The mM of 10 μ, 153 μ of X PS Buffer 1 2.5 dNTP, the cDNA of 2.0 μ 1, the PrimeSTAR of 1.0 μ 1,10 μM of primer SEQ ID NO:10 standing grain P SEQ ID NO:11 each 2.0 μ 1 and
30 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension lmin), 72 °C of 10 min of extension.
Pcr amplification product adds A tails：The absolute ethyl alcohol of 2.5 times of volumes is added in PCR primer, -20 °C are placed 10 minutes, centrifugation is removed supernatant, dried, and is then dissolved with 21 μ distilled waters.Then the mM of 2.5 0.5 μ of μ Ι Ο Χ Ε χ Buffer 15 dATP, the Ex Taq of 1.0 μ 1 is added thereto.Reaction condition：70 °C are reacted 30 minutes.Obtained about 900bp DNA fragmentation is reclaimed（Omega QIAquick Gel Extraction Kits）, and be connected on pGEM T-easy carriers（Obtain GhIPT2-pGEM plasmids）, then converting JM109, (method is ibid）.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/ml ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup.With SEQ ID NO:10 and SEQ ID NO:11 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 7 positive colonies are obtained, wherein 4 positive colonies is chosen and delivers to Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO:2, the amino acid sequence of its protein encoded is SEQ ID NO: 1.
The amino acid sequence of IPT2 albumen： SEQ ID NO: 1
1 MTVSMSMCKE MVSLLDISSG
21 VLKSDFFSPR RHKEKWILM
41 GATGTGKSRL S IDLATRFPA
61 EI INSDKIQV HEGLDIVTNK
81 ISEEERCGI P HHLLGVISPN
101 IDFTVTNFVD MASRATAS IV
121 SRSQLPI IAG GSNSYIEALV
141 DDERSRFRSK YECCFLWVDV
161 AMPVLHQYVS ERVDK VENG
181 MVDEARSFFD YNANYSKGIR
201 KAIGVPEFDR YFRAEPFLDK
221 RQRAKLLQEA IQEIKRNTCR
241 LACRQLEKIQ RLRNKKNWTI
261 HRLDATEVFL RRGKGADEAW
281 EELVADPSTE IVAQFLCDIS
301 SGALLSTSPA IEFLW*
The nucleotide sequence SEQ ID NO of GMPT2 encoding genes: 2
1 ATGACAGTTT CAATGTCAAT GTGCAAAGAA ATGGTGTCTT TACTGGACAT AAGTTCTGGG
61 GTGCTGAAAT CGGACTTCTT TAGTCCTAGG AGACATAAAG AAAAGGTTGT GATCCTAATG
121 GGAGCAACCG GAACAGGAAA ATCGAGGCTC TCCATTGATC TCGCAACCCG ATTCCCAGCA
181 GAAATTATAA ATTCGGATAA GA ACAAGTC CACGAAGGGC TTGACATAGT ACCAACAAG
241 ATCAGTGAGG AAGAACGGTG TGGGATTCCG CACCATTTGT TAGGTGTAAT AAGTCCCAAC
301 ATAGATTTTA CAGTGACGAA TTTCGTGGAC ATGGCTTCGC GTGCCACAGC TTCGATCGTG
361 TCCCGCAGCC AGCTTCCGAT CATAGCCGGC GGCTCGAATT CATACATTGA GGCTTTGGTC
421 GATGACGAAA GGTCTAGATT CCGGTCGAAA TATGAATGTT GTTTCCTTTG GGTAGATGTG
481 GCAATGCCAG TGTTACATCA GTATGTATCA GAGAGAGTAG ATAAGATGGT TGAAAATGGG
541 ATGGTTGATG AGGCAAGAAG CTTCTTTGAT TACAATGCAA ATTATTCAAA GGGGATTAGG
601 AAAGCTATCG GAGTCCCCGA ATTTGACCGG TACTTCCGAG CCGAACCGTT TTTAGACAAG
661 AGACAACGTG CTAAACTGCT GCAAGAAGCA A CCAAGAAA TCAAAAGAAA CACTTGCAGA
721 TTAGCTTGTC GTCAATTGGA GAAGATTCAG CGGCTGAGGA ACAAGAAGAA TTGGACGATA
781 CACAGGCTCG ACGCCACCGA AGTGTTCCTC AGGCGTGGAA AAGGAGCTGA TGAGGCTTGG
841 GAAGAGCTTG TAGCTGATCC AAGTACAGAA ATAGTAGCAC AATTTCTATG TGATATCAGC
The GMPT2 gene plant expression vector establishments of 901 TCAGGAGCAC TGCTCAGTAC TAGTCCTGCC ATTGAGTTCC TTGTGGTATA A embodiments 3
Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoter that Ν Ρ Τ Π genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.The rd29A promoters and terminator Tnos of induction type are selected respectively as the promoter and terminator of GMPT2 genes.
With primer SEQ ID NO:12 and SEQ ID NO:13 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector pBI121）For template amplification Pnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 y l PCR reaction systems：The X PS Buffer of 10 l 5, the mM of 3 μ 1 2.5 dNTP, the PrimeSTAR of 1.0 μ, 1 121 1.0 μ of ρ Β Ι 1,10 μ Μ primer SEQ ID NO:12 standing grain P SEQ ID NO:13 each μ of 2.0 μ 1 and 31 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 56 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30.(promega, T4 ligase box is connected as PCR primer of EcoRI, Bglll digestion by obtained by）PCAMBIA2300-l is obtained to pCAMBIA2300.
SEQ ID NO: 12
GCACGAATTC ggcgggaaac gacaatctga
SEQ ID NO: 13
ATCCAGATCTAGATCCGGTGCAGATTATTTG
With primer SEQ ID NO:14 and SEQ ID NO:15 using pBI121 as template amplification Tnos, using TaKaRa
PrimeSTARHS archaeal dna polymerases.50 μ PCR reaction systems：10 l5XPSBuffer, the mM of 3 μ 12.5 dNTP, the Prime STAR of 1.0 μ, 1 pBI121,1.0 μ 1,10 μM of primer SEQ ID NO:14 and SEQ ID NO:15 each μ of 2.0 μ 1 and 31 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30s, 58 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30.PCAMBIA2300-2 is obtained to pCAMBIA2300-l as PCR primer connection (promegaT4 ligase box) of SacI, EcoRI digestion by obtained by.
SEQ ID NO: 14；
AAGGAGCTCGAATTTCCCCGATCGTTCAA.A
SEQ ID NO: 1.5：
TCAGAATTCCCAGTGAATICCCGATCTAGTA primer SEQ ID NO:16 and SEQ ID NO:17 with arabidopsis（Colombia's type, purchased from www.arabidopsis.org) DNA be template amplification arabidopsis rd29A promoters（With reference to Zeng J., et al.2002, Preparation of total DNA from " recalcitrant plant taxa ", Acta Bot. Sin., 44 (6):Method in 694-697 extracts arabidopsis DNA).Using TaKaRa PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1：10 yl5XPSBuffer, the mM of 3 μ 12.5 dNTP, the arabidopsis DNA of 1.0 μ 1, the PrimeSTAR of 1.0 μ 1,10 μ Μ primer SEQ ID NO:16 and SEQ ID NO:17 each distilled waters of 2.0 μ, 1 and 31 μ 1.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30.Connected as PCR primer of HindIII, Pstl digestion by obtained by（Connection method is ibid）PCAMBIA2300-3 is obtained to pCAMBIA2300-2.
SEQ ID. NO; 16；
ACTAAGCTTGCTTCTIGACATCAITCAATTTTA SEQ ID; HO: 17-
TGACTGCAGTCCAAAGATTTTTTICTTTCCAATAG primer SEQ ID NO:18 and SEQ ID NO:The full length sequence of 19 amplification GMPT2 encoding genes（Template is that embodiment 2 obtains positive GhIPT2-pGEM plasmids）, using TaKaRa PrimeSTARHS DNA polymerases.50 lPCR reaction systems：10 l 5XPS Buffer, the mM of 3 μ 12.5 dNTP, the PrimeSTAR of 1.0 μ, 1 GhIPT2-pGEM, 1.0 μ 1,10 μ Μ primer SEQ ID NO:18 standing grain P SEQ ID NO:19 each μ distilled waters of 2.0 μ 1 and 31.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 V are denatured 30 s, 58 °C of annealing 30 s, 72V extension 2min), 72V extends 10 min.Connected as PCR primer of Pstl, Sad digestion by obtained by（Connection method is ibid）To pCAMBIA2300-3, obtain plant expression and carry
Body rd29A- GhIPT2-2300.
SEQ ID; NO : IS :
TGACTGCAG ATGACAGTTT CAATGTCAAT GT SEQ ID NO : 19；
The rd29A-GhIPT2-2300 expression vectors of AAGGAGCTC TTATACCACA AGGAACTCAA TGG embodiments 4 convert Agrobacterium
The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：Agrobacterium LBA4404 was drawn to single spot inoculation on the LB solid mediums containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins in 1-2 days in advance, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in LB fluid nutrient mediums of 5 ml containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins, and overnight incubation (about 12-16 h) is shaken under 28 °C to OD6..It is worth for 0.4, formation seed bacterium solution.Take the bacterium solution after 5 ml activation（1 :20 ratio）It is inoculated in LB fluid nutrient mediums of 100 ml containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins, 28 °C are shaken culture 2-2.5 h to OD6QQ=0.8.The min of ice bath bacterium solution 10, shakes up once every 3 min, makes the bacterium even into resting state.10 min are centrifuged in 4000 g under 4 °C, supernatant is abandoned；Add 4000 g under 10% glycerine resuspension thalline of a certain amount of ice precooling, 4 °C and centrifuge 10 min, collect precipitation；Ice-cold 10% glycerine repeats to wash 3-4 times；Then adding 10% glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, is dispensed, is saved backup in -70 °C with 40 μ/pipe.
Convert Agrobacterium：Melt described competent cell on ice, 1 μ 1 plasmid, the min of ice bath about 10 after mixing are added into 40 μ 1 competent cell.The mixture of competent cell and rd29A-GhIPT2-2300 DNAs is transferred to the electric shock cup of ice precooling with liquid-transfering gun（Purchased from bio-md) in, rapping makes suspension reach electric shock cup bottom, has been careful not to bubble.The electric shock cup is put on the slideway of electroporation chamber, promotes slideway to put electric shock cup to electroporation chamber base electrode.Using 0.1cm specifications electric shock cup when, MicroPulser (be purchased from bio-rad) program is set to " Agr ", and electric shock is once.Electric shock cup is taken out immediately, adds the LB culture mediums of 28 °C of preheatings.It is quickly soft to be beaten cell with liquid-transfering gun.Suspension is transferred to 1.5 ml centrifuge tube, 1 h of culture is shaken in 28 °C of 225 rpm.100-200 μ 1 bacterium solution is taken to be coated on corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 μ g/ml rifampins, 50 μ g/ml streptomysins, 50 μ g/ml kanamycins）, 28 °C of cultures.Positive transformants clone is screened, and its bacterium solution is saved backup in -70 °C.Embodiment 5 obtains transgene tobacco using Agrobacterium-medialed transformation method
With 75% alcohol-pickled tobacco seed（Countries tobacco mid-term storehouse, obtains unit：Tobacco institute of the Chinese Academy of Agricultural Sciences, storehouse numbering I5A00660) 30 s, are then washed twice with sterilizing distilled water.Again 8 min, Ran Houyong are soaked with 0.1% mercuric chloride
Sterilizing distilled water is washed twice, completes surface sterilizing.The tobacco seed of surface sterilizing is placed in MS solid mediums（Contain 18.78 mM KN03 1.25 mM KH2P04、 20.6 mM H4N03 1.5 mM MgS04、 3.0 mM CaCl2、 50 μ M KI、 100 μ M H3B03、 100 μ M MnS04、 30 μ M ZnS04、 1 μ M Na2Mo04、 0.1 μ M CoCl2 100 μ M Na2EDTA 100 μ M FeS04, 7.4 g/1 fine jades month purports, 30 g/1 sucrose）On under aseptic condition germinate, prepare aseptic seedling.Take tests for sterility to be cut into the leaf dish of the mm sizes of 5 mm X 5, with the min of During Agrobacterium leaf dish 10 of the rd29A-GhIPT2-2300 containing expression vector in exponential phase, blot bacterium solution, co-cultured 2 days under dark condition（MS solid mediums）.Blade is gone into differentiation solid medium（The MS+1 mg/1 basic elements of cell division（BA)+0.1 mg/1 methyl α-naphthyl acetates（NAA) the mg/1 cephalosporins of+50 mg/1 kanamycins+500）On, daily with 2000 Lx illumination 16h, cultivate 45 days or so, cut after bud is grown up and be transferred to rooting solid culture medium（The mg/1 cephalosporins of MS+50 mg/1 kanamycins+500）Middle culture 30 days or so, preservation is numbered after being transferred to seedling after well developed root system on the MS solid mediums only added with 500 mg/1 cephalosporins.
The blade for the transgenic tobacco plant that clip is obtained, extracts DNA (arabidopsis DNA extraction methods in be the same as Example 3）, with SEQ ID NO:10 and SEQ ID NO:11 enter performing PCR amplification identification（The PCR reaction systems of 50 μ 1：The mM of 5 μ, 13 μ of the Ι Ο Χ Ε χ BufFer 1 2.5 Ex Taq of 1.0 μ of dNTP 2.0 μ, 1 DNA 1,10 μM of primer SEQ ID NO:10 and SEQ ID NO:11 each μ 1 of 2.0 μ 1 and 35 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 2 min of extension), 72 °C of 10 min of extension), PCR is accredited as positive plant numbering and is T0Q1-T0Q20 and preserves.Embodiment 6 is overexpressed drought resisting simulated experiments of the GhIPT2 transgene tobaccos T1 for plant
Sterilized vermiculite is impregnated with 1/2MS culture mediums.T0Q1-T0Q20 transgene tobaccos and the seed of control tobacco are sowed on vermiculite respectively, and 15 seeds, 25 °C, optical culture/10 hour light culture circulation in 14 hours are sowed per basin.A 1/2MS is poured weekly, after cultivating 25 days, SEQ ID NO:10 and SEQ ID NO:11 do PCR detections, remove negative plant.Choose transgene tobacco of the same size and control tobacco is cooked drought-enduring experiment, per 4-5 more consistent seedling of basin reservation size.Transgene tobacco, arid 14 days (not the watering) of control tobacco, 25 °C, optical culture/10 hour light culture circulation in 14 hours.T1 is for transfer-gen plant（The plant that T0 grows up to for the seed of transfer-gen plant）Identification of Drought show that adjoining tree is all wilted seriously, and tri- strains of T1Q4, T1Q7, T1Q9 show obvious drought resistance（See Fig. 3 a and 3b, with T1Q4, T1Q7, T1Q9 result with it is similar, be not shown here）.Embodiment 7 verifies IPT2 protein expressions on transcriptional level
The good T1 of the medium drought resistant of embodiment 6 is for randomly selecting 8 in transfer-gen plant（It is belonging respectively to above three drought resisting strain）, adjoining tree randomly selects 4 in embodiment 6, and each clip arid g of blade 0.05 of 14 days uses plant RNA extraction kit（Invitrogen total serum IgE) is extracted.Ultraviolet spectrophotometry total serum IgE is in 260 nm
With 280 nm absorbance, each RNA concentration is calculated.Reverse transcription is carried out according to method shown in invitrogen reverse transcription reagent box Superscript III Reverse Transcriptase（1 μ g total serum IgEs are used as template, reverse transcription primer SEQ ID NO: 11 ).Pass through SEQ ID NO:10 standing grain P SEQ ID NO:20 amplification GhIPT2, detect IPT2 albumen relative expression's situations.Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of reverse transcription.50 y l PCR reaction systems：10 μ 15 X PS Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR, 10 μM of primer SEQ ID NO:10 standing grain P SEQ ID NO:20 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,29 circulations（94 V are denatured 30 s, 58 °C of annealing 30 s, 72 V extension lmin), 72 V extend 10 min.Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-8 be drought-resistant T1 for transgenic tobacco plant, 9 be plasmid PCR positive control（Rd29A-GhIPT2-2300 plasmids）, 10-13 is not drought-resistant control tobacco plant.Stripe size shown in figure and GMPT2's is in the same size（About 930bp).As a result show, drought-resistant T1 is stronger for the transcription of GMPT2 in transgenic tobacco plant there is no GMPT2 transcriptions in not drought-resistant control tobacco plant.
SEQ ID NO: 20:
GGCAGGACTA GTACTGAGCA GT
Claims (10)
- Claims1. the encoding gene of a prenyltransferase for cotton, is named as GhIPT2, its nucleotide sequence such as SEQ ID NO:Shown in 2.2. a kind of recombinant expression carrier, it contains the gene described in claim 1 and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector.3. the carrier described in claim 2, it is the rd29A-GhIPT2-2300 carriers shown in accompanying drawing 2.4. a kind of recombinant cell, it contains the recombinant expression carrier described in gene or Claims 2 or 3 described in claim 1；Preferably, the recombinant cell is restructuring agrobatcerium cell.5. a kind of method of improvement plant drought resistance, including：Recombinant expression carrier described in gene or claim 2 or 3 described in claim 1 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is tobacco.6. a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or Claims 2 or 3 described in claim 1 or plant tissue are cultivated under conditions of plant is effectively produced.7. the method described in claim 6, wherein the plant is tobacco.8. the recombinant expression carrier described in gene, Claims 2 or 3 described in claim 1 or the recombinant cell described in claim 4 are used to improve plant drought resistance and the purposes for plant breeding.9. the purposes described in claim 8, wherein the plant is tobacco.10. the albumen of the gene code described in claim 1, its amino acid sequence such as SEQ ID NO:Shown in 1.
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| WO2012007919A2 (en) * | 2010-07-15 | 2012-01-19 | Technion Research & Development Foundation Ltd. | Nucleic acid construct for increasing abiotic stress tolerance in plants |
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| Y.D. LIU ET AL.: "Improved salt tolerance and delayed leaf senescence in transgenic cotton expressing the Agrobacterium IPT gene", 《BIOLOGIA PLANTARUM》 * |
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| CN108753749A (en) * | 2018-06-20 | 2018-11-06 | 西南大学 | Silkworm tRNA isopentenyl transferase genes and its recombinant vector and application |
| CN108753749B (en) * | 2018-06-20 | 2021-06-08 | 西南大学 | Silkworm tRNA isopentenyl transferase gene, recombinant vector and application thereof |
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