CN100569948C - Paddy rice zinc finger protein gene and stress tolerance genetic engineering thereof are used - Google Patents
Paddy rice zinc finger protein gene and stress tolerance genetic engineering thereof are used Download PDFInfo
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- CN100569948C CN100569948C CNB2007101352633A CN200710135263A CN100569948C CN 100569948 C CN100569948 C CN 100569948C CN B2007101352633 A CNB2007101352633 A CN B2007101352633A CN 200710135263 A CN200710135263 A CN 200710135263A CN 100569948 C CN100569948 C CN 100569948C
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Abstract
The invention discloses paddy rice zinc finger protein gene ZFP207 and stress tolerance genetic engineering thereof and use, belong to biological technical field.The cDNA sequence of ZFP207 is seen SEQ ID NO.1.ZFP207 is positioned endonuclear transcription factor, and the mRNA expression analysis shows that this gene is subjected to significantly inducing of dormin, high salt, low temperature and arid.The structure of the extraction by total RNA, the clone of paddy rice zinc finger protein gene ZFP207, plant expression vector and the T2 generation of transgenic tobacco plant and adjoining tree carry out the resistance of reverse analysis and obtain the resistance of reverse transgenic tobacco plant respectively.Transgenic experiments proves that the overexpression of this gene has improved the salt tolerance of transgene tobacco, resistance to cold and drought tolerance.ZFP207 can be used as goal gene and imports plant, improves the comprehensive resistance of reverse of transgenic plant.
Description
Technical field
The present invention relates to paddy rice zinc finger protein gene ZFP207 and stress tolerance genetic engineering thereof and use, belong to the genetically engineered field, relate to the zinc finger transcription factor that the high salt of plant, low temperature, drought stress are replied specifically.
Technical background
TFIIIA type zinc finger protein is present in each kind of plant widely, and grow and the environment-stress of involved in plant replied (Huang Ji etc., 2007).In plants such as petunia, Arabidopis thaliana, wheat, cotton first rear clone nearly 40 TFIIIA type zinc finger protein genes, result of study shows that it has mainly participated in the growth course of plant at generative growth phase, or participates in abiotic stress reactions such as high salt, low temperature, arid.This type of zinc finger protein of soybean SCOF-1 studies show that, overexpression SCOF-1 gene has significantly strengthened cold inducible protein expression of gene in Arabidopis thaliana and tobacco, thereby has improved the resistance to cold (Kim et al, 2001) of plant.The overexpression of Arabidopis thaliana zinc finger protein gene STZ/ZAT10 in transfer-gen plant then improved drought-enduring and salt tolerance (Sakamoto et al., 2004).Therefore plant TFIIIA type zinc finger protein has important use value for improveing the resistance of plant to abiotic stress.
Paddy rice is important crops and monocotyledonous model plant, about the functional description of paddy rice TFIIIA type zinc finger protein gene also very limited (Huang et al., 2007).The present invention has identified the 1st TFIIIA type zinc finger protein gene ZFP207 relevant simultaneously with high salt, low temperature and drought stress of monocotyledons from paddy rice, and studied itself and various relation of coercing, proposed to utilize the ZFP207 gene to carry out the genetically engineered modification method that plant is improved comprehensive resistance of reverse.
Summary of the invention
Technical problem the objective of the invention is to open paddy rice zinc finger protein gene ZFP207 and stress tolerance genetic engineering is used, and this gene can be used as goal gene and imports plant, improves the comprehensive resistance of reverse of plant, to carry out plant species improvement.
Technical scheme
Paddy rice zinc finger protein gene ZFP207, its sequence is: SEQ ID NO.1.
The stress tolerance genetic engineering of paddy rice zinc finger protein gene ZFP207 is used, and comprising:
1) extraction of total RNA
Select rice varieties " leek green grass or young crops " for use, treat that rice seedling grows to 3 leaf after dates, handles with 150mM NaCl, get blade after 12 hours immediately and place the freezing preservation of liquid nitrogen, get partial blade, grind, add the 1.5mLEP pipe that fills lysate with mortar, fully after the vibration, move into again in the glass homogenizer, move in the 1.5mL EP pipe extracted total RNA after the homogenate, identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then;
2) clone of paddy rice zinc finger protein gene ZFP207
According to the complete encoding sequence of paddy rice zinc finger protein gene ZFP207, design two ends primer:
Upstream primer: CCTAATACACACACACAAAG
Downstream primer: CTACAAACTTATAACCGCAG
The total RNA that obtains with step 1) is a template, behind synthetic cDNA first chain of reverse transcription, carry out pcr amplification, the PCR program is as follows: 94 ℃ of pre-sex change 4 minutes, 94 ℃ of sex change 30s, 55 ℃ of renaturation 1min, 72 ℃ are extended 2min, after 33 circulations, and 72 ℃ of 10min, the PCR product is cloned into the pMD18-T carrier, and the acquisition of order-checking back has the cDNA sequence SEQ ID NO.1 of the paddy rice zinc finger protein gene ZFP207 of complete coding region;
3) structure of plant expression vector
According to the cDNA sequence of paddy rice zinc finger protein gene ZFP207, design amplifies the primer that complete coding is read frame, and introduces BamH I restriction endonuclease sites on upstream primer, and the downstream primer sequence is with step 2):
Upstream primer: GGATCCTTCTACTCCTAATACACAC
Downstream primer: CTACAAACTTATAACCGCAG
With step 2) in the pcr amplification product that obtains be template, behind pcr amplification, the cDNA of ZFP207 is cloned into intermediate carrier pMD18-T, further be cloned into binary expression vector pBI121.
4) acquisition of transfer-gen plant
The expression vector that step 3) is obtained changes Agrobacterium over to, further change tobacco over to, the transfer-gen plant that obtains is carried out PCR, carry out the resistance of reverse evaluation of plant after Southern hybridization and the RT-PCR checking, the T2 generation and the adjoining tree of transfer-gen plant carry out the resistance of reverse analysis.
The acquisition of salt tolerant transfer-gen plant: transfer-gen plant seedling and adjoining tree are carried out the lasting processing of 400mM NaCl, after salt is handled 5 days, transfer to continued growth on the wet filter paper that does not contain NaCl, handle the back and show the salt tolerant transfer-gen plant (Fig. 2) that the transgenic tobacco plant that obviously is better than control group is acquisition;
The acquisition of resistance to cold transfer-gen plant: with the seedling of transfer-gen plant and adjoining tree in 4 ℃ handle 5d after, be positioned in 25 ℃ of incubators and recover 12h, the plant that recovers obviously to be better than control group is resistance to cold enhanced transfer-gen plant (Fig. 3);
The acquisition of drought tolerance transfer-gen plant: transfer-gen plant and adjoining tree are handled the plant that growth after 30 days is better than control group be drought tolerance enhanced transfer-gen plant (Fig. 4) in 300mM N.F,USP MANNITOL.
Beneficial effect
1, the invention discloses a kind of paddy rice TFIIIA type zinc finger protein gene (ZFP207 gene) and coded protein thereof.This gene can be used as goal gene and imports plant from paddy rice (Oryza sativa L.), improves plant salt endurance, resistance to cold and drought tolerance, and to carry out plant species improvement, coded protein has anti-reverse function.
2, the ZFP207 gene function that provides of the inventor is high salt, low temperature and the drought stress responsing reaction of involved in plant, and quantitatively inverse transcription polymerase chain reaction (Real-time RT-PCR) analysis revealed ZFP207 gene is expressed enhancing under high salt (150mM NaCl), low temperature (4 ℃) and simulating drought (20%PEG6000) inductive condition.
3, ZFP207 gene of the present invention is from paddy rice, has the optimizing codon that monocotyledonss such as being suitable for paddy rice is expressed, its genetically engineered recipient plant is except dicotyledons, as being more suitable for monocotyledonss such as paddy rice, corn, wheat outside tobacco, soybean, the cotton etc.
4, utilize ZFP207 gene of the present invention to make up plant expression vector as goal gene, wherein available any promotor is cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promotor or other promotor for example, can comprise enhanser in case of necessity in this expression vector, no matter be transcriptional enhancer or translational enhancer.Can use selected marker for the evaluation of simplifying transformant and comprise enzyme antibiotics resistance, also can utilize the enzyme of the compound that colour-change (for example β-glucuronidase GUS) or luminous (for example luciferase) discern, also available unmarked selection.Used expression vector can use Ti-plasmids, Ri plasmid, plant viral vector etc.Method for transformation can use agrobacterium-mediated transformation, particle bombardment, pollen tube passage method or other method to transform plant.
Description of drawings
The Subcellular Localization of Fig. 1, ZFP207.
(a) (b) transient expression of pBI-GFP (contrast) in onion epidermis cell.(c) (d) transient expression of pBI-ZFP207-GFP in onion epidermis cell.(a) (c) observation under laser confocal microscope; (b) (d) observation under visible light.By the observation of laser confocal microscope, find that ZFP207-GFP mosaic gene product is positioned nucleus interior (white arrow place) (c) (d), shows that paddy rice ZFP207 is for being positioned endonuclear transcription factor.
Fig. 2, salt tolerance enhanced transfer-gen plant.
ZFP207 transgene tobacco and contrast tobacco plant are transferred on the filter paper that does not contain NaCl and were recovered 3 days after handling 5 days on the filter paper that contains 400mM NaCl.(a) (b) for handling transgene tobacco afterwards; (c) be the contrast non-transgenic tobacco after handling.
Fig. 3, resistance to cold enhanced transfer-gen plant.
ZFP207 transgene tobacco and contrast tobacco plant are handled after 5 days in 4 ℃ of illumination boxs, transfer to 25 ℃ and recover 12h.(a) (b) for handling transgene tobacco afterwards; (c) be the contrast non-transgenic tobacco after handling.
Fig. 4, drought tolerance (the N.F,USP MANNITOL simulating drought is coerced) enhanced transfer-gen plant.
ZFP207 transgene tobacco and contrast tobacco plant were handled 30 days in 300mM N.F,USP MANNITOL simulating drought is coerced.(a) (b) for handling transgene tobacco afterwards; (c) be the contrast non-transgenic tobacco after handling.
Embodiment
Embodiment 1
Select rice varieties " leek green grass or young crops " (Taihu Lake basin japonica rice local variety are strong salt tolerant kind) for use, treat that rice seedling grows to 3 leaf after dates, handle, get blade after 12 hours immediately and place the freezing preservation of liquid nitrogen with 150mM NaCl.Get partial blade, grind, add the 1.5mL EP pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to after the homogenate in the 1.5mL EP pipe, and extracted total RNA (TRIzol Reagents, Invitrogen, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.With total RNA of obtaining is template, and the specification sheets of the reverse transcription test kit that provides according to U.S. Promega company carries out reverse transcription, synthetic cDNA first chain.Conserved structure with plant TFIIIA type zinc finger protein is the rice genome database that probe sequence (Probesequence) search is positioned at GenBank, process EST comparison and sequence assembly obtain the sequence of the paddy rice TFIIIA type zinc finger protein gene of a total length 605bp, design two ends primer: CCTAATACACACACACAAAG respectively according to this sequence, CTACAAACTTATAACCGCAG, adopt the RT-PCR method to carry out the cDNA clone, the PCR response procedures is: 94 ℃ of pre-sex change 4 minutes, 94 ℃ of sex change 30s, 55 ℃ of renaturation 1min, 72 ℃ are extended 2min, after 33 circulations, 72 ℃ of 10min, to the pMD18-T carrier, the acquisition of order-checking back has the cDNA sequence SEQ ID NO.1 of the paddy rice zinc finger protein gene ZFP207 of complete coding region with the PCR product cloning.
ZFP207 total length 605bp, its open reading frame is positioned at the 31-597 place.The DNAssist software analysis shows ZFP207 188 amino acid of encoding altogether, wherein contain 1 typical C ys2/His2 type zinc fingers, being mainly the section that acidity or hydrophobic amino acid form by 12 between Cys-Xaa-Cys and the His-Xaaa-His district separates, coordination structure adds Phe and the formed hydrophobic core of Leu, has determined basic system and shape that zinc refers to.The zinc fingers of ZFP207 comprises plant specific QALGGH conserved structure.Use BioXM software (version 2 .6) to estimate its iso-electric point pI=10.28, molecular weight MW=20.7KDa.With existing rice genome (comprising japonica rice and long-grained nonglutinous rice) database among the ZFP207 search GenBank, discovery ZFP207 is a single copy gene.The gene that the result of BLAST and 3D-PSSM database analysis result proof newly obtain from paddy rice really is a coding TFIIIA type zinc finger protein gene.
Embodiment 2
ZFP207 two ends primer with design in the example 1 carries out the expression of real-time quantitative RT-PCR analysis ZFP207 gene in rice seedling, with paddy rice actin gene Rac1 (McElroy et al, 1990) be expressed as confidential reference items, the result shows, the following 2h of 150mM NaCl processing that is expressed in of ZFP207 significantly strengthens, reach the highlyest during 24h, its expression amount is about 6 times of contrast; Coerce down at low temperature (4 ℃), being expressed in after 0.5h handles of ZFP207 is that expression amount increases, and expression amount reaches the highest during 6h, and expression amount descends gradually subsequently, but the expression amount of ZFP207 still is higher than contrast when handling 24h; Under simulating drought (20%PEG6000) was handled, the expression of ZFP207 significantly increased when handling 12h, and expression amount is about 10 times of contrast, and expression amount reaches 13 times of contrasts when handling 24h.Paddy rice zinc finger protein gene ZFP207 of the present invention is the reported first in the paddy rice, simultaneously also be TFIIIA type zinc finger protein gene relevant with high salt, low temperature and drought stress when finding first in the monocotyledons, be expected to be applied to the especially monocotyledonous comprehensive resistance genetic improvement of plant.
Embodiment 3
The full length sequence of the ZFP207 that obtains according to embodiment 1, design amplifies the primer that complete coding is read frame, and at downstream primer introducing BamH I restriction endonuclease sites, upstream primer: CCTAATACACACACACAAAG, downstream primer: GATGGATCCTTAACCGCAGGCTCAAG.The amplified production that obtains with embodiment 1 is a template, behind pcr amplification, the cDNA of ZFP207 is cloned into intermediate carrier pMD18-T, further be cloned into amended binary expression vector pBI121 (β-glucuronidase reporter gene is wherein replaced to the green fluorescent protein reporter gene), make up the plasmid vector pBI-ZFP207-GFP that comprises the ZFP207-GFP mosaic gene.The pBI-ZFP207-GFP plasmid is transformed behind the Agrobacterium further transient expression in onion epidermis cell, observation by laser confocal microscope, find that ZFP207-GFP mosaic gene product is positioned in the nucleus, shows that paddy rice ZFP207 is for being positioned endonuclear transcription factor (Fig. 1).
Embodiment 4
The expression vector that step 3) is obtained changes Agrobacterium over to, further changes tobacco over to, and the transfer-gen plant that obtains is carried out PCR, and the resistance of reverse analysis is carried out to the T2 generation and the adjoining tree of transfer-gen plant in Southern hybridization and RT-PCR checking back.
The acquisition of salt tolerant transfer-gen plant: transfer-gen plant seedling and adjoining tree are carried out the lasting processing of 400mM NaCl, after salt is handled 5 days, transfer to continued growth on the wet filter paper that does not contain NaCl, handle the back and show the salt tolerant transgene tobacco (Fig. 2) that the transgenic tobacco plant that obviously is better than control group is acquisition;
The acquisition of resistance to cold transfer-gen plant: with the seedling of transfer-gen plant and adjoining tree in 4 ℃ handle 5d after, be positioned in 25 ℃ of incubators and recover 12h, the plant that recovers obviously to be better than control group is resistance to cold enhanced transfer-gen plant (Fig. 3);
The acquisition of drought tolerance transfer-gen plant: transfer-gen plant and adjoining tree are handled the plant that growth after 30 days is better than control group be drought tolerance enhanced transfer-gen plant (Fig. 4) in 300mM N.F,USP MANNITOL (simulating drought).
The foregoing description shows the paddy rice zinc finger protein gene ZFP207 that clones among the present invention, is clone time and high salt, low temperature and arid relevant paddy rice TFIIIA type zinc finger protein gene first in the paddy rice.Embodiment 2 shows that high salt, low temperature and the drought stress of this gene and plant is closely related.Embodiment 4 can improve the salt tolerant of transfer-gen plant, cold-resistant and drought tolerance after showing this gene and overexpression, has the function that improves the comprehensive resistance of reverse of plant.This genoid has the codon that monocotyledons is optimized owing to derive from monocotyledons, except dicotyledonss such as suitable tobaccos, also is suitable for the resistance genetic improvement of monocot cropss such as paddy rice, corn, wheat.
Sequence that the present invention relates to and mark apportion are as follows:
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 605bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: Nucleotide
(iii) sequence description: SEQ ID NO.1
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 188a.a
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: protein
(iii) sequence description: SEQ ID NO.2
Sequence table
＜110〉Agricultural University Of Nanjing
＜120〉paddy rice zinc finger protein gene and stress tolerance genetic engineering thereof are used
<130>000
<140>000
<141>2007-11-09
<160>2
<170>PatentIn?version?3.1
<210>SEQ?ID?NO.1
<211>605
<212>DNA
＜213〉paddy rice (Oryza sativa)
<220>
<221>CDS
<222>(31)..(597)
<223>
<220>
<221>mRNA
<222>(1)..(605)
<223>
<400>1
cctaatacac?acacacaaag?agagacagga?atg?gag?aga?tca?cct?gaa?gtg?gtg 54
Met?Glu?Arg?Ser?Pro?Glu?Val?Val
1 5
caa?cac?aag?gca?gca?aac?cga?gct?gcc?gag?gat?gat?cac?acc?acc?tcg 102
Gln?His?Lys?Ala?Ala?Asn?Arg?Ala?Ala?Glu?Asp?Asp?His?Thr?Thr?Ser
10 15 20
att?cca?tgg?cta?aag?ctg?ggt?gtc?gtc?gat?gca?ctg?aca?gca?gaa?gca 150
Ile?Pro?Trp?Leu?Lys?Leu?Gly?Val?Val?Asp?Ala?Leu?Thr?Ala?Glu?Ala
25 30 35 40
ggt?aag?ctt?ccc?gag?tcg?aat?ccg?aag?ccg?gcc?gtg?gcg?gcg?ccg?cat 198
Gly?Lys?Leu?Pro?Glu?Ser?Asn?Pro?Lys?Pro?Ala?Val?Ala?Ala?Pro?His
45 50 55
aga?acc?ttc?tcc?tgc?aac?tac?tgc?atg?agg?aag?ttc?ttc?agc?tcg?cag 246
Arg?Thr?Phe?Ser?Cys?Asn?Tyr?Cys?Met?Arg?Lys?Phe?Phe?Ser?Ser?Gln
60 65 70
gcg?ctg?ggt?ggg?cac?cag?aac?gcc?cac?aag?agg?gag?agg?tgc?gcg?ccg 294
Ala?Leu?Gly?Gly?His?Gln?Asn?Ala?His?Lys?Arg?Glu?Arg?Cys?Ala?Pro
75 80 85
agg?aag?tcg?cac?ggc?ttc?cag?cag?cag?cat?ctc?atg?gtt?ggg?ctc?tcc 342
Arg?Lys?Ser?His?Gly?Phe?Gln?Gln?Gln?His?Leu?Met?Val?Gly?Leu?Ser
90 95 100
ccg?act?gca?ccg?tcg?tcg?ttt?ctt?cac?cac?atg?agg?gtt?aac?cct?cac 390
Pro?Thr?Ala?Pro?Ser?Ser?Phe?Leu?His?His?Met?Arg?Val?Asn?Pro?His
105 110 115 120
gcc?acg?atc?ctg?aag?gtg?aac?cgt?ggc?tat?tct?tct?gct?gat?ggt?gtt 438
Ala?Thr?Ile?Leu?Lys?Val?Asn?Arg?Gly?Tyr?Ser?Ser?Ala?Asp?Gly?Val
125 130 135
gtc?gtg?gcg?aaa?ttc?cat?gga?ggc?cag?atg?agt?agc?agc?tgg?gtg?cct 486
Val?Val?Ala?Lys?Phe?His?Gly?Gly?Gln?Met?Ser?Ser?Ser?Trp?Val?Pro
140 145 150
ttt?gcg?gtt?gaa?cat?ggt?aga?ggg?tca?gtc?tgg?cct?ggg?agc?ttc?aaa 534
Phe?Ala?Val?Glu?His?Gly?Arg?Gly?Ser?Val?Trp?Pro?Gly?Ser?Phe?Lys
155 160 165
gca?agc?tct?cag?gag?cag?aag?aag?cgc?acg?gag?gaa?gat?ctt?gac?ttg 582
Ala?Ser?Ser?Gln?Glu?Gln?Lys?Lys?Arg?Thr?Glu?Glu?Asp?Leu?Asp?Leu
170 175 180
agc?ctg?cgg?tta?taa?gtttgtag 605
Ser?Leu?Arg?Leu
185
<210>SEQ?ID?NO.22
<211>188
<212>PRT
＜213〉paddy rice (Oryza sativa)
<400>2
Met?Glu?Arg?Ser?Pro?Glu?Val?Val?Gln?His?Lys?Ala?Ala?Asn?Arg?Ala
1 5 10 15
Ala?Glu?Asp?Asp?His?Thr?Thr?Ser?Ile?Pro?Trp?Leu?Lys?Leu?Gly?Val
20 25 30
Val?Asp?Ala?Leu?Thr?Ala?Glu?Ala?Gly?Lys?Leu?Pro?Glu?Ser?Asn?Pro
35 40 45
Lys?Pro?Ala?Val?Ala?Ala?Pro?His?Arg?Thr?Phe?Ser?Cys?Asn?Tyr?Cys
50 55 60
Met?Arg?Lys?Phe?Phe?Ser?Ser?Gln?Ala?Leu?Gly?Gly?His?Gln?Asn?Ala
65 70 75 80
His?Lys?Arg?Glu?Arg?Cys?Ala?Pro?Arg?Lys?Ser?His?Gly?Phe?Gln?Gln
85 90 95
Gln?His?Leu?Met?Val?Gly?Leu?Ser?Pro?Thr?Ala?Pro?Ser?Ser?Phe?Leu
100 105 110
His?His?Met?Arg?Val?Asn?Pro?His?Ala?Thr?Ile?Leu?Lys?Val?Asn?Arg
115 120 125
Gly?Tyr?Ser?Ser?Ala?Asp?Gly?Val?Val?Val?Ala?Lys?Phe?His?Gly?Gly
130 135 140
Gln?Met?Ser?Ser?Ser?Trp?Val?Pro?Phe?Ala?Val?Glu?His?Gly?Arg?Gly
145 150 155 160
Ser?Val?Trp?Pro?Gly?Ser?Phe?Lys?Ala?Ser?Ser?Gln?Glu?Gln?Lys?Lys
165 170 175
Arg?Thr?Glu?Glu?Asp?Leu?Asp?Leu?Ser?Leu?Arg?Leu
180 185
Claims (1)
1, the stress tolerance genetic engineering of paddy rice zinc finger protein gene ZFP207 is used, and comprising:
1) extraction of total RNA
Select rice varieties " leek green grass or young crops " for use, treat that rice seedling grows to 3 leaf after dates, handles with 150mM NaCl, get blade after 12 hours immediately and place the freezing preservation of liquid nitrogen, get partial blade, grind, add the 1.5mL EP pipe that fills lysate with mortar, fully after the vibration, move into again in the glass homogenizer, move in the 1.5mL EP pipe extracted total RNA after the homogenate, identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then;
2) clone of paddy rice zinc finger protein gene ZFP207
According to the full length sequence of paddy rice zinc finger protein gene ZFP207, design two ends primer:
Upstream primer: CCTAATACACACACACAAAG
Downstream primer: CTACAAACTTATAACCGCAG
The total RNA that obtains with step 1) is a template, behind synthetic cDNA first chain of reverse transcription, carry out pcr amplification, the PCR program is as follows: 94 ℃ of pre-sex change 4 minutes, 94 ℃ of sex change 30s, 55 ℃ of renaturation 1min, 72 ℃ are extended 2min, after 33 circulations, and 72 ℃ of 10min, the PCR product is cloned into the pMD18-T carrier, and the acquisition of order-checking back has the cDNA sequence SEQIDNO.1 of the paddy rice zinc finger protein gene ZFP207 of complete coding region;
3) structure of plant expression vector
According to the cDNA sequence of paddy rice zinc finger protein gene ZFP207, design amplifies the primer that complete coding is read frame, and introduces BamH I restriction endonuclease sites on upstream primer, and the downstream primer sequence is with step 2):
Upstream primer: GGATCCTTCTACTCCTAATACACAC
Downstream primer: CTACAAACTTATAACCGCAG
With step 2) in the pcr amplification product that obtains be template, behind pcr amplification, the cDNA of ZFP207 is cloned into intermediate carrier pMD18-T, further be cloned into binary expression vector pBI121;
4) acquisition of transfer-gen plant
The expression vector that step 3) is obtained changes Agrobacterium over to, further change tobacco over to, the transfer-gen plant that obtains is carried out PCR, carry out the resistance of reverse evaluation of plant after Southern hybridization and the RT-PCR checking, the T2 generation and the adjoining tree of transfer-gen plant carry out the resistance of reverse analysis.
The acquisition of salt tolerant transfer-gen plant: transfer-gen plant seedling and adjoining tree are carried out the lasting processing of 400mM NaCl, after salt is handled 5 days, transfer to continued growth on the wet filter paper that does not contain NaCl, handle the back and show the salt tolerant transfer-gen plant that the transgenic tobacco plant that obviously is better than control group is acquisition;
The acquisition of resistance to cold transfer-gen plant: with the seedling of transfer-gen plant and adjoining tree in 4 ℃ handle 5d after, be positioned in 25 ℃ of incubators and recover 12h, the plant that recovers obviously to be better than control group is resistance to cold enhanced transfer-gen plant;
The acquisition of drought tolerance transfer-gen plant: transfer-gen plant and adjoining tree are handled the plant that growth after 30 days is better than control group be drought tolerance enhanced transfer-gen plant in 300mM N.F,USP MANNITOL.
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| US20120102588A1 (en) * | 2009-04-08 | 2012-04-26 | Shanghai Institutes For Biological Sciences, Cas | Rice zinc finger protein transcription factor dst and use thereof for regulating drought and salt tolerance |
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| CN102517305B (en) * | 2011-12-13 | 2014-05-07 | 南京农业大学 | Drought resistance gene engineering application of rice OsCUT1 gene |
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| CN107164404B (en) * | 2017-06-30 | 2020-10-20 | 浙江农林大学 | Application of E.grandis EgrZFP6 in improving stress adaptation of plants under osmotic stress |
| CN109777805B (en) * | 2017-11-13 | 2021-01-29 | 华中农业大学 | Petunia zinc finger protein gene PhZFP1 and application thereof in improving cold resistance of plants |
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