CN108866056B - Promoter for specific expression of IbCBF3 gene abiotic stress of sweet potato and application thereof - Google Patents
Promoter for specific expression of IbCBF3 gene abiotic stress of sweet potato and application thereof Download PDFInfo
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Abstract
The invention relates to a promoter for abiotic stress specific expression, in particular to a promoter for the abiotic stress specific expression of IbCBF3 genes of sweet potatoes and application thereof, belonging to the field of plant genetic engineering. The invention provides a promoter for the specific expression of IbCBF3 abiotic stress of sweet potatoes, wherein the nucleotide sequence of the promoter is shown in SEQ ID NO. 1; the DNA fragment is a promoter of a key transcription factor IbCBF3 in a sweet potato low-temperature stress pathway, and the promoter contains a plurality of adversity and hormone response elements. Through the analysis of a double-luciferase system in tobacco leaves, the promoter is rapidly combined with an upstream gene IbICE1 under low-temperature stress to induce the expression condition of a downstream gene, and can be used for the research of the low-temperature resistance of sweet potatoes and the transgenic breeding.
Description
Technical Field
The invention relates to a promoter for abiotic stress specific expression, in particular to a promoter for the abiotic stress specific expression of IbCBF3 genes of sweet potatoes and application thereof, belonging to the field of plant genetic engineering.
Background
Temperature is one of the most important environmental factors during plant growth and development. The low temperature not only affects the growth and the yield of crops, but also restricts the cultivation time and the geographical distribution of the crops. According to the latest data statistics of Food and Agriculture Organization (FAO) of the United Nations, the sweet potato planting area in China is 337 hectares, which accounts for 40% of the world planting area and is the largest sweet potato planting country; the total yield is 713 ten thousand tons, which accounts for 73 percent of the world sweet potato yield. Sweet potatoes are widely applied to daily consumption of people, animal feed and industrial fuel due to barren resistance, easy management, high yield and rich nutritive value, and are the fourth largest grain product after rice, wheat and corn. However, sweet potatoes like rice, corn and cotton like sweet potatoes which are warm and cold-resistant are susceptible to low temperature, resulting in reduced yield. Sweet potatoes are very sensitive to low temperatures whether they are grown in the seedling stage or stored after harvesting. When the temperature is reduced to 15 ℃, the growth of the sweet potatoes is stopped, and when the temperature is lower than 9 ℃, the sweet potatoes are gradually rotten due to cold damage; the stem and leaf of the overground part lose the activity quickly after frost and die. The development of the sweet potato industry is severely restricted by the non-low temperature resistance of the sweet potatoes.
In order to deal with various abiotic stresses such as low temperature and the like, the plants form a complex regulation network to sense, respond and adapt to the change of the external environment. The ICE-CBF transcription factor approach is the most deep research on the cold-resistant mechanism of plants. In many plants, over-expression of either ICE or CBF can improve the cold resistance of the plant. In arabidopsis thaliana, the CBF3 transcription factor not only regulates the expression of a number of downstream stress-resistant genes, but also its own expression is affected by upstream genes. Its upstream transcription factor ICE1 can bind to the MYC element (or ICE box) of CBF3 promoter to activate expression of CBF3 in response to low temperature stress.
In the invention, a promoter sequence of the IbCBF3 gene is obtained by designing a primer through a chromosome walking method according to the IbCBF3 gene sequence cloned from the main cultivar Xushu 18 of sweet potato. The sequence was analyzed using plant care software in PLACE and the predicted results for promoter elements showed: besides the TATA-box and GATA-box which are necessary for transcription, CAAT-box and other conserved elements in the IbCBF3 promoter, some elements sensing environmental stress, light, moisture, hormone response and growth and development regulation exist, such as response to ACE, ATCT-motif, GATT-motif, G-box, I-box and the like which are involved in light regulation; abscisic acid-responsive factor ABRE; ethylene response factor ERE and methyl jasmonate response factor CGTCA-motif and the like. In addition, the promoter also comprises a plurality of MYCRE cis-acting elements CANNTG and the like which can be combined with an upstream regulatory factor ICE gene. And a double-fluorescence expression system is utilized, and the transient expression of tobacco leaves is utilized to verify that the promoter can be combined with an upstream gene under the low-temperature condition to induce the expression of the gene related to the low-temperature stress.
Disclosure of Invention
The invention aims to provide a promoter for the abiotic stress specific expression of IbCBF3 genes of sweet potatoes and application thereof, the promoter belongs to a promoter of a key transcription factor CBF3 in a low-temperature response path, the content relates to the analysis of a cDNA full-length sequence and a functional domain of the promoter, and the promoter can be combined with an upstream gene IbICE1 under the induction of low-temperature stress to induce the expression of a downstream gene.
The first purpose of the invention is to provide a promoter for the specific expression of IbCBF3 abiotic stress of sweet potatoes, wherein the nucleotide sequence of the promoter is shown in SEQ ID NO. 1.
The primer pair for amplifying any fragment of the IbCBF3 promoter of the sweet potato is shown as SEQ ID NO. 2-11.
The primer pair for amplifying the full length of the sweet potato IbCBF3 promoter is shown as SEQ ID No. 12-13.
An upstream gene IbCBF3 of sweet potato IbICE1, which is characterized in that the upstream gene IbCBF3 is a cDNA fragment shown as SEQ ID NO.14 in a sequence table.
The primer pair for amplifying the full length of the sweet potato IbICE1 promoter is shown in SEQ ID No. 15-16.
The embodiment of the invention provides a method for constructing a promoter for the abiotic stress specific expression of IbCBF3 genes of sweet potatoes,
(1) cloning the IbCBF3 gene promoter of the sweet potato, and carrying out PCR amplification by taking the cDNA of the sweet potato as a template to obtain the full length of the IbCBF3 gene promoter sequence of the sweet potato;
(2) constructing an upstream gene IbCBF3 of a sweet potato IbCBF 1, connecting the upstream gene IbICE1 to a T-blunt vector, transforming the upstream gene IbCBF 1 into an escherichia coli DH5 alpha competent cell, and obtaining recombinant vectors IbCBF3 pro-GUS and CaMV35S-IbICE 1;
(3) the recombinant vector plasmids are respectively transformed into agrobacterium strain GV3101, and single colony genome is extracted from YEP culture medium for PCR verification.
The second purpose of the invention is to provide a recombinant vector containing the promoter.
In one embodiment of the invention, the recombinant vector is IbCBF3 pro-GUS and CaMV35S-IbICE 1.
The invention also provides construction of a tobacco bifluorase expression system, which contains a recombinant vector, a recombinant bacterium, a transgenic cell line or an expression cassette.
The DNA fragment containing the DNA fragments shown in SEQ ID NO.1 and SEQ ID NO. 14.
The third purpose of the invention is to utilize the promoter for the low-temperature induction specific expression of the sweet potato in the genetic breeding of plants.
The promoter is a nucleic acid sequence of 2118 basic group in a nucleotide sequence, and the nucleic acid sequence encodes a transcription factor which is induced by low temperature and can regulate and control cold-induced genes.
The promoter region comprises 4 ABRE, 1 ERE, 1 CGTCA-motif and other cis-acting elements and can specifically respond to abscisic acid, ethylene, methyl jasmonate and other hormones. The promoter also contains 5 MYCRE cis-acting elements CANNTG which can be combined with an upstream regulatory factor IbICE1 gene.
The gene promoter and the upstream regulatory gene IbICE1 are used for constructing a dual-luciferase report system vector, and an agrobacterium-mediated technology is used for injecting tobacco leaves. Experiments show that when tobacco is stressed at low temperature, IbICE1 can be strongly combined with the gene promoter to induce the expression of a reporter gene (GUS gene).
The invention has the beneficial effects that:
the DNA fragment is a promoter of a key transcription factor IbCBF3 in a sweet potato low-temperature stress pathway, and the promoter contains a plurality of adversity and hormone response elements. Through the analysis of a double-luciferase system in tobacco leaves, the promoter is rapidly combined with an upstream gene IbICE1 under low-temperature stress to induce the expression condition of a downstream gene, and can be used for the research of the low-temperature resistance of sweet potatoes and the transgenic breeding.
Drawings
FIG. 1 is a schematic diagram of a dual fluorescent expression vector
FIG. 2 shows that the promoter is induced by low temperature and combined with the upstream gene in tobacco leaves
Detailed Description
Example 1 cloning of IbCBF3 Gene promoter from sweetpotato
According to the cDNA sequence of a transcription factor IbCBF3 important in the sweet potato low-temperature stress signal transduction pathway, 3 specific primers (SEQ ID NO.2: SP1: 5'-GAAGTTGAGACAGGCGGAGCAG-3'; SEQ ID NO.3 SP2: 5'-GTCTCCCGAAACTTCTTCCTCCC-3'; SEQ ID NO.4 SP3: 5'-CCAACAACACCTCTTCATCCGACA-3', the design direction is the direction of an unknown region needing amplification, the position of SP2 is positioned on the inner side of SP1, and SP3 is positioned on the inner side of SP 2) are designed by using the obtained full-length sequence of an IbCBF3 gene according to the specification of a genome walker kit of Clontech corporation, and four uniquely designed degenerate primers with lower annealing temperatures, namely AP1, AP2, AP3 and AP4, are used as nested primers for carrying out nested PCR amplification. And (5) sequencing the amplification result after gel recovery. Designing SEQ ID NO. 5-6 (SEQ ID NO.5: 5' -TGATAGTGAGTTGGGTTAGC-3; SEQ ID NO.6: 5'-TCCATATACGGCGGTACTT-3'), carrying out PCR amplification on the sequence by taking sweet potato cDNA as a template, carrying out gel recovery on an amplification product, sequencing, and verifying the sequence. According to the sequencing result, a specific primer (SEQ ID NO.7 SP4: 5'-GGAGTGTGATAAGGAGACAG-3'; SEQ ID NO.8 SP5: 5'-CGCTACTGAAAGAGACAACA-3'; SEQ ID NO.9 SP6: 5'-CCTTCACGGTGCAGAATG-3', SP6 is positioned at the inner side of SP5, and SP5 is positioned at the inner side of SP 4) of second genome walking and four uniquely designed degenerate primers with lower annealing temperature, namely AP1, AP2, AP3 and AP4, provided in the kit are designed as primers to carry out nested PCR amplification, and the amplification result is subjected to gel recovery and then sequenced. Designing SEQ ID NO. 10-11 (SEQ ID NO.10: 5'-CATCTCGCCTAACTGTCAA-3'; SEQ ID NO.11: 5'-GCTAACCCAACTCACTATCA-3'), carrying out PCR amplification on the sequence by taking sweet potato cDNA as a template, carrying out gel recovery on an amplification product, then sequencing, and verifying the sequence. And splicing the results of two genome walking to obtain the full length of the IbCBF3 gene promoter sequence of the sweet potato.
Example 2 sequence analysis of IbCBF3 Gene promoter of sweetpotato
The sequence was analyzed by plant care software in PLACE and the results showed: besides the conservative elements such as TATA-box, GATA-box and CAAT-box which are necessary for transcription, some elements sensing environmental stress, light, moisture, hormone response and growth and development regulation exist in the IbCBF3 promoter, such as response to ACE, ATCT-motif, GATT-motif, G-box and I-box which are involved in light regulation; abscisic acid-responsive factor ABRE; ethylene response factor ERE and methyl jasmonate response factor CGTCA-motif and the like. In addition, the promoter also comprises a plurality of MYCRE cis-acting elements CANNTG capable of being combined with an upstream regulatory factor ICE gene.
EXAMPLE 3 construction of Dual fluorescent expression vectors
The cDNA of Xuchi 29 is taken as a template, the full-length sequence of a promoter of IbCBF3 is amplified by primers SEQ ID NO. 12-13 (SEQ ID NO. 125 '-GATTGGGAAGAAAGTAT-3'; SEQ ID NO. 135 '-ATACGGCGGTACTTAGTAAAC-3') and the IbICE1 gene is amplified by primers SEQ ID NO. 15-16 (SEQ ID NO. 155 '-ATGTTATCAAGAGTGACAAGC'; SEQ ID NO. 165 '-GATCATCCCGTTGAAGCTAG'), is connected to a T-blunt vector, and is transformed into a competent cell of Escherichia coli DH5 alpha. Adding kanamycin into an LB culture medium, obtaining a single colony through plate screening, extracting a genome of the single colony for PCR verification, and shaking the single colony through the verified single colony to extract a plasmid for sequencing. The IbCBF3 promoter (IbCBF 3pro for short) and IbICE1 genes are respectively constructed into the vector pBI101 and pCAMBIA1200 according to the requirements of an In-fusion kit of TAKARA company, and recombinant vectors IbCBF3 pro-GUS and CaM35S-IbICE1 are obtained.
Example 4 analysis of binding of promoter to upstream Gene induced by Low temperature in tobacco leaves
The recombinant vector plasmids IbCBF3 pro-GUS and CaM35S-IbICE1 are respectively transformed into an agrobacterium strain GV3101, kanamycin or chloramphenicol is added into a YEP culture medium, a single colony is obtained through plate screening, and a single colony genome is extracted for PCR verification. A single colony of agrobacterium is selected and inoculated in a liquid YEP culture medium, and cultured overnight at 30 ℃. Transferring 100 μ l overnight culture solution into fresh YEP culture medium containing 100 μ M acetosyringone (As), culturing at 30 deg.C for 4-6 hr until OD600 of the culture solution reaches 0.8-1.0, and centrifuging to collect thallus. With a staining solution (50 mM Mes-KOH, 50mM MgCl)2 And 200. mu.M As) were resuspended and the final concentration OD was adjusted600After =1.5, tobacco leaves were injected and left at room temperature for 2 days of recovery. The tobacco was transferred to 4 ℃ for 3 hours and a single control group (treated at 25 ℃ for 3 hours) was designed and subjected to GUS staining analysis.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Xuzhou agricultural science research institute of Xuhuai district of Jiangsu Xuhuai (Xuzhou sweet potato research center of Jiangsu)
Promoter for specific expression of IbCBF3 gene abiotic stress of <120> sweet potato and application thereof
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atctattttc ctggtaccac ggaatttgaa tttcatttcc ttcaaatatt ttctagcaac 600
caaacgaact gttacttttc cacacaaatc ttgatataga tacttatatt gaaatctaca 660
tgaaaaaata gcttattagt gtccgtcata tcaagaaata aaatgtccat gtagtagata 720
tattggtaat aattgattct agaaaaagta atgtagtatt gtcacaaatt tggtacaata 780
cgacaagttg atactgagat ttgtgaatct ccagccttcc gtacaggtgt actaggtggt 840
agtaggtacc taggcgatga aagggacaaa ggagtagaga tttggtggtt ccttgacaag 900
atttgggcgg ggcatgggat gaagccatta ggcatgtggc tcacgcgttt ccgtggaaaa 960
accaccggcg ttttactgct tccaaccaat catcaatcct aataatgttt tttttttttt 1020
tgagaaatca atcctaataa tgttggcggt tgtaaaacta acatctcgcc taactgtcaa 1080
ccgcgtctat catctccatc tttcatcctc caattaactg tcacctcgct ccaaacaatc 1140
cttattctgt cctttcttcc atccatccat ctccaatcct taatctatac tatatgtttt 1200
ctgtactcta tgtattgctg attatatatt tattttaaag aaaattaata taaaatttga 1260
aattttacgt agccgtataa aaatcttgtg tgagaccgtt ttacgaatct taatctattt 1320
tggtttgtaa taatcttcag ataataaata tagttctcaa caagaattac atcaaaataa 1380
aaaatagtag gtcttgtatt aggacaatat aatcctcaaa tcttattttt taaaaaatag 1440
tattagtaag tataaaaagg tatttctaga aaataatatt tttattaaat tatatcaaac 1500
tagggaatta aaattaaatt gatagtgaaa ctgaagaggg actgagtata ctgtaacgtg 1560
tgtgcatggc ggaaaagtca tagtatctta ctcgtagtta tggaaatatg aagtacggaa 1620
gatattatga tagtgagttg ggttagctga actttgactt gatatattca gcattctgca 1680
ccgtgaaggt ccgtggaatc ccccgtaaac aagggggcgg gccccattca tgaagtgaaa 1740
gcacacgtgt cgggtacaca acttacgata ccttttaatt tcatgttgtc tctttcagta 1800
gcgtgaccca aatccagttg gcagtttgaa tgaccccacc acgtacagct ggctattcac 1860
ttcatcatat tttccatatt tacctcaatt cccagttcct tcctcctagg ctgtctcctt 1920
atcacactcc gtgttttttc gcgtctccaa cgtaagctta tcaccttaca cttatcgaac 1980
actccccccc tcacccaccc atacgcatac atatatatat aactcccatt ttaggtagtg 2040
tgttaattac ttcatacata cacactccat tttagctttt ccatatatat atatatactt 2100
tactaagtac cgccgtat 2118
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaagttgaga caggcggagc ag 22
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtctcccgaa acttcttcct ccc 23
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccaacaacac ctcttcatcc gaca 24
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgatagtgag ttgggttagc 20
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tccatatacg gcggtactt 19
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggagtgtgat aaggagacag 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cgctactgaa agagacaaca 20
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccttcacggt gcagaatg 18
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
catctcgcct aactgtcaa 19
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gctaacccaa ctcactatca 20
<210> 12
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gattgggaag aaagtat 17
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atacggcggt acttagtaaa c 21
<210> 14
<211> 1638
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgttatcaa gagtgacaag catggtttgg atggatggga aagaggaaga acaagcaggt 60
tcttgggtac agaacaacaa tggcggcggt ggggccggag gaggaggatt agcgggcaag 120
gaggaaatgg agatggcaac aatcaagtcc atgctggaag ctgaggaagt ggagtggtac 180
atggctaata atcagcatag ccacaacaat ggtgcaccca tgcaaggcca tgggggcatt 240
tctttctcta caaatttctc tgagcctgac aacaatctga tcttgcaccc tgtggattcc 300
tcttcctctt gctctccttc ctctgcttct gttttcaatg ctcttgaccc ttctcaggtt 360
cactattttt tgccccacaa ggctgccatg atgagccatc ccttggatca gggtgggttt 420
gatttggggt gtgagagtgg gtttcttgaa actcaagccc tgagtggttt gagtagaggg 480
ggaggggttt tgggtggtgg gtttggtgat ttgagctgtc agaacttctt gggggctccc 540
aacttgagct ctgttcctca atttggttca acccatttgc tgcaacttcc acacaatggt 600
ggaggagggg ggtttggtcc actagggttt ggagagggct atgtgaatgt gaatgagaat 660
gagaatgaga atgctttgtt tcttaatagg tccaagttgt taaagccact tgataatttt 720
gcttcaattg gggcacagcc tactctcttt caaaagaggg ctgctcttag gaagaatctt 780
ggcaattcta gtggaaattt agcacttttg ggtggtgaaa ttggccacac tgatagcagc 840
tttgataaga agagtgaagt gaatgagagg aagaggaaag ggagcaatgg gggggatgaa 900
ttggaggatg tgagcattga tggctccaac ttgaactatg actcagatga gcttgtcgaa 960
aacagtggca aagttgatga aagtgtgaag aatggtggaa ttagctcctc caatgccact 1020
gggggtgacc aaaaggggaa gaagaaaggg cttccagcca agaacttgat ggccgaaagg 1080
aggcgtagga agaagctcaa cgacaggctt tacatgttga ggtctgttgt cccgaagatt 1140
agtaagatgg acagagcttc gattttaggg gatgcaattg aatacttgaa ggaacttctg 1200
cagaaaatca atgacctcca caatgaactc gagtctactc ctccttgctc cgcattaacc 1260
cctaattcga gtttctaccc gttgacacca actgcatctg ccctaccctg ccgtatcaaa 1320
gaagaaatca gtccaactgc atttgcaagc ccgctgtcta gtccaactgg acagcctgca 1380
agggttgaag taagggttag agaaggaaga gcggtgaata tccatatgtt ttgtagccgc 1440
aaacccggcc tattactttc aacaatgaag gctcttgaca accttggtat agacatccaa 1500
caggctgtta tcagctgctt caacgggttt gccttggata ttttccgagc agagcaatgc 1560
aaggaaggcc aagacatcca tccagatcaa atcaaagctg tacttatgga ttccgctagc 1620
ttcaacggga tgatctaa 1638
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgttatcaa gagtgacaag c 21
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gatcatcccg ttgaagctag 20
Claims (5)
1. The promoter for the abiotic stress specific expression of the IbCBF3 sweet potato is characterized in that: the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
2. The promoter for the abiotic stress-specific expression of sweetpotato IbCBF3 according to claim 1; the method is characterized in that: the primer pair for amplifying any fragment of the IbCBF3 promoter of the sweet potato is shown as SEQ ID NO. 2-11.
3. The promoter for the abiotic stress-specific expression of sweetpotato IbCBF3 according to claim 2; the method is characterized in that: the primer pair for amplifying the full length of the sweet potato IbCBF3 promoter is shown as SEQ ID No. 12-13.
4. A construction method of a dual-fluorescence expression vector is characterized in that:
(1) cloning the IbCBF3 gene promoter of the sweet potato, and carrying out PCR amplification by taking the cDNA of the sweet potato as a template to obtain the full length of the IbCBF3 gene promoter sequence of the sweet potato;
(2) constructing an upstream gene IbCBF3 of a sweet potato IbCBF 1, connecting the upstream gene IbICE1 to a T-blunt vector, transforming the upstream gene IbCBF 1 into an escherichia coli DH5 alpha competent cell, and obtaining recombinant vectors IbCBF3 pro-GUS and CaM35S-IbICE 1;
(3) the recombinant vector plasmids are respectively transformed into agrobacterium strains GV3101, and single colony genomes are extracted from YEP culture medium for PCR verification; the nucleotide sequence of the promoter of the IbCBF3 gene of the sweet potato is shown in SEQ ID NO. 1.
5. The use of the promoter for the abiotic stress specific expression of sweetpotato IbCBF3 as claimed in claim 1 in the genetic breeding of plants.
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Application publication date: 20181123 Assignee: Xuzhou Youda Qingyuan Agricultural Technology Development Co.,Ltd. Assignor: JIANGSU XUHUAI DISTRICT XUZHOU AGRICULTURAL Research Institute (JIANGSU XUZHOU SWEET POTATO RESEARCH CENTER) Contract record no.: X2023980049637 Denomination of invention: Promoter of IbCBF3 gene in sweet potato and its application for non biological stress-specific expression Granted publication date: 20210518 License type: Common License Record date: 20231205 |