CN101058811A - Plant salt resistant gene - Google Patents
Plant salt resistant gene Download PDFInfo
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- CN101058811A CN101058811A CN 200610124983 CN200610124983A CN101058811A CN 101058811 A CN101058811 A CN 101058811A CN 200610124983 CN200610124983 CN 200610124983 CN 200610124983 A CN200610124983 A CN 200610124983A CN 101058811 A CN101058811 A CN 101058811A
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- plant salt
- salt
- salt tolerance
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Abstract
The invention relates to a plant salt tolerance gene sequence in the gene engineering technique field, wherein the gene length is 725bp, open reading frame work encodes 166 amino acids from the 26th -526th nucleic acid. The research that the gene expression is intimidated by 1. 7% after natrium chloratum after 24 hours with semi-quantitative PCR finds that expression quantity of the gene (plant salt tolerance gene) increases to more than 5 times. The clone of the gene increased the gene resources of plant salt tolerance research, which improves more abundant fine candidate gene.
Description
Technical field
The present invention is a kind of plant salt tolerance gene of alternanthera philoxeroides, belongs to gene engineering technology field.
Background technology
At present, the salt tolerance that applying transgene technique improves plant has become common recognition, but isolating plant salt tolerance gene kind and quantity are also very limited, and is mostly that the clone obtains from the salt sensitive plant.The alternanthera philoxeroides salt resistance ability is very strong (referring to people such as David J.Longstreth., vegetable cell, tissue and organ culture, 78:225-230 (2004).), the strong gene of a series of salt resistance abilities should be arranged in its body, be the good material of carrying out resistant gene of salt research.
But since paddy rice and alternanthera philoxeroides all normal growth in fresh water environment, with come from the salt sensitive plant especially the resistant gene of salt of terrestrial plant compare, will change paddy rice over to from the resistant gene of salt of alternanthera philoxeroides may be more effective.Therefore the resistant gene of salt that separates alternanthera philoxeroides for the utilization genetic engineering technique improves especially paddy rice salt tolerance of plant, provides abundant more good candidate gene.
Summary of the invention
The present invention has cloned a kind of plant salt tolerance gene of alternanthera philoxeroides.This mrna length is 725bp, and open reading frame is from Nucleotide 26-526 position, 166 amino acid of encoding.
Embodiment
Adopt sandy culture by pouring Hoagland nutritive medium the indoor cultivation alternanthera philoxeroides, 25 ℃ of 30 ℃ of evenings on daytime, relative air humidity 70%, illumination 12h, light intensity is 400umol/m2/s1.When growing the fibrous root of about 5cm length on the stipes of alternanthera philoxeroides, applying analytical pure sodium-chlor makes its final concentration reach 1.7%, clip fibrous root after 24 hours extracts fibrous root RNA according to INVITR0GEN Trizol specification sheets, removes remaining DNA among the RNA according to TAKARA DNaseI specification sheets again.Carry out reverse transcription with the total RNA of 5 μ g according to the M-MLV Reverse Transcriptase specification sheets of Promega company and obtain cDNA.According to nucleotide sequence disclosed by the invention (SEQ ID NO.1), design upstream primer 5 '-GATCAAGTCCCCCACCTA-3 ' and downstream primer 5 '-CGGCAATGATTCAAATATATT-3 ' carries out pcr amplification.The PCR reaction system is as follows: 5ul10 * buffer, and 4 ul dNTP (2.5mmol.L-1), 1ul upstream primer (10 μ mol.L-1), 1ul downstream primer (10 μ mol.L-1), 0.25ul (5U.ul-1) Taq enzyme, 2ulcDNA replenishes aseptic ddH20 to 50ul.The PCR reaction conditions is as follows, 94 ℃ of pre-sex change 1min, 94 ℃ of 30sec, 54 ℃ of 30sec, 72 ℃ of 1min, 35 circulations, 72 ℃ of 10min.After the PCR product being carried out glue recovery purifying, connect the T carrier and transform order-checking.
Detect it by the sxemiquantitative round pcr and handle the back expression at 1.7% sodium-chlor.Vegetable material is cultivated the same, and just material is divided into two portions, is respectively applied for contrast and salt and handles.In order to eliminate the influence of cDNA concentration, select for use house-keeping gene actin to contrast as confidential reference items.The amplification method of Actin is that the cDNA before and after handling with salt is a template, with upstream primer 5 ' TGAACTTCGTGTTGCTCCAGA-3 ' and downstream primer 5 '-AACCCTCGTAGATTGGCACAG-3 ' carries out pcr amplification.The PCR reaction system is as follows: 5ul10 * buffer, and 4ul dNTP (2.5mmol.L-1), 1ul upstream primer (10 μ mol.L-1), 1ul downstream primer (10 μ mol.L-1), 0.25ul (5U.ul-1) Taq enzyme, 2ulcDNA replenishes aseptic ddH20 to 50ul.PCR response procedures: 94 ℃ of 3min; 94 ℃ of 20sec, 55 ℃ of 20sec, 72 ℃ of 20sec, 72 ℃ of 10min, the amplified production size is 230bp.Again according to nucleotide sequence disclosed by the invention (SEQ ID NO.1), design upstream primer 5 '-TCGCCCTCATCCCACCAT-3 ' and downstream primer 5 '-GCAGTCACCTGGGTTCTTCG-3 ', the cDNA before and after handling with salt is that template is carried out pcr amplification.The PCR reaction system is as follows: 5ul10 * buffer, and 4ul dNTP (2.5mmol.L-1), 1ul upstream primer (10 μ mol.L-1), 1ul downstream primer (10 μ mol.L-1), 0.25ul (5U.ul-1) Taq enzyme, 2ulcDNA replenishes aseptic ddH20 to 50ul.PCR response procedures: 94 ℃ of 3min, 94 ℃ of 10sec, 59 ℃ of 10sec, 72 ℃ of 10sec, 72 ℃ of 10min.The amplified production size is 102bp.For pcr amplification was carried out in exponential phase, actin and above-mentioned resistant gene of salt all carry out 25,30,35,40 round-robin amplifications.Index amplification phase PCR product is carried out electrophoresis, then handle fibrous root front and back actin and the brightness of above-mentioned resistant gene of salt PCR product band with gel imaging system observation analysis 1.7% sodium-chlor.After salt was handled, resistant gene of salt was expressed and is strengthened more than 5 times.
Submit the resistant gene of salt sequence that obtains to Genbank, show that through the Blast comparison this gene and pythium spp secreted protein gene homology do not have homology up to 95% with other species.This type of gene function does not appear in the newspapers.Our this resistant gene of salt of above-mentioned evidence expression amount behind salt stress increases by 500 above, is a kind of new resistant gene of salt.The clone of this gene has enlarged the genetic resources of plant salt tolerance research, for the utilization genetic engineering technique improves especially paddy rice salt tolerance of plant, provides abundant more good candidate gene.
<110〉Central China University of Science and Technology
<120〉a kind of plant salt tolerance gene
<140>2006101249835
<141>2006-11-09
<160>1
<170>PatentIn?Version?3.3
<210>1
<211>725
<212>DNA
<213〉alternanthera philoxeroides (Alternanthera philoxeroides)
<400>1
gatcaagtcc?cccacctaca?gcacaatgta?cgccaagacc?ctcctcgtcg?ccgccgtggc 60
cgccctcgct?gccgtcaacg?ccgccccatg?tgacatgctc?actgaggtca?ccaagctgac 120
ccctctcatc?tcggacccaa?acgttgctaa?gtgcagcacc?caggaggagt?cgggcggctt 180
cgccctcatc?ccaccatcgg?gcctgccaac?ccctgaccag?tacaagaaga?tgtgtgtcag 240
cgacgcctgt?aagaaggtca?ttgaggctgt?tgcctcgaag?aacccaggtg?actgcgacct 300
caccgtcggc?tcggtcaccc?tcaacgtgaa?gcagcttgtg?tcgaacttcc?caacggagtg 360
cgccaagtac?acgacgccag?ccaccacggc?tccagcccca?accacgactg?cgccagctcc 420
atcgtcgggc?ccagccccat?cgtcgtcggc?tccagcccca?accggtcaga?ccaccccagc 480
cccaactggc?cagagcaccc?ctgccccaac?caaggctcag?tgctaagtta?tttcgcatct 540
gactcgcggt?cgatggttac?tgctgctcac?tgagtagtgg?tgtcggccca?gtactctcat 600
cgccttcagc?gttttgtcag?tgtccgcgaa?tcaggagctc?atgagcgtcg?ggaggtggtc 660
tatttccctt?cggtcctgct?gaaccgtggt?cgtgtgctgg?aatatatttg?aatcattgcc 720
gtcac 725
Claims (1)
- A kind of plant salt tolerance gene order is characterized in that having one of following nucleotide sequence: 1) a kind of isolating polynucleotide, it has nucleotide sequence shown in the SEQ IDN0.1.2) a kind of isolating polynucleotide, its have with SEQ ID N0.1 in show at least 70% homology from the nucleotides sequence of Nucleotide 26-526 position.
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CNB2006101249835A CN100465275C (en) | 2006-11-09 | 2006-11-09 | Plant salt resistant gene |
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CNB2006101249835A CN100465275C (en) | 2006-11-09 | 2006-11-09 | Plant salt resistant gene |
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CN101058811A true CN101058811A (en) | 2007-10-24 |
CN100465275C CN100465275C (en) | 2009-03-04 |
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CNB2006101249835A Expired - Fee Related CN100465275C (en) | 2006-11-09 | 2006-11-09 | Plant salt resistant gene |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103739080A (en) * | 2014-01-13 | 2014-04-23 | 黑龙江省科学院大庆分院 | Plant water treatment method of municipal wastewater at high altitude |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1239704C (en) * | 2003-11-25 | 2006-02-01 | 中国农业大学 | Gene associated with plant salt resistance and drought resistance, encoded protein and application thereof |
CN100362104C (en) * | 2004-12-21 | 2008-01-16 | 华中农业大学 | Using gene of transcriptional factor OSNACX of paddy to increase drought resistance and salt tolerant abilities of plants |
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2006
- 2006-11-09 CN CNB2006101249835A patent/CN100465275C/en not_active Expired - Fee Related
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103739080A (en) * | 2014-01-13 | 2014-04-23 | 黑龙江省科学院大庆分院 | Plant water treatment method of municipal wastewater at high altitude |
CN103739080B (en) * | 2014-01-13 | 2016-01-13 | 黑龙江省科学院大庆分院 | A kind of Plant water treatment method of municipal wastewater at high altitude |
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CN100465275C (en) | 2009-03-04 |
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