CN1687417A - Gene sequence in enrichment protein of late stage embryo in third group of vinca rosea - Google Patents
Gene sequence in enrichment protein of late stage embryo in third group of vinca rosea Download PDFInfo
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- CN1687417A CN1687417A CNA2005100099380A CN200510009938A CN1687417A CN 1687417 A CN1687417 A CN 1687417A CN A2005100099380 A CNA2005100099380 A CN A2005100099380A CN 200510009938 A CN200510009938 A CN 200510009938A CN 1687417 A CN1687417 A CN 1687417A
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Abstract
The third group late embryo protein of a kind of Changchun flower and coding product amino acid sequence, it relates to a kind of new gene sequence. Its character lies in as follows, the length of gene is 829bp, it has the beginning code ATG at 127bp, it has the ending code TGA at 618bp, it has the accessional code polyA at 806bp. The sequence has no son, and has intact opening knowing code frame at 492bp, creating 163amino acid code. The nucleotide acid sequence and carrot sequence have the same source, which is up to 69%. The nucleotide sequence and chick bean group late cembryo protein have the same source, which is up to 51%.It includes six basis sequence, which has eleven amino acid residue. This invention also provides a kind of a way which can get the growing gene rapidly. First it uses measurable drought dealing condition, to make the bearable drought gene expression rich. Second according to the conduction library cDNA and detect sequence, in a shortest time it can get the growing gene relating to bearable drought. This kind of gene conduction on the real nucleus expression carrier particle pCMV, it can detect sequence using t3 and t7.Meanwhile,it can filter real nucleus biology expression directly. This invention departs the third group late cembryo protein growing gene from the plant cDNA, The gene has a good bearable drought ability. Meanwhile, it has other good effect when faced to terrible circumstance, such as cold and salty. The copy of the gene expands the gene source of bearable drought research. It can enhance bearable drought ability using gene project technique, and provides richer and better selecting gene for the excellent character.
Description
Technical field
The invention belongs to field of biological genes, be specifically related to the gene order of Vinca anti-drought gene enrichment protein of late stage embryo.
Background technology
(late embryogenesis abundant Protein LEA) extensively is present in the higher plant enrichment protein of late stage embryo LEA, has stronger drought-resistant ability.Dissimilar LEA expresses in the embryogenetic different steps embryo of higher plant seed or expresses under the drought stress condition.They can be induced by ABA (abscisic acid).Simultaneously, also bringing into play important effect aspect lea protein, the salt tolerance cold-resistant plant.
Lea protein common constitutional features is that bias amino acid is formed the highly hydrophilic polypeptide of formation.Most of lea proteins lack halfcystine and tyrosine residues, but are rich in Methionin and glycine.According to homology of their aminoacid sequences and the Sequence of Primitive Elements that some are special, lea protein can be divided into 6 groups.
The 3rd group of lea protein contains the Sequence of Primitive Elements (TAQAAKEKAGE) of 11 amino acid compositions of multiple copied usually.This group lea protein difference in size is very big, what the copy number of contained Sequence of Primitive Elements was few has only 5, and as cotton LEA D7 albumen, many then has tens, as rape LEA 76 albumen (13), soybean cDNApGmPM8 contains with the pGmPM10 proteins encoded and surpasses 30 Sequence of Primitive Elements that link to each other.This histone can have or not specific amino acid extension of section can be divided into two types according to the carboxyl terminal of its Sequence of Primitive Elements, one class is that the repeating group metasequence that 11 amino acid are formed is separated by sequence similarly, claim the 3rd group of lea protein (I), as the HVA1 of barley and the PMA2005 of wheat, DC3 of Radix Dauci Sativae and the D7 of cotton; Another kind of is to be separated by the appearance of specific amino acids extension of section at C-terminal, claims the 3rd group of lea protein (II), as the PMA1949 of wheat, and the pGmPM2 of Radix Dauci Sativae DC8 and soybean etc.
The ionic concn of enchylema can raise rapidly in arid dehydration.High-intensity ionic concn can cause the irreversible injury of cell.The Sequence of Primitive Elements that 11 amino-acid residues in the 3rd group of lea protein are formed can form facultative α
2Spirane structure provides the water-wetted surface in hydrophobic district of a tool, and the hydrophobic surface of spiral can form homodimer, but is in spissated ion in the charged group chelating cell dehydration process of water-wetted surface, as Na
+And PO
3 -4In addition, forming these proteic most of amino-acid residues is alkalescence, hydrophilic amino acid, no halfcystine and tryptophane, the amino-acid residue of these high electric charges can redirect intracellular water molecules, the constraint salt ion, thereby the caused damage of accumulation of high concentration ion in the cell when avoiding drought stress also can prevent the excessive tissue dehydration simultaneously.
At present, in various plants all relevant for the research of the 3rd group of lea protein or gene report.The osmotic stress resistance positive correlation of these expression of gene or albumen accumulation and plant.3 days barley seedlings of rudiment all has high-caliber HVA1 mRNA and protein to occur after 011mmol/LABA or arid processing in all organs of seedling, it can be by environment-stress and ABA abduction deliverings such as arids.Xu etc. import paddy rice with HVA1 cDNA complete sequence, have obtained the transgenic paddy rice of drought resisting, anti-salt, thereby have confirmed that directly the 3rd group of LEA gene may have arid defencive function.
By retrieval, do not find any gene order and aminoacid sequence that discloses or reported the Vinca lea protein.
Summary of the invention
First purpose of the present invention is to provide a kind of nucleotide sequence that comes from the 3rd group of lea protein of Vinca, it is characterized in that, it comprises: coding is according to the functional zone sequence that the lea protein of the 3rd group of LEA (late embryo generation Abundant protein) is arranged, and it comprises 6 Sequence of Primitive Elements that are made of 11 amino-acid residues.Described nucleotide sequence and Radix Dauci Sativae LEA DC3 have 69% homology, reach 51% with chicken beans lea protein homology.
Second purpose of the present invention is to provide a kind of quick acquisition to coerce the method for genes involved full length sequence.
CDNA molecule of the present invention is to obtain by following method:
At first seek the treatment condition that the plant moderate is coerced by measuring the plant leaf flow of water.Make and express proteic abundance enrichment under the plant drought stress.
Secondly, adopt the suitableeest processing material to carry out the extraction of total RNA and the purifying of mRNA, carry out the structure of full-length cDNA expression library with mRNA.Make up back cDNA and be on the eukaryotic expression plasmid pCMV, can directly clone the screening of the expression library of cDNA.
At last, adopt 3730 sequenators, the clone of random choose is checked order.The EST label is surveyed logical total length to relevant clone after comparing with ncbi database, obtain the complete encoding sequence of complete LEA gene.
Significant application value of the present invention is:
This gene has the following advantages in the drought resisting process: lea protein is as the dehydration protection agent, one side is had an effect with other albumen in the born of the same parents, make their structure keep stable, be hydrophilic αLuo Xuanjiegou on the other hand, provide one in conjunction with lining matter for intracellular irreducible water, make cell in dehydration, avoid persecution; And existing periodic charged ion space binding site, charged ionic bond improves the cell damage that causes in these sites thereby cushioned because of dehydration makes ionic strength when dehydration; In addition, it is that the part of plant osmoregulation mechanism is the adjusting albumen of the synthetic and transportation of osmoregulation materials such as sugar alcohol, proline(Pro), trimethyl-glycine, thereby lea protein has stronger drought-resistant ability, the drought resisting mechanism of difference and the single adjusting of other genes.
Description of drawings
Fig. 1 is the homology comparison chart of different plant origin lea proteins
Fig. 2 is the homology comparison chart and the Sequence of Primitive Elements of Vinca and Radix Dauci Sativae lea protein
Embodiment:
Embodiment 1 Vinca anti-drought gene enrichment protein of late stage embryo Cloning of Entire Gene
Step 1, vegetable material is handled
Vinca seedling sowing is cultivated in illumination box in perlite, and water every day, gets Vinca plant dehydration naturally in light training case of 1 month seedling age, every one hour survey leaf water potential and osmotic potential, with reach-1.0MPa is that standard is drawn materials.Get the blade liquid nitrogen flash freezer, put into the Ultralow Temperature Freezer of-70 degree immediately.
Step 2, the extraction of total RNA and the purifying of mRNA
1. utilize Trizol (Gibco) single stage method to extract total RNA, carry out according to test kit specification sheets step.To total RNA quantitatively and carry out the mass analysis of sex change electrophoresis.
2.mRNA purifying: utilize Oligotex mRNA Purification Kit (Qiagen) purified mRNA and carrying out quantitatively from total RNA.
Step 3, λ ZAP
ExpressThe structure in cDNA library (Stratagene)
1.cDNA first chain is synthetic
(1) set up following system on ice:
5.0 μ L 10 * buffer 3.0 μ LdNTP Mix, 2.0 μ L Link-Primer, 12.5 μ L DEPC-H
2O, 1.0 μ LRNAsin, mixing adds mRNA 5 μ g, adds 1.5 μ L ThermoScript II.
(2) mixing gently, 42 ℃ of incubation 1hr.
2.cDNA second chain is synthetic
(1) order adds in the first chain reaction system: 20 μ L, 10 * buffer, 6.0 μ L dNTP Mix, 116 μ LDEPC-H
2O, 2.0 μ L RNAseH (1.5U/ μ L), 11.0 μ L DNA polymerase I (9.0U/ μ L)
(2) put 16 ℃ of water bath heat preservation 2.5hr.
3.cDNA end-filling
(1) adds following reagent on ice: 23 μ L dNTP Mix, 2.0 μ L pfu DNA pol (2.5U/ μ L).
(2) 72 ℃ of incubation 30min.
(3) once, room temperature high speed centrifugation 2min changes supernatant in another new pipe with 200 μ L phenol/chloroform extractings.
(4) once, change supernatant in another new pipe with the extracting of equal-volume chloroform.
(5) add 20 μ L 3mol/LNaAc, 400 μ L, 100% ethanol, mixing ,-20 ℃ are spent the night.
(6) 4 ℃ of centrifugal 60min of 16000g collect the cDNA precipitation.
(7) adding 500 μ L, 70% ethanol washes gently.
(8) the centrifugal 2min of full speed vacuumizes under the room temperature and makes the precipitation drying.
(9) precipitation is resuspended among the 9.0 μ L EcoR I adapters.4 ℃ of insulations are to dissolving fully.
4.EcoR the connection of I adapter
(1) order adds: 1.0 μ L, 10 * buffer (Ligase Buffer).
1.0μL?10mmol/L?rATP
1.0μL?T
4DNA?ligase(4U/μL)
4 ℃ connect 2 days.
(2) 70 ℃ of water-bath 30min are with deactivation Ligase.
5.EcoR the phosphorylation of I connexon end.
(1) the centrifugal 2s of room temperature and place 5min adds:
1.0μL?10×Ligase?Buffer
2.0μL?10mmol/L?rATP
5.0μL?sterile?H
2O
2.0μL?T4?Kinase(5U/μL)
37 ℃ of insulation 30min.
(2) 70 ℃ of heating 30min deactivation Kinase
6.Xho I digestion
Add following ingredients:
28.0μL?Xho?I?buffer
3.0μL?Xho?I(40U/μL)
37 ℃ of insulation 1.5hr.
7. carrier connects
7.1 use Wizard SV Gel and PCR CLEAn System kit (Promega) to reclaim the cDNA fragment, it is quantitative that the cDNA that reclaims is carried out EB.
7.2 cDNA is connected with ZAP Express Vector
In the 1.5ml centrifuge tube, add:
2.0 the resuspended cDNA of μ L
0.5μL?10×ligase?buffer
0.5μL?10mM?rATP
1.0μL?ZAP?Express?Vector
0.5μL?ddH
2O
T4?DNA?ligase(4U/μL)0.5μL，
4 ℃ connect 2 days.
8. the external packing of λ recombinant DNA
(1) adds target DNA 100ng to packaging protein, mixing, the centrifugal pipe end that is compiled in.
Behind (2) 22 ℃ of incubation 2hr, add 500 μ L SM Buffer, adding 20 μ L chloroform, mixings gently.
9. the transfection of lambda particles phage
9.1 the mensuration of phage library titre
(1) streak culture XL1-blue MRF ', 37 ℃ are spent the night, and substratum is LB (containing 12.5 μ g/ml Tet).
(2) be seeded to LB broth, 30 ℃, 200rpm spends the night and shakes (OD＜1), and the centrifugal collection thalline of 1000 * g adds 10mmol/L MgSO
4Resuspended thalline is to OD
600=0.5.
(3) in above-mentioned packaging system, respectively get 1.0 μ L and do a series of dilutions, promptly 1 → 10
-2→ 10
-4, respectively get 1.0 μ L diluents in the host bacterium, behind the absorption 15min, add the 3mL top-agar, be layered on rapidly and educate temperature in advance to 37 ℃ bottom-layer agar, wait to solidify back 37 ℃ and spend the night to be inverted and cultivate.
(3) treat that plaque grows after, calculate titre.After testing, the storage capacity in elementary library reaches 1 * 10
6Pfu/ml.
9.3 the amplification of phage library
(1) preparation of host bacterium is the same.
(2) completing behind the plate 37 ℃ spends the night to be inverted and cultivates.Plaque is no more than 1～2mm.
(3) with the 4mL SM Buffer bacterium plate of coating, 4 ℃ of storages are spent the night.
(4) sucking-off SM Buffer is in sterile tube, washes the bacterium plate once adding 1mL SM Buffer, is drawn onto to add chloroform in the pipe to final concentration 5%, and mixing is put incubated at room temperature 15min, and the centrifugal 10min of 500g removes cell debris.
(5) change supernatant in another pipe, add chloroform to final concentration and deposit for 0.3%, 4 ℃.
(6) survey titre.Through amplification, the titre in library reaches 1 * 10
8Pfu/ml
Shear in the body of 10 phage libraries
(1) streak culture XL1-blue MRF ' and XLOLR bacterium are in LB substratum (containing 12.5 μ g/ml Tet). choose single colony inoculation to 50mL LB broth, 30 ℃, 200rpm incubated overnight.
The centrifugal collection of (2) 1000 * g XL1-blue MRF ' and XLOLR cells use 10mM MgSO
4Resuspended to OD
600=1.
(3) in centrifuge tube, add 100 times to the phagocytosis scale of construction in elementary storehouse.Add host bacterium XL1-blue by 10: 1 (cells-to-lambdaphage) and adding helper phage by (10: 1 helper phage-to-cells ratio).
(4) 37 ℃ of absorption 15min add 20ml NZY broth with supplements, and 37 ℃ are shaken training 3hr, 65 ℃ of heat shock 20min, and the centrifugal 10min of 1000 * g changes supernatant in another pipe.
(5) survey titre: get supernatant 1.0 μ L and be added to respectively in the 200 μ L XLOLR bacterium, 37 ℃ of absorption 15min add 37 ℃ of 200 μ L NZY broth again and shake training 45min.
(6) (50 μ g/ml, kana) 37 ℃ of incubated overnight obtain bacterium colony therefrom to take out 100 μ L shop LB.
11 use the PCR reaction system detects the average segmental length of inserting
Get the carrier primer, choose mono-clonal and carry out PCR checking insertion segment, the PCR reaction conditions is:
95.0 ℃ pre-sex change 5min, 94.0 ℃ of sex change 30s then, 50.0 ℃ of template annealing 30s, 72.0 ℃ are extended 2min, and totally 30 circulations are last, 72.0 ℃ of final 7min that extend, reaction is got the PCR product and carry out electrophoresis detection on 1% sepharose after finishing.Through check, on average insert the long 1.2kb of being of segment.
Step 4 adopts 3730 sequenators check order (ABI)
Adopt the T7 primer to carry out unidirectional order-checking, each reaction can reach 800-1200bp.The est sequence that obtains enters NCBI compare after, drought stress genes involved plasmid is surveyed logical, obtain full-length gene order.
Sequence information and the homology analysis of embodiment 2 Vinca LEA
With the lower section is the nucleotide sequence of clone's Vinca lea protein:
ggcacgaggc?aaaatcaaag?tcgaatcgat?cgagtttaat?attcttcagc?aagtaagttt 60
ggattagtta?attaattact?catagttaaa?ttaacagtag?agagagaaag?taagcgcagt 120
gcaaaaatgg?catcccacga?gcagagctat?aaagctggtg?aagccaaggg?tcaagctcag 180
gaaaaaactg?gccaaatgat?gggcgacgta?aaggagaaga?ctcaacaagc?caaagacaag 240
gcttccgaaa?cagcccagtc?ggcacaaggc?cgtgcgcagg?agaagaaaga?tcaaaccggc 300
agttacgtct?cggacaaagc?cggcgctgca?aaggacaagc?tttcagagac?tactcaatct 360
gcaaaagaga?gagcttctgg?agcagctgaa?gctacaaagc?aaaaggcatc?ggaaatggca 420
caatcgggca?aagagacagc?agaggcaggg?aaagagaaga?caggtgggtt?tctgcagaag 480
acaggtgaac?aagttaaagg?catggctcaa?ggtgctgctg?atgctgtgaa?acataccttt 540
ggtatggctg?aaagtgagga?aggtttcgaa?aataagggcg?gagcggcaga?agccacgggg 600
aggaagcgta?taacttgagt?tattttgggg?agaaatggtg?ttcgatttcc?ttggtttctt 660
tgttgatttc?tgggttaatt?tcggtatttc?ttgatgttta?tgcacctgaa?tgatgatacc 720
tcatctgttt?cctttgaggt?attgctgtac?gttgtatgaa?taaatactga?gttctcttgg 780
taataatatt?gtagaatttc?tattcaaaaa?aaaaaaaaaa?aaaaaaaaa 829
Whole coding sequence is atg to 618 tga from 127.The 806th has the polyA additional signal.Its amino acid sequence coded is:
MASHEQSYKAGEAKGQAQEKTGQMMGDVKEKTQQAKDKASETAQSAQGRA
QEKKDQTGSYVSDKAGAAKDKLSETTQSAKERASGAAEATKQKASEMAQSG
KETAEAGKEKTGGFLQKTGEQVKGMAQGAADAVKHTFGMAESEEGFENKGG
AAEATGRKRIT
LEA is the conservative territory of supposition not, and the Sequence of Primitive Elements that is made of 11 amino-acid residues is the constitutional features of the 3rd group of lea protein.This Sequence of Primitive Elements is that hydrophilic lipophilic is held concurrently, and is α-Luo Xuanjiegou, is the basis that forms the 3rd group of lea protein higher structure, is its basis with drought resisting function.The homology compare of analysis is the result indicate, with Radix Dauci Sativae (Daucus carota) full-length gene Lea Dc3 homology up to 69%, reach 51% with chicken beans (Cicer arietinum) lea protein homology.
The present invention separates this gene from Vinca, promptly separate this gene from new species, thereby have novelty; Drought-resistant, the saline alkali tolerant plant of Vinca, thereby predict this gene stronger drought resisting, salt tolerant alkali function are arranged, the practical value of renewal is arranged.
Claims (3)
- A coding Vinca anti-drought gene enrichment protein of late stage embryo (Late Embryogenesis Abundant Protein, nucleotide sequence LEA) is characterized in that it has following nucleotide sequence and amino acid sequence corresponding:ggcacgaggc?aaaatcaaag?tcgaatcgat?cgagtttaat?attcttcagc?aagtaagttt 60ggattagtta?attaattact?catagttaaa?ttaacagtag?agagagaaag?taagcgcagt 120gcaaaaatgg?catcccacga?gcagagctat?aaagctggtg?aagccaaggg?tcaagctcag 180gaaaaaactg?gccaaatgat?gggcgacgta?aaggagaaga?ctcaacaagc?caaagacaag 240gcttccgaaa?cagcccagtc?ggcacaaggc?cgtgcgcagg?agaagaaaga?tcaaaccggc 300agttacgtct?cggacaaagc?cggcgctgca?aaggacaagc?tttcagagac?tactcaatct 360gcaaaagaga?gagcttctgg?agcagctgaa?gctacaaagc?aaaaggcatc?ggaaatggca 420caatcgggca?aagagacagc?agaggcaggg?aaagagaaga?caggtgggtt?tctgcagaag 480acaggtgaac?aagttaaagg?catggctcaa?ggtgctgctg?atgctgtgaa?acataccttt 540ggtatggctg?aaagtgagga?aggtttcgaa?aataagggcg?gagcggcaga?agccacgggg 600aggaagcgta?taacttgagt?tattttgggg?agaaatggtg?ttcgatttcc?ttggtttctt 660tgttgatttc?tgggttaatt?tcggtatttc?ttgatgttta?tgcacctgaa?tgatgatacc 720tcatctgttt?cctttgaggt?attgctgtac?gttgtatgaa?taaatactga?gttctcttgg 780taataatatt?gtagaatttc?tattcaaaaa?aaaaaaaaaa?aaaaaaaaa 829MASHEQSYKAGEAKGQAQEKTGQMMGDVKEKTQQAKDKASETAQSAQGRAQEKKDQTGSYVSDKAGAAKDKLSETTQSAKERASGAAEATKQKASEMAQSGKETAEAGKEKTGGFLQKTGEQVKGMAQGAADAVKHTFGMAESEEGFENKGGAAEATGRKRIT
- 2. a nucleotide sequence as claimed in claim 1 is characterized in that the 127bp place has initiator codon ATG, and the 618bp place has terminator codon TGA, has the polyA additional signal at the 806bp place.This sequence intronless has the complete opening code-reading frame of 492bp, 163 amino acid of encoding.
- 3. the cloning process of the nucleotide sequence of a Vinca enrichment protein of late stage embryo as claimed in claim 1 is characterized in that: at first use the arid treatment condition of appropriateness, make the foliage combat drought gene expression enrichment; By making up the cDNA library of total length, reach order-checking then, in the shortest time, obtain the relevant full-length intimidated gene of drought resisting.The anti-drought gene that obtains directly is structured on the carrier for expression of eukaryon pCMV plasmid, can express directly by eukaryote and screen.Gene can use t3, t7 universal primer to check order.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937590A (en) * | 2017-12-12 | 2018-04-20 | 山东省花生研究所 | Clone primer pair and its application of peanut embryonic development late period Abundant protein gene |
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2005
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937590A (en) * | 2017-12-12 | 2018-04-20 | 山东省花生研究所 | Clone primer pair and its application of peanut embryonic development late period Abundant protein gene |
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