CN1281754C - Cold resisting application of glycan aldehydic acid enzyme arrestin gene AtPGIP1 of Arabidopsis thalianum - Google Patents
Cold resisting application of glycan aldehydic acid enzyme arrestin gene AtPGIP1 of Arabidopsis thalianum Download PDFInfo
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- CN1281754C CN1281754C CN 200410052293 CN200410052293A CN1281754C CN 1281754 C CN1281754 C CN 1281754C CN 200410052293 CN200410052293 CN 200410052293 CN 200410052293 A CN200410052293 A CN 200410052293A CN 1281754 C CN1281754 C CN 1281754C
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Abstract
The present invention discloses the cold resistant application of glycan aldehydic acid enzyme of Arabidopsis thalianum to inhibit protein gene AtPGIP1. The gene AtPGIP1 of the present invention, which has the cold resistant function, is obtained from an information resource TAIR database of Arabidopis thalianum by the comparison with a carrot PGIP sequence via a BLAST method; the gene AtPGIP1 is found in an Arabidopis thalianum cDNA library treated by drought, cold and salt induction by screening, and the number of the gene AtPGIP1 in GenBank is AT3G55530. The gene AtPGIP1 has CDS with the length of 993 bp, 5' UTR with the length of 23 bp, 3' UTR with the length of 153 bp, and protein with a code of 330 aa. The present invention verifies that the gene can improve the cold resistant capability of plants; the application of the gene is capable of reducing crop loss caused by low temperature and chilling injury globally every year and has favorable economic value and an application prospect.
Description
Technical field
The present invention relates to the genetically engineered field, relate in particular to the application of a kind of Arabidopis thaliana saccharan aldehydic acid enzyme arrestin gene A tPGIP1.
Background technology
Plant has enough adaptability and the anti-ability of supporting to the change of circumstances and poor environment, and this resistance was controlled by phylogenetic gene both, is subjected to again that physiological ecological restricts in the ontogeny.Illustrating of plant cold resistance mechanism not only has important scientific theory meaning, and has important use value.The whole world is annual causes the loss of farm crop up to hundreds billion of dollars because of the low temperature cold damage.Therefore, for a long time, existing multiple subject as plant physiology, morphology, genetic thremmatology, biological chemistry, biophysics, cytobiology and molecular biology etc., has carried out extensive studies to plant cold resistance mechanism from many aspects.
Weiser thinks that plant genetic expression in adapting to chilling process changes.Guy etc. discover that cold domestication can induce and increase some expression of gene, several genes is expressed changed.The product of these cold inducible gene expressions can be divided into two classes, and the one, modulin is regulated and control cold signal conduction, cold-resistant genetic expression and Coldproof protein activity; The 2nd, functional protein, directly related with the raising of plant cold resistance.Along with the continuous development of science and technology, people successfully utilize transgenic technology that acquired cool tone joint gene is transferred in the not cold-resistant plant, and result of study proves that cool tone joint gene has the function that improves plant cold resistance power really.But also exist some transfer-gen plant cold hardinesses to improve unconspicuous example simultaneously.Claudia etc. utilize agrobacterium-mediated transformation that the CAP160 in the spinach, CAP85 gene are changed in the tobacco, found that the LT50 of transfer-gen plant and wild-type do not have significant difference, illustrate that two albumen are little to the raising influence of plant cold resistance power.This proves that also plant cold resistance power is the accumulation proterties by many groups gene regulating simultaneously, utilizes the term single gene transfer techniques to improve plant cold resistance power, and its effect is very limited, has only a plurality of cool tone joint genes to cooperate jointly and just can reach the purpose that strengthens plant cold resistance power.Just present gene transformation technology utilizes polygene to transform and improves plant cold resistance power, still need treat with time.
People are when research polar region fish and insect, and finding has a kind of antifreeze protein (AFP) in their bodies.This albumen can cause heat stagnation and can restrain recrystallization, thereby improves the patience of these biologies to cold.But people never find antifreeze protein in plant.1998, Worrall etc. found antifreeze protein in Radix Dauci Sativae, and it can restrain recrystallization, and the sequence height homology of restraining albumen (PGIPs) with polygalacturonase.PGIPs belongs to a protein family---be rich in leucic repeating structure (Leu-rich repeat, LRR) protein family.The characteristics of LRR are conserved sequence GxIPxxLGxLxxLxxLxLxxNxLx, and it participates in inhibition, cell adhesion, cell transportation and the mammalian cell early development of hormone receptor reaction, enzyme.PGIPs can be special the polygalacturonase activity of inhibition fungi, but do not suppress plant or bacterium, thereby cause the defensive raction of plant to fungi.The researchist is devoted to seek the gene that can improve the plant cold resistance ability, and this has far-reaching economic worth.
Summary of the invention
The object of the present invention is to provide the application of a kind of Arabidopis thaliana saccharan aldehydic acid enzyme arrestin gene A tPGIP1 aspect the raising plant cold resistance.
Technical scheme of the present invention is as follows: the AtPGIP1 gene of cold-resistant function of the present invention is the method with BLAST, by with the comparison of Radix Dauci Sativae PGIP sequence, from Arabidopis thaliana information resources TAIR database, obtain; Screening finds that its numbering in GenBank is AT3G55530 in the Arabidopis thaliana cDNA library arid, that cold-peace salt is induced processing, the long 993bp of the CDS of this gene, the long 23bp of 5 ' UTR, the long 153bp of 3 ' UTR, the albumen of coding 330aa.
With SMART programanalysis prediction AtPGIP1 albumen, this albumen contains four LRR structural domains and a segment signal peptide.
The AtPGIP1 gene order is the method with BLAST, by with the comparison of Radix Dauci Sativae PGIP sequence, from Arabidopis thaliana information resources TAIR database, obtain.Come out by pcr amplification the cDNA library of the Arabidopis thaliana (environmental Landsberg eracta) that the AtPGIP1 gene makes up from we oneself with following primer: 5 '-CGGGATCCATGGATAAGACAG-3 ' and 5 '-CGGGATCCTAGAGAT AAGCTT-3 ', cut by the BamHI enzyme then and be connected into pBluescript and make up recombinant vectors pBS-PGIP1, and change intestinal bacteria over to and increase.Recombinant vectors pBS-PGIP1 cuts through XbaI and SalI enzyme, carrier pBA002 cuts through XbaI and XhoI enzyme, and SalI and XhoI are isocaudarners, can produce identical sticky end after enzyme is cut, fragment is connected with carrier and changes intestinal bacteria over to and increase, and obtains recombinant vectors pBA002-PGIP1.Change pBA002-PGIP1 over to agrobacterium tumefaciens, carry out enlarged culturing, use the vacuum method arabidopsis thaliana transformation.The seed process of results is screened, and identifies with blade PCR, obtains the transgenic plant of high expression level AtPGIP1.
Beneficial effect of the present invention: the present invention has decoded the function of Arabidopis thaliana saccharan aldehydic acid enzyme arrestin gene A tPGIP1, has confirmed that this gene can improve the cold tolerance of plant.The application of this gene can reduce the loss of the annual farm crop of causing because of the low temperature cold damage in the whole world, and huge economic and application prospect are arranged.
Description of drawings
Fig. 1 is AtPGIP1 and AtPGIP2 position on genome and the structural domain of the AtPGIP1 of supposition;
Fig. 2 is the northern result of AtPGIP1 and AtPGIP2 expression amount after 0 ℃ of processing;
Fig. 3 is the Nothern blot result of AtPGIP1 transgenic plant expression amount;
Fig. 4 is subzero treatment wt, the result who changes P35S-PGIP1 plant and mutant pgip1 plant.
Wherein, A is AtPGIP1 and the position of AtPGIP2 on genome among Fig. 1, and arrow is partly represented opening code-reading frame, and intron is all arranged in the middle of two genes; B is the structural domain of the AtPGIP1 of supposition; AtPGIP1 contains four LRR structural domains and a segment signal peptide.Among Fig. 3,7.2,8.1,16.2,21.4 for changeing P
Ubiquitin-PGIP1 plant, 1,4,5,6,13,17 for changeing P
35s-PGIP1 plant.
Embodiment
The ecotype that is used for doing wild-type (wt), mutant (mutant) and genetically modified Arabidopis thaliana is Columbia.Sprout on the flat board of MS seed sterilization back, and 4 ℃ of vernalization 3 days is grown in 22 ℃ the greenhouse photoperiod of 16 hours every days then.Move in the soil behind the fortnight, under same condition, grow.
The plant of wild-type is handled different time at 0 ℃, carry out Northern then and analyze, the result finds that the expression amount of AtPGIP1 and AtPGIP2 changes different as shown in Figure 2.This explanation AtPGIP1 can be induced by cold, and AtPGIP2 can not be induced by cold, thus the AtPGIP1 gene with cold induce relevant.
Embodiment 2
The AtPGIP1 gene is cloned into respectively in the expression vector of the promotor 35S that contains monocotyledonous promotor ubiquitin and dicotyledons, the agriculture bacillus mediated wild-type Arabidopis thaliana that changes over to obtains genetically modified plant.We have selected 9 to do Nothern blot analysis altogether, and the result finds the AtPGIP1 expression amount difference of different plants as shown in Figure 3, and a small amount of expression is arranged in the wild-type plant, and does not express in the mutant plant.And commentaries on classics P
UbiquitinThe overall expression level of-PGIP1 plant is than changeing P
35sIt is high that-PGIP1 plant is wanted.
Embodiment 3 cold tests
The present invention handles wt, changes P under cold condition
35S-PGIP1 plant and mutant pgip1 plant.Three kind of plant were handled two months at 4 ℃, handled two weeks for 10 ℃, handled two months at 15 ℃ afterwards.The result as shown in Figure 4, the plant maximum of transgenic plant AtPGIP1, and the plant minimum of mutant pgip1, deepfreeze is to changeing P
35SThe growth effect minimum of-PGIP1 plant.This description of test, PGIP1 expression of gene can improve the tolerance of plant to cold.
Embodiment 4 RNA extract and Northern blot analyzes
Extract big wt of about two weeks respectively, change P with hot phenol method
35STotal RNA of-PGIP1 plant and pgip1 mutant plant carries out Northern blot and analyzes.The probe of Northern blot is prepared with the test kit of Redi-prime.Nothern blot result shows with the wild-type plant and compares, changes P
35SThe expression amount of AtPGIP1 increases to some extent in the-PGIP1 plant, does not then express in the mutant.
A kind of plan ~ 1.TXT
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Claims (1)
1, a kind of Arabidopis thaliana saccharan aldehydic acid enzyme arrestin gene A tPGIP1 is in the application that improves on the plant cold resistance ability, it is characterized in that described gene A tPGIP1 derives from Arabidopis thaliana cDNA library, the numbering of gene A tPGIP1 in GenBank is AT3G55530, gene A tPGIP1 nucleotide sequence is shown in SEQ ID NO:1,330 amino acid of encoding, aminoacid sequence is shown in SEQ ID NO:2.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410052293 CN1281754C (en) | 2004-11-19 | 2004-11-19 | Cold resisting application of glycan aldehydic acid enzyme arrestin gene AtPGIP1 of Arabidopsis thalianum |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410052293 CN1281754C (en) | 2004-11-19 | 2004-11-19 | Cold resisting application of glycan aldehydic acid enzyme arrestin gene AtPGIP1 of Arabidopsis thalianum |
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| Publication Number | Publication Date |
|---|---|
| CN1609218A CN1609218A (en) | 2005-04-27 |
| CN1281754C true CN1281754C (en) | 2006-10-25 |
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| CN 200410052293 Expired - Fee Related CN1281754C (en) | 2004-11-19 | 2004-11-19 | Cold resisting application of glycan aldehydic acid enzyme arrestin gene AtPGIP1 of Arabidopsis thalianum |
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| CN112662679B (en) * | 2020-12-14 | 2021-11-05 | 宁波大学 | Peach fruit polygalacturonase inhibitor protein PpPGIP1 gene and cloning method and application thereof |
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