CN101736030B - Acyl-acyl carrier protein thioesterase gene RNAi carrier in poplar and application thereof - Google Patents
Acyl-acyl carrier protein thioesterase gene RNAi carrier in poplar and application thereof Download PDFInfo
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Abstract
The invention relates to an acyl-acyl carrier protein thioesterase gene RNAi carrier in poplar and an application thereof in silencing target gene. The carrier is constructed by inserting a PtFATB gene segment in a plant expression carrier in a positive direction and a negative direction, and the obtained carrier is a transgenic plant obtained by Agrobacteria transformation. The comparison of the transgenic plant with a wild type plant shows that the transcript levels of the PtFATB gene of the transgenic plant is reduced by above 60% compared with that of the wide type plant; and the content of leaf saturated fatty acid is reduced by above 50% when leaf total fatty acid of the transgenic plant is compared with that of the wide type plant.
Description
Technical field
The present invention relates to a kind of willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier; Also relate to the host cell and the application of this carrier in reticent willow FATB gene that contain willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier, belong to the plant gene engineering technology field.
Background technology
Vegetable fatty acid synthesizes (fatty acid synthesis; FAS) main position is a chloroplast(id); Part chloroplast(id) synthetic lipid acid is participated in inner metabolism in chloroplast(id), generate materials such as glyceryl phosphatide, and this process is called as protokaryon approach (prokaryotic pathway); Another part chloroplast(id) synthetic lipid acid is transported to participates in multiple bio-chemical pathway in the endoplasmic reticulum, these processes are called eucaryon approach (euokaryoticpathway).Kind and quantity that chloroplast(id) outwards transports lipid acid receive acetyl-acetyl carrier proteins thioester enzyme (FATs) control; Acetyl-acetyl carrier proteins thioester enzyme (FATs) hydrolysis acetyl-acetyl carrier proteins (acyl-ACP), thus lipid acid and acetyl carrier proteins produced.In plant, find to exist two types of acetyl-acetyl carrier proteins thioester enzyme (FATs), be respectively FATA and FATB, wherein FATA is external the highest to oleic acid acetyl carrier proteins activity, and is low to sfas acetyl carrier proteins activity; FATB is active high to sfas acetyl carrier proteins, and oleic acid acetyl carrier proteins is also had action activity.Make in the arabidopsis mutant body of FatB inactivation owing to T-DNA inserts, palmitinic acid in the triglyceride level in the blade (16:0) content has reduced 42% than wild-type, in spending, has reduced 56%, in root, has reduced 48%, in seed, has reduced 56%; And Triple Pressed Stearic Acid (18:0) content in blade has also reduced 50%, in seed, has reduced 30%; Two mutants has reduced 50% than the wild-type fresh weight in growth during 4 weeks; The seedling increment has only 1/2nd of wild-type; And the seed viability of two mutants has also reduced, and on the seedling form that becomes to live change has taken place also, and FATB physiological function in Arabidopis thaliana is quite important.
Cortex Populi Tomentosae acetyl-acetyl carrier proteins thioester enzyme gene PtFATB clones; But PtFATB studies in willow and is still waiting to carry out; Because lipid acid especially unsaturated fatty acids plays a significant role in increasing biomembranous flowability; Its content and body in microbial film has extremely strong dependency to the resistance of low temperature adverse circumstance, so the research of fatty acid metabolism genes involved just seems particularly important.
Summary of the invention
The object of the present invention is to provide a kind of willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier; Simultaneously, also comprise the host cell that contains this carrier, said host cell is Bacillus coli cells, agrobatcerium cell or vegetable cell.
The present invention also aims to provide a kind of willow acetyl-application of acetyl carrier proteins thioester enzyme gene RNAi carrier in reticent destination gene expression.
Technical program of the present invention lies in adopting a kind of willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier; This carrier is to be inserted to make up in the plant expression vector with positive and negative both direction by the PtFATB gene fragment to form; The intron that wherein comprises an AtFAD2 gene between two PtFATB gene fragments; Described PtFATB gene is a kind of in Cortex Populi Tomentosae acetyl-acetyl carrier proteins thioester enzyme gene, and described AtFAD2 gene is an Arabidopis thaliana oleate desaturase gene.
Described plant expression vector is the pBI121-1 plasmid; Said plasmid vector comprises plant screening mark gene NPT-II.
Described PtFATB gene fragment, wherein reverse fragment is the pGEM-T-PtFATB plasmid is cut the 1kb of acquisition through EcoR I enzyme a PtFATB gene fragment; The forward fragment is the pGEM-T-PtFATB plasmid is cut the 1.5kb of acquisition through Not I enzyme a PtFATB gene fragment.
Technical program of the present invention also lies in adopting a kind of host cell that contains willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier, described host cell comprises Bacillus coli cells, agrobatcerium cell or vegetable cell.
Technical program of the present invention also lies in adopting a kind of willow acetyl-application of acetyl carrier proteins thioester enzyme gene RNAi carrier in reticent destination gene expression; Get the Bacillus coli cells that contains willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier and extract plasmid; Transform agrobatcerium cell then; Infect poplar leaf through Agrobacterium, and finally obtain transfer-gen plant.Detected result shows that the PtFATB genetic transcription amount of transgenic line reduces more than 60% than wild-type plant PtFATB genetic transcription amount; Transgenic line blade total fatty acids is compared with contrast wild-type plant, and leaf portion sfas (22:0) content descends all more than 50%.
Certainly, for the used gene of the present invention, use other restriction enzyme site to carry out enzyme and cut; And with the constructed RNAi carrier of gene fragment that obtains; And the application in reticent destination gene expression, all belong to the replacement that is equal to of the present invention, also within protection scope of the present invention.
The constructed RNAi carrier of the present invention can be used for reticent PtFATB gene; This carrier passes through Agrobacterium-mediated Transformation; RNAi structure on the carrier is incorporated in the willow genome, forms transfer-gen plant, through transfer-gen plant and wild-type plant are compared; The result shows that the PtFATB genetic transcription amount of transgenic line reduces more than 60% than wild-type plant PtFATB genetic transcription amount; In the greenhouse 18-25 ℃, behind the growth 180d, the plant height of transgenic line has only 50% of contrast wild-type plant height under the normal daylight condition; Transgenic line blade total fatty acids is compared with contrast wild-type plant, and leaf portion sfas (22:0) content descends more than 50%.
Description of drawings
Fig. 1 is the physical map of plasmid pHurricane;
Fig. 2 is the physical map of plasmid pGEM-T-PtFATB;
Fig. 3 is the physical map of plasmid pBI121-1;
Fig. 4 cuts the evaluation electrophorogram for the enzyme of intermediate carrier pHurricane-PtFATB RNAi; 1 is 100bp DNA Ladder; 2 is the electrophoretic band behind the PtFATB gene cDNA pcr amplification; 3 is the electrophoretic band of pHurricane-PtFATB RNAi plasmid behind Not I single endonuclease digestion, and 4 is the electrophoretic band of pHurricane-PtFATB RNAi plasmid behind EcoR I single endonuclease digestion, and 5 is the electrophoretic band of pHurricane-PtFATBRNAi plasmid behind BamH I/Sac I double digestion; 6 is the electrophoretic band of pHurricane-PtFATBRNAi plasmid, and 7 is 1kb DNA Ladder;
Fig. 5 is the physical map of plasmid pHurricane-PtFATB RNAi;
Fig. 6 is the restriction enzyme digestion and electrophoresis figure of pBI121-PtFATB RNAi plasmid; 1 is 100bp DNALadder; 2 is the electrophoretic band behind the PtFATB gene cDNA pcr amplification; 3.pBI121-PtFATBRNAi the electrophoretic band of plasmid behind Not I single endonuclease digestion, 4 is the electrophoretic band of pBI121-PtFATB RNAi plasmid behind EcoR I single endonuclease digestion, and 5 is the electrophoretic band of pBI121-PtFATB RNAi plasmid behind Sma I/Sac I double digestion; 6 is the electrophoretic band of pBI121-PtFATB RNAi plasmid, and 7 is 1kbDNA Ladder;
Fig. 7 is the physical map of plasmid pBI121-PtFATB RNAi;
Fig. 8 is transfer-gen plant southern blotting technique hybridization figure ,+be NPT-II gene 729bp PCR product (positive control) hybridization banding pattern, ck is adjoining tree (negative control) hybridization banding pattern; 1 and 2 is pBI121-PtFATB RNAi transfer-gen plant hybridization banding pattern;
Fig. 9 is transfer-gen plant and adjoining tree PtFATB gene expression amount Real-time pcr analysis result's a bar graph, and 1 and 2 are respectively transgenic line 1,2, and ck is the wild-type plant;
Figure 10 is transfer-gen plant and adjoining tree growing height statistical graph, and 1 and 2 are respectively transgenic line 1,2, and ck is the wild-type plant;
Figure 11 is transfer-gen plant and adjoining tree blade fatty acid component analysis, and 1 and 2 are respectively transgenic line 1,2, and ck is the wild-type plant.
Embodiment
Experiment material used in the present invention is originated as follows: it is Time Technology ltd that coli strain (Escherichia coli) DH5 α buys from sky, Beijing; Agrobacterium strains GV3101 buys from Shanghai Bioisystech Co., Ltd; Plasmid pHurricane (plasmid map is seen Fig. 1) is so kind as to give by Canberra, AUS CSIRO plant industrial research center doctor Liu Qing; The paper " the cloning and expression analysis of Cortex Populi Tomentosae acetyl-acetyl carrier proteins thioester enzyme gene PtFATB " that the gene source of plasmid pGEM-T-PtFATB (plasmid map is seen Fig. 2) and plasmid construction were delivered on " Acta Genetica Sinica " referring to Zhou Zhou etc. in 2007; Plasmid pBI121-1 (plasmid map is seen Fig. 3) is named through improvement on the pBI121 basis by me; Increased more restriction enzyme site, preserved by this laboratory.Employed enzyme, reagent and test kit etc. are the commercially available prod.
Present embodiment willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi construction of carrier may further comprise the steps
(1) structure of intermediate carrier pHurricane-PtFATB
Enzyme is cut: the pGEM-T-PtFATB plasmid with EcoR I single endonuclease digestion, is produced three fragments, reclaim the PtFATB gene fragment of 1kb, this fragment contains the ATG initiator codon; Use EcoR I single endonuclease digestion pHurricane plasmid simultaneously, reclaim the big fragment of linear carrier 4.5kb;
Connect: connect with the T4DNA ligase enzyme; Linked system is the big fragment of pHurricane plasmid (0.3pmol) the 1.0 big fragments of μ L+PtFATB 1kb (0.3pmol) 3.0 μ L+2 * reactionbuffer 5.0 μ L+T4DNA ligase enzymes 1 μ L, and 4 ℃ of ligations are spent the night;
Transform: get connection product transformed into escherichia coli DH5 α, screening contains the bacterial plaque of r plasmid on the LB flat board that contains penbritin (60mg/L);
Checking: 1. PCR checking: the bacterial plaque upgrading grain of antagonism screening carries out the PCR checking; Checking the primer sequence is PF 5 '-GTAAAACGACGGCCAGT-3 '; Forward primer is according to the carrier sequences Design; PR 5 '-CATGGTCATAGCTGTTTCC-3 '; Reverse primer is according to PtFATB gene 1kb fragment (be above-mentioned pGEM-T-PtFATB plasmid obtain through EcoR I single endonuclease digestion 1kb fragment) sequences Design, filters out the bacterial plaque that PtFATB gene 1kb fragment is oppositely inserted the pHurricane plasmid; 2. enzyme is cut checking: can amplify the segmental plasmid of 1kb to PCR, with EcoR I single endonuclease digestion and SmaI/SacI double digestion identification and analysis, select the correct vector plasmid of enzyme slitting band, with its called after pHurricane-PtFA TB;
(2) structure of intermediate carrier pHurricane-PtFATB RNAi
Enzyme is cut: the pGEM-T-PtFATB plasmid with Not I single endonuclease digestion, is reclaimed the PtFATB gene fragment of 1.5kb; Use Not I single endonuclease digestion pHurricane-PtFATB plasmid simultaneously, reclaim the big fragment of linear carrier 5.5kb;
Connect: connect with the T4DNA ligase enzyme; Linked system is the big fragment of pHurrican e-PtFA TB plasmid (0.3pmol) 1.0 μ L+PtFATB 1.5kb fragments (0.3pmol) 3.0 μ L+2 * reaction buffer 5.0 μ L+T4DNA ligase enzymes 1 μ L, and 4 ℃ of ligations are spent the night;
Transform: get connection product transformed into escherichia coli DH5 α, screening contains the bacterial plaque of r plasmid on the LB flat board that contains penbritin (60mg/L);
Checking: 1. PCR checking: the bacterial plaque upgrading grain of antagonism screening carries out the PCR checking; Checking the primer sequence is PF 5 '-GGAAACAGCTATGACCATG-3 '; Forward primer is according to the carrier sequences Design; PR 5 '-GTTGAGCTGCTCTGCCTCTC-3 ', reverse primer filters out the bacterial plaque that PtFATB gene 1.5kb fragment forward inserts the pHurricane-PtFATB plasmid according to the design of PtFATB gene cDNA sequence; 2. enzyme is cut checking: can amplify the segmental plasmid of 1.5kb to PCR; Carry out enzyme with Not I single endonuclease digestion, EcoR I single endonuclease digestion, BamHI/Sac I double digestion and cut identification and analysis (electrophoresis result after enzyme is cut is seen Fig. 4); Select the correct vector plasmid of enzyme slitting band, with its called after pHurricane-PtFATB RNAi (plasmid map is seen Fig. 5);
(3) structure of RNAi carrier pBI121-PtFATB RNAi
Enzyme is cut: with the pBI121-1 plasmid with Sma I/Sac I double digestion; Reclaim the big fragment of pBI121-1 plasmid; Simultaneously with intermediate carrier pHurricane-PtFATB RNAi plasmid with Sma I/Sac I double digestion, reclaim PtFATBRNAi structure fragment (this fragment comprises the AtFAD2 gene intron of 1.2kb);
Connect: connect with the T4DNA ligase enzyme; Linked system is the big fragment of pBI121 plasmid (0.3pmol) 1.0 μ L+PtFATB RNAi structure fragments (0.3pmol) 3.0 μ L+2 * reactionbuffer 5.0 μ L+T4DNA ligase enzymes 1 μ L, and 16 ℃ of ligations are spent the night;
Transform: get connection product transformed into escherichia coli DH5 α, screening contains the bacterial plaque of r plasmid on the LB flat board that contains penbritin (60mg/L);
Checking: the bacterial plaque that antagonism filters out is extracted plasmid; And the plasmid that is extracted used Not I single endonuclease digestion, EcoR I single endonuclease digestion, Sma I/Sac I double digestion identification and analysis (electrophoresis result after enzyme is cut is seen Fig. 6) respectively; Select the correct vector plasmid of enzyme slitting band, with its called after pBI121-PtFATB RNAi (plasmid map is seen Fig. 7).
Present embodiment is the application example of pBI121-PtFATBRNAi carrier, and concrete steps are following
(1) pBI121-PtFATB RNAi plasmid is transformed Agrobacterium; After the empirical tests; The Agrobacterium that use contains pBI121-PtFATB RNAi plasmid is infected the silver-colored gland poplar (blade of clone 84K of P.alba * P.glandulossa); Method through tissue culture obtains PtFATB RNAi transgenic resistant plant, the transfer-gen plant that the PCR preliminary identification obtains, the transfer-gen plant intermediate house that detected result is positive; Use the southern blotting technique hybridization analysis further to verify (detected result is as shown in Figure 8), the result shows that the PtFATBRNAi gene fragment has been incorporated in the genome of silver-colored gland poplar.
(2) transfer-gen plant FATB genetic expression component analysis
Through the Realtime pcr analysis; In the pBI121-PtFATB-RNAi transgenic lines 1 and 2 PtFATB genetic transcription amount compare with wild-type plant PtFATB genetic transcription amount average reduced by 60% and 35% (as shown in Figure 9; Wherein the X axle is represented the different tests plant, and the Y axle is represented the gene relative expression quantity).
(3) transfer-gen plant growth and blade fatty acid component are analyzed
In the greenhouse 18-25 ℃, behind the growth 180d, the plant height of transgenic line has only 50% (shown in figure 10, wherein the X axle is represented the different tests plant, and the Y axle is represented the growing height of each plant) of contrast wild-type plant height under the normal daylight condition.
Get transgenic line 1 and 2 plant tops the 3rd or the 4th blade, the about 3cm of its width carries the blade total fatty acids, and gas chromatographic analysis after esterification; Compare with contrast wild-type plant, leaf portion sfas (22:0) content in transgenic line 1 and 2 blades descends all (shown in figure 11, wherein the X axle is represented the different tests plant, and the Y axle is represented the fatty acid content of each plant) more than 50%.
Claims (5)
1. willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier is characterized in that: this carrier be by
PtFATBGene fragment is inserted to make up in the plant expression vector with positive and negative both direction and is formed wherein two
PtFATBComprise one between the gene fragment
AtFAD2The intron of gene, described
PtFATBGene is Cortex Populi Tomentosae acetyl-acetyl carrier proteins thioester enzyme gene, and is described
AtFAD2Gene is an Arabidopis thaliana oleate desaturase gene; Wherein,
PtFATBThe reverse fragment of gene fragment is pGEM-T-
PtFATBThe plasmid warp
EcoRThe I enzyme is cut the 1kb of acquisition
PtFATBGene fragment, the forward fragment is pGEM-T-
PtFATBThe plasmid warp
NotThe I enzyme is cut the 1.5kb of acquisition
PtFATBGene fragment.
2. a kind of willow acetyl according to claim 1-acetyl carrier proteins thioester enzyme gene RNAi carrier, it is characterized in that: said carrier comprises plant screening mark gene
NPT-II.
3. host cell that contains claim 1 or 2 described willow acetyl-acetyl carrier proteins thioester enzyme gene RNAi carrier.
4. the host cell of a kind of willow acetyl according to claim 3-acetyl carrier proteins thioester enzyme gene RNAi carrier, it is characterized in that: this cell is Bacillus coli cells, agrobatcerium cell.
5. the willow acetyl-application of acetyl carrier proteins thioester enzyme gene RNAi carrier in reticent destination gene expression according to claim 1 or claim 2, described goal gene does
PtFATBGene.
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Citations (4)
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|---|---|---|---|---|
| CN1655669A (en) * | 2002-03-21 | 2005-08-17 | 孟山都技术有限公司 | Nucleic acid constructs and methods for producing altered seed oil compositions |
| CN1724669A (en) * | 2005-03-17 | 2006-01-25 | 东北师范大学 | Method in increasing oleic acid content of soybean and peanut seed by applying gene silent technology |
| CN101421406A (en) * | 2006-02-13 | 2009-04-29 | 孟山都技术有限公司 | Nucleic acid constructs and methods for producing altered seed oil compositions |
| CN101516181A (en) * | 2006-07-14 | 2009-08-26 | 联邦科学技术研究组织 | Altering the fatty acid composition of rice |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1655669A (en) * | 2002-03-21 | 2005-08-17 | 孟山都技术有限公司 | Nucleic acid constructs and methods for producing altered seed oil compositions |
| CN1724669A (en) * | 2005-03-17 | 2006-01-25 | 东北师范大学 | Method in increasing oleic acid content of soybean and peanut seed by applying gene silent technology |
| CN101421406A (en) * | 2006-02-13 | 2009-04-29 | 孟山都技术有限公司 | Nucleic acid constructs and methods for producing altered seed oil compositions |
| CN101516181A (en) * | 2006-07-14 | 2009-08-26 | 联邦科学技术研究组织 | Altering the fatty acid composition of rice |
Non-Patent Citations (2)
| Title |
|---|
| Zhou Zhou等.Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr..《Journal of Genetics and Genomics》.2007,第34卷(第3期),第267-274页. * |
| 张承妹等.利用高效农杆菌遗传转化系统将ACP反义基因片段导入油菜.《上海农业学报》.2003,第19卷(第2期),第5-8页. * |
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