CN1888064A - Gene engineering method of obtaining low erucic acid rape variety and its application - Google Patents
Gene engineering method of obtaining low erucic acid rape variety and its application Download PDFInfo
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- CN1888064A CN1888064A CN 200610019696 CN200610019696A CN1888064A CN 1888064 A CN1888064 A CN 1888064A CN 200610019696 CN200610019696 CN 200610019696 CN 200610019696 A CN200610019696 A CN 200610019696A CN 1888064 A CN1888064 A CN 1888064A
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Abstract
The present invention relates to bioengineering technology, and is especially gene engineering method of obtaining low erucic acid rape variety and its application. The method includes the following steps: 1. cloning Fae1 gene on rape material; 2. constituting RNAi vector with Fae1 gene as target sequence; 3. transforming low erucic acid rape or high erucic acid rape; 4. screening the transformed plants; 5. detecting the composition of fatty acid in the transformed plant seeds; and 6. hybridizing the transformed plant and non-transgenic plant and detecting the composition of fatty acid in the filial generation seed. The present invention can transform high erucic acid rape to obtain low erucic acid character and obtain low erucic acid rape hybrid.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a kind of RNAi of utilization method carrier construction and transform the method that rape obtains dominance low erucic acid proterties; It is used based on this method and produces dominance low erucic acid kind and keep the low erucic acid characteristic of variety seeds not change because of scurrying powder hybridization.
Background technology
Rape is one of main oil crops of China.The Vegetable oil lipoprotein of the national consumption of China has 1/3rd from Semen Brassicae campestris.The quality quality of rapeseed oil is directly connected to the healthy of China its people.
Erucic acid is distinctive lipid acid in the cress seed (22 a carbon monoenoic acid), is the anti-nutrition component in the rapeseed oil.Content of erucic acid is the major objective of rapeseed breeding in the reduction rapeseed oil.Through the effort of recent two decades, China has cultivated large quantities of low erucic acid kinds and popularizing planting aborning.Problem is still very outstanding but content of erucic acid exceeds standard, and middle and lower reach of Yangtze River erucic acid findings of the survey show that China's average content of erucic acid in rape producing region is 16%, the international low erucic acid standard considerably beyond 1%.
Low erucic acid rape variety content of erucic acid in the popularization process rises, even forfeiture low erucic acid characteristic, major cause is that the low erucic acid characteristic of present low erucic acid rape variety belongs to recessive character, in a single day the low erucic acid rape variety has the pollination of high erucic acid pollen when blooming, the content of erucic acid of seed raises immediately.China takes in the rural area management mode of single household, flower arrangement plantation, miscegenation, mixes that to receive phenomenon serious, is easy to cause the production of " two low (low erucic acid, low-sulfur are for glucoside) " rape, and does not have " two low " rape product situation up to standard.Traditional breeding method can't address this problem.
The biosynthesizing of erucic acid (C22:1) prolongs combined enzyme agent catalysis by the oleoyl on the microbody membrane in the cell.Oleoyl prolongs combined enzyme agent by four kinds of enzymes: 3-ketone acyl-CoA synthetase (being called for short KCS), and 3-ketone acyl-CoA reductase enzyme, 3-OH acyl-CoA dehydratase, trans 2,3-alkene acyl-CoA-reductase is formed; 4 successive reactions of difference catalysis: condensation, reduction, dehydration, alkene acyl-coenzyme A reductase.As the first step of reaction, KCS is the key rate-limiting enzyme that carbochain prolongs.Fatty acid prolonging enzyme gene Fae1 (fatty acid prolonging enzyme 1) is responsible for encoded K CS enzyme, and it is synthetic that the expression of blocking-up Fae1 can interrupt erucic acid, obtains the low erucic acid characteristic.
The immunology detection of KCS enzyme is found in the seed development process, the protein of a kind of 57kDa of Fae1 genes encoding, and this protein only is present in the high erucic acid kind.It is synthetic to make the low erucic acid rape recover erucic acid the Fae1 gene importing low erucic acid rape that extracts, and proves that the low erucic acid mutant is the generation because the structure gene of KCS enzyme or regulatory gene are undergone mutation.To import tobacco and yeast from the Fae1 gene of Arabidopis thaliana and all obtain the longer chain fatty acid expression, Fae1 gene overexpression in Arabidopis thaliana obviously raises content of erucic acid, proves that the Fae1 gene is the key gene of control fatty acid prolonging.In Arabidopis thaliana and rape, confirmed that the Fae1 gene is a kind of gene that is subjected to the seed-specific expression of specific promoter regulation and control, nourishing body and the lipid acid in the floral organ tissue that the sudden change of this gene can not influence plant constitute.
In February, 1998, the double-stranded RNA (dsRNA) of the discovery nematodes such as Mello of masschusetts, u.s.a medical college of university can efficiently be blocked the expression of goal gene specifically.This group is called RNA with this phenomenon and disturbs (being called for short RNAi).Subsequently, the RNAi phenomenon is found in the various biotic populations from the microorganism to the mankind, proves a kind of ubiquitous genetic mechanism.
After double-stranded RNA enters cell, under the effect of Dicer enzyme, be cracked into siRNA on the one hand, after self increases under the effect of RdRP, become siRNA by the Dicer enzymatic lysis more on the other hand.The two strands of SiRNA is untied and is become strand, and forms mixture with some albumen, and this mixture is with combining with siRNA complementary mRNA, under the effect of RNA enzyme, make the mRNA cracking on the one hand, on the other hand with SiRNA as primer, be template with mRNA, under the RdRP effect, synthesize the complementary strand of mRNA.MRNA has also become double-stranded RNA as a result, and it also is cracked into siRNA under the effect of Dicer enzyme.These newly-generated siRNA also have the effect of bringing out RNAi, and by this polymerase chain reaction, intracellular siRNA increases greatly, has significantly increased the inhibition to genetic expression.The siRNA of from 21 to 23 Nucleotide can both bring out RNAi to the double-stranded RNA of a hundreds of Nucleotide, but the effect that long double-stranded RNA blocking gene is expressed obviously is better than short double-stranded RNA.Compare with Antisense RNA Technique, siRNA has higher inhibition and renders a service, and suppresses effect and can reach 100%, and positive rate can reach 80-100%.Compare with other several technology of carrying out afunction or reducing sudden change, the RNAi technology has tangible advantage, and it is more effective that it suppresses technology altogether than Antisense RNA Technique and homology, and easier inducing function forfeiture or function reduce.But by combining, can be used for vegetable fatty acid genetically engineered purpose very effectively in the different times of growing or Different Organs inhibition of gene expression selectively with cell specificity promotor and inducible system.
Summary of the invention
Purpose of the present invention just is to overcome the above-mentioned shortcoming and defect that prior art exists, and a kind of method and application thereof that utilizes genetic engineering technique to obtain dominance low erucic acid rape variety is provided; The low erucic acid rape variety low erucic acid characteristic of utilizing this method to obtain can not change because of the scurrying powder hybridization of external high erucic acid pollen, and, this kind all is a low erucic acid as hybrid strain and any incross, makes the possibility of breeding high-yield combination increase greatly.
The object of the present invention is achieved like this:
1, the method for utilizing genetic engineering technique to obtain dominance low erucic acid rape variety comprises the following steps:
(1) is material with the rape, clones its Fae1 gene;
(2) making up with the Fae1 gene is the RNAi carrier (as Fig. 1) of target sequence;
(3) transform high erucic acid or low erucic acid rape variety;
(4) screening transformant (as Fig. 2);
(5) composition (as Fig. 3) of lipid acid in the detection transformant seed;
(6) transformant and non-transgenic plant hybridization, the composition of detection filial generation seed lipid acid.
2, the application of present method comprises the following steps:
(1) be material with the rape, clone its Fae1 gene, making up with the Fae1 gene is the RNAi carrier of target sequence;
(2) utilizing with the Fae1 gene is the RNAi carrier conversion high erucic acid rape acquisition low erucic acid proterties of target sequence;
(3) utilizing with the Fae1 gene is the stable transformed variety of RNAi carrier conversion low erucic acid rape acquisition low erucic acid proterties of target sequence;
(4) be parent's cross-breeding with low erucic acid rape material.
Compared with prior art, the advantage and the effect that have of the present invention is as follows:
(1) the present invention can transform high erucic acid rape and obtain the low erucic acid proterties;
(2) transformed variety of the present invention's acquisition is done the parent and can be obtained the low erucic acid hybrid with any parent (comprising the high erucic acid parent) hybridization;
(3) transformed variety of the present invention's acquisition does not influence the low erucic acid characteristic when foreign pollen scurries powder.
Description of drawings
Fig. 1-FAE1 gene RNAi carrier figure, wherein:
Promoter is a plant promoter;
The target fragment of fae1 for needing to suppress;
Spacer is an intervening sequence;
Terminater is a terminator.
Fig. 2-transformant PCR result detects figure, wherein:
The 1-15 sample is a transfer-gen plant;
0 is the wild plant of contrast;
M is molecular weight Marker.
The result shows: all transfer-gen plant has all successfully imported the RNAi construct.
Fig. 3-transformant seed gas chromatogram;
The result shows: in the transformant seed lipid acid, no erucic acid detects.
Specific embodiments
The present invention intends taking RNAi strategy and agriculture bacillus mediated transgenic method to realize re-set target.Concrete grammar is as follows:
1, Fae1 gene order clone
(1) according to the Fae1 gene order of having delivered at gene pool, (BLASTN CLUSTALX), utilizes computer software PRIMER3 to carry out design of primers on the basis of comparison cress kind Fae1 gene order.
(2) extraction of rape genomic dna
Get the 0.5g young leaflet tablet, add and left standstill 30 minutes after 2ml extracts the damping fluid grinding, centrifugal (10000rpm/min) gets supernatant liquor and adds 600 μ l chloroforms and primary isoamyl alcohol mixed solution (24: 1), the centrifuging and taking supernatant liquor adds 500 μ l Virahols, make the DNA precipitation, centrifugal supernatant discarded, 70% washing with alcohol precipitation adds the dissolving of 100 μ lTE damping fluids (containing the RnaseA enzyme) after the vacuum-drying.
(3) polymerase chain reaction (PCR)
From the rape genomic dna, amplify the homologous sequence of Fae1 gene, promotor, terminator, intron according to Taq polysaccharase specification sheets.The PCR reaction system is: 1 μ l DNA of plants, 2 μ l, 10 x PCR buffer, 1.5 μ l MgCl2,1.3 μ l dNTP (each 2mM), 0.5 μ l F primer (10mM), 0.5 μ l R primer (10mM), 0.2 μ l Tap enzyme (5U/ μ l) add sterilization distilled water to 20 μ l; Increase with PE9700 type amplification instrument, reaction conditions is: circulation of 94 ℃ of pre-sex change 4min; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 1min30s, totally 30 circulations.
(4) Fae1 dna homolog sequence reclaims and purifying
With above-mentioned PCR product gel electrophoresis, utilize gel to reclaim test kit QiAquick Gel Extractionkit the electrophoresis product is reclaimed purifying, be the DNA of Fae1 dna homolog sequence.
2, order-checking
(1) homologous sequence with the Fae1 gene is connected to sequencing vector
The DNA that gel is reclaimed is connected with cloning vector, the ligation system is: 1.5 μ l 10x Fast-LinkLigation buffer, 0.75 μ l 10mM ATP, 1 μ l PGEM-T Vector, 9 μ l reclaim purify DNA, 1 μ l Fast-Link DNA ligase, add sterilization distilled water to 15 μ l, and 16 ℃ connect 1h.
(2) transformed into escherichia coli obtains recombinant clone
Preparation E.coli DH10B electricity transformed competence colibacillus cell.Above-mentioned connection product 1 μ l and competent cell 50 μ l are carried out the electricity conversion on electric exciter.Add 1ml SOC.Get 100 μ l electricity converted product and coat on the LB solid medium that contains Amp and Lac Z screening, be inverted for 37 ℃ and cultivate 12-16h.Select white colony on the substratum, carry out bacteria PCR and screen positive recombinant clone, to selected to the clone carry out enlarged culturing, with the plasmid extraction test kit to mono-clonal culture extracting plasmid.
(3) homologous sequence of Fae1 gene order-checking
With SphI, PstI double digestion plasmid, identify that plasmid has or not chimerism, then check order.
3, conversion carrier makes up
(1) utilizes computer software blastN and clustalX etc. that the sequence that order-checking produces is compared, find out the height of content of erucic acid and the relation of sequence, produce the molecule marker of the content of erucic acid prediction of a cover PCR-based reaction; According to the RNAi principle of design, determine that one group of 500-1500bp dna fragmentation is used to make up the RNAi recombinant vectors;
(2) above-mentioned homologous fragment is connected with relevant necessary assemblies such as promotor, intron, terminators by reverse repetition.It is the double-stranded RNA (as Fig. 1) of target that the transcript of this member will form with Fae1.
4, conversion and tissue regeneration
With the rape is acceptor, utilizes methods such as agrobacterium-mediated transformation and particle bombardment to carry out genetic transformation.
5, screening transformant
Selecting to screen transformant on the substratum.Get blade after waiting to grow to a certain size, with above the method that provided extract genomic dna, carry out PCR screening verification (as Fig. 2) according to the genetically modified crops examination criteria.
6, transformant functional verification
Transformant seed lipid acid is formed, content carries out (as Fig. 3) with vapor-phase chromatography.
The screening content of erucic acid is 0 transformant, selfing or with the high erucic acid mixing breed.Plantation detects filial generation seed lipid acid and forms.
7, application method
(1) the method according to this invention structure is the RNAi carrier of target sequence with the Fae1 gene.
(2) utilize the present invention to transform high erucic acid rape and obtain the low erucic acid proterties.
(3) transformed variety that utilizes the present invention to obtain is done the parent and can be obtained the low erucic acid hybrid with any parent (comprising the high erucic acid parent) hybridization.
(4) the transformed variety foreign pollen that utilizes the present invention to obtain scurries powder does not influence content of erucic acid.
Claims (2)
1, utilizes genetic engineering technique to obtain the method for dominance low erucic acid rape variety, it is characterized in that comprising the following steps:
(1) is material with the rape, clones its Fael gene;
(2) making up with the Fael gene is the RNAi carrier of target sequence;
(3) transform high erucic acid or low erucic acid rape;
(4) screening transformant;
(5) composition of lipid acid in the detection transformant seed;
(6) transformant and non-transgenic plant hybridization, the composition of detection filial generation seed lipid acid.
2, utilize genetic engineering technique to obtain the application of the method for dominance low erucic acid rape variety, it is characterized in that comprising the following steps:
(1) be material with the rape, clone its Fael gene, making up with the Fael gene is the RNAi carrier of target sequence;
(2) utilizing with the Fael gene is the RNAi carrier conversion high erucic acid rape acquisition low erucic acid proterties of target sequence;
(3) utilizing with the Fael gene is the stable transformed variety of RNAi carrier conversion low erucic acid rape acquisition low erucic acid proterties of target sequence;
(4) be parent's cross-breeding with low erucic acid rape material.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101869049A (en) * | 2010-05-11 | 2010-10-27 | 西南大学 | Preparation method of cabbage type rape high erucic acid material |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869049A (en) * | 2010-05-11 | 2010-10-27 | 西南大学 | Preparation method of cabbage type rape high erucic acid material |
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