CN100339481C - Method in increasing oleic acid content of soybean and peanut seed by applying gene silent technology - Google Patents
Method in increasing oleic acid content of soybean and peanut seed by applying gene silent technology Download PDFInfo
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- CN100339481C CN100339481C CNB2005100166328A CN200510016632A CN100339481C CN 100339481 C CN100339481 C CN 100339481C CN B2005100166328 A CNB2005100166328 A CN B2005100166328A CN 200510016632 A CN200510016632 A CN 200510016632A CN 100339481 C CN100339481 C CN 100339481C
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Abstract
The present invention belongs to the plant breeding field, particularly to the biology engineering breeding field to improve soybean qualities and earthnut qualities. The present invention using a PCR method clones specific gene fragments of seeds from soybean and peanut gene groups and constructs the gene fragments to a reverse repeating sequence structure according to the gene silence principle; the reverse repeating sequence gene fragments of the specific gene of the soybean and the peanut seeds are constructed to a carrier containing a promoter and a terminator containing the specific expression of the seeds; the genes are transferred and tissues are cultivated and selected by the method of agrobacterium-mediated transformation; the expression of target genes, and the contents of oleic acid are detected by the methods of PCR and Southern blot; the soybean and the peanut materials with the stable high contents of the oleic acid are determined by the selection of a system to improve the soybean qualities and the earthnut qualities.
Description
Technical field
The invention belongs to field of plant breeding, particularly the biotechnology breeding field of soybean and peanut quality improvement.Application double-stranded RNA gene silent technology is reconstructed the seed Δ 12 fatty acid dehydrogenase gene FAD2-1 of soybean and peanut, by transgenic technology the fatty acid content of soybean and peanut seed is carried out the specificity genetic improvement, and then high soybean and the peanut material of seed selection oleic acid content.
Background technology
The double-stranded RNA gene silencing is meant has double-stranded RNA to participate in instructing in vivo, serves as the target of degrading with external source and endogenous mRNA, the genetic phenomenon that causes specific gene not express.The double-stranded RNA gene silent technology is that present controlling gene is expressed effective and the most advanced technology, is applied in the research field of plant genetic engineering widely.Δ 12 fatty acid dehydrogenases are that catalysis oleic acid forms linoleic key enzyme in the fatty acid metabolism, the gene silencing principle of utilizing the RNA inverted repeats to cause is carried out genetic modification to soybean and peanut seed Δ 12 fatty acid dehydrogenase gene FAD2-1, cause this gene silencing, Δ 12 fatty acid dehydrogenases are formed be obstructed and the catalytic activity reduction, thereby reduce linoleic synthesizing, accumulate a large amount of oleic acid.Oleic acid content is about 20% in the general soybean seeds grease, and peanut is 40-50%.Oleic acid is a kind of monounsaturated fatty acids (18: 1), and stability is high, can reduce total cholesterol and blood fat in the human body, reduces deleterious low density lipoprotein cholesterol (LDC), but keeps the level of useful high density lipoprotein cholesterol (HDL).Benefit health owing to oleic acid is stable, improve soybean and the oleic content of peanut seed, become the important content of present soybean and peanut quality improvement to improve its nutritive value and oxidative stability.
The double-stranded RNA gene silencing is meant has double-stranded RNA to participate in instructing in vivo, serves as the target of degrading with external source and endogenous mRNA, causes specific gene can not express or reduce the genetic phenomenon of expression.Since the importance of double-stranded RNA in gene silencing was found, many in the world laboratories were used for plant gene function research and plant quality improvement research with the double-stranded RNA gene engineering.This technology is that the genetic expression on the most effective present rna level suppresses means, and its gene inhibition effect is far above traditional antisense antisense gene silent technology.Hair fastener type double-stranded RNA of the present invention (has the part intron, hpRNA-intron) the gene silencing structure is most effective in the gene silencing type of being studied at present, can reach 100%, and the reticent efficient that just sense and antisense antisense suppress is 10% and 15%, the reticent efficient that does not have the hair fastener type doubly-linked RNA structure (hpRNA-loop) of intron is 69%, and these results are confirmed (Smith et al.2001 Nature 407:319) by a lot of experiment.
High oleic acid soybean: the high oleic acid soybean varieties of the DUPOND company of the U.S. (G94-1, G94-19, and G168), applied for a patent in 1996, obtained drugs approved by FDA and can come into the market in 1997 by commercialization.The goal gene of the high oleic acid soybean material that they obtained and source suppress (sense) for the justice of the gmFad2-1 gene of coded delta-12 oleic acid dehydrogenase.Adopt the design of double-stranded RNA gene silencing and make up the activity suppress soybean oil acidohydrogenase (Δ-12 oleic acid dehydrogenase) gmFAD2-1 with us, so synthetic with to accumulate a large amount of oleic principle of work different fully.
Summary of the invention
The objective of the invention is to use the gene silent technology that double-stranded RNA causes, the special conversion carrier of design and structure soybean and peanut seed Δ 12 fatty acid dehydrogenase gene FAD2-1, pass through genetic engineering means, the silence that causes this gene, promote oleic accumulation in the seed, obtain high oleic soybean and peanut material.By clone and reconstruction to soybean and peanut seed Δ 12 fatty acid dehydrogenase gene FAD2-1, make up the inverted repeats structure of FAD2-1, and the promotor and the terminator of application FAD2-1 seed specific expression, its specific genes vector construction can cause the FAD2-1 gene silencing, and then reaches synthetic a large amount of oleic purposes.Promptly the purpose of this invention is to provide a kind of method that the double-stranded RNA gene silent technology improves soybean and peanut seed oleic acid content of using.
1. application PCR method, the Δ 12 fatty acid dehydrogenase gene fragments of clone's seed specific from soybean and peanut genome, and this gene fragment is built into the inverted repeats structure according to double-stranded RNA gene silencing principle;
2. the inverted repeats gene fragment of the Δ 12 fatty acid dehydrogenase gene FAD2-1 that soybean and peanut seed is special is building up in the carrier of the promotor that contains seed-specific expression and terminator;
3. carry out transgenosis by the agrobacterium mediation converted method, tissue culture and screening operation, the strain system of the high oleic acid content of screening in its transgenic progeny; Adopt PCR and Southern blot method testing goal gene, detect oleic acid content,, establish stable soybean of high oleic acid content and peanut material by systematic breeding with gas-chromatography (GC).
The present invention can shorten the cycle, raises the efficiency, and increases substantially oleic acid content, and this will promote the soybean of China and the research and development of peanut quality improvement breeding.Soybean and peanut seed Δ 12 fatty acid dehydrogenase gene inverted repeats structure Design and structures, can improve the expression efficiency of goal gene, by the soybean of foundation and the high oleic acid elite clone of genetic transformation system seed selection of peanut, for further high oleic acid soybean of seed selection and peanut varieties provide the basis.
Embodiment
Improving the oleic content of peanut seed with applying gene silent technology is example:
1. material therefor
The peanut material: peanut varieties is richly to spend No. 1, standing grain to spend No. 1 and 8130.
Bacterial strain and plasmid: intestinal bacteria E.coli-DH5 α, Agrobacterium LBA4404; Cloning vector pGEM-Teasy, pBI121 and pGLe-10.
Enzyme and chemical reagent: Plant Genome is extracted test kit; Plasmid extraction kit, DNA restriction enzyme, T4DNA ligase enzyme, T4DNA polysaccharase, CIAP calf intestines alkaline phosphatase enzyme, pfu polysaccharase and DNA ladder; Microbiotic (kantlex etc.) and other biochemical reagents.
2. technological method
The clone of FAD2-1 gene segment and expression vector establishment
The total DNA of preparation carries out polymerase chain reaction (PCR) as template from the peanut material.With reference to two groups of PCR primers of the design of the peanut FAD2-1 gene (AF272950) among the GeneBank, primer is given birth to worker company by Shanghai and is synthesized.
The 1st group of primer sequence is:
Primer1：GAGCTGGAGGGCGTGTCACTAAGAT；
Primer2：CAAGTACAAGGGCCATCCTAGTGTG
The 2nd group of primer sequence is:
Primer1：AGGGCGTGTCACTAAGATTGAAGCT；
Primer2：AATGGGTATGGAAGCTTGTGGAAAT
The PCR reaction system comprises 10 μ l PCR buffer, 10 μ l dNTP (each 2.5nmol/L), and 2 μ l Tag enzymes, 2 μ l primers (each 20nmol/ μ l), 2 μ l template DNAs add sterilization distilled water to 100 μ l.Gene-amplificative instrament is the PerinElmer9600 type, and the PCR time-program(me) is: 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, 59 ℃ of 1min, 72 ℃ of 90s, totally 35 circulations.
These two groups of PCR products (fragment 1 and fragment 2) are connected into the pGEM-Teasy carrier respectively, carry out dna sequence analysis then.Sequence is carried out homology relatively among sequencing result and the Genebank.
2 usefulness EcoRI downcut from pGEM-Teasy with fragment, mend with the T4DNA polysaccharase on the SacII site of the pGEM-Teasy that is reversely connected to fragment 1 place after flat (flush end connection method), formed the inverted repeats (homing sequence of the sequence of fragment 2 and fragment 1 is identical) of FAD2-1 gene fragment.
Utilize the soybean agglutinin gene promoter of seed specific expression and the structure that terminator carries out plant expression vector.The pLecSma (including the Smal restriction enzyme site) that produces with carrier pBI121 and pGle-10 reorganization is as kalamycin resistance and have the lectin promotor of soybean seeds specifically expressing and the binary vector of terminator.The FAD2-1 inverted repeats that builds is discharged from pGEM-Teasy with Ncol and Pstl, pfu polysaccharase flush endization, with after the Smal linearizing and the dephosphorylized carrier pLecSma of CIAP be connected, construct expression vector pPNHO-1 (see figure 1).
The clone of soybean FAD2-1 gene fragment is identical with peanut with construction of carrier, and the expression vector of structure is the pSBHO-1 (see figure 2).
The conversion of peanut and plant regeneration:
Peanut conversion and tissue culture are with reference to Kiran Sharma ﹠amp; Vanamala Anjaiah (Plant Science 159 (2000) 7-19) method is carried out.Promptly binary vector pPNHO-1 is imported agrobacterium tumefaciens lba4404 by tri-parent conjugation method, with this agroinfection peanut cotyledon explant, cultivated altogether under the illumination three days, go to the bud inducing culture (20uMBA+10uM2 that contains the 250mg/L cephamycin then, 4-D+MS) going up induced bundle sprouts, the bud of will growing thickly after two weeks goes to the bud inducing culture (20uMBA+10uM2 that contains 125mg/L kantlex and 250mg/L cephamycin, 4-D+MS) enterprising row filter, two Zhou Houzai go on the stem elongation medium (2uMBA+MS) that contains 125mg/L kantlex and 250mg/L cephamycin, about 12 weeks of this process, when treating that bud grows to 3-4 centimetre high, change no any antibiotic root media (5uMNAA+MS) over to, cultivation through 4 weeks, transformed plant is taken root and is grown up to complete plantlet, through hardening, transplant to soil and grow up and blossom and bear fruit.Described bud inducing culture based formulas is 20uMBA+10uM2,4-D+MS, and the stem elongation medium is 2uMBA+MS, root media is 5uMNAA+MS.
The evaluation of transgenosis peanut and the mensuration of oleic acid content:
PCR detects: extract the total DNA of regeneration plant, as template, carry out the PCR reaction in order to NPTII and soybean lectin gene 5 ' and 3 ' end parts sequences Design primer.
NPTII Gene Partial aligning primer:
Primer1:5-TGAGAATATCACCGGAATTG-3; Primer2:5-GGAAGAACAGTATGTCGAGCTA-3; Soybean lectin promoter gene partial sequence primer:
Primer1：5-CATGTGACAGATCGAAGGAA-3；Primer2：5-ATCTAATTATTCTATTCAGAC-3。
Southern hybridization: the total DNA of regeneration plant (10ug) carries out Southern hybridization behind the complete enzymolysis of HindIII.Dna probe is the full sequence of soybean lectin promoter gene, carries out mark by TaKaRa company random primer labelling test kit specification sheets.
T1 and T2 measure for the seed oil acid content: milling process extracts the total grease of seed, detects oleic acid content with vapor-phase chromatography after the esterification.Detected by test center of Northeast Normal University and Australian CSIRO plant research.
3. result
Utilize agrobacterium mediation method that the inverted repeats of ahFAD2-1 gene is imported the peanut genome, spend No. 1, standing grain to spend on No. 1 and 8130 and succeed in that three kinds are rich, obtain strain more than 100 and transformed the peanut seedling, had good repeatability, set up peanut transformation system efficiently.
The PCR of transgenosis peanut detects: adopt marker gene NPTII, goal gene promotor (soybean lectin promotor) gene order design primer carries out PCR and detects.The PCR detected result shows, detects in 88 strains that in the plant 65 strains to be arranged be the PCR positive, amplified the band with marker gene and promoter gene sequence fragment same molecular amount, and adjoining tree do not amplify respective strap.
The Southern hybridization analysis of transgenosis peanut: with of the DNA hybridization of goal gene promotor one soybean lectin promoter gene sequence as probe and transgenic regenerated plant, in the middle of the PCR positive plant, the transgenosis verity of most of plant confirms with the Southern hybridization analysis, contains one to multiple hybrid belt.
T1 measures for the seed oil acid content: milling process extracts the total grease of seed, detects oleic acid content with gas-chromatography, Megabore chromatography column after the esterification.2004 and 2005 are measured through Institute of Analysis of Northeast Normal University and Australian CSIRO plant research, transgenosis peanut T1 for single-strain seed under the constant condition of total fat content, adjoining tree oleic acid content average out to 40%, 4061 (tentatively titled) average out to 77% is spent in transgenosis peanut east, and its oleic acid content average specific contrast has improved 37 percentage points.
Description of drawings
Accompanying drawing 1 is for being used to transform the carrier mode chart of peanut
Accompanying drawing 2 is for being used for the carrier mode chart of soybean transformation
Accompanying drawing 3 is a gene silencing type efficient synoptic diagram
Claims (4)
1, a kind of applying gene silent technology improves the method for the oleic acid content of peanut seed, it is characterized in that:
(1). use PCR method, from the peanut genome, clone the Δ 12 fatty acid dehydrogenase gene fragments of seed specific, and this gene fragment is built into the inverted repeats structure according to double-stranded RNA gene silencing principle,
The clone of FAD2-1 gene segment and expression vector establishment: the total DNA of preparation from the peanut material, carry out polymerase chain reaction as template, F272950 designs two groups of PCR primers with peanut FAD2-1 gene A:
The 1st group of primer sequence is:
Primer1：GAGCTGGAGGGCGTGTCACTAAGAT；
Primer2：CAAGTACAAGGGCCATCCTAGTGTG
The 2nd group of primer sequence is:
Primer1：AGGGCGTGTCACTAAGATTGAAGCT；
Primer2：AATGGGTATGGAAGCTTGTGGAAAT
The PCR reaction system comprises 10 μ l PCR buffer, 2.5nmol/L dNTP10 μ l, 2 μ l Tag enzymes, the primer 2 μ l of 20nmol/ μ l, 2 μ l template DNAs, add sterilization distilled water to 100 μ l, gene-amplificative instrament is PerinElmer 9600 types, and the PCR time-program(me) is: 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, 59 ℃ of 1min, 72 ℃ of 90s, totally 35 circulations;
With this two groups of PCR product fragments 1 and fragment 2, be connected into the pGEM-Teasy carrier respectively, carry out dna sequence analysis then, sequence is carried out homology relatively among sequencing result and the Genebank;
2 usefulness EcoRI downcut from pGEM-Teasy with fragment, mend with the T4DNA polysaccharase on the SacII site of the pGEM-Teasy that is reversely connected to fragment 1 place after flat, have formed the inverted repeats of FAD2-1 gene fragment;
(2). the inverted repeats gene fragment of the Δ 12 fatty acid dehydrogenase gene FAD2-1 that peanut seed is special is building up in the carrier of the promotor that contains seed-specific expression and terminator;
(3). carry out transgenosis by the agrobacterium mediation converted method, tissue culture and screening operation, the strain system of the high oleic acid content of screening in its transgenic progeny,
The conversion of peanut and plant regeneration: binary vector pPNHO-1 is imported agrobacterium tumefaciens by tri-parent conjugation method, with this agroinfection peanut cotyledon explant, cultivated altogether under the illumination about three days, going on the bud inducing culture that contains the 250mg/L cephamycin induced bundle then sprouts, the bud of will growing thickly after two weeks goes to the enterprising row filter of bud inducing culture that contains 125mg/L kantlex and 250mg/L cephamycin, two Zhou Houzai go on the stem elongation medium that contains 125mg/L kantlex and 250mg/L cephamycin, about 12 weeks of this process, when treating that bud grows to 3-4 centimetre high, change no any antibiotic root media over to, cultivation through about 4 weeks, transformed plant is taken root and is grown up to complete plantlet, transplants then to soil through hardening;
(4). adopt PCR and Southern blot method testing goal gene, detect oleic acid content,, establish the stable peanut material of high oleic acid content by systematic breeding.
2, improve the method for the oleic acid content of peanut seed according to the applying gene silent technology of claim 2, it is characterized in that: described bud inducing culture based formulas is 20uMBA+10uM2,4-D+MS, the stem elongation medium is 2uMBA+MS, root media is 5uMNAA+MS.
3, improve the method for the oleic acid content of peanut seed according to the applying gene silent technology of claim 2, it is characterized in that: described peanut varieties is richly to spend No. 1, standing grain to spend No. 1 and 8130.
4, improve the method for the oleic acid content of peanut seed according to the applying gene silent technology of claim 2, it is characterized in that: described agrobacterium tumefaciens bacterial classification is LBA4404.
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| CN101210242B (en) * | 2006-12-29 | 2011-07-20 | 河南农业大学 | Highly effective expression method for peanut delta12 fatty acid dehydrogenase gene |
| CN101736030B (en) * | 2010-01-08 | 2012-05-23 | 河南科技大学 | Acyl-acyl carrier protein thioesterase gene RNAi carrier in poplar and application thereof |
| CN101914534B (en) * | 2010-05-05 | 2012-12-19 | 东北农业大学 | Seedling and seed specific expression GmPLPA gene promoter for soybean and application thereof |
| AR085892A1 (en) * | 2011-01-14 | 2013-11-06 | Univ Missouri | METHOD FOR DEVELOPING SOYA WITH A HIGH CONTENT OF OIL ACID USING CONVENTIONAL DEVELOPMENT TECHNIQUES |
| CN105246324B (en) * | 2013-03-15 | 2018-08-28 | 塞尔克蒂斯股份有限公司 | Soybean oil component is improved by targeting knock out FAD2-1A/1B genes |
| US10113162B2 (en) | 2013-03-15 | 2018-10-30 | Cellectis | Modifying soybean oil composition through targeted knockout of the FAD2-1A/1B genes |
| EP3410843A1 (en) | 2016-02-02 | 2018-12-12 | Cellectis | Modifying soybean oil composition through targeted knockout of the fad3a/b/c genes |
| CN107217070A (en) * | 2017-05-22 | 2017-09-29 | 广东省农业科学院作物研究所 | One kind is based on TALENs gene editing peanut breeding methods |
| CN108456679B (en) * | 2018-02-03 | 2021-07-30 | 吉林省农业科学院 | High-oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof |
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