CN101519660B - Method for improving content of amylose in rice by using RNA interference - Google Patents
Method for improving content of amylose in rice by using RNA interference Download PDFInfo
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Abstract
The invention discloses a method for improving content of amylose in rice by using RNA interference in the technical field of biology. The method comprises: cloning a Q enzyme RBE3 genetic fragment from the rice as an RNA interference fragment; starting an expression by using an endosperm specific promoter; constructing a siRNA expression vector of RBE3 gene; and obtaining transgenic rice interfering the RBE3 gene by using a method of Agrobacterium-mediated transformation. RT-PCR and Southern are hybridized to check the integration and expression conditions of a target gene; an iodine development process for measuring the content of endosperm amylose of transgenic rice strains shows that the average improvement magnitude of the amylose of the transgenic plants is 140 percent compared with non-transgenic strains, and reaches 238 percent to the maximum; and after screening, the transgenic rice strains of which seed amylose is remarkably improved are obtained. The method for improving thecontent of the amylose in the rice lays a foundation for producing the amylose on a large scale by using the transgenic rice.
Description
Technical field
The present invention relates to the method for amylose content in a kind of raising rice paddy seed of biological technical field, relate in particular to a kind of method that improves amylose content in the rice paddy seed of interfering with RNA.
Background technology
Starch is the main storage form of carbohydrate in the higher plant, also is the main composition of food crop product.The storage starch of plant mainly comprises two kinds of compositions: amylose starch and amylopectin.The ratio of amylose starch and amylopectin is determining the purposes of starch, and amylopectin is mainly used in grocery trade, and amylose starch has a purposes widely industrial, relates to more than 30 fields such as food, medical treatment, weaving, papermaking, environmental protection.Amylose starch, the amylose starch function after especially modifying through physics and chemistry is further strengthened, as amylose starch is dissolved, with hydrogen bonded, can form the opaque colloid of rigidity, this characteristic is used for candy industry, can make shape that candy is maintained fixed and complete moulding; Amylose starch also is used for the Drug coating of the excessive oil suction branch of intensifier, fixing agent, fried potato prevention of grocery trade; Utilize the amylose starch substituted polystyrene to produce degradable plastics, this plastics have the packaging industry of being widely used in, agricultural film processing industry and fundamentally solve the potential of white pollution problems.
The synthetic process generation that is amylosynthease via a series of complexity of plant amylum.Think that at present the biosynthesizing of starch relates generally to four fermentoids---ADPG pyrophosphorylase, amylosynthease, Q-enzyme and debranching enzyme enzyme.They are formation, the extension of dextran chain and the formation of branched chain of catalysis ADP-glucose respectively.Amylose starch is directly catalytic by particle mating type amylosynthease I (granule-bound starch synthase I, GBSS I or WAXY); Amylopectin then is Q-enzyme (starch branching enzyme, SBE), Zulkovsky starch synthetic enzyme (soluble starch synthase, SSS) and starch debranching enzyme (debranching enzyme, DBE) coefficient product, wherein SBE isoform RBE3 is considered to play the effect of most critical in forming the amylopectin process.Therefore, utilize the strategy of genetic engineering means modified starch quality to concentrate on the content or the rapport between several enzyme of regulating certain enzyme in the starch building-up process.
In cereal crop research, the breakthrough that obtains high-caliber amylose starch while and not obvious minimizing starch total content is that nineteen fifty-two Vineyard and Bear have found to be positioned at ae (amylose extender) gene on the 5th karyomit(e) of corn.Genetic research shows high amylose starch mainly by the ae Gene Handling, and the synergy of ae gene and modifying factor thereof can make in the W-Gum content of amylose starch bring up to 50-80%.Be positioned at ae gene on the 5th linkage group at present by the SSR linked marker, cloned the ae gene from high starch maize, this gene has 23449bp, and its acceptance in Genebank number is AF072725.These transfer and utilizations for this proterties provide important germ plasm resource and genetic resources, but because this gene is bigger, not easy to operate, and this genetic expression simultaneously follows that other economical character becomes bad, grain yield is low, and application is restricted.In addition, because the heredity of high amylose starch is very complicated, adopt breeding methods such as conventional hybridization, backcross transformation and recurrent selection, it is enough big that required colony wants, and the cycle is long, analytic sample is many, therefore drops into also more.
Genetically engineered changes starch quality and concentrates on GBSS, in the operation of SSS and three kinds of enzymes of SBE, and main use technology:
1, justice and sense-rna transgenic technology, promptly be by importing the just structure or the antisense construct of certain enzyme gene, influence the content or the activity of desmo enzyme, cause the overexpression of goal gene or make its expression by inhibitation system, thereby reach control starch structure.People such as Visser (1991) utilize Antisense RNA Technique, import the gbss gene that oppositely connects in potato, cause gbss gene content and active decline, and then cause that amylose content falls sharply in the potato tuber (reducing 70%-100%).Similarly utilize Antisense RNA Technique, in plants such as cassava (Salehuzzman, 1993), paddy rice, also obtained the transformant of low (or nothing) amylose starch.International monopoly WO9722703A2 has reported and has used the research of Antisense RNA Technique with the full-length cDNA maize transformation of corn sbe2b gene that the result has obtained the higher transgenic corns of amylose content in the seed starch.Yet there is certain limitation in the application of antisense technology, a little less than its inhibition to endogenous gene expression, often produces cambic phenotype, can hinder the accurate judgement to the goal gene function.Its retarding effect genetic stability is relatively poor, can not reliablely and stablely reduce the expression of goal gene.
2, the genetic modification technology of gene knockout technology and DNA/RNA chimeric molecule mediation, but this method once can only be studied a gene, can not knock out multigene family effectively.Some have the gene of keying action in the animals and plants growth course, if adopt gene knockout or sudden change to carry out heritable modification at dna level, then gene silencing prematurely produces the phenotype that causes death, thereby can't further investigate.
Summary of the invention
Technical problem solved by the invention provides the content that is difficult to improve the rice grain amylose starch in the present conventional breeding process of a kind of customer service, improve the content of amylose in rice by the method that adopts RNA to interfere, make it suppress the synthetic expression of key enzyme SBEII b gene in seed endosperm of rice fecula, thereby improve the method for amylose content in the rice paddy seed with the RNA interference technique.
The present invention is achieved by the following technical solutions: a kind of method that improves content of amylose in rice of interfering with RNA, clone RBE3 gene fragment is as the RNA interference fragment from paddy rice, start expression with endosperm specificity promoter, structure contains the positive and negative segmental expression vector of RBE3 gene, with Agrobacterium tumefaciens mediated, the positive antistructure of RBE3 gene is changed in the paddy rice, the integration and the expression of RT-PCR and Southern hybridization check goal gene, transgenic rice plant endosperm amylose content is adopted the iodine determination of color, and the screening back obtains the transgenic rice plant that the seed amylose content significantly improves.
Specifically comprise following key step:
(1) according to the sequence of the synthetic key enzyme RBE3 gene of rice fecula, pass through sequence alignment, determine the specific RNA interference sequence of amylose in rice enzyme gene,, carry out the segmental specific amplification of interference of the synthetic key enzyme RBE3 gene of rice fecula according to interfering the sequences Design primer;
(2) start expression with endosperm specificity promoter, structure contains the positive and negative segmental plant expression vector of RBE3 gene;
(3) transform and to contain purpose and interfere segmental carrier, and interfere fragment to be incorporated in the rice genome purpose;
(4) identify transgenic paddy rice by RT-PCR and Southern hybridization equimolecular detection method;
(5) utilize the iodine development process to detect the seed amylose content, screening obtains the transgenic paddy rice that amylose content improves.
In described step (1), by homology search and sequence alignment, determine that this RNA interference specific fragment is positioned at 66197～66391 positions of rice genome, long 195bp, comprise the 12nd exon (135bp) sequence of RBE3 gene, and the intron sequences of 60bp is arranged.
In described step (2), as endosperm specificity promoter, the positive and negative fragment of RBE3 gene is placed after the specific promoter the specific RBE3 expression of gene that in endosperm, suppresses with high molecular weight glutenin (HMW) promotor.
In described step (4), described Molecular Detection is respectively with the pcr amplified fragment of the Auele Specific Primer of selection markers bar gene and makes probe, adopts Southern hybridization to verify that accurately purpose interference fragment is incorporated in the rice genome and copy number; With the special primer of design RBE3 Gene RT-PCR primer special and paddy rice actin gene, RT-PCR is carried out in transgenic paddy rice and contrast respectively then, adopts sxemiquantitative pcr analysis RBE3 expression of gene difference.
Compared with the prior art, beneficial effect of the present invention is embodied in:
1, the present invention utilizes the RNA interference technique to carry out the rice fecula quality-improving.This is that the RNA interference technique is studied in this field first and used, and also is original creation part of the present invention.The RNA interference technique has high efficiency, specificity, inheritability, characteristics such as simple to operate, and this is that traditional gene knockout technology and Antisense RNA Technique is incomparable.
2, on acceptor selection, the acceptor that traditional gene knockout technology and Antisense RNA Technique will be studied generally is complete gene order, for the indefinite acceptor of gene order, has tangible limitation.And utilize the RNA interference technique, only need know and the gene fragment of receptor homolog that just can acceptor be studied, therefore, research range is comparatively extensive.
3, on working method, what Antisense RNA Technique generally will make up is the big fragment that contains thousands of base pairs, the comparatively difficulty that operates, and the carrier that will make up in the RNA interference technique only is to contain tens small segments to a hundreds of base pair, simple, convenient, be easy to utilization.In addition, a gene can select one or more to interfere fragment, can carry out analysis, the optimization of interference effect to these fragments or combination, to obtain the most effective fragment.
4, on genetic stability, utilize traditional Antisense RNA Technique, because of full gene transformation influences big to acceptor, can cause acceptor to produce bigger variation, and genetic stability is relatively poor, is difficult to obtain good genetically modified crops, but the RNA that adopts this technical transform interferes fragment little, little to other economical character influence of acceptor, the genetic stability height is convenient to obtain goal gene is interfered and the good transfer-gen plant of other economical character.
Embodiment
Below embodiments of the present invention are specified.
1, the segmental acquisition of RBE3 gene specific RNAi
(1), utilize the CTAB method to carry out the extraction of oryza sativa genomic dna.
Get 0.5g paddy rice young leaflet tablet, add liquid nitrogen and pulverize, add 2 * CTAB extract of 2%65 ℃ of insulations of 2mL, mixing, 65 ℃ of insulation 30～60min.Add isopyknic chloroform/primary isoamyl alcohol (24: 1), the light and slow mixing of putting upside down, 10000r/min, centrifugal 5min.Get supernatant, add 65 ℃ CTAB/NaCl solution of 1/10 volume (about 0.2mL), put upside down mixing.With isopyknic chloroform/primary isoamyl alcohol (24: 1) extracting, 10000r/min, centrifugal 5min.Get supernatant, add (just in time) isopyknic CTAB precipitated liquid, put upside down mixing, as precipitate as seen, continue to do down the step, otherwise, 65 ℃ of insulation 30min.4 ℃, 2700r/min, centrifugal 5min.Remove supernatant, with the TE buffer resuspended (0.25～0.5mL) of high salt.(can 65 ℃ insulation 30min, to most of dissolving).The isopropanol precipitating nucleic acid that adds 0.6 volume, abundant mixing, 4 ℃, 10000r/min, centrifugal 15min.Remove supernatant, 80% washing with alcohol precipitation, drying is with the least possible TE buffer resuspended (0.025～0.05mL).
(2), the pcr amplification of RBE3 gene fragment.
With reference to the rice fecula branching enzyme gene RBE3 that delivered (accession number: D16201) and the nucleotide sequence of rice genome, the RBE3 gene fragment that selection is suitable for RNAi is used for the structure of carrier, this fragment is positioned at 66197～66391 positions of rice genome, long 195bp, comprise the 12nd exon (135bp) sequence of RBE3 gene, and the intron sequences of 60bp is arranged.5 ' end at primer has added BamH I and Sal I restriction enzyme site respectively, the design primer: 5 ' end primer is 5 '-GCGGATCCGGGAAGTAGCGATTAACGTGTT-3 ', and 3 ' end primer RBE3i-R is 5 '-GCGTCGACATAGCTTTACCTTTGCCCCTT-3 '.Reaction system (50 μ L) is dNTP (10mM) 1.0 μ L, each 1.0 μ L of primer (10pmol/ μ L), oryza sativa genomic dna template (1 μ g/ μ L) 1.0 μ L, 10 * pfu Buffer (+Mg
2+) 5.0 μ L, pfu DNA polymase (5U/ μ L) 1.0 μ L complement to 50 μ L with the sterilization distilled water.PCR reaction conditions: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 56 ℃ of annealing 45sec, 72 ℃ are extended 30sec, totally 35 circulations; Extend 10min at 72 ℃ at last.
2, the foundation of RBE3 gene siRNA expression carrier
(1), RNAi (2RBE3i) intermediate carrier makes up.
To interfere fragment RBE3i to scale off with the RBE3 that pcr amplification obtains and clones on the pGM-T carrier with BamH I and Sal I enzyme, be connected with the pUCCRNAi carrier of cutting with same enzyme, (RNAi (RBE3i) primers designed 5 ' end primer pUCC1 is 5 '-GGACCGTACTACTCTATTCGTTTC-3 ' with colony polymerase chain reaction (PCR) method preliminary screening recon earlier, 3 ' end primer is RBE3i-R), cut checking with Pst I enzyme again, obtain recombinant plasmid RNAi (RBE3i); Then cut recombinant plasmid RNAi (RBE3i) with Xho I and Bgl II enzyme, BamH I/Sal I endonuclease bamhi with RBE3i carries out ligation again, form the reverse repeating structure that contains intron of about 600bp, (RNAi (2RBE3i) primers designed 5 ' end primer is RBE3i-F with colony polymerase chain reaction (PCR) method preliminary screening recon earlier, 3 ' end primer pUCC2 is 5 '-GAAACGAATAGAGTAGTACGGTCC-3 '), cut the checking recon with Pst I enzyme, be built into cloning vector RNAi (2RBE3i).
(2), the structure of siRNA expression vector p1300 (2RBE3i).
RNAi (2RBE3i) carrier is cut with Pst I and Sal I enzyme, reclaim enzyme and cut the dna fragmentation of about 600bp in the product, be connected with the HMWGUS carrier that Sal I enzyme is cut then with Pst I, cut the checking recon, be built into HMW (2RBE3i) carrier with Pst I and Sal I enzyme.According to the complete sequence design primer of HMWGUS promotor to terminator, 5 ' end at primer adds restriction enzyme site Sac I and Xba I respectively, 5 ' end primer HMW-F is 5 '-CCGGAGCTCGCAAATATGCAACATAATTTCC-3 ', and 3 ' end primer HMW-R is 5 '-CCCTCTAGATGATCTTGAAAGATCTTT-3 '.DNTP (10mM) 1.0 μ L are arranged, each 1.0 μ L of primer (10pmol/ μ L), dna profiling (HMW (2RBE3i) plasmid 1 μ g/ μ L) 1.0 μ L, 10 * pfu Buffer (+Mg in 50 μ L reaction systems
2+) 5.0 μ L, pyrobest DNA polymase (5U/ μ L) 1.0 μ L complement to 50 μ L with the sterilization distilled water.PCR reaction conditions: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 61 ℃ of annealing 45sec, 72 ℃ are extended 3min, totally 35 circulations; Extend 10min at 72 ℃ at last.Obtain the goal gene dna fragmentation of about 2.6kb.This purpose fragment purification is reclaimed and is cloned into pGM-T, the correct back of order-checking scales off with Sac I and Xba I enzyme, carry out ligation with the pCAMBIA1300 that cuts with Sac I and Xba I enzyme, obtain siRNA expression vector p1300 (2RBE3i), use Sac I and Xba I enzyme to cut the checking recon.
Present embodiment utilizes vector construction gene engineering method commonly used, with the forward and reverse potato GA20-oxydase intron 1 fragment both sides that are connected in of RBE3 gene fragment, with the promotor startup of wheat HMW glutenin Glu-1D-1 gene.P1300 (2RBE3i) expression vector that makes up, this expression vector can utilize gene engineering method to improve content of amylose in rice.
3, agrobacterium tumefaciens transforms and obtains candidate's transgenic paddy rice
(1), expression vector imports Agrobacterium.
The single colony inoculation 5ml of picking LBA4404 or EHA105 contains in the YEB liquid nutrient medium of Rifampin 100ug/ml, and 28 ℃, 200r/min concussion were cultivated about 48 hours.Get 2ml bacterium liquid and change 50ml YEB liquid nutrient medium over to, continue to be cultured to about OD600 to 1.0.Change aseptic centrifuge tube over to, ice bath 30min.The centrifugal 5min of 5000r/min removes supernatant.The resuspended thalline of 20mmol/L precooling CaCL2 that adds the new preparation of 2ml.Get the centrifuge tube after the sterilization, every pipe is at packing 200ul on ice.Get p1300 (2RBE3i) plasmid of 2ug, join in the Agrobacterium competence of 200ul.) ice bath 5min, change freezing 1min in the liquid nitrogen over to, then 37 ℃ of water-bath 5min.The YEB liquid culture that adds 800ul, 28 ℃, 200r/min concussion were cultivated about 5 hours.Draw bacterium liquid 300ul and be coated on (Rifampin and the kantlex that contain 100ug/ml) on the YEB solid medium.The picking positive colony carries out bacterium colony PCR and extracts plasmid and identify.
(2), agriculture bacillus mediated rice genetic transforms.
Callus induction is spent 11 ratarias in adopting, and the rataria of getting about pollination 10d is cultivated on inducing culture, about inducing culture 20d; Get the callus that induces and carry out about succeeding transfer culture 30d, subculture medium isogeneous induction substratum; Prepare Agrobacterium and callus then and carry out common cultivation, culture temperature is 21 ℃ altogether, and the 3d solid is substratum altogether: NB minimum medium+2,4-D2.0mg/L+ inositol 2.0g/L+AS 100 μ M/L (liquid is cultivated altogether and do not added 2,4-D and agar powder); Aseptic then washing bacterium changes screening culture medium over to and cultivates 40d:NB minimum medium+2,4-D 2.0mg/L+cef.500mg/L+Hyg 50mg/L (or 20mg/L PPT); Kanamycin-resistant callus tissue illumination cultivation after the picking screening is about 1 month, division culture medium: NB minimum medium+NAA0.25mg/L+6-BA 2mg/L+KT 0.5mg/L+cef.500mg/L+Hyg 50mg/L (or 20mg/L PPT); Picking differentiation seedling carries out root culture, root media: NB minimum medium (N6 reaches sucrose in a large number and reduces by half)+MET 1mg/L+NAA1mg/L.N6 is a large amount of+and MS-Fe salt+B5 is organic+proline 500mg/L+glutamine 500mg/L+ caseinhydrolysate 300mg/L+ sucrose 30g/L+ agar powder 2.6g/L.
Present embodiment adopts agriculture bacillus mediated genetic transformation, utilizes the rataria evoked callus about paddy rice 10d, by cultivating altogether and the PPT screening, has obtained resistant plant.This plant will carry out Molecular Identification and detection, provide material for transgenosis improves content of amylose in rice.
4, the evaluation of transgenic paddy rice
(1), the Molecular Detection of candidate's transgenosis resistant plant
Southern is hybridized detection
Get the spire of transgenosis resistant plant, extract DNA with the CTAB method.Press bar gene order design primer, 5 ' end primer bar-F is 5 '-ATGAGCCCAGAAGACG-3 ', and 3 ' end primer bar-R is 5 '-TCAGATCTCGGTGACGG-3 '.Use the standard reaction system.Amplification condition is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec, 70 ℃ of annealing 30sec, 72 ℃ of extensions 1min, totally 35 circulations; Last 72 ℃ are extended 10min.The plant of PCR tests positive carries out Southern hybridization again and detects.Southern is hybridized the bar gene (550bp) of used probe for carrying out with above-mentioned Auele Specific Primer (bar-F and bar-R) reclaiming behind the pcr amplification.Probe mark, hybridize and wash and the method that all provides such as touch and carry out by Promega company Prime-a-Gene LabelingSystem test kit.Whether occur judging whether to be transgenic paddy rice by Southern hybridization band.
RT-PCR detects candidate's transgenosis resistant plant
Extract the total RNA in wild-type and the transfer-gen plant immature seed (spending 2 weeks of back) respectively, according to the paddy rice Actin1 gene order of having delivered (accession number: AY212324) design primer, 5 ' end primer Act-F is 5 '-CCCTTGTGTGTGACAATGGAACT-3 ', and 3 ' end primer Act-R is 5 '-GACACGGAGCTCGTTGTAGAAGG-3 '; According to paddy rice RBE3 gene order design RT-PCR primer, 5 ' end primer RBE3-F is 5 '-ATGAGTTCGGACATCCTGAATGG-3 ', and 3 ' end primer RBE3-R is 5 '-CATTCCGCTGGAGCATAGACAAC-3 '.The reverse transcription test kit uses the First-Strand cDNA Synthesis Kit of Shanghai Shenergy Biocolor BioScience ﹠ Technology Company.As confidential reference items, the PCR response procedures is 94 ℃ of pre-sex change 2min with the Actin1 gene; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 30s, totally 25 circulations, last 72 ℃ are extended 10min, with the gel imaging quantified system analysis amplified production agarose electrophoresis bands of a spectrum are carried out quantitative analysis.Detect the acquisition transgenic paddy rice through Southern hybridization analysis and RT-PCR.The Molecular Detection of transfer-gen plant.
(2), the genetic analysis of transgenosis strain.
Some seeds of picked at random and contrast and spend 11 seed in the unconverted paddy rice from all paddy rice transgenic lines are sprouted in clear water.After treating that seedling grows to 2-3cm, change clear water, use 20mg/L PPT aqueous solution pouring screening instead, the resistance seedling that filters out is planted in the land for growing field crops, and observes its proterties separation case of check, filters out the isolating strain in 3: 1 of compound mendelian inheritance to be.
5, the amylose content determination of transgenic paddy rice
The endosperm of seed partly is used for the mensuration of amylose content, and the seed of 30 transparency change of every strain picked at random, and be divided into three groups is at random spent also 30 of picked at random and be divided into three groups at random of 11 seed in the unconverted paddy rice of control group.Adopt the iodine development process, the mensuration of amylose starch in the sample: sample was pulverized 60 mesh sieves, used ether defatting, took by weighing about degreasing sample 0.1g (being accurate to 1mg), placed the 50ml volumetric flask.Add 0.5mol/L KOH solution 10ml, in boiling water bath, heat 10min, take out, be settled to 50ml if there is foam to adopt ethanol to eliminate, leave standstill with distilled water.Draw two parts of 2.5ml of sample liquid (being sample determination liquid and blank solution), all adding distil water 30ml transfers to pH about 3.5 with 0.1mol/LHCI solution, adds iodine reagent 0.5ml in the sample, and blank solution does not add iodine reagent, all is settled to 50ml then.Leaving standstill 20min, is contrast with sample blank liquid, uses the 1cm cuvette, records absorbancy separately respectively, and obtains corresponding amylose content by regression equation, averages as the endosperm amylose content of this strain.The result is that the transfer-gen plant amylose content is 140% than the average increase rate of non-transgenic strain, is up to 238%, can obtain the transgenic rice plant that the seed amylose content significantly improves after the screening.
The present invention clones the 195bp specific fragment of Q-enzyme RBE3 gene as the RNA interference fragment from paddy rice, with the high molecular weight glutenin promotor as endosperm specificity promoter, oppositely repeat to be connected among the plant expression vector pCAMBIA1300, made up the siRNA expression vector of RBE3 gene, the callus that utilizes agrobacterium mediation method rice transformation immature embryo to induce has obtained to interfere the transgenic paddy rice of RBE3 gene.After PCR and Southern hybridization evaluation, detection shows that RBE3 expression of gene amount is starkly lower than contrast to its sxemiquantitative RT-PCR.Transgenic rice plant endosperm amylose content iodine determination of color is shown that the transfer-gen plant amylose content is 140% than the average increase rate of non-transgenic strain, is up to 238%.Thereby a kind of method that improves amylose content in the paddy rice is provided, has laid a good foundation for utilizing transgenic paddy rice scale operation amylose starch.
Sequence table
＜110〉Agricultural University Of Anhui
＜120〉interfere the method that improves content of amylose in rice with RNA
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<160>1
<170>PatentIn?version?3.3
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<211>589
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＜213〉presequence
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The RBE3 gene fragment forward sequence of＜221〉being cloned
<222>(1)..(195)
<220>
＜221〉the GA20 intron in the PUCRNAi carrier
<222>(196)..(394)
<220>
The RBE3 gene fragment reverse sequence of＜221〉being cloned
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aaccgttcaa?cccatctagg?gtattatttg?acgcatggct?gcaaaaggaa?aaaaaagggg 180
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agataaacaa?tttgatatag?aggcactata?tatatacata?ttctcgatta?tatatgtaaa 360
tgagttaacc?tttttttcca?cttaaattat?atagtatcga?aatggaaacg?gggaaaaaaa 420
aggaaaacgt?cggtacgcag?tttattatgg?gatctaccca?acttgccaaa?actaccatgt 480
ctatgcgtaa?tgaaagtatc?accaagtgcg?ccggtagtaa?cctacaccct?aagagcggaa 540
aagttgatac?ccttaaccct?tcattccttg?tgcaattagc?gatgaaggg 589
Claims (3)
1. method that interfere to improve content of amylose in rice with RNA, it is characterized in that: clone RBE3 gene fragment is as the RNA interference fragment from paddy rice, start expression with endosperm specificity promoter, structure contains the positive and negative segmental expression vector of RBE3 gene, with Agrobacterium tumefaciens mediated, the positive antistructure of RBE3 gene is changed in the paddy rice, the integration and the expression of RT-PCR and Southern hybridization check goal gene, transgenic rice plant endosperm amylose content is adopted the iodine determination of color, and the screening back obtains the transgenic rice plant that the seed amylose content significantly improves; Comprise following key step:
(1) according to the sequence of the synthetic key enzyme RBE3 gene of rice fecula,, determines that the RBE3 specific RNA of amylose in rice enzyme gene is interfered sequence,, carry out specific amplification according to interfering the sequences Design primer by sequence alignment;
(2) start expression with endosperm specificity promoter, structure contains the RBE3 specific RNA and interferes the positive and negative segmental plant expression vector of sequence;
(3) transform the carrier that contains RBE3 specific RNA interference sequence, and interfere sequence to be incorporated in the rice genome RBE3 specific RNA;
(4) identify transgenic paddy rice by RT-PCR and Southern hybridization equimolecular detection method;
(5) utilize the iodine development process to detect the seed amylose content, screening obtains the transgenic paddy rice that amylose content improves;
In described step (1), by homology search and sequence alignment, determine that this RBE3 specific RNA interference sequence is positioned at 66197～66391 positions of rice genome, long 195bp, comprise the 12nd exon sequence 135bp of RBE3 gene, and the intron sequences of 60bp is arranged.
2. the method that improves content of amylose in rice of interfering with RNA according to claim 1, it is characterized in that: in described step (2), with high molecular weight glutenin HMW promotor as endosperm specificity promoter, interfere the RBE3 specific RNA the positive and negative fragment of sequence to place after the HMW promotor the specific RBE3 expression of gene that in endosperm, suppresses.
3. the method that improves content of amylose in rice of interfering with RNA according to claim 2, it is characterized in that: in described step (4), described Molecular Detection is respectively with the pcr amplified fragment of the Auele Specific Primer of selection markers bar gene and makes probe, adopts Southern hybridization to verify that accurately RBE3 specific RNA interference sequence is incorporated in the rice genome and copy number; With the special primer of design RBE3 Gene RT-PCR primer special and paddy rice actin gene, RT-PCR is carried out in transgenic paddy rice and contrast respectively then, adopts sxemiquantitative pcr analysis RBE3 expression of gene difference.
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| CN102206639B (en) * | 2010-03-30 | 2013-11-20 | 中国科学院上海生命科学研究院 | Gene adjusting starch anabolism of plant seeds and application thereof |
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| CN110387371A (en) * | 2019-07-31 | 2019-10-29 | 上海市农业科学院 | The method for cultivating soft rice using activating transcription factor sample effector nucleic acid zymotechnic |
| CN114740166A (en) * | 2022-03-15 | 2022-07-12 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Stable and convenient sweet potato amylose content determination method |
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| CN1248292A (en) * | 1997-02-21 | 2000-03-22 | 丹尼斯科有限公司 | Antisense intron inhibition of starch branching enzyme expression |
| CN1652681A (en) * | 2002-05-09 | 2005-08-10 | 联邦科技产业研究组织 | Barley with altered branching enzyme activity and starch and starch containing products with an increased amylose content |
| CN1886507A (en) * | 2003-10-27 | 2006-12-27 | 联邦科技产业研究组织 | Rice and products thereof having starch with an increased proportion of amylose |
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