CN1623370A - Method of improving cereal crop seed starch quality using transgene - Google Patents
Method of improving cereal crop seed starch quality using transgene Download PDFInfo
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Abstract
A process for improving the quality of starch in the seeds of agricultural crops by transgenic method includes such steps as configuring the starch branching enzyme gene and the gene RNA interference expression carrier of one or more metabolic enzymes taking part in plant glycolysis and TCA cycle, integrating the interference fragment in plant genome, and specifically suppressing the gene activity of said starch branching enzyme and said metabolic enzymes. It can increase the ratio of straight-chain starch and the content of starch in seed.
Description
Technical field:
The present invention relates to improve the method for cereal crop seed starch quality using, more specifically say so and interfere the method for transgenic technology improvement cereal crop seed starch quality using by RNA.
Background technology:
Cereal crop such as corn are exactly starch as the topmost composition of the raw material of industry, because corn starch processing is not subject to seasonal restrictions, starch quality is better, content of starch reaches 64%-78% in the seed, and it is high and be easy to characteristics such as recovery than potato starch to have comprehensive utilization benefit, so 81.8% of starch market, the world is corn starch, the starch of U.S.'s starch processing industry 95% is corn starch.Usually said corn starch is the mixture of amylose (amylose) and amylopectin (amylopectin).At present, the corn variety contents of starch mostly is about 60% of seed dry weight both at home and abroad, and wherein amylose is about 22%, and amylopectin is about 78%.And what be used widely is amylose industrial, relates to more than 30 fields such as food, medical treatment, weaving, papermaking, environmental protection.Amylose, the amylose function after especially modifying through physics and chemistry is further strengthened, as amylose is dissolved, combine with hydrogen bond, 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 also is used for the seed coating medicine of the excessive oil suction branch of intensifier, fixative, chips prevention of grocery trade; Utilize the amylose substituted polystyrene to produce degradation plastic, this plastics have the packaging industry of being widely used in, agricultural film processing industry and fundamentally solve the potential of white pollution problems.
In cereal crop research, the breakthrough that obtains high-caliber amylose 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 chromosome of corn.Genetic research shows high amylose starches mainly by the ae Gene Handling, and the synergy of ae gene and modifier gene thereof can make in the corn starch content of amylose 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 gene 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 starches 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.
Gene engineering changes starch quality and concentrates on GBSS, in the operation of SSS and three kinds of enzymes of SBE, and main operation technique:
1, justice and antisense 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 desmoenzyme, cause the overexpression of genes of interest 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 tubers (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.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 genes of interest function.Its depression effect genetic stability is relatively poor, can not reliablely and stablely reduce the expression of genes of interest.
2, the gene conversion 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 key effect 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:
The present invention is for avoiding above-mentioned existing in prior technology weak point, provides that a kind of simple and effective, genetic stability are good, the method for specificity and workable improving cereal crop seed starch quality using transgene.
The technical scheme that technical solution problem of the present invention is adopted is:
The characteristics of the inventive method are that the gene RNA that makes up one or more metabolic enzymes of starch branch enzyme gene and involved in plant glycolysis and TCA circulation is respectively interfered expression vector, interfere purpose fragment to be incorporated in the Plant Genome by agriculture bacillus mediated or particle gun conversion, suppress the gene activity of one or more metabolic enzymes of plant amylum branching enzyme and involved in plant glycolysis and TCA circulation specifically, thereby improve the ratio and the total contents of starch of seed of amylose in the crop seed starch.
Compared with the prior art, advantage applies of the present invention exists:
1, the present invention utilizes the RNA interference technique to carry out the improvement of cereal crop starch quality.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, hereditability, 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 genetic fragment of receptor homolog that just can acceptor be studied, therefore, research range is comparatively extensive.
3, on method of operating, 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 fragments 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 genetic transformation influences big to acceptor; usually 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 genes of interest is interfered and the good transfer-gen plant of other economical character.
Description of drawings:
Fig. 1 is a pcr amplified fragment electrophoresis detection collection of illustrative plates.
Among the figure, the M:DNA standard molecular weight; The specific fragment that 1-6 is respectively is former 92,52106, prosperous 72 from the corn variety Shandong, increase the genomic DNA of Zheng 58,543, connection 87.
Fig. 2 cuts purifying figure behind checking and the BamH I, Sal I double digestion for pUCCRNAi plasmid Pst I enzyme.
Among the figure, the M:DNA standard molecular weight; 1-2:pUCCRNAi plasmid Pst I enzyme is cut checking; The fragment of 3-4:PCR product B amHI, Sal I double digestion purifying; The fragment of 5-6:pUCCRNAi plasmid BamH I, Sal I double digestion purifying.
Fig. 3 cuts the checking electrophoresis pattern for RNAi+1F plasmid Pst I enzyme.
Among the figure, M
1: Marker (λ DNA/Hind III), M
2: DL2000 DNA Marker, swimming lane 1 is contrast pUCCRNAi/pstI product; 2,3,4,5,6 and 7 is (RNAi+1F)/pstI product, wherein the 1F specific fragment that is that Shandong behind the purifying is former 92,52106, prosperous 72, increases in the genomic DNA of Zheng 58,543, connection 87
Fig. 4 cuts the checking electrophoresis pattern for .RNAi+2F plasmid Pst I enzyme.
Specific embodiments:
Operating procedure:
A, sequence according to one or more metabolic enzyme genes of crop starch branch enzyme gene or involved in plant glycolysis and TCA circulation, by sequence alignment, determine that the specific RNA of starch branch enzyme gene in the crop interferes sequence to be: the specific RNA of one or more metabolic enzyme genes of ttcatgacatctgat caccagtata tttcccggaa acatgaggag gataaggtga ttgtgttcga aaagggagat ttggtatttg tgttcaacttccactgcaac aacagctatt ttgactaccg tattggttgt cgaaagcctg gggtgtataa and involved in plant glycolysis and TCA circulation interferes sequence to be: tcaatgtgg ccgtcatggt tggtggattccccaggaagg agggaatgga aaggaaagat gttatgtcga aaaatgtttc aatctacaaa tcccaagcat ctgcccttgaagcccatgca gctcccaact gcaaggttct ggtggttgcc aacccagcaa acaccaatgc tcttatcttg aaagaatttgctccatctat tccagag;
B, according to the sequences Design primer of target gene fragment, carry out the specific amplification that purpose is interfered fragment;
C, structure RNA interfere expression vector, comprise the forward and reverse plant introne two ends that are connected in of purpose interference fragment, and its upstream connects endosperm specificity promoter, downstream connection termination;
D, conversion contain the carrier that purpose is interfered fragment, and interfere fragment to be incorporated in the Plant Genome purpose;
E, by screening of selected marker or reporter gene and Molecular Detection, the transfer-gen plant of the seed starch quality that is improved.
In step a, by autoploidy search and sequence alignment, the specific RNA sequence of determining corn starch branching enzyme gene is the 13rd exon sequence of corn sbe2b gene, i.e. the 2086-2240 fragment of total length sbe2b gene, 155bp altogether.
The gene of the metabolic enzyme in concrete the enforcement is a NAD-malate dehydrogenase gene.
The concrete operations example:
1, according to the sequence of one or more metabolic enzyme NAD-malate dehydrogenase genes of crop starch branch enzyme gene and involved in plant glycolysis and TCA circulation, carry out sequence alignment by Clustal W software, determine that the specific RNA of starch branch enzyme gene and NAD-malate dehydrogenase gene in the crop is interfered sequence.
For example: by autoploidy search and sequence alignment, determine that it is the 13rd exon sequence of sbe2b gene that specific RNA is interfered sequence, be the 2086-2240 fragment of total length sbe2b gene (Genebank Accession Number:AF072725), be total to 155bp, its sequence is as follows:
ttcat gacatctgat caccagtata?tttcccggaa acatgaggag gataaggtga
ttgtgttcga aaagggagat?ttggtatttg tgttcaactt ccactgcaac
aacagctatt ttgactaccg?tattggttgt cgaaagcctg gggtgtataa
seq1 -------------------TTCATGACATCTGATCACCAGTATATTTCCCGGAAACATGA 41
seq2 CCTTGAGGGAAAATATGAATTCATGACATCTGATCACTCATATGTATCACGGAAGCATGA 2160
****************** ***?*?**?*****?*****
seq1 GGAGGATAAGGTGATTGTGTTCGAAAAGGGAGATTTGGTATTTGTGTTCAACTTCCACTG 101
seq2 GGAGGATAAGGTGATCATCTTTGAGAGAGGAGATTTGGTCTTCGTGTTCAACTTCCACTG 2220
*************** *?**?**?* ***********?**?*****************
seq1 CAACAACAGCTATTTTGACTACCGTATTGGTTGTCGAAAGCCTGGGGTGTATAA------ 155
seq2 GAGCAATAGCTATTTTGACTATCGCGTTGGTTGTTTCAAGCCTGGGAAGTACAAGATCGT 2280
*?***?**************?** ******** ********* ***?**
seq1：sbe2b?gene?DNA?sequence?2086-2240?fragment?(155bp)
seq2：sbe2a?mRNA，partial?cds.ACCESSION?NUMBER：U65948
seq1 -------------TTCATGACATC--TGATCACCAGTATATTTCCCGGAAACATGAGGAG 45
seq3 GAAGCTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGGACCATACGGGGAG 660
*?* *?** * *** ** *?***?* *?*?*?****
seq1 GATAAGGTGATTGTGTTCGAAAAGGGAGATTTGGTATTTGTGTTCAACTTCCACTGCAAC 105
seq3 GACGTCGTGTTCGTCTGCAACGACTGGCACACCGGCCCTCTCTCGTGCTACCTCAAGAGC 720
** ***?*?**?*?*?* * * * * *?*?* **?**?* *?*
seq1 AACAGCTATTTTGACTACCGTATTGGTTGTCGAAAGCCTGGGGTGTATAA---------- 155
seq3 AACTACCAGTCCCACGGCATCTACAGGGACGCAAAGACCGCTTTCTGCATCCACAACATC 780
*** *?*?* ** * * ****?*?* *?* *
seq1：sbe2b?gene?DNA?sequence?2086-2240?fragment(155bp)
seq3：Waxy(maize)mRNA；ACCESSION?NUMBER：M24258
2, interfere fragments sequence according to selected genes of interest RNA, utilization Primer Premier 5.0 softwares design primer carries out the specific amplification that purpose is interfered fragment.
Adopt the T of primer design design of primers program to primer
mParameters such as value, the hairpin structure that may form, dimer and primer mispairing evaluate and optimize.
For example: increase specifically and separate the 13rd exon sequence of corn sbe2b gene, it is the 2086-2240 fragment of total length sbe2b gene, define strict amplification condition, make design of primers want faithful to original sequence, to take into account the carrying out of follow-up test simultaneously, in amplification procedure, not change to guarantee this 155bp sequence.When the design primer, we have added BamH I and Sal I point respectively at 5 ' end of primer, adopt hand-designed, and utilize the design of primers program to carry out assistant analysis.The primer sequence of design is as follows:
5’PRIMER?Sequence： 5′-GCGGATCCTTCATGACATCTGATCACC-3′
3’PRIMER?Sequence： 5′-GCGTCGACTTATACACCCCAGGCTTTC-3′
Because target DNA segment GC content is higher, and the Tm value of primer is also higher, purpose fragment to be amplified is shorter, and is so amplification procedure adopts two-step method PCR, as follows behind its reaction condition optimization: (1) 94 ℃ of pre-sex change, 5 minutes, (2) 35 circulations, each circulation two-step reaction: 94 ℃ 1 minute, 62 ℃ 1 minute, (3) 72 ℃ were extended (4) 4 ℃ of insulations 5 minutes.
3, make up RNA and interfere expression vector, comprise the forward and reverse plant introne two ends that are connected in of purpose interference fragment, its upstream connects endosperm specificity promoter, downstream connection termination.
For example: different cultivars corn starch branching enzyme SBEIIb gene RNA is interfered the structure of expression vector HMW+2F.
(1) acquisition of purpose fragment
For the purpose RNA that obtains each experimental cultivar more specifically interferes fragment, be template with each experimental cultivar genomic DNA respectively, carry out pcr amplification by the response procedures after optimizing.After pcr amplification finishes, carry out 1.4% agarose gel electrophoresis and take pictures, the result obtains a specific fragment about 150bp, and master tape is obvious, and is identical with the expection clip size, as shown in Figure 1.
(2) purpose fragment and interference vector is connected
A, double digestion pUCCRNAi plasmid and PCR product
After the pUCCRNAi plasmid is cut checking with the PstI enzyme, itself and PCR product are used BamHI, SalI double digestion respectively, cut glue recovery, purifying then, electrophoresis detection is taken pictures as shown in Figure 2.
The structure of b, RNAi+1F and checking thereof
As shown in Figure 3, cut the product electrophoresis result, cut control plasmid pUCCRNAi with PstI from enzyme, produce a 250bp bright band, cut recon RNAi+1F plasmid with PstI, but produced the bright band of a treaty 400bp, show that purpose segment (about 155bp) correctly is connected among the plasmid pUCCRNAi.
The structure of c, RNAi+2F and checking thereof
As shown in Figure 4, cut the product electrophoresis result from enzyme, cut control plasmid pUCCRNAi with PstI, produce a 250bp bright band, cut recon RNAi+1F plasmid with PstI, but produced the bright band of a treaty 550bp, shown that forward and reverse purpose segment (about 155bp) correctly is connected among the plasmid pUCCRNAi.Show successfully forward and reverse purpose fragment is connected on the pUCCRNAi carrier, for the later structure of expression vector lays the foundation.(wherein among the swimming lane 1-6 2F be respectively that Shandong behind the purifying is former 92,52106, prosperous 72, forward and reverse fragment of the aim sequence of Zheng 58,543, connection 87.)
(3) HMW+2F interferes the structure of expression vector
The RNAi+2F carrier restriction enzyme site collection of illustrative plates of analyzing the HMW.GUS carrier and having built, carry out PstI, SalI double digestion respectively, and in contrast with its SalI single endonuclease digestion, then electrophoresis, cut glue, purifying and reclaim target fragment HMW (after removing GUS) and 2F fragment, then carry out the enzyme of the two coupled reaction, conversion, screening and recon and cut evaluation.
4, will contain purpose by methods such as agriculture bacillus mediated converted in-situ or particle guns interferes the plasmid that has built of fragment to be transformed in the Plant Genome.
5, by selected marker or reporter gene screening and Molecular Detection, the transfer-gen plant of the seed starch quality that is improved.Utilize agriculture bacillus mediated converted in-situ that the RNA interference expression vector of above-mentioned structure is transformed former 92 corn inbred lines in Shandong, utilize hygromycin (55mg/L) (T in the present age
1Generation) 15000 seeds carry out resistance screening, and its resistance rate is 8.6 ‰.Albuminous cell and blastocyte PCR positive rate are respectively 5.3 ‰ and 3.6 ‰.
6, the RNA interference effect of transgenosis seed is analyzed.Analyze by the seed amylose content to the albuminous cell PCR positive, its amylose content accounts for the ratio of total starch content between 35.26%～50.60%, and mean value is 41.54%, and the ratio of contrast amylose content is 22.46%.This shows that this method can significantly improve amylose content of corn seeds, has improved the corn starch quality.
In the present embodiment, the NAD-malate dehydrogenase gene RNA of involved in plant glycolysis and TCA circulation interferes the structure of expression vector (to comprise the comparison of specific RNA interference sequence, design primer and purpose are interfered the specific amplification of fragment), containing purpose interferes the plasmid of fragment to be integrated in Plant Genome, the NAD-malate dehydrogenase gene activity that suppresses involved in plant glycolysis and TCA circulation specifically, by selected marker or reporter gene screening and Molecular Detection, the concrete steps that obtain a series of processes such as transfer-gen plant of improvement seed starch quality all interfere step identical with the RNA of starch branch enzyme gene, and the main distinction only is that both interference sequences there are differences.Analyze by the RNA interference effect to the transgenosis seed, the result shows that transgenosis strain seed total starch content contrast (acceptor) improves 6.2%, and therefore, this method can significantly improve the total contents of starch of crop kernel.
Claims (4)
1, the method for improving cereal crop seed starch quality using transgene, the gene RNA that it is characterized in that making up respectively one or more metabolic enzymes of starch branch enzyme gene and involved in plant glycolysis and TCA circulation is interfered expression vector, interfere purpose fragment to be incorporated in the Plant Genome by agriculture bacillus mediated or particle gun conversion, suppress the gene activity of one or more metabolic enzymes of plant amylum branching enzyme and involved in plant glycolysis and TCA circulation specifically, thereby improve the ratio and the total contents of starch of seed of amylose in the crop seed starch.
2, according to the described method of claim 1, it is characterized in that operating as follows:
A, sequence according to one or more metabolic enzyme genes of crop starch branch enzyme gene or involved in plant glycolysis and TCA circulation, by sequence alignment, determine that the specific RNA of starch branch enzyme gene in the crop interferes sequence to be: the specific RNA of one or more metabolic enzyme genes of ttcatgacatctgat caccagtata tttcccggaa acatgaggag gataaggtga ttgtgttcga aaagggagat ttggtatttg tgttcaacttccactgcaac aacagctatt ttgactaccg tattggttgt cgaaagcctg gggtgtataa and involved in plant glycolysis and TCA circulation interferes sequence to be: tcaatgtgg ccgtcatggt tggtggattccccaggaagg agggaatgga aaggaaagat gttatgtcga aaaatgtttc aatctacaaa tcccaagcat ctgcccttgaagcccatgca gctcccaact gcaaggttct ggtggttgcc aacccagcaa acaccaatgc tcttatcttg aaagaatttgctccatctat tccagag;
B, according to the sequences Design primer of target gene fragment, carry out the specific amplification that purpose is interfered fragment;
C, structure RNA interfere expression vector, comprise the forward and reverse plant introne two ends that are connected in of purpose interference fragment, and its upstream connects endosperm specificity promoter, downstream connection termination;
D, conversion contain the carrier that purpose is interfered fragment, and interfere fragment to be incorporated in the Plant Genome purpose;
E, by screening of selected marker or reporter gene and Molecular Detection, the transfer-gen plant of the seed starch quality that is improved.
3, method according to claim 2, it is characterized in that in described step a, by autoploidy search and sequence alignment, the specific RNA interference sequence of determining corn starch branching enzyme gene is the 13rd exon sequence of corn sbe2b gene, be the 2086-2240 fragment of total length sbe2b gene, be total to 155bp.
4, method according to claim 1 and 2, the gene that it is characterized in that described metabolic enzyme are NAD-malate dehydrogenase gene.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101029314B (en) * | 2007-02-02 | 2010-12-01 | 吉林农业大学 | Corn starch branching enzyme gene sirna expression carrier |
| CN101213943B (en) * | 2006-05-16 | 2011-04-13 | 布鲁诺布二世有限公司 | Hybrid corn plant and seed |
| CN102174565A (en) * | 2010-11-12 | 2011-09-07 | 安徽农业大学 | Method for fast genetic transformation of rice |
| CN102533851A (en) * | 2012-01-31 | 2012-07-04 | 安徽农业大学 | Corn gene normal position transformation method mediated by high throughput agrobacteria |
| CN114807163A (en) * | 2022-03-28 | 2022-07-29 | 河南农业大学 | Application of TaSBE I gene in promoting wheat starch synthesis |
| CN116064567A (en) * | 2022-07-26 | 2023-05-05 | 济南大学 | Corn small kernel mutant and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2239979C (en) * | 1995-12-20 | 2007-09-25 | E.I. Du Pont De Nemours And Company | Novel starches via modification of expression of starch biosynthetic enzyme genes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101213943B (en) * | 2006-05-16 | 2011-04-13 | 布鲁诺布二世有限公司 | Hybrid corn plant and seed |
| CN101029314B (en) * | 2007-02-02 | 2010-12-01 | 吉林农业大学 | Corn starch branching enzyme gene sirna expression carrier |
| CN102174565A (en) * | 2010-11-12 | 2011-09-07 | 安徽农业大学 | Method for fast genetic transformation of rice |
| CN102533851A (en) * | 2012-01-31 | 2012-07-04 | 安徽农业大学 | Corn gene normal position transformation method mediated by high throughput agrobacteria |
| CN114807163A (en) * | 2022-03-28 | 2022-07-29 | 河南农业大学 | Application of TaSBE I gene in promoting wheat starch synthesis |
| CN114807163B (en) * | 2022-03-28 | 2023-08-25 | 河南农业大学 | Application of TaSBE I Gene in Promoting Wheat Starch Synthesis |
| CN116064567A (en) * | 2022-07-26 | 2023-05-05 | 济南大学 | Corn small kernel mutant and application thereof |
| CN116064567B (en) * | 2022-07-26 | 2024-05-17 | 济南大学 | Corn small kernel mutant and application thereof |
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