CN1958799A - Expression vector of duplicate inverted waxy gene, preparation method, and application - Google Patents
Expression vector of duplicate inverted waxy gene, preparation method, and application Download PDFInfo
- Publication number
- CN1958799A CN1958799A CN 200510110049 CN200510110049A CN1958799A CN 1958799 A CN1958799 A CN 1958799A CN 200510110049 CN200510110049 CN 200510110049 CN 200510110049 A CN200510110049 A CN 200510110049A CN 1958799 A CN1958799 A CN 1958799A
- Authority
- CN
- China
- Prior art keywords
- gene
- waxy gene
- expression vector
- waxy
- promotor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
This invention discloses a method for preparing an expression vector containing double antisense waxy genes and its application. The structure and characteristics of the expression vector are shown in figure (d). The expression vector comprises promoter sequence, antisense DNA fragment of the target gene, terminator sequence, and endonuclease-sensitive sequence or marker gene sequence. All the sequences are doubled along the same direction. The expression vector can be used for breeding Gramineae plants such as rice without resistance-screening gene and with low amylase content.
Description
Technical field
The invention belongs to the genetically engineered field, relate more specifically to plant genetic engineering field.
Background technology
In recent years, along with the raising of living standards of the people and the developing of foreign trade, rice food flavor quality causes people's attention day by day, the focus in the research of the rice quality of rice food flavor quality-improving research having become nowadays.According to the literature, in influencing rice food flavor quality, rice grain amylose content is a most important index (Li Xiulan etc., Chinese agronomy circular, 2002,18 (3): 61-64,119; Zhang Xiaoming etc., rice in China science, 2002,16 (2): 157-161; Luo Zhixiang etc., Chinese agronomy circular, 2002,18 (6): 18-21).The rice that amylose content is high more, food flavor are poor more; The rice bloated property of cooking of low amylose content is little, and food flavor is better relatively.
The research (Xuan Wenmin, agricultural and technology, 2002 (3): 67-69,74) of the soft rice of a kind of novel rice----that is nowadays rising in the world.Rice matter circle of soft rice is between glutinous property and viscosity.The maximum characteristics of natural soft rice are that rice grain amylose content is lower, are generally less than 10% (Zhao Zesheng etc., Chinese extraordinary rice, Shanghai science tech publishing house, 1995:49; Song Wenchang etc., extraordinary rice growing and utilization, Science Press, 1997:11-12), so soft rice is the raw material of making snack food product such as rice dumpling.
Amylose starch in the rice be by (GBSS) waxy gene (Waxy) catalysis synthetic of coding " being incorporated into the amylosynthease on the starch small grain " (Okagaki etc., Genetics, 1988,120:1137-1143).Currently reported importing waxy gene (Waxy) the antisense fragment that shows can effectively reduce rice grain amylose content (Shimada H, et al, Theor ApplGenet, 1993,86 (6): 665-672; Terada R, et al, Plant Cell Physiol, 2000,41 (7): 881-888; Wang Xinqi etc., Shanghai Agricultural journal, 2002,18 (supplementary issue): 69-73; Chen Xiuhua etc., Science Bulletin, 2002,47 (9): 684-688; Liu Qiaoquan etc., Transgenic Res, 2003,12:71-82; Li Jianyue etc., Science Bulletin, 2004,49 (24): 2556-2561; Shen Gezhi etc., Mol.Biol., 2004,30 (6): 637-643).
Although it is existing more about utilizing antisense waxy gene to reduce the report of rice grain amylose content, but analyze these existing reports, in all transgenic progeny, it is all lower that rice grain amylose content is lower than the ratio that 10% offspring or rice grain amylose content obviously reduce the offspring.This is because the conversion carrier of people's use at present all is that a waxy gene antisense fragment is structured on the T-DNA section (Fig. 1), utilization contains the transgenic progeny of the Agrobacterium tumefaciens mediated rice transformation acquisition of these carriers, and the offspring that single often copy is integrated is more than the progeny size of multi-copy integration.
Li Jianyue etc. studies show that, adopt the p13W4 carrier to transform to obtain plant T1 that two copies integrate for seed brown rice amylose content average out to 6.8%, to T4 generation the isozygoty rice grain amylose content of transgenic progeny still can be stabilized in less than in 10% the scope.
On the other hand, along with the continuous appearance of commercialization plant transgene kind, the safety issue of Genetic engineering food more and more causes people's attention.The Genetic engineering food safety issue mainly produces in the transgenic plant owing to resistance selectable marker genes such as antibiotics resistant or herbicide-resistant retain in, so, the expression vector that acquisition does not have the resistance selectable marker gene carries out transgeneic procedure, and is very important for applying of genetically modified crops.Adopt the cotransformation method to address this problem.Currently reportedly will contain resistance selectable marker gene and gus reporter gene carrier respectively and only contain the agrobacterium tumefaciens cotransformation paddy rice of a antisense waxy gene carrier (p13W8), can cultivate the selectable marker-free gene and the transgenic progeny (Li Jianyue etc. of rice grain amylose content comparison photograph reduction, Science Bulletin, 2004,49 (24): 2556-2561).
Yet, utilize Agrobacterium tumefaciens mediated cotransformation method to cultivate transgenic progeny, the likelihood ratio that obtains two copy transfer-gen plants is less, even if obtain the transfer-gen plant that two copies are integrated, because two copies are incorporated into the different loci of rice genome, it is also relatively more difficult screen the transgenic progeny that two T-DNA integration sites all isozygoty.
So the extraordinary paddy rice that rice grain amylose content can obviously reduce in order more effectively to utilize the cotransformation method that antisense waxy gene is imported rice cultivating selectable marker-free gene need be structured in one and can make the easier expression vector that obtains containing duplicate inverted waxy gene of transformant.
Summary of the invention
Technical problem to be solved by this invention provides a kind of expression vector and method for making and purposes of duplicate inverted waxy gene, contain the resistance selectable marker gene or contain the promotor that comes from virus to overcome existing expression vector, be unfavorable for the security control of genetically engineered plants; And only containing a antisense waxy gene, the transformant of acquisition and offspring's related gene expression thereof reduce significantly, be difficult to obtain the defective of the transformant that two T-DNA integration sites all isozygoty.
Technical scheme
First aspect of the present invention provides a kind of expression vector of double inverted defined gene, comprises following sequence:
(1) promotor;
(2) the some or all of antisense DNA fragment of target gene;
(3) terminator;
(4) responsive sequence of restriction endonuclease or marker gene sequence;
Wherein, sequence (1), (2) and (3) are double.
The expression vector of above-mentioned double inverted defined gene, the order of double sequence wherein are (1), (2), (3) and (1), (2), (3) that same direction is arranged.
A kind of special case of the expression vector of duplicate inverted waxy gene provided by the invention is that said target gene is the waxy gene of coding amylosynthease.
The another kind of preferred version of the expression vector of duplicate inverted waxy gene provided by the invention is, said promotor is the promotor of paddy rice waxy gene, and terminator is a CaMV 35S gene terminator.
Another preferred version of the expression vector of duplicate inverted waxy gene provided by the invention is to also have intron in the promotor downstream.
Do not have the resistance selectable marker gene in the expression vector of duplicate inverted waxy gene of the present invention,, do not contain the 35S promoter that comes from cauliflower mosaic virus yet as antibiotics resistant or herbicide-resistant gene.
Second aspect of the present invention provides the preparation of expression vectors method of duplicate inverted waxy gene, comprises the steps:
(1) adopt the PCR method amplification to obtain waxy gene promotor and waxy gene antisense DNA fragment;
(2) the PCR product that will contain waxy gene promotor and waxy gene antisense DNA is connected with other carrier sequences with terminator, obtains intermediate carrier;
(3) designing a pair of primer, with terminator downstream and the pairing of waxy gene promotor upstream, is that template increases with the intermediate carrier respectively;
(4) pcr amplification product is inserted between the waxy gene promotor upstream of intermediate carrier and the T-DNA right margin and be built into the duplicate inverted waxy gene carrier.
A kind of special case of the preparation of expression vectors method of above-mentioned duplicate inverted waxy gene is to also have the first intron fragment between waxy gene promotor that the said employing PCR method amplification of step (1) obtains and the waxy gene antisense DNA fragment.
The another kind of special case of the preparation of expression vectors method of above-mentioned duplicate inverted waxy gene is that the said pcr amplification product of step (4) inserts the waxy gene promotor upstream of intermediate carrier and the restriction endonuclease sites in the multiple clone site between the T-DNA right margin.This restriction enzyme site can be KpnI.
A kind of special case of the preparation of expression vectors method of above-mentioned duplicate inverted waxy gene is, the PCR product that said step (2) will contain waxy gene promotor and first intron and waxy gene antisense DNA is connected this step with terminator with other carrier sequences be to realize by resistant gene and the promotor thereof that substitutes in the carrier with the PCR product.
The 3rd aspect of the present invention provides the application of the expression vector of duplicate inverted waxy gene, comprises with the transforming gramineous plant of said expression vector; Also comprise with said expression vector rice transformation, wheat, barley, corn, Chinese sorghum or highland barley.
A kind of special projects of the application of the expression vector of above-mentioned duplicate inverted waxy gene is, with the expression vector rice transformation acquisition low amylose starch paddy rice of duplicate inverted waxy gene.
The expression vector of duplicate inverted waxy gene of the present invention transforms rice grain amylose content and is about the new variety that 15% paddy rice obtains, and the amylose content of its rice is less than 10%.
Double target gene provided by the invention is the expression vector of the waxy gene part antisense DNA of coding amylosynthease, be a kind of special case, said target gene can also be the functional gene in synthesizing of storage protein, plant hormone, resistance protein, heat shock protein or amylopectin or the mediation process.
Beneficial effect
(1) prior art reduces amylose content of rice, all is to utilize antisense waxy gene p13W4 or two kinds single part antisense waxy gene carrier of p13W8 (Fig. 1).The expression vector of duplicate inverted waxy gene provided by the invention, have the complete promotor of two copies of equidirectional arrangement, the part antisense DNA fragment and the terminator (Fig. 3) of target gene, can reduce the expression of the amylose starch of rice more effectively than single part of antisense paraffin paper genophore.
(2) two kinds of carriers of p13W4 or p13W8 all are that a waxy gene antisense fragment is structured on the T-DNA section, the rice that obtains behind the rice transformation, in its all transgenic progeny, the offspring that single copy is integrated is more than the progeny size of multi-copy integration, it is all lower to cause offspring's rice grain amylose content to be lower than the ratio of 10% scope, but keeps a high proportion of low amylose starch rice among the duplicate inverted waxy gene render transgenic offspring that the present invention makes up on a T-DNA section.
(3) utilizing the cotransformation method to contain resistance selectable marker gene carrier respectively and only to contain antisense waxy gene carrier (p13W8 at present, Fig. 1 b) rice transformation, can cultivate the amylose content comparison according to the transgenic progeny that reduces, but the likelihood ratio that obtains two copy transfer-gen plants is less, even if obtain the transfer-gen plant that two copies are integrated, because two copies are incorporated into the different loci of rice genome, it is also relatively more difficult screen the transgenic progeny that two T-DNA integration sites all isozygoty.Duplicate inverted waxy gene carrier provided by the invention can obtain the transfer-gen plant that duplicate inverted waxy gene is integrated effectively, and duplicate inverted waxy gene is incorporated into the same karyomit(e) of rice genome, and is close linkage, is convenient to screen homozygote.
(4) expression vector of duplicate inverted waxy gene provided by the invention does not have resistance selectable marker genes such as antibiotics resistant or herbicide-resistant, in breeding process, avoided the puzzlement of Genetic engineering food safety issue, very important for applying of genetically modified crops.
Term used herein " waxy gene (Waxy) " is meant coding " be incorporated into the amylosynthease on the starch small grain " (GBSS) gene.
Term used herein " PCR method " is meant the gene amplification method of polymerase chain reaction.
Term used herein " CaMV 35S gene terminator " is meant the 35S terminator that comes from cauliflower mosaic virus.
Term used herein " intermediate carrier " is meant the carrier that obtains in the destination carrier preparation process.
Term used herein " T-DNA " is meant in the agrobacterium tumefaciens Ti-plasmids genome transferable and be incorporated into the specific DNA fragments that host plant cell karyomit(e) gets on, that is a kind of transposable element.
Description of drawings
Fig. 1 is two kinds and can be used in the carrier p13W4 (a) of reduction rice grain amylose content and the structure iron in p13W8 (b) T-DNA district.Among the figure, 232Wx-pro, 232INT.l: the promotor of paddy rice 232Waxy gene and first intron; Wx frag: the antisense DNA fragment (756bp) of paddy rice 232Waxy gene; The border, the left and right sides of LB, RB:T-DNA; Nos-ter:Nos gene terminator; 35S-pro, 35S-ter:CaMV 35S gene promoter and terminator; Hpt: hygromycin gene; Gus: beta-glucuronic acid Glycosylase gene; Xh:XhoI; E:EcoRI; Sa:SacI; B:BamHI; X:XbaI; S:SalI; P:PstI; H:HindIII.
Fig. 2 is the structure iron in single part of antisense waxy gene intermediate carrier p13AW-1 (c) the T-DNA district of non-resistant mark.Among the figure, Wx-pro, Int.l: the promotor of paddy rice Waxy gene and first intron; Wx frag: the antisense DNA fragment of paddy rice Waxy gene; The border, the left and right sides of LB, RB:T-DNA; Terminator: gene terminator; Xh:XhoI; E:EcoRI; B:BamHI; K:kpnI; P:PstI.
Fig. 3 is the structure iron in duplicate inverted waxy gene carrier p13AW-2 (d) the T-DNA district of non-resistant mark.Among the figure, Wx-pro, Int.l: the promotor of paddy rice Waxy gene and first intron; Wx frag: the antisense DNA fragment of paddy rice Waxy gene; The border, the left and right sides of LB, RB:T-DNA; Terminator: gene terminator.
Fig. 4 is the structure iron in pCAMBIA1300 plasmid T-DNA district.Among the figure, the border, the left and right sides of LB, RB:T-DNA; 35S-pro, 35S-ter:CaMV 35S gene promoter and terminator; Hpt: hygromycin gene; Xh:XhoI; Bs:BstXI; E:EcoRI; Sa:SacI; K:KpnI; S:SmaI; B:BamHI; X:XbaI; Sal:SalI; P:PstI; H:HindIII.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.After having read above-mentioned teachings of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
Present embodiment is to form for the structure of the method that the expression vector that makes up the double inverted defined gene is described and the carrier that obtains.
Method main reference " molecular cloning experiment guide " (Sa nurse Brooker J etc., molecular cloning experiment guide (second edition), Science Press, 1992) such as make up conventional pcr amplification, the digestion with restriction enzyme that relates in the operation of this expression vector and be connected.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, or the condition of advising according to manufacturer.
From the fine rice genome of disclosed Japan, search one and be approximately the dna sequence dna that does not have SmaI and XhoI restriction enzyme site in the 100bp nucleotide sequence in BamHI restriction enzyme site two ends length, and according to this sequences Design primer, the primer two ends respectively carry SmaI restriction enzyme site and XhoI restriction enzyme site.Utilize conventional PCR this sequence that from the fine rice total dna of Japan, increases, the PCR product is connected with the T carrier obtains the T-1 carrier, and the transformed competence colibacillus intestinal bacteria.Utilize ordinary methods such as the molecular biology enzyme is cut, PCR to detect the T-1 carrier.
From publication number is that the CN1298021A patent specification is searched 232 indica type paddy rice waxy gene promotor nucleotide sequences, before about 2.6kb place, distance translation starting point ATG upstream and translation starting point ATG, design primer with reference to this sequence respectively, respectively carry SmaI restriction enzyme site and BamHI restriction enzyme site at the primer two ends of design.Utilize conventional PCR from 9311 indica type rice total dnas, to increase and obtain the about 2.6kb waxy gene promotor and first intron, the PCR product is connected acquisition T-2 carrier with the T carrier, and the transformed competence colibacillus intestinal bacteria.Utilize ordinary methods such as the molecular biology enzyme is cut, PCR to detect the T-2 carrier equally.
Be that the CN1298021A patent specification is searched 232 indica type paddy rice waxy gene nucleotide sequences from publication number again, respectively design one couple of PCR primers with reference to this sequence at the 2333rd bit base to 3087 bit base two ends, introduce XhoI and downstream primer introducing BamHI restriction enzyme site at upstream primer simultaneously.Utilize conventional PCR from total DNA of 9311 indica type paddy rice, to increase and obtain about 0.750kb part waxy gene fragment, the PCR product is connected acquisition T-3 carrier, also transformed competence colibacillus intestinal bacteria with the T carrier.Utilize ordinary methods such as the molecular biology enzyme is cut, PCR to detect the T-3 carrier equally.
Extract T-1 and T-2 carrier respectively from intestinal bacteria, utilize two kinds of digestion with restriction enzyme T-1 of SmaI and BamHI and T-2 carrier, the small segment after big fragment behind the electrophoresis recovery T-1 carrier double digestion and T-2 carrier enzyme are cut utilizes T
4Ligase enzyme is finished connection procedure, obtains the T-4 carrier.
With BamHI and XhoI T-3 and T-4 carrier are carried out double digestion, electrophoresis reclaims the small segment of T-3 and the big fragment of T-4, the big or small fragment of these two recovery is connected obtain T-5 carrier, transformed competence colibacillus intestinal bacteria.Utilize SmaI and XhoI that the T-5 carrier is carried out double digestion again, electrophoresis reclaims and contains waxy gene promotor, first intron and the segmental large fragment DNA of waxy gene part antisense.
Utilize Bst XI enzymic digestion pCAMBIA1300 plasmid (Fig. 4 comes from Chinese Academy of Sciences's Shanghai plant physiology and the Wang Zongyang researcher of ecological Studies institute) back to adopt T
4Archaeal dna polymerase is mended flat sticky end, adopts the linear pCAMBIA1300 DNA after XhoI enzymic digestion benefit is put down again, and agarose gel electrophoresis has three dna fragmentations, reclaims maximum fragment.The big fragment of this fragment with as above T-5 carrier S maI and the recovery of XhoI double digestion electrophoresis is connected, forms intermediate carrier p13AW-1 (Fig. 2 c).
According to the pCAMBIA1300 sequence, terminator downstream and waxy gene promotor upstream at p13AW-1, each designs a primer, two primers are all introduced the KpnI restriction enzyme site, with the p13AW-1 intermediate carrier is that template increases, the PCR product is connected acquisition T-6 carrier, also transformed competence colibacillus intestinal bacteria with the T carrier.Utilize ordinary methods such as the molecular biology enzyme is cut, PCR, order-checking to detect the T-6 carrier equally.With KpnI respectively enzyme cut T-6 and p13AWY-1 carrier, the big fragment that the T-6 electrophoresis is reclaimed is inserted p13AW-1 and is gone up KpnI site in the multiple clone site of waxy gene promotor upstream, is built into p13AW-2 carrier (Fig. 3 d).As above the waxy gene antisense fragment length in two kinds of expression vectors is approximately 0.75kb, and the promotor and the first introne DNA length that are positioned at before the waxy gene antisense fragment are about 2.6kb.
Embodiment 2
Present embodiment is for the expression vector rice transformation of the duplicate inverted waxy gene that how to utilize structure is described.
The agrobacterium tumefaciens cultivation that relates in cultivating transgenic paddy rice, rice conversion and resistant calli screening and plant regeneration mainly carry out (Liu Qiaoquan etc., plant physiology journal, 1998,24 (3): 259-271) with reference to the method for Liu Qiaoquan.
Utilize Agrobacterium tumefaciens mediated cotransformation method (Li Jianyue etc., Science Bulletin, 2004,49 (24): 2556-2561) the p13AW-2 expression vector import is gone up Normal University No. 2 or rice grain amylose contents such as super 2-10 are approximately 15% rice strain or kind, can obtain the selectable marker-free gene, can be at area, the middle and lower reach of Yangtze River novel germplasm plantation, low amylose content.
Claims (10)
1. the expression vector of a double inverted defined gene comprises following sequence:
(1) promotor;
(2) the some or all of antisense DNA fragment of target gene;
(3) terminator;
(4) responsive sequence of restriction endonuclease or marker gene sequence;
It is characterized in that sequence (1), (2) and (3) are double.
2. the expression vector of duplicate inverted waxy gene according to claim 1 is characterized in that, the order of said double sequence is (1), (2), (3) and (1), (2), (3) that same direction is arranged.
3. the expression vector of duplicate inverted waxy gene according to claim 1 and 2 is characterized in that, said target gene is the waxy gene of coding amylosynthease.
4. the expression vector of duplicate inverted waxy gene according to claim 1 and 2 is characterized in that, said promotor is the promotor of paddy rice waxy gene, and terminator is a CaMV 35S gene terminator.
5. the expression vector of duplicate inverted waxy gene according to claim 1 and 2 is characterized in that, also has intron in the promotor downstream.
6. the preparation of expression vectors method of a duplicate inverted waxy gene comprises the steps:
(1) adopt the PCR method amplification to obtain waxy gene promotor and waxy gene antisense DNA fragment;
(2) the PCR product that will contain waxy gene promotor and waxy gene antisense DNA is connected with other carrier sequences with terminator, obtains intermediate carrier;
(3) designing a pair of primer, with terminator downstream and the pairing of waxy gene promotor upstream, is that template increases with the intermediate carrier respectively;
(4) pcr amplification product is inserted between the waxy gene promotor upstream of intermediate carrier and the T-DNA right margin and be built into the duplicate inverted waxy gene carrier.
7. the preparation of expression vectors method of duplicate inverted waxy gene according to claim 6, it is characterized in that the said PCR product that will contain waxy gene promotor and waxy gene antisense DNA is connected this step with terminator with other carrier sequences be to realize by resistant gene and the promotor thereof that substitutes in the carrier with the PCR product.
8. the application of the expression vector of a duplicate inverted waxy gene comprises with the transforming gramineous plant of said expression vector.
9. the application of the expression vector of duplicate inverted waxy gene according to claim 8 comprises with said expression vector rice transformation, wheat, barley, corn, Chinese sorghum or highland barley.
10. the application of the expression vector of duplicate inverted waxy gene according to claim 9 is characterized in that, the indica type or easier obviously new variety of reduction of rice grain amylose content of obtaining of Japonica rice with the expression vector conversion of duplicate inverted waxy gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101100493A CN100489103C (en) | 2005-11-04 | 2005-11-04 | Expression vector of duplicate inverted waxy gene, preparation method, and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101100493A CN100489103C (en) | 2005-11-04 | 2005-11-04 | Expression vector of duplicate inverted waxy gene, preparation method, and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1958799A true CN1958799A (en) | 2007-05-09 |
| CN100489103C CN100489103C (en) | 2009-05-20 |
Family
ID=38070655
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005101100493A Expired - Fee Related CN100489103C (en) | 2005-11-04 | 2005-11-04 | Expression vector of duplicate inverted waxy gene, preparation method, and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100489103C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667306A (en) * | 2012-08-31 | 2014-03-26 | 天津农学院 | Sorghum wax synthesis regulation gene and application in Arabidopsis plant |
| CN107604087A (en) * | 2017-08-09 | 2018-01-19 | 四川农业大学 | The method for detecting wheat Wx B1 variations |
| CN110964743A (en) * | 2019-12-30 | 2020-04-07 | 浙江大学 | Method for editing and creating rice amylose content variation by using promoter |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100516226C (en) * | 2003-06-02 | 2009-07-22 | 上海师范大学 | Method for producing rice seed with protein concentration improvement |
-
2005
- 2005-11-04 CN CNB2005101100493A patent/CN100489103C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667306A (en) * | 2012-08-31 | 2014-03-26 | 天津农学院 | Sorghum wax synthesis regulation gene and application in Arabidopsis plant |
| CN103667306B (en) * | 2012-08-31 | 2016-12-21 | 天津农学院 | The application in Arabidopsis plant of one Sorghum vulgare Pers. waxiness synthesis regulation gene |
| CN107604087A (en) * | 2017-08-09 | 2018-01-19 | 四川农业大学 | The method for detecting wheat Wx B1 variations |
| CN110964743A (en) * | 2019-12-30 | 2020-04-07 | 浙江大学 | Method for editing and creating rice amylose content variation by using promoter |
| CN110964743B (en) * | 2019-12-30 | 2021-07-20 | 浙江大学 | Method for editing and creating rice amylose content variation by using promoter |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100489103C (en) | 2009-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103382468B (en) | Site-directed modification method of rice genome | |
| CN101993906B (en) | Transgenic plant of expressing CIVPS or intein modified proteins and preparation method thereof | |
| CN103554238B (en) | Plant starch synthesis-related protein FLO6 and encoding gene and applications thereof | |
| CN1333834A (en) | Vectors for transforming plants | |
| Luo et al. | Editing of the starch branching enzyme gene SBE2 generates high-amylose storage roots in cassava | |
| CN103865862A (en) | Bacillus licheniformis strain loaded with serine acetyl transferase gene as well as building method and application thereof | |
| CN101063139A (en) | Seed specificity highly effective promoter and its application | |
| CN103981216A (en) | Backbone plasmid vector and application thereof | |
| CN100489103C (en) | Expression vector of duplicate inverted waxy gene, preparation method, and application | |
| CN1193047A (en) | C3 plants expressing photosynthetic enzyme of C4 plants | |
| CN107254475B (en) | A method of based on ZmMIKC2a Gene regulation Seed Starch Content in Maize | |
| CN1292826A (en) | Transgenic crops accumulating fructose polymers and methods for their production | |
| CN101429520A (en) | Epiphyte genome conformity plasmid pUNFIN without selection mark, construction method and uses thereof | |
| CN1782074A (en) | Method for amplifying rice T-DNA insertion site flanking sequence | |
| CN104788549A (en) | Rice disease resistance related protein RIR1, coding gene and applications thereof | |
| CN101962657A (en) | Plant expression vector | |
| CN105039345A (en) | Clone of miRNA for improving salt resistance capacity of mulberries and application thereof | |
| CN1151262C (en) | Method for expression of foreign genes and vectors therefor | |
| CN101250550B (en) | Expression vector changing distribution of rice storage protein as well as preparation and use thereof | |
| CN109486819B (en) | Cassava U6 promoter gene and application thereof | |
| CN1244585A (en) | Method for leading-in petunia transcription factor Pet SPL 2 gene to shorten inflorescence internode | |
| CN106883291B (en) | Plant type related protein PROG2 and encoding gene and application thereof | |
| CN1239702C (en) | Rice root system phosphorus starvation induction specific expression promoter and its plant culture method | |
| CN105061570B (en) | Plant amylum synthesis associated protein IbSSI and its encoding gene and application | |
| CN107058321B (en) | Application of miR156 and precursor thereof in regulation and control of wheat tillering number |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090520 Termination date: 20111104 |