CN103882019B - A kind of nematode regulated genes ced-4 evoked promoter, paddy rice expression vector and preparation method thereof - Google Patents
A kind of nematode regulated genes ced-4 evoked promoter, paddy rice expression vector and preparation method thereof Download PDFInfo
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- CN103882019B CN103882019B CN201410067025.3A CN201410067025A CN103882019B CN 103882019 B CN103882019 B CN 103882019B CN 201410067025 A CN201410067025 A CN 201410067025A CN 103882019 B CN103882019 B CN 103882019B
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Abstract
The invention discloses the preparation method of a kind of nematode regulated genes ced-4 evoked promoter wn-2, paddy rice expression vector pCA-Wn-2-CED-4 and pCA-Wn-2-CED-4 expression vector. In W-box series startup that utilization of the present invention has been in the news, induce the core sequence of best results, add therein the shared TATA-box core sequence of animal-plant gene, innovative design nematode body cell apoptogene paddy rice express new wn-2 sequence. The intrinsic promoter of wn-2 and paddy rice expression vector is merged, be built into new paddy rice expression vector pCA-Wn-2-CED-4 expression vector, there is the superior effect that induction genes of interest ced-4 expresses in paddy rice, screen and connected correct carrier cloning, and be successfully transformed into rice varieties: Japanese warm and fine Lijiang xintuanheigu.
Description
Technical field
The application belongs to biological technical field, is specifically related to a kind of nematode regulated genes ced-4 evoked promoter, paddy riceExpression vector and preparation method thereof.
Background technology
Due to elegans cell regulated genes, ced-4 does not exist in paddy rice body, and can ced-4 gene open smoothly in paddy riceMove and successful expression destination protein, most important to the science design of promoter; The gene that is animal body carries out at plantExpress, must solve the compatibility issue of promoter between nematode and paddy rice, but also need to be that pathogen infects induction and opensMoving gene expression, the technical goal of Here it is this patent.
From domestic and international up-to-date correlative study document, infect promoter and the table thereof of induction in unifacial leaf paddy rice by pathogenReach carrier, have no play-by-play.
Evoked promoter W-box studies and infects successfully abduction delivering at pathogen in tobacco etc. dicotyledon, butThat its startup induces the expression vector of genes of interest ced-4 not yet to have report in paddy rice.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, and a kind of nematode regulated genes ced-4 evoked promoter, water are providedRice expression vector and preparation method thereof.
In order to achieve the above object, technical scheme provided by the invention is:
The present invention has designed a kind of nematode regulated genes ced-4 evoked promoter wn-2, described elicitor wn-2 sequenceAs shown in SEQIDNO.1, be specially:
TTCTAGCCACCAGATTTGACCAAACCATCAACTCATCTGTATATAATA。
The present invention has designed a kind of paddy rice expression vector, and described paddy rice expression vector comprises wn-2 promoter, described paddy rice tableReach the sequence of carrier as shown in SEQIDNO.2, called after pCA-Wn-2-CED-4 expression vector.
The present invention also provides the preparation method of pCA-Wn-2-CED-4 expression vector, and described preparation method is as follows:
(1) by promoter wn-2 and P35S(SEQIDNO.7) be cloned into pCAMBIA1305.1 carrier (pCAMBIA1305.1Carrier is commercially available) (wn-2 is positioned at before P35S), obtaining intermediate carrier pCA-Wn-2, amplification Wn-2-P35S merges productThe primer of thing is as follows:
Wn-2-P35S-FP:
AAAGAATTC-TTCTAGCCACCAGATTTGACCAAACCATCAACTCATCTGTATATAATATGGAGTCAAAGATTCAAATAGAGGACC（SEQIDNO.3）；
Wn-2-P35S-RP:
AAAACTAGTCTGCAGAGTCCCCCGTGTTCTCTCCA（SEQIDNO.4）；
Detailed process is: be inserted into EcoRI and the SpeI(p35S in pCAMBIA1305.1 carrier by EcoRI and SpeI siteBefore promoter and GUS fore-end), obtain intermediate carrier pCA-Wn-2;
(2) genes of interest ced-4 is cloned into pCA-Wn-2 carrier (by PstI and PmlI site), obtains described paddy rice expression and carryBody, called after pCA-wn-2-CED-4 expression vector;
The primer of amplification CED-4 is as follows:
Ced-4FP:
AAACTGCAGATGCTCTGCGAAATCGAATGC（SEQIDNO.5）；
Ced-4RP:
AAAACTAGTTTAACAGCATGCAAAATTTTTGAGGG（SEQIDNO.6）。
Concrete clone's mode of the inventive method design and enzyme the step such as are cut and are this area normal experiment operation.
Below the present invention is further explained:
Because CED-4 is the albumen of the main regulate gene expression of elegans cell apoptosis, therefore must consider this controlling gene asWhere in paddy rice, start expressing protein.
In order to solve the compatibility issue of promoter in nematode and paddy rice, pass through design and experiment screening many times and testCard, on the W-box basis of forefathers' report, the promoter TATA-box order that the present invention has added animal and plant all to shareRow, start genes of interest ced-4 smoothly to scheme induction on paddy rice, express elegans cell apoptotic proteins CED-4. Through fromIn the W-TATA-box promoter of multiple transformations, the evoked promoter screening: wn-2 promoter, induction genes of interest ced-4Express CED-4 successful in paddy rice.
The present invention has built taking wn-2 promoter as basic expression vector, and rice transformation obtains transgenic paddy rice, transforms waterRice seedling root can be expressed CED-4 albumen by inducing paddy rice in the time that parasitic nematode infects.
The transformation seedlings of expression vector rice transformation of the present invention is successfully induced ced-4 gene in the time that paddy rice parasitic nematode infectsExpress.
At present, research both at home and abroad shows that TIR domain protein only finds to exist in dicotyledon, its express also have correspondingPromoter, as expressed at arabidopsis; But in unifacial leaf paddy rice, not yet find that there is the expression of TIR domain protein. NematodeApoptosis TIR domain protein gene, in expression vector, before genes of interest, 32-45 base places add w-boxInduced sequence, in the time that pathogenic nematode infects, genes of interest promoter can be replied when parasitic nematode is infected to wound and be started and react,Induction destination gene expression albumen strengthens. Therefore the design of, carrying out this class evoked promoter is in theoretical research and practical applicationIn all there is very large value.
In a word, in W-box series startup that utilization of the present invention has been in the news, induce the core sequence of best results, at itIn add the shared TATA-box core sequence of animal-plant gene, innovative design nematode body cell apoptogene at paddy rice tableThe new wn-2 sequence reaching. The intrinsic promoter of wn-2 and paddy rice expression vector is merged, be built into new paddy rice and expressedCarrier pCA-Wn-2-CED-4 expression vector, has the superior effect that induction genes of interest ced-4 expresses in paddy rice, sievesChoose and connect correct carrier cloning, and be successfully transformed into rice varieties: Japanese warm and fine Lijiang xintuanheigu.
Brief description of the drawings
Fig. 1 is pCA-Wn-2-CED-4 expression vector cleavage map;
Fig. 2 is the electrophoretogram that protein hybridization detects paddy rice expression destination protein CED-4; In figure: swimming lane 1,3 opens for inducingMover wn2 carrier transforms the fine inoculation nematode of Japan, and 2,4 do not inoculate nematode for wn2 carrier conversion Japan is fine; M is albumenMarker, 9 inoculate nematode for Wn2 carrier transforms Lijing seedling.
Detailed description of the invention
Embodiment 1
Described wn-2 promoter sequence is as SEQIDNO.1;
By promoter wn-2 and P35S(SEQIDNO.7) (wn-2 is positioned at P35S to be cloned into pCAMBIA1305.1 carrier, obtain intermediate carrier pCA-Wn-2 above), the primer of amplification Wn-2-P35S fusion product is as follows:
Wn-2-P35S-FP:
AAAGAATTC-TTCTAGCCACCAGATTTGACCAAACCATCAACTCATCTGTATATAATATGGAGTCAAAGATTCAAATAGAGGACC（SEQIDNO.3）；
Wn-2-P35S-RP:
AAAACTAGTCTGCAGAGTCCCCCGTGTTCTCTCCA（SEQIDNO.4）；
Detailed process is: be inserted into EcoRI and the SpeI(p35S in pCAMBIA1305.1 carrier by EcoRI and SpeI siteBefore promoter and GUS fore-end), obtain intermediate carrier pCA-Wn-2;
Genes of interest ced-4 is cloned into pCA-Wn-2 carrier (by PstI and PmlI site), obtains described paddy rice expression and carryBody, called after pCA-wn-2-CED-4 expression vector, pCA-wn-2-CED-4 expression vector sequence is as SEQIDNO.2 instituteShow;
The primer of amplification CED-4 is as follows:
Ced-4FP:
AAACTGCAGATGCTCTGCGAAATCGAATGC（SEQIDNO.5）；
Ced-4RP:
AAAACTAGTTTAACAGCATGCAAAATTTTTGAGGG（SEQIDNO.6）。
The checking of embodiment 2 paddy rice expression vectors
Build paddy rice expression vector, various processing by experiment, experimental design and inducing action experimental procedure thereof and result asUnder:
(1) the latent root nematode of transgenic paddy rice seedling Inoculated Rice.
(2) detect rice root apoptosis in epidermal cell and allergic reaction phenotype, see staining examine result statistical form.
(3) transgenosis destination gene expression PROTEIN C ED-4 detects, and sees Figure of description.
Rice root cell allergic reaction method is measured in dyeing:
Rice root dyes respectively after 15min and fully rinses and remove not combination dye with flowing water, at 50 DEG C with 20mL containing 50%The dyestuff 30min that the solution extracting of methyl alcohol and 1%SDS is combined with dead cell, measures extract absorbance value, and bromophenol blue is dyedThe mensuration wavelength of look is 595nm.
The concrete content of measuring is:
(a) root tissue of accurate weighing different disposal paddy rice and control treatment, each processing repeats 3 times, and each sample is got at randomArticle 10, rice root, gets 10 roots of paddy rice of same breed wild type, and methyl alcohol is processed 15min rice root cell, uses respectively phaseWith measuring after the dyeing of concentration bromophenol blue, measure relatively different disposal and root epidermis is occurred thin after Inoculated Rice parasitic nematodeThe impact of born of the same parents' apoptosis; The dive impact of root nematode of Inoculated Rice (each is processed respectively the Inoculated Rice root nematode of diving, 22 DEG C of normal temperatureIllumination box, root is after 500 great-hearted latent root nematode aqueous solution inoculations 96 hours), with the sample of not inoculating nematodeProduct are staining examine comparison respectively;
(b) by methyl alcohol or Ethanol Treatment, or boil and process after rice cell different time, then dye and measure by 0.03% bromophenol blue,Relatively different disposal (wild type, induction carrier, non-induction carrier; Inoculate nematode and do not inoculate nematode) rice root epidermisThe difference of cell generation apoptosis quantity;
(c) use visible-ultraviolet specrophotometer (UV-6100 and PerkinElmerLambda35) to different disposal bromophenol blueAfter dyeing and wash-out, carry out wavelength (595nm) scanning and detect, record result analysis.
(d) the rice root sample of these same quantity is dyeed, measure the quantity of apoptosis in epidermal cell, calculate each processingThe incidence of apoptosis in epidermal cell.
(e) pressing formula calculates: Apoptosis incidence (%)=(OD595 processing-OD595 contrast)/(100-OD595 contrasts correctionValue) × 100% calculating.
(f) known wild type paddy rice epidermis living cells, cellular damage occurs in nematode infection processs all can increase dyeing mensurationEnd value.
(g) different paddy rice (Japan is fine) are processed the inspection of processing in the epidermal cell dyeing of not inoculating in nematode, inoculation nematode situationSurvey result is as follows:
The testing result of nematode is not inoculated in the each processing of table 1
The each testing result of processing the latent root nematode of Inoculated Rice of table 2
Claims (2)
1. a nematode regulated genes ced-4 evoked promoter wn-2, is characterized in that, described evoked promoter wn-2 orderRow are as shown in SEQIDNO.1.
2. a paddy rice expression vector, is characterized in that, described paddy rice expression vector comprises promoter claimed in claim 1,The sequence of described paddy rice expression vector is as shown in SEQIDNO.2.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000029592A2 (en) * | 1998-11-12 | 2000-05-25 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Chimeric promoters capable of mediating gene expression in plants upon pathogen infection and uses thereof |
| WO2001027298A2 (en) * | 1999-10-14 | 2001-04-19 | Pioneer Hi-Bred International, Inc. | Sclerotinia-inducible genes and promoters and their uses |
| WO2001036464A2 (en) * | 1999-11-18 | 2001-05-25 | Pioneer Hi-Bred International, Inc. | SUNFLOWER RhoGAP, LOX, ADH, AND SCIP-1 POLYNUCLEOTIDES AND METHODS OF USE |
| CN103409423A (en) * | 2012-12-11 | 2013-11-27 | 湛江师范学院 | Method for constructing plant pathogen induced artificial promoters |
-
2014
- 2014-02-26 CN CN201410067025.3A patent/CN103882019B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000029592A2 (en) * | 1998-11-12 | 2000-05-25 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Chimeric promoters capable of mediating gene expression in plants upon pathogen infection and uses thereof |
| WO2001027298A2 (en) * | 1999-10-14 | 2001-04-19 | Pioneer Hi-Bred International, Inc. | Sclerotinia-inducible genes and promoters and their uses |
| WO2001036464A2 (en) * | 1999-11-18 | 2001-05-25 | Pioneer Hi-Bred International, Inc. | SUNFLOWER RhoGAP, LOX, ADH, AND SCIP-1 POLYNUCLEOTIDES AND METHODS OF USE |
| CN103409423A (en) * | 2012-12-11 | 2013-11-27 | 湛江师范学院 | Method for constructing plant pathogen induced artificial promoters |
Non-Patent Citations (4)
| Title |
|---|
| 人工病原物诱导启动子调控nprl的载体构建及转化辣椒的初步研究;彭舒;《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑D048-29》;20140115;全文 * |
| 植物WRKY转录因子的分子生物学功能;陈峰 等;《湖南农业科学》;20101231;第19卷;全文 * |
| 秀丽新小杆线虫ced3/ced4在拟南芥及其寄生线虫的表达调控;邹一平 等;《中国植物病理学会2008年学术年会论文集》;20080701;全文 * |
| 细胞凋亡基因ced3/ced4在水稻和拟南芥的转化与表达调控;邹一平 等;《中国细胞生物学学会第九次会员代表大会暨青年学术大会论文摘要集》;20071101;全文 * |
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