CN102154295B - Specific promoter and method for culturing disease-resistant transgenic plant - Google Patents
Specific promoter and method for culturing disease-resistant transgenic plant Download PDFInfo
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Abstract
The invention discloses a specific promoter and a method for culturing disease-resistant transgenic plant. The promoter provided by the invention is DNA (deoxyribonucleic acid) shown as a sequence 2 in a sequence table. The method provided by the invention is as follows: the promoter and the coding gene of TiERF1 protein are introduced to a starting plant; the promoter is used to start the expression of the coding gene of the TiERF1 protein to obtain a transgenic plant the disease resistance of which is stronger than that of the starting plant. The amino acid sequence of the TiERF1 protein is shown as the sequence 4 in a sequence table. The transgenic wheat obtained through the method disclosed by the invention has enhanced resistance on banded sclerotial blight and uninfluenced agronomic trait, thus having important practical value.
Description
Technical field
The method that the present invention relates to a kind of special-purpose promotor and cultivate disease-resistant transgenic plant.
Background technology
Wheat hypochnus claims that again wheat point eye spot (wheat sharp eyespot) is the important disease of harm China Wheat Production; mainly cause (Chen Yanxi by Rhizoctonia cerealis (Rhizoctonia cerealis) CAG-1; Tang Wenhua; Zhang Dunhua; letter eaglet .A preliminary study on etiology of sharp eye-spot of wheat inChina. plant protection journal 1986,13 (1): 39-44).Banded sclerotial blight is destroyed the stem stalk section phloem tissue of wheat, not only causes plant easily to lodge, and the nutritive substance transportation is not smooth, makes seed shrivelled, even causes withered dead ears, causes the underproduction.Banded sclerotial blight generally can make wheat yield 10%-30%, and the serious plot underproduction is more than 50%, even No kernels or seeds are gathered, as in a year of scarcity.In addition improving constantly of Wheat Production level in recent years, the change of cropping system, and the impact of the factor such as Global warming, make wheat hypochnus be the trend (Shi Jianrong that rises year by year, Wang Yuzhong, Shen Suwen, Chen Huai paddy .Pathogenicity of Rhizoctonia cerealis to wheat in Jiangsu province. Jiangsu agricultural journal, 1997,13 (3): 188-190).Report according to National Agricultural technology popularization master station; 2005-2008; China suffers the wheatland area of wheat hypochnus harm greatly about ten thousand hectares of 670-800 every year; financial loss is at (Zhao Meiqi more than 1,000,000,000 yuan; Wei Kaifeng; but finish political affairs .Studieson the epidemic prediction of wheat sharp eyespot. plant protection journal, 1997,24 (4): 303-308).Cultivate and use anti-banded sclerotial blight new variety of wheat and control beyond doubt the most economical and the most effective approach of this disease.
The most important condition that the anti-disease wheat germplasm that is easy to utilize and disease-resistant gene are breeding for disease resistance.Domestic scholars is carried out in the Disease Resistance Identification nearly thousand parts of wheat lines, there is not discovery to the wheat breed (being) of banded sclerotial blight immunity, deficient (the Shi Jianrong of the material of high resistance, Wang Yuzhong, Chen Huaigu, Shen Suwen. the screening of wheat hypochnus varietal resistance authenticate technology and disease-resistant resource and analysis. plant protection journal, 2000,27 (2): 107-112; Yang Lijun, Yang Xiaojun, explain clear greatly, .Resistance evaluation of wheat cultivars (lines) the to Rhizoctonia cerealis Varder Hoeven and screening of its resistance resources. plant protection of Wang Shao south, 2001,27 (2): 4-7; Wan Yingxiu, Wang Wenxiang, Zhang Pingzhi, Cao Wenxin, Zhao Li .Identification Techniques and Screening of Sharp Eyespot Resistance of Wheat. China agronomy circular, 2009,25 (7): 223-226).Genetics of resistance analysis and disease-resistant proterties Locus Analysis in Shoots result to some anti-sources of wheat hypochnus show, wheat sharp eyespot resistance is by key-gene and the coefficient quantitative character of minor gene, and there is a complicated interaction of genes (hamlet, Li Sishen, Zhao Xinhua, the .Genetic Analysis on Resistance to Sharp Eyespot by Using Fifteen Populations of Recombinant Inbred Lines in Wheat. of Li Rui army wheat crops journal, 2004,24 (3): 13-16; The soup Ting, Ren Lijuan, Cai Shibin, Wu Jizhong, Lu Weizhong, Chen Jianmin, Ma Hongxiang .Study on QTL mapping of sharp eyespot resistance (Rhizoctonia cerealis) in wheat ARz. wheat crops journal, 2004,24 (4): 11-16; Cai Shibin, Ren Lijuan, Yan Wei, Wu Jizhong, Chen Huaigu, Wu Xiaoyou, Zhang Xianyi .Germplasm development and mapping of resistance to sharp eyespot (Rhizoctonia cerealis) in wheat. Scientia Agricultura Sinica, 2006,39 (5): 928-934).Wheat is to the complex inheritance mode of sharp eyespot resistance, so that the cycle of conventional breeding is long, foresight is poor etc., directly affected the progress of wheat sharp eyespot resistance improvement.Transgenic engineering has become the new tool of raising crop disease-resistant ability because the advantage such as the cycle is short, with strong points.Such as the Zhang Zengyan of Institute of Crop Science, Chinese Academy of Agricultural Science problem, clone and isolate pathogeny evoked Thinopyrum intermedium ERF-transcription factor TiERF1 gene, prove that it is activated form ERF transcription factor (the Liang H X that can be combined with the GCC-Box cis element, Lu Y, Liu H X, Wang FD, Xin Z Y, Zhang Z Y.A novel activator-type ERF of Thinopyrum intermedium, TiERF1, positively regulates defence responses.Journal of Experimental Botany, 2008,59 (11): 3111-3120).Adopt transgenic technology, the genes such as TiERF1 gene and antibacterial peptide Rs-AFP2 are imported wheat commercial variety to be raised in the wheat 12, obtained transgenic wheat new germ plasm (the Chen Liang that sharp eyespot resistance improves, Zhang Zeng-Yan, Liang Hong-Xia, Liu Hong-Xia, Du Li-Pu, Xu Hui-Jun, Xin Zhi-Yong.Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat.Journal of Experimental Botany, 2008,59 (15): 4195-4204; The road is beautiful, Zhang Zengyan, Ren Lijuan, Liu Baoye, Liao Yong, Xu Huijun, Du Lipu, Ma Hongxiang, Ren Zhenglong, the well study of the Chinese classic, the brave .Molecular Analyses of hot will on Rs-AFP2Transgenic Wheat Plants and Their Resistance to RhizoctonicI cerenl, 2009,35 (4): 640-646).
At present, employed promotor great majority are composition type expression promoter in transgenic technology, as in dicotyledonous transgenic plant, using cauliflower mosaic virus (CaMV) 35S promoter, the main promotors such as corn ubiquitin ubiquitin and rice actin actin of using in monocotyledons.The genetic expression of these composition type expression promoter regulation and control is not subjected to inducing of space-time restriction and external environment, namely plants in a organized way in equal overexpression goal gene products, its result can cause the unnecessary waste of transgenic plant substance in vivo and energy, causes some side effects.Raise wheat 12 positive plants such as the Zhang Zengyan problem by what particle bombardment had obtained to turn the Ubi::TiERF1 gene, proof TiERF1 crosses expression and can improve wheat to resistance (the Chen Liang of banded sclerotial blight, Zhang Zeng-Yan, Liang Hong-Xia, LiuHong-Xia, Du Li-Pu, Xu Hui-Jun, Xin Zhi-Yong.Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat.Journal of Experimental Botany, 2008,59 (15): 4195-4204).But AP2/ERF transcription factor overexpression is when improving the transfer-gen plant resistance of reverse, also can produce the detrimentally affects such as growth retardation, plant dwarfing, production declining (characters cut in relief dragon, Liu Jingmei, Liu Qiang, public affairs are spread out, Zhao Nanming .Isolation and characterization of a DREB-like transcription factor gene from tall fescue. nuclear agricultural science newspaper, 2006,20 (3): 187-192).
Summary of the invention
The method that the purpose of this invention is to provide a kind of special-purpose promotor and cultivate disease-resistant transgenic plant.
Dna molecular provided by the invention is following (1) or (2) or (3) described dna molecular:
(1) dna molecular shown in the sequence 2 in the sequence table;
(2) the DNA hybridization that under stringent condition, limits with (1) and the dna molecular with promoter function;
(3) DNA that limits with (1) has at least 90% above homology and has the dna molecular of promoter function.
Described stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain described promotor all belong to protection scope of the present invention.
Described recombinant vectors specifically can be following (I) or (II) or (III):
(I) SphI of pAHC20 plasmid and the small segment between the Bgl II restriction enzyme site are replaced with the recombinant plasmid that dna molecular obtains shown in the sequence 1;
(II) dna molecular shown in the sequence 1 that Sph I and the small segment between the Bgl II restriction enzyme site of pAHC20 plasmid is replaced with sequence table, the small segment between BamH I and the Sac I restriction enzyme site replace with the recombinant plasmid (being the promotor shown in the sequence 2 of sequence table between the SphI of recombinant plasmid and the BamH I restriction enzyme site) that the encoding gene of TiERF1 albumen obtains;
(III) with restriction enzyme Bgl II and BamH I double digestion pAHC20 carrier, reclaim small segment; Cut the recombinant plasmid first with the restricted enzyme BamH I enzyme of cutting, reclaim carrier framework; With described carrier framework be connected small segment and connect the recombinant plasmid obtain; Described recombinant plasmid first is for being inserted into the dna molecular shown in the sequence 1 of sequence table the recombinant plasmid that obtains between the HindIII of pAHC20 vector plasmid and the BamH I site;
The aminoacid sequence of described TiERF1 albumen is following (a) or (b):
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 4;
(b) with the aminoacid sequence of sequence 4 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant resistance to environment stress protein of being derived by sequence 4.
The encoding gene of described TiERF1 albumen is following 1) to 3) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 3 in the sequence table;
2) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance-associated protein;
3) with 1) or 2) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant resistance-associated protein.
Described stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The method of cultivation provided by the invention transgenic plant comprises the steps: to import in the plant that sets out with described promotor with by the encoding gene of the described TiERF1 albumen of described promoters driven, obtains the transgenic plant that disease resistance is better than the described plant that sets out.
The encoding gene of described promotor and described TiERF1 albumen specifically can import the described plant that sets out by recombinant plasmid pA20-Ubi::TiERF1.Described recombinant plasmid pA20-EnRSS1P::TiERF1 inserts the recombinant plasmid that the encoding gene of described promotor and described TiERF1 albumen obtains in the multiple clone site of pAHC20 plasmid.Described recombinant plasmid pA20-EnRSS1P::TiERF1 is preferably the small segment between the Sph I of pAHC20 plasmid and the Bgl II restriction enzyme site is replaced with dna molecular shown in the sequence 1 of sequence table, and the small segment between BamH I and the Sac I restriction enzyme site replaces with the recombinant plasmid (being the promotor shown in the sequence 2 of sequence table between the Sph I of recombinant plasmid and the BamH I restriction enzyme site) that the encoding gene of TiERF1 albumen obtains.
The described plant that sets out can be dicotyledons or monocotyledons.Described monocotyledons specifically can be wheat (as raising wheat 12).
The described disease-resistant anti-wheat hypochnus that can be.Described wheat hypochnus specifically can be the wheat hypochnus that is caused by Rhizoctonia cerealis (Rhizoctonia cerealis).
The described disease-resistant disease that anti-Rhizoctonia cerealis (Rhizoctonia cerealis) causes that specifically can be.
The invention provides a kind of enhancement type paddy rice sucrose synthase gene promotor (EnRSS1P), be tissue-specific promoter.The promotor of transgenic plant can be divided into constitutive promoter and tissue-specific promoter's two large classes.The foreign gene that constitutive promoter drives all can be expressed in all periods of transgenic plant and tissue, has caused the waste of energy.So, for the research of tissue-specific promoter with use the attention that just day by day is subject to vast transgenic breeding person.Tissue-specific promoter not only can express in the specific tissue of transfer-gen plant by induction exogenous gene, but also can be by its abduction delivering element that contains, under the different times of plant or different condition, express, thereby avoided the waste of energy, the energy that guarantees plant more carries out the accumulation of yield traits, also the food safety question of transgenic plant is had important meaning.
The present invention has also made up the recombinant plasmid pA20-EnRSS1P::TiERF1 that drives TiERF1 gene organization specifically expressing with EnRSS1P.Do not contain the bar gene in the recombinant plasmid, thereby overcome the possibility that there is environmentally safe impact in the bar gene.Utilize particle bombardment to change this recombinant plasmid over to wheat, obtained not only sharp eyespot resistance to be strengthened and the impregnable transgenic wheat of economical character.With promoters driven TiERF1 genetic expression of the present invention, not only can effectively prevent and treat root, stem's diseases such as wheat hypochnus, and can solve the edible safety problem of transgenic wheat seed.The transgenic wheat that the Ubi promotor is induced is then because its constitutive expression characteristic, some disadvantageous changes have occured at aspects such as plant heights in the render transgenic wheat, and with promoters driven TiERF1 genetic expression of the present invention, do not affect the fundamental characteristics of acceptor wheat breed, further specifying organizing specific type promotor EnRSS1P has more advantage with respect to constitutive promoter aspect transgenic breeding.Therefore, EnRSS1P is applied to genetically engineered, can more effectively prevent and treat root, stem's diseases such as wheat hypochnus, has important practical value.
Description of drawings
Fig. 1 is pA20-EnRSS1P::TIERF1 Vector construction synoptic diagram among the embodiment.
Fig. 2 is the coloration result of gus gene.
Fig. 3 is part T
1PCR for plant identifies; WT is for raising wheat 12 (negative control), and P is recombinant plasmid pA20-EnRSS1P::TiERF1B (positive control), and 1-15 is T
1For plant, wherein 4 is that RSTI-3 strain, 5 is that RSTI-39 strain, 6 is that RSTI-62 strain, 7 is that RSTI-124 strain, 13 is the RSTI-127 strain.
Fig. 4 is semi-quantitative RT-PCR analysis transgenic wheat T
1Expression amount for TiERF1 among different tissues and the Yang Mai 12.
Fig. 5 is fluorescence quantitative PCR detection transgenic wheat T
1Relative expression quantity for TiERF1 among different tissues and the Yang Mai 12.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Rice varieties " Japan is fine ": seed is available from Institute of Crop Science, Chinese Academy of Agricultural Science's germplasm resource bank.
Wheat breed is raised wheat 12: available from the Lixiahe District Institute of agricultural sciences.
The wheat hypochnus pathogenic bacterium are Rhizoctonia cerealis (Rhizoctonia cerealis): available from the Jiangsu Province Agriculture Science Institute.
PAHC20 plasmid: Christensen A H, Quail P H.Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.Transgenic Research, 1996,5:213-218.
The clone of embodiment 1, organizing specific type promotor RSS1P
Design a pair of Auele Specific Primer (RSS1P-F1 and RSS1P-R1) according to existing paddy rice sucrose synthase gene 5 ' end sequence, target sequence is 5 ' terminal non-coding region upstream sequence (1715bp).
RSS1P-F1：5′-CTCCTTCATTTTCAGTGCAAATGTG-3′；
RSS1P-R1：5′-CCAATGGTGGTCAGAGACG AG-3′。
Extract the genomic dna of rice varieties " Japan is fine " spire according to improved method of CTAB.
Take genomic dna as template, (RSS1P-F1/RSS1P-P1) carries out pcr amplification with Auele Specific Primer, obtains pcr amplification product.Pcr amplification adopts Taq plus archaeal dna polymerase (Takara); Reaction parameter is: 94 ℃ of denaturation 5min; 94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 3min circulate 30 times; Extend 10min after 72 ℃.The PCR product is cloned in carrier pMD18-T (Takara) through the low melting-point agarose gel purification, obtains plasmid pMD18-RSS1P, send Sinogenomax Co., Ltd.'s order-checking.
Sequencing result shows, inserted the foreign DNA shown in the sequence 1 of sequence table in carrier pMD18-T, with the RSS1P of DNA called after shown in the sequence 1.
The acquisition of the structure of embodiment 2, recombinant expression vector (pA20-EnRSS1P::TiERF1) and enhancement type RSS1P (EnRSS1P)
One, the structure of recombinant expression vector pA20-EnRSS1P::TiERF1
Make up as shown in Figure 1 recombinant expression vector pA20-EnRSS1P::TiERF1.
1, according to improved method of CTAB (Murray M G, Thompson W F.Rapid isolation of high molecular weight plant DNA.Nucleic Acids Research, 1980,8 (19): the 4321-4325) genomic dna of extraction rice varieties " Japan is fine " spire.
2, the genomic dna that extracts take step 1 is as template, and the primer that forms with RSS1P-F2/RSS1P-R2 obtains pcr amplification product to carrying out pcr amplification.
RSS1P-F2:5 '-AGA
CTCCTTCATTTTCAGTGCAAATG-3 ' (being Sph I recognition sequence in the frame);
RSS1P-R2:5 '-ATA
CCAATGGTGGTCAGAGAC-3 ' (being BamH I recognition sequence in the frame).
3, with the pcr amplification product of restriction enzyme Sph I and BamH I double digestion step 2, reclaim enzyme and cut product (about 1720bp).
4, with restriction enzyme Sph I and Bgl II double digestion pAHC20 plasmid, reclaim skeleton carrier (about 4130bp).
5, the enzyme of step 3 is cut skeleton carrier that product is connected with step and connected with the T4 ligase enzyme that (3 moles of enzymes are cut product: 1 mole of skeleton carrier), obtain recombinant plasmid, recombinant plasmid is checked order.Sequencing result shows, obtained recombinant plasmid pA20-EnRSS1P::bar, its skeleton carrier is pAHC20, with the Ubi promotor between Sph I and the Bgl II restriction enzyme site, replacement is for the RSS1P shown in the sequence 1 of sequence table, and the Exon of RSS1P and Ubi and Intron sequence have consisted of the long enhancement type RSS1P of 2787bp (EnRSS1P) that is.The nucleotide sequence of EnRSS1P is seen the sequence 2 of sequence table.
6, synthetic TiERF1 gene (nucleotide sequence of TiERF1 gene shown in the sequence 3 of sequence table, the TiERF1 albumen of sequence 4 expressions of code sequence tabulation).
7, take the synthetic TiERF1 gene of step 6 as template, the primer that forms with TiERF1-F/TiERF1-R obtains pcr amplification product to carrying out pcr amplification.
TiERF1-F:5 '-TTC
ATGCTGCTGAACCCGGCC-3 ' (being BamH I recognition sequence in the frame);
TiERF1-R:5 '-TCT
CTAGCTGACCAGCTGCTG-3 ' (being Sac I recognition sequence in the frame).
8, with the pcr amplification product of restriction enzyme BamH I and Sac I double digestion step 7, reclaim enzyme and cut product (about 891bp).
9, with restriction enzyme BamH I and Sac I double digestion recombinant plasmid pA20-EnRSS1P::bar, reclaim the carrier framework (about 5375bp) of removing bar.
10, the enzyme of step 8 is cut carrier framework that product is connected with step and connected that (3 moles of enzymes are cut product: 1 mole of skeleton carrier), obtain recombinant plasmid, recombinant plasmid is checked order, sequencing result shows, obtained recombinant plasmid pA20-EnRSS1P::TiERF1 (about 6260bp), its skeleton carrier is the pAHC20 plasmid, between Sph I and BamH I restriction enzyme site, be the EnRSS1P shown in the sequence 2 of sequence table, between BamH I and Sac I restriction enzyme site, be the TiERF1 gene shown in the sequence 3 of sequence table; EnRSS1P promotor control TiERF1 genetic expression.
Two, the structure of recombinant expression vector pA20-Ubi::TiERF1
1, synthetic TiERF1 gene (nucleotide sequence of TiERF1 gene shown in the sequence 3 of sequence table, the TiERF1 albumen shown in the sequence 4 of code sequence tabulation).
2, take the synthetic TiERF1 gene of step 1 as template, the primer that forms with TiERF1-F/TiERF1-R obtains pcr amplification product to carrying out pcr amplification.
3, with the pcr amplification product of restriction enzyme BamH I and Sac I double digestion step 2, reclaim enzyme and cut product (about 891bp).
4, with restriction enzyme BamH I and Sac I double digestion pAHC20 plasmid, reclaim carrier framework.
5, the enzyme of step 3 is cut carrier framework that product is connected with step and connected with the T4 ligase enzyme that (3 moles of enzymes are cut product: 1 mole of skeleton carrier), obtain recombinant plasmid, recombinant plasmid is checked order.Sequencing result shows that (skeleton carrier is the pAHC20 plasmid, is the TiERF1 gene shown in the sequence 3 of sequence table between BamH I and Sac I restriction enzyme site to have obtained recombinant plasmid pA20-Ubi::TiERF1; Ubi promotor control TiERF1 genetic expression).
The specific activity of embodiment 3, RSS1P, EnRSS1P and Ubi promotor
One, the preparation of recombinant plasmid
The gus gene expression cassette that has the Ubi promoters driven in the pAHC20 plasmid.
RSS1P shown in the sequence 1 of composition sequence table.RSS1P is inserted between the HindIII and BamH I site of pAHC20 vector plasmid, obtains recombinant plasmid first (pRss1p:GUS).In the recombinant plasmid first, RSS1P starts the expression of gus gene.
With restriction enzyme Bgl II and BamH I double digestion pAHC20 carrier, reclaim small segment (UBI-INTRON fragment); Cut the recombinant plasmid first with the restricted enzyme BamH I enzyme of cutting, reclaim large fragment (carrier framework); With described large fragment be connected small segment and connect, obtain recombinant plasmid second (pEnRss1p::GUS).In the recombinant plasmid second, EnRSS1P starts the expression of gus gene.
Two, transient expression experiment
Utilize (the Xu H-J (Xu Huijun) such as monarch Xu Hui, Pang J-L (Pang Junlan), Ye X-G (leaf is made the country prosperous), Du L-P (Du Lipu), Li L-C (Li Liancheng), Xin Z-Y (Xin Zhiyong), Ma Y-Z (horse is strong-willed), Chen J-P (Chen Jianping), ChenJ (Chen Jiong), Cheng S-H (Cheng Shunhe), Wu Y-H (Wu Hongya) .Study on the gene transferring of Nib8 into wheat for it ' s resistance to the yellow mosaic virus by bombardment.Acta Agronomica sinca (Acta Agronomica Sinica), 2001,27:684-689 (in Chinese with English abstract)) particle bombardment of report is with recombinant plasmid (recombinant plasmid first, recombinant plasmid second or pAHC20 plasmid) change the rataria callus of raising wheat 12 over to, carry out the dyeing of gus gene after 72 hours.
The results are shown in Table 1 and Fig. 2.
The coloration result of the dyeing of table 1GUS gene
The result shows that RSS1P can not start the expression (or expression amount is not enough to form the blue dot of GUS) of gus gene, and EnRSS1P and Ubi promotor can efficiently start gus gene to be expressed, and the startup of EnRSS1P and Ubi promotor is renderd a service substantially suitable.
Embodiment 4, turn the acquisition of EnRSS1P::TiERF1 plant (wheat)
One, turns the acquisition of EnRSS1P::TiERF1 plant
1, T
0Acquisition for plant
Utilize (the Xu H-J (Xu Huijun) such as monarch Xu Hui, Pang J-L (Pang Junlan), Ye X-G (leaf is made the country prosperous), Du L-P (Du Lipu), Li L-C (Li Liancheng), Xin Z-Y (Xin Zhiyong), Ma Y-Z (horse is strong-willed), Chen J-P (Chen Jianping), ChenJ (Chen Jiong), Cheng S-H (Cheng Shunhe), Wu Y-H (Wu Hongya) .Study on the gene transferring of Nib8 into wheat for it ' s resistance to the yellow mosaic virus by bombardment.Acta Agronomica sinca (Acta Agronomica Sinica), 2001,27:684-689 (in Chinese with English abstract)) particle bombardment of report changes recombinant plasmid pA20-EnRSS1P::TiERF1 over to raise wheat 12 rataria callus, obtains to turn the T of EnRSS1P::TiERF1 wheat
0For plant (103 strain).
Extract T according to improved method of CTAB
0For the total DNA of the blade of plant, take total DNA as template.Adopt the special primer of EnRSS1P::TiERF1 gene and the method evaluation of nest-type PRC to turn the EnRSS1P::TiERF1 wheat.Amplified reaction adopts the upstream primer EnRSS1P-2678U19 be positioned on the EnRSS1P promoter sequence (5 '-GCTCTGCCTTCATACGCTA-3 ') and is positioned at downstream primer TIET-3505L on the TiERF1 sequence (5 '-CCTCTCTTCCTTCTTTTCGG-3 ').PCR reaction system (20ul) comprises 10 μ l, 2 * GC Buffer (Takara), the 50ng genomic dna, 2 μ l dNTPs (2.5mM/L), each 0.5 μ l (10 μ m/L) of upstream and downstream primer, 0.2 μ l r-Taq polysaccharase (5U/ μ l).PCR reaction parameter: 95 ℃ of denaturation 3min; 94 ℃ of 45s, 59 ℃ of 45s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min extensions fill.The plant that pcr amplification obtains the amplified production of about 847bp is positive plant.
2, T
1Acquisition for plant
It (is T that results step 1 is accredited as positive plant selfing acquisition seed
1For seed).With T
1Be that plant (is T for cultivating seeds
1For plant) after identify that respectively method is with the authentication method of step 1.
Part T
1Electrophorogram for the pcr amplification product of plant is seen Fig. 3.
If T
0For plant and T thereof
1All be accredited as the positive for plant, can judge that this plant is for turning the EnRSS1P::TiERF1 plant.Bombard altogether 1200 rataria callus, obtain 103 strain T
0For plant, therefrom screening obtains 29 strains and turns EnRSS1P::TiERF1 plant (namely obtained 29 and turned the EnRSS1P::TiERF1 strain), and transformation efficiency is 2.42%.
Two, turn the acquisition of empty carrier plant
Replace recombinant plasmid pA20-EnRSS1P::TiERF1 to transform with the pAHC20 plasmid and raise the rataria callus of wheat 12, obtain to turn empty carrier plant (the same step 1 of method), as the contrast that turns the EnRSS1P::TiERF1 plant.
Three, turn the acquisition of Ubi::TiERF1 plant
Replace recombinant plasmid pA20-EnRSS1P::TiERF1 to transform with recombinant plasmid pA20-Ubi::TiERF1 and raise the rataria callus of wheat 12, obtain to turn Ubi::TiERF1 plant (the same step 1 of method), as the contrast that turns the EnRSS1P::TiERF1 plant.
Embodiment 5, the tissue expression that turns TiERF1 in the EnRSS1P::TiERF1 wheat plant are active
From turn the EnRSS1P::TiERF1 plant, choose at random 5 strains (RSTI-3, RSTI-39, RSTI-62, RSTI-124 and RSTI-127), from turn the Ubi::TiERF1 plant, choose at random 4 strains (UBTI-1, UBTI-2, UBTI-3 and UBTI-4), from turn the empty carrier plant, choose at random 2 strains (DZ-1 and DZ-2), raise wheat 12 in contrast.With T
1Carry out active detection of tissue expression of TiERF1 for plant.
1, in the wheat milk stage, get respectively total RNA of each tissue (root, stem, leaf and immature seed), behind the purifying, press the synthetic article one chain cDNA of RNA kit ver.3.0cDNA synthetic agent box (Takara) specification sheets.
2, take article one chain cDNA as template, with wheat Actin gene specific primer (ActA:5 '-CACTGGAATGGTCAAGGCTG-3 ' and ActB:5 '-CTCCATGTCATCCCAGTT G-3 ') each sample is increased, so that template cDNA homogenization, the response procedures that Actin detects is: 95 ℃ of denaturation 3min; 94 ℃ of 30s; 58 ℃ of 35s; 72 ℃ of 35s, 26 circulations.
3, take article one chain cDNA as template, with the TiERF1 special primer (TIE-317U:5 '-CGTCGTGCTTCGGTTTCCT-3 ', TIE-743L:5 '-TCGCCTCTCTT CCTTCTTTT-3 ') carry out semi-quantitative RT-PCR analysis; TiERF1 detection reaction condition is: 95 ℃ of denaturation 3min; 94 ℃ of 30s, 58 ℃ of 35s, 72 ℃ of 35s, 10 circulations; 94 ℃ of 30s, 56 ℃ of 35s, 72 ℃ of 35s, 25 circulations; Extend 5min after 72 ℃.
Sxemiquantitative RT-PCR detected result shows: 5 strain results that turn the EnRSS1P::TiERF1 plant are consistent, and 4 strain results that turn the Ubi::TiERF1 plant are consistent, and two strain results that turn the empty carrier plant are consistent; The EnRSS1P promotor can drive TiERF1 gene effective expression in the root of wheat, stem, leaf; The TiERF1 genetic expression intensity of EnRSS1P promoters driven is the strongest in root and stem stalk, and blade takes second place, and is minimum in the seed; Ubi promoters driven TiERF1 gene is organized all at each and is expressed, and intensity does not have significant difference; It is very low to turn in each tissue of empty carrier plant and Yang Mai 12 the TiERF1 gene expression amount, and sxemiquantitative RT-PCR almost can detect out.Raise the RT-PCR analytical results of each tissue of the root of wheat 12 (WT) and RSTI-39 and see Fig. 4.
4, take article one chain cDNA as template, with the TiERF1 special primer (TIE-317U:5 '-CGTCGTGCTTCGGTTTCCT-3 ', TIE-743L:5 '-TCGCCTCTCTT CCTTCTTTT-3 '), carry out Q-RT-PCR at ABIPRIS-MR7000 real-time fluorescence quantitative PCR instrument (American AB I company) and analyze (take Actin as interior mark).Reaction system 25uL is according to SYBR Premix ExTaq (TaKaRa) reactive system, by detecting the purpose that reaches the gene expression detection amount in conjunction with the SYBR GREEN I intensity of double-stranded DNA.Reaction conditions is 94 ℃ of denaturation 2min; Enter 94 ℃ of sex change 15s, 60 ℃ of annealing 31s, 41 circulations.Each reaction all has independently repeated authentication 3 times.Adopt 2
-Δ Δ CTMethod (Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method.Methods, 2001,25:402-408) calculating turns the EnRSS1P::TiERF1 dna triticum is raised the TiERF1 gene of wheat 12 with respect to acceptor relative expression quantity.
Raise the Q-RT-PCR analytical results of each tissue of the root of wheat 12 (WT) and RSTI-39 and see Fig. 5.Turn in EnRSS1P::TiERF1 wheat leaf blade, root and the stem stalk, the relative expression quantity of TiERF1 gene is respectively 6.5 times, 12.3 times and 10.1 times of root that transgenosis is not raised wheat 12, and the relative expression quantity in the seed is 1.3 times, and is basic identical with the root of raising wheat 12.
Embodiment 6, turn the evaluation of EnRSS1P::TiERF1 wheat
From turn the EnRSS1P::TiERF1 plant, choose at random 5 strains (RSTI-3, RSTI-39, RSTI-62, RSTI-124 and RSTI-127), from turn the Ubi::TiERF1 plant, choose at random 4 strains (UBTI-1, UBTI-2, UBTI-3 and UBTI-4), from turn the empty carrier plant, choose at random 2 strains.Respectively with the T1 of each strain for plant (each strain 30 strain), raise wheat 12 (30 strain), CI 12633 (the anti-disease wheat kind is as disease-resistant contrast) and Yangmai No.158 (susceptible contrast) and carry out every evaluation of embodiment 5.
One, sharp eyespot resistance is identified
Adopt report (the Cai S-B (Cai Shibin) such as Cai Shibin, Ren L-J (Ren Lijuan), Yan W (Yan Wei), Wu J-Z (Wu Jizhong), Chen H-G (Chen Huaigu), Wu X-Y (Wu Xiaoyou), Zhang X-Y (Zhang Xianyi) .Germplasmdevelopment and mapping of resistance to sharp eyespot (Rhizoctonia cerealis) in wheat.Scientia Agricultura Sinica (Scientia Agricultura Sinica), 2006,39 (5): the toothpick bacterium inoculation method of 928-934 (inChinese with English abstract) carries out sharp eyespot resistance to be identified: at wheat during jointing stage, cover with the toothpick section of rhizoctonia cerealis mycelia with the disinfecting forceps gripping, embed lightly in the wheat seeding leaf sheath, each leaf sheath embeds one bacterium toothpick section, moisturizing 7 days, in wheat harvest individual plant is carried out disease-resistant investigation, the sick level of individual plant is divided into the 0-4 level, calculates its disease index (PI) according to the average sick level (IT) of each strain wheat.
PI={ (0 * X
0+ 1 * X
1+ 2 * X
2+ 3 * X
3+ 4 * X
4)/[(X
0+ X
1+ X
2+ X
3+ X
4)] * 4} * 100, X
0, X
1, X
2, X
3And X
4Represent respectively 0 grade of banded sclerotial blight, 1 grade, 2 grades, 3 grades, 4 grades stem stalk number.
Average sick level and the disease index of each strain wheat see Table 2.
Average sick level and the disease index of each strain wheat of table 2
| Strain | Average sick level (IT) | Disease index (%) |
| RSTI-3 | 1.3 | 25.8** |
| RSTI-39 | 1.3 | 26.2** |
| RSTI-62 | 1.4 | 28.0* |
| RSTI-124 | 1.3 | 27.6** |
| RSTI-127 | 1.3 | 26.6** |
| UBTI-1 | 1.2 | 25.3** |
| UBTI-2 | 1.3 | 26.4** |
| UBTI-3 | 1.3 | 27.3** |
| UBTI-4 | 1.3 | 25.6** |
| DZ-1 | 1.9 | 38.9 |
| DZ-2 | 1.9 | 39.7 |
| |
1.9 | 39.6 |
| Yangmai No.158 | 2.2 | 44.6 |
| CI12633 | 1.1 | 22.2 |
* be respectively at P=0.05 and P=0.01 with * *, raise the disease-resistant degree difference significance of wheat 12 with contrast.
By as seen from Table 2: the disease index of wild-type plant (raising wheat 12) is 39.6%, the disease index of disease-resistant contrast CI12633 is 22.2%, the disease index of susceptible contrast Yangmai No.158 is 44.6%, turn the disease-resistant index of EnRSS1P::TiERF1 plant between 25.8%-28.0%, turn the disease-resistant index of Ubi::TiERF1 plant between 25.3%-27.3%.Compare with the wild-type plant, great majority turn the EnRSS1P::TiERF1 plant to the resistance of banded sclerotial blight, and are basic identical with the resistance that turns the Ubi::TiERF1 plant.The EnRSS1P promotor can start the TiERF1 effective expression, improves plant to the resistance of banded sclerotial blight, is improving on the disease resistance, and suitable with the effect of Ubi promotor, the quantity statistics analysis does not have notable difference yet.The result shows, the TiERF1 gene of EnRSS1P promoters driven is substantially suitable the expression efficiency of rhizome section and Ubi promotor.
Two, economical character investigation
When harvesting wheat, plant height, spike length, the grain number per spike of wheat are investigated, the results are shown in Table 3.
The Other Main Agronomic Characters of each strain wheat of table 3
| Strain | Plant height | Spike number | Spike length | Spikelet number |
| RSTI-3 | 78.9 | 4.6 | 7.9 | 17.6 |
| RSTI-39 | 78.9 | 4.5 | 7.8 | 17.6 |
| RSTI-62 | 77.3 | 4.5 | 7.6 | 17.4 |
| RSTI-124 | 80.7 | 4.3 | 7.7 | 17.7 |
| RSTI-127 | 78.6 | 4.4 | 7.8 | 17.6 |
| UBTI-1 | 65.5** | 4.6 | 7.7 | 17.0 |
| UBTI-2 | 63.5** | 4.8 | 8.0 | 17.2 |
| UBTI-3 | 68.8* | 5.0 | 7.6 | 17.2 |
| UBTI-4 | 63.5** | 4.8 | 7.7 | 17.4 |
| DZ-1 | 78.1 | 4.5 | 8.0 | 17.4 |
| DZ-2 | 77.6 | 5.6 | 7.9 | 17.6 |
| |
78.3** | 4.6 | 8.0 | 17.5 |
* be respectively at P=0.05 and P=0.01 with * *, with the disease-resistant degree difference significance that turns the Ubi::TiERF1 plant.
The plant height and the wild-type plant (raising wheat 12) that turn the EnRSS1P::TiERF1 plant are basic identical, and turning the Ubi::TiERF1 plant has dwarfism at plant height, obviously short in the wild-type plant with turn the EnRSS1P::TiERF1 plant.Turn the Ubi::TiERF1 plant seedling stage obviously than wild-type plant with turn EnRSS1P::TiERF1 plant reduction, the wild-type plant with turn the EnRSS1P::TiERF1 plant and do not have notable difference.The reason of analyzing above-mentioned phenotypic difference is as follows: Ubi promotor (constitutive promoter) drives ethene transcription factor TiERF1 in each organizationally efficient expression of whole strain, EnRSS1P promotor (specificity promoter) drives TiERF1 and efficiently expresses at the phloem tissue specificity, so the side effects such as plant height reduction that the overexpression because of whole strain TiERF1 causes do not occur.
Claims (9)
1. a dna molecular is the dna molecular shown in the sequence in the sequence table 2.
2. the recombinant vectors, expression cassette or the recombinant bacterium that contain the described dna molecular of claim 1.
3. recombinant vectors as claimed in claim 2, it is characterized in that: described recombinant vectors is following recombinant vectors corresponding to recombinant plasmid:
With restriction enzyme BglII and BamHI double digestion pAHC20 carrier, reclaim small segment; Dna molecular shown in the sequence 1 of sequence table is inserted between the HindIII of pAHC20 vector plasmid and the BamHI site obtains the recombinant plasmid first; Cut the recombinant plasmid first with restriction enzyme BamHI enzyme, reclaim carrier framework; With described carrier framework be connected small segment and connect the recombinant plasmid obtain.
4. method of cultivating transgenic plant, the encoding gene that comprises the steps: the TiERF1 albumen that drives with dna molecular claimed in claim 1 with by described dna molecular imports in the plant that sets out, and obtains the transgenic plant that disease resistance is better than the described plant that sets out;
Described TiERF1 albumen is by protein corresponding to amino acid shown in the sequence in the sequence table 4.
5. method as claimed in claim 4, it is characterized in that: the encoding gene of described TiERF1 albumen is the dna molecular shown in the sequence 3 in the sequence table.
6. such as claim 4 or 5 described methods, it is characterized in that: the encoding gene of dna molecular claimed in claim 1 and described TiERF1 albumen imports the described plant that sets out by recombinant plasmid pA20-EnRSS1P::TiERF1;
Described recombinant plasmid pA20-EnRSS1P::TiERF1 is dna molecular shown in the sequence 1 that Sph I and the small segment between the Bgl II restriction enzyme site of pAHC20 plasmid replaced with sequence table, and the small segment between BamH I and the Sac I restriction enzyme site replaces with the recombinant plasmid that the encoding gene of described TiERF1 albumen obtains.
7. such as claim 4 or 5 described methods, it is characterized in that: the described plant that sets out is wheat.
8. method as claimed in claim 7 is characterized in that: the described disease-resistant anti-wheat hypochnus that is caused by Rhizoctonia cerealis (Rhizoctonia cerealis) that is.
9. method as claimed in claim 4 is characterized in that: the withered line disease of the described disease-resistant wheat that causes for anti-Rhizoctonia cerealis (Rhizoctonia cerealis).
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Non-Patent Citations (2)
| Title |
|---|
| 庄洪涛等.TiERF1基因组织特异型表达载体的构建及其转化小麦的研究.《2009中国作物学会学术年会》.2009,92. * |
| 庄洪涛等.巢式PCR检测转基因小麦的研究.《安徽农业科学》.2009,第38卷(第19期),9972,9992. * |
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