CN103014061A - Root system recombined expression vector of brassinosteroid inactivation gene BAS1 and establishment method and application thereof - Google Patents
Root system recombined expression vector of brassinosteroid inactivation gene BAS1 and establishment method and application thereof Download PDFInfo
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- CN103014061A CN103014061A CN201310015909XA CN201310015909A CN103014061A CN 103014061 A CN103014061 A CN 103014061A CN 201310015909X A CN201310015909X A CN 201310015909XA CN 201310015909 A CN201310015909 A CN 201310015909A CN 103014061 A CN103014061 A CN 103014061A
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Abstract
The invention provides establishment of a root system recombined expression vector of brassinosteroid inactivation gene BAS1. According to the establishment, a tobacco root system promoter TobRB7 is cloned, a root system specific expression vector pCAMBIA2301-TobRB7-rbc is established, the brassinosteroid inactivation gene BAS1 is integrated to the root system specific expression vector so as to obtain a recombined vector pCAMBIA2301-TobRB7-BAS1; and the recombined vector is transferred so as to obtain the transgenic tobacco. By utilizing the root system recombined expression vector, the brassinosteroid gene is directionally expressed in a root system, and at the same time possible ways for plant root system improvement are provided.
Description
Technical field
The present invention relates to plant genetic engineering field, specifically, relate to structure and the application of brassinolide gene root system recombinant expression vector.
Background technology
Brassinolide (Brassinosteroid, BR) is the sterols hormone, and the content in plant materials is less, but activity is higher, the many aspects of growth and development of plants are had significant physiological effect, and it can strengthen the resistance of plant.Studies show that the environment stress condition such as saline and alkaline, arid is lower outside executes the resistance of reverse that brassinolide can improve plant.But the height of brassinolide level mainly is that expression and regulation and control by original position brassinolide metabolism related gene decide in the plant materials.A Cytochrome P450 of BAS1 genes encoding (CYP72B1), overexpression causes brassinolide C-26 hydroxyl hydroxylation, but the brassinolide biological activity behind the C-26 hydroxylation is far below normal brassinolide, therefore BAS1 is an important brassinolide inactivation gene as the C-26-hydroxylase.And the level of the synthetic precursor rape element sterone of brassinolide and 6-deoxidation rape element sterone also is suppressed in the bas1 mutant, thinks that therefore the BAS1 gene also works in the metabolic process of the synthetic precursor of brassinolide.
Crop root is the pillar of plant, is the major organs of Crop nutrient and moisture, or the organ that at first acts on of the environment stress such as saline and alkaline, arid, and root system is movable to have influence on growing and even output formation of plant overground part thereby adverse circumstance is by affect.Therefore, keeping the physiological function of root system, strengthen root system to the receptivity of moisture and nutrient, is the key that improves the crop resistance of reverse.But because root growth is underground, the complicacy of root district environment and the limitation of root system research ways and means relatively lag behind the research of root traits and improvement.Therefore, the present invention utilizes the root system specific promoter to make up the root system recombinant expression vector, provides approach for utilizing the brassinolide gene to carry out the root system improvement.
Tobacco TobRB7 gene is the root tissue different expression gene, known promoter region segment length is 1878bp, and this promotor has the two-way startup subfunction ,-823～+ this promotor both forward and reverse directions of 70bp zone inserts and all shows the specific expressed adjusting function of stronger root tissue.Therefore, the present invention take TobRB7 gene promoter-823～+ 70bp zone is as main survey region.
The present invention is exactly for above background technology, by clone's tobacco root-specific expression promotor from the tobacco gene group, make up the root system specific expression carrier, from Arabidopis thaliana, obtain brassinolide inactivation gene BAS1, and be integrated on the root system expression vector, obtain the root system recombinant expression vector of brassinolide inactivation gene, make the brassinolide gene at the root system of plant specifically expressing.This Vector construction can be the anti-contrary improvement of crop new way is provided.
Summary of the invention
The purpose of this invention is to provide a kind of tobacco root-specific promoter that utilizes makes the brassinolide gene in the method for root system specifically expressing.The technical problem that solves is to make goal gene at the root system specifically expressing.
The present invention utilizes tobacco root-specific promoter to make brassinolide inactivation gene in the method for root system orientation expression, comprise the clone of root system specific promoter, the structure of root system expression vector, the structure of brassinolide inactivation gene cloning, brassinolide gene root system recombinant expression vector finally obtains the transfer-gen plant of brassinolide gene root system specifically expressing.
The technical solution used in the present invention is as follows:
A kind of root system of plant expression vector pCAMBIA2301-TobRB7-rbc take the pCAMBIA2301-35S-rbc carrier as skeleton, is cloned into the promoter fragment of tobacco TobRB7 at EcoR I and Sac I restriction enzyme site.
The construction process of above-mentioned root system of plant expression vector pCAMBIA2301-TobRB7-rbc,) take tobacco DNA as template, take TobRB7-F1:5'-GGAATTCGGCATACTTTTAGAATGCGT-3'(SEQ ID NO.1) and TobRB7-R1:5'-CGAGCTCTTCTCACTAGAAAAATGCCC-3'(SEQ ID NO.2) as primer, promoter region by pcr amplification TobRB7, with EcoR I and Sac I PCR product and pCAMBIA2301-35S-rbc carrier are carried out double digestion simultaneously, connect into pCAMBIA2301-TobRB7-rbc with T4ligase.
A kind of root system of plant recombinant expression vector pCAMBIA2301-TobRB7-BAS1 is take described pCAMBIA2301-TobRB7-rbc carrier as skeleton, is cloned into the full length fragment of Arabidopis thaliana BAS1 gene coding region at Xba I and Kpn I restriction enzyme site.
The construction process of above-mentioned root system of plant recombinant expression vector pCAMBIA2301-TobRB7-BAS1, be that brassinolide gene BAS1 is incorporated on the root system expression vector pCAMBIA2301-TobRB7-rbc of claim 1, obtain the root system recombinant expression vector pCAMBIA2301-TobRB7-BAS1 of brassinolide gene BAS1.Specifically: take Arabidopis thaliana RNA as template, with BAS1-F1:5'-TGCTCTAGAAAGAAACCAAACTCGC AAAG-3'(SEQ ID NO.3); BAS1-R1:5'-CGGGGTACCTTCCAAGTCCGGAACAAA-3'(SEQ IDNO.4) is primer, amplification comprises the full length fragment of BAS1 coding region, with Xba I and Kpn I PCR product and pCAMBIA2301-TobRB7-rbc carrier are carried out double digestion simultaneously, connect into pCAMBIA2301-TobRB7-BAS1 with T4ligase.
Above-mentioned plant expression vector pCAMBIA2301-TobRB7-BAS1 is used for the agrobacterium mediation converted of tobacco, obtains the transfer-gen plant of brassinolide inactivation gene root system specifically expressing.
The invention also discloses kind of the transgene tobacco of brassinolide inactivation gene root system specifically expressing, is to obtain by the agrobacterium mediation converted tobacco with plant expression vector pCAMBIA2301-TobRB7-BAS1.
The present invention has cloned tobacco root promotor TobRB7, made up root system specific expression carrier pCAMBIA2301-TobRB7-rbc, again brassinolide inactivation gene BAS1 is incorporated on the above-mentioned root system specific expression carrier, obtain recombinant vectors pCAMBIA2301-TobRB7-BAS1, again this recombinant vectors transformation of tobacco is obtained transgene tobacco.This root system recombinant expression vector can make the brassinolide gene at the root system orientation expression, provides possible approaches for the root system of plant improvement simultaneously.
Description of drawings
Fig. 1 is that the enzyme of plant expression vector pCAMBIA2301-TobRB7-rbc is cut as a result M:DL2000DNA marker, and 1,2,3 are the pCAMBIA2301-TobRB7-rbc carrier of structure.
Fig. 2 is as a result M:DL2000DNA marker of brassinolide inactivation gene BAS1 pcr amplification.
Fig. 3 is the construction step of the root system recombinant expression vector pCAMBIA2301-TobRB7-BAS1 of brassinolide inactivation gene BAS1.
Fig. 4 is the physical map of the root system recombinant expression vector pCAMBIA2301-TobRB7-BAS1 of brassinolide inactivation gene BAS1.
Fig. 5 is that the enzyme of carrier pCAMBIA2301-TobRB7-BAS1 cuts as a result that M is DL2000, and 1,2,3 are the pCAMBIA2301-TobRB7-BAS1 carrier of structure.
Fig. 6 is that the PCR of transfer-gen plant detects M:DL2000DNA marker; 1-10 is transfer-gen plant."+" is the pCAMBIA2301-TobRB7-BAS1 plasmid, "-" negative contrast.
Fig. 7 is that the upgrowth situation 1 of transfer-gen plant is the transgene tobacco of overexpression BAS1 gene, 2 transgene tobaccos for root system expression BAS1 gene.
Fig. 8 is that the RT-PCR analysis CK of transfer-gen plant is the non-transgenic plant, and 1 is the transgene tobacco of overexpression BAS1 gene, 2 transgene tobaccos for root system expression BAS1 gene.
Embodiment
Among the following embodiment, the PTG19-T carrier is purchased from Shanghai Jierui Biology Engineering Co., Ltd; Plasmid pCAMBIA2301-35s-rbc(Zheng Qing, the research [D] of resisting I-type diabetes gene transformation tobacco, tomato, the Master degree candidate of Yangzhou University Diplomarbeit, 2010) preserved by Commercial Crop Inst., Jiangsu Prov. Academy of Agricultural Sciences cotton transgenic technology experiment chamber.
1, the acquisition of tobacco TobRB7 promoter sequence
Take tobacco as experiment material, extract its genomic dna and take it as template, (the GenBank accession number: S45406) promoter region design primer, pcr amplification TobRB7 promoter sequence of TobRB7 gene from the tobacco gene group database of announcing.Introduce respectively EcoR I and Sac I restriction enzyme site at the primer two ends, primer sequence is as follows: TobRB7-F1:5'-G
GAATTCGGCATACTTTTAGAATGCGT-3' introduces EcoR I restriction enzyme site; TobRB7-R1:5'-C
GAGCTCTTCTCACTAGAAAA ATGCCC-3' introduces Sac I restriction enzyme site.The PCR response procedures is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 40s, 56 ℃ of renaturation 40s, 72 ℃ are extended 1min, totally 35 circulations; 72 ℃ are extended 10min again.
Pcr amplification product is carried out agarose gel electrophoresis to be detected, downcutting purpose bar zone purification reclaims, be connected to amplified production on the PTG19-T carrier and transform the bacillus coli DH 5 alpha competent cell, the mono-clonal that picking grows in the Amp resistant panel, extract plasmid, enzyme is cut evaluation and is checked order, with the plasmid called after p-TobRB7 of TobRB7 promoter sequence.By finding behind the nucleotide sequencing that the promoter fragment total length that increases is 892bp, carry out the nucleotide homology retrieval with blast program, result's demonstration: the promoter fragment that experiment obtains and known TobRB7(S45406) promotor subregion sequence is basically identical, research after can be used for.
2, carry the structure of the root system of plant specific expression carrier of TobRB7 promotor
Plasmid p-TobRB7 and plasmid pCAMBIA2301-35s-rbc are used respectively EcoR I, Sac I double digestion 3h under 37 ℃ of conditions, enzyme is cut product and is carried out respectively 1% agarose gel electrophoresis analysis, large fragment after the promoter sequence of the TobRB7 of recovery 892bp and the plasmid pCAMBIA2301-35s-rbc enzyme of 12kb are cut is spent the night with 4 ℃ of connections of T4DNA ligase enzyme.Connect product and transform the bacillus coli DH 5 alpha competent cell, the mono-clonal that picking grows in kantlex (Kan) resistant panel extracts plasmid, carries out PCR evaluation and enzyme and cuts evaluation.With the recombinant expression vector called after pCAMBIA2301-TobRB7-rbc that obtains.Fig. 1 is that the pCAMBIA2301-TobRB7-rbc enzyme is cut the result, illustrates that TobRB7 successfully is building up on the pCAMBIA2301-35s-rbc plasmid.
3, the clone of brassinolide inactivation gene BAS1
Take wild-type Arabidopis thaliana plant as experiment material, extract total RNA, take the cDNA of reverse transcription as template.According to reporting Arabidopis thaliana BAS1 gene (GenBank No.NM_128228.3) mRNA primers, the full length fragment of pcr amplification BAS1 coding region.Introduce respectively Xba I and Kpn I restriction enzyme site at the primer two ends, primer sequence is as follows: BAS1-F1:5'-TGC
TCTAGAAAGAAACCAAACTCGCAAAG-3' introduces Xba I restriction enzyme site; BAS1-R1:5'-CGG
GGTACCTTCCAAGTCCGGAACAAA-3' introduces Kpn I restriction enzyme site.The PCR response procedures is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 40s, 52 ℃ of renaturation 40s, 72 ℃ are extended 2min, totally 35 circulations; 72 ℃ are extended 10min again.
Pcr amplification product is carried out agarose gel electrophoresis detect (Fig. 2), downcutting purpose bar zone purification reclaims, be connected to amplified production on the PTG19-T carrier and transform the bacillus coli DH 5 alpha competent cell, the mono-clonal that picking grows in the Amp resistant panel, extraction plasmid, enzyme are cut evaluation and are checked order, with the plasmid called after p-BAS1 of BAS1 gene.By finding behind the nucleotide sequencing that the sequence total length that increases is 1770bp, carry out the nucleotide homology retrieval with blast program, result's demonstration: the gene fragment that experiment obtains and known BAS1(NM_128228.3) open reading frame (ORF) the nucleotide sequence consistence of gene is 99.94%, and consensus amino acid sequence is 99.81%.Research after can be used for.
4, the structure of the root system recombinant expression vector of brassinolide inactivation gene BAS1
Plasmid p-BAS1 and plasmid pCAMBIA2301-TobRB7-rbc are used respectively Kpn I, Xba I double digestion 3h under 37 ℃ of conditions, enzyme is cut product and is carried out respectively 1% agarose gel electrophoresis analysis, large fragment after the BAS1 gene order of recovery 1770bp and the plasmid pCAMBIA2301-TobRB7-rbc enzyme of 12kb are cut is spent the night with 4 ℃ of connections of T4DNA ligase enzyme.Connect product and transform the bacillus coli DH 5 alpha competent cell, the mono-clonal that picking grows in the Kan resistant panel extracts plasmid, carries out PCR evaluation and enzyme and cuts evaluation.Root system recombinant expression vector called after pCAMBIA2301-TobRB7-BAS1 with the brassinolide inactivation gene BAS1 that obtains.
Fig. 3 is the construction step of the root system recombinant expression vector pCAMBIA2301-TobRB7-BAS1 of brassinolide inactivation gene BAS1.Fig. 4 is the physical map of the recombinant expression vector pCAMBIA2301-TobRB7-BAS1 of brassinolide inactivation gene BAS1.Fig. 5 is that the enzyme of carrier pCAMBIA2301-TobRB7-BAS1 is cut the result, illustrates that the root system recombinant expression vector pCAMBIA2301-TobRB7-BAS1 of brassinolide inactivation gene BAS1 successfully constructs.
5, BAS1 gene specifically expressing in Tobacco Root of TobRB7 promoters driven
(1) recombinant expression vector pCAMBIA2301-TobRB7-BAS1 transformation of tobacco
The recombinant expression vector pCAMBIA2301-TobRB7-BAS1 that makes up in 4 is transformed the Agrobacterium competent cell, and single bacterium colony that picking is grown in Rif and Kan resistant panel carries out PCR to be identified.The Agrobacterium that will contain recombinant expression vector pCAMBIA2301-TobRB7-BAS1 adopt leaf disc transformation method (Wang Guanlin and the Fang Hong skin of bamboo. plant genetic engineering [M] .1998) transformation of tobacco, contemporary transfer-gen plant (T0 generation) is carried out PCR detects.After the results T1 is being contained the positive seedling of MS substratum screening of Kan for seed, and carrying out RT-PCR and analyze.
(2) detection of transgene tobacco
Take T0 for the total DNA of transfer-gen plant as template, with the positive contrast of plasmid, with the negative contrast of total DNA of unconverted plant, with BAS1 gene primer (BAS1-F2:5'-AAGAAACCAAACTCGCAAAG-3'(SEQ ID NO.5) and BAS1-R2:5'-TTCCAAGTCCGGAACAAA-3'(SEQIDNO.6)) carry out pcr amplification.Positive control and transfer-gen plant amplify the purpose fragment of about 1770bp, and negative control is without amplified band (Fig. 6).Proof brassinolide gene BAS1 has been incorporated in the tobacco gene group.
Fig. 7 is the transfer-gen plant upgrowth situation, can find out that downgrading appears in the transgenic tobacco plant of constitutive expression, but the growth of the transgene tobacco of root system specifically expressing is normal.Extract T1 for total RNA of tobacco leaf, root, take transgenic tobacco plant cDNA as template, with the negative contrast of the cDNA of unconverted tobacco plant, carry out the semi-quantitative expressed analysis of BAS1 gene.CDNA sequences Design RT-PCR primer according to the BAS1 gene, primer is BAS1-F3:5'-ACGGTG AAGTTGAGGTAGA-3'(SEQ ID NO.7) and BAS1-R3:5'-AACATAAGGACGGTAGGTG-3'(SEQ ID NO.8), take actin gene (GenBank No:EU938079) as internal reference gene (469bp), primer is actin-F:5 '-CCTCTTAACCCGAAGGCTAA-3 ' (SEQ IDNO.9), actin-R:5 '-GAAGGTTGGAAAAGGACTTC-3 ' (SEQ ID NO.10).The semi-quantitative expressed result (Fig. 8) of BAS1 gene confirms that further the root system specific expression carrier makes the BAS1 gene only at the root system specifically expressing, does not substantially express in leaf.
Claims (6)
1. a root system of plant expression vector pCAMBIA2301-TobRB7-rbc is characterized in that, the pCAMBIA2301-35S-rbc carrier is skeleton, is cloned into the promoter fragment of tobacco TobRB7 at EcoR I and Sac I restriction enzyme site.
2. the construction process of root system of plant expression vector pCAMBIA2301-TobRB7-rbc claimed in claim 1, it is characterized in that: take tobacco DNA as template, take TobRB7-F1:5'-GGAATTCGGCATACTTTTAGAATGCGT-3' and TobRB7-R1:5'-CGAGCTCTTCTCACTAGAAAAATGCCC-3' as primer, promoter region by pcr amplification TobRB7, with EcoR I and Sac I PCR product and pCAMBIA2301-35S-rbc carrier are carried out double digestion simultaneously, connect into pCAMBIA2301-TobRB7-rbc with T4 ligase.
3. root system of plant recombinant expression vector pCAMBIA2301-TobRB7-BAS1, it is characterized in that, take the described pCAMBIA2301-TobRB7-rbc carrier of claim 1 as skeleton, be cloned into the full length fragment of Arabidopis thaliana BAS1 gene coding region at Xba I and Kpn I restriction enzyme site.
4. the construction process of a root system of plant recombinant expression vector pCAMBIA2301-TobRB7-BAS1, it is characterized in that, brassinolide gene BAS1 is incorporated on the root system expression vector pCAMBIA2301-TobRB7-rbc of claim 1, obtains the root system recombinant expression vector pCAMBIA2301-TobRB7-BAS1 of brassinolide gene BAS1.
5. method according to claim 4 is characterized in that: take Arabidopis thaliana RNA as template, with BAS1-F1:5'-TGCTCTAGAAAGAAACCAAACTCGCAA AG-3'; BAS1-R1:5'-CGGGGTACCTTCCAAGTCCGGAACAAA-3' is primer, amplification comprises the full length fragment of BAS1 coding region, with Xba I and Kpn I PCR product and pCAMBIA2301-TobRB7-rbc carrier are carried out double digestion simultaneously, connect into pCAMBIA2301-TobRB7-BAS1 with T4 ligase.
6. the transgene tobacco of a brassinolide inactivation gene root system specifically expressing is characterized in that, is to obtain by the agrobacterium mediation converted tobacco with plant expression vector pCAMBIA2301-TobRB7-BAS1.
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| WO2014166012A1 (en) * | 2013-04-09 | 2014-10-16 | 中国农业科学院棉花研究所 | Plant type related protein, and coding gene and application thereof |
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| CN1546672A (en) * | 2003-12-09 | 2004-11-17 | 江苏省农业科学院 | Construction method of recombinant expression vector of plant gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014166012A1 (en) * | 2013-04-09 | 2014-10-16 | 中国农业科学院棉花研究所 | Plant type related protein, and coding gene and application thereof |
| US10041085B2 (en) | 2013-04-09 | 2018-08-07 | Cotton Research Institute, Chinese Academy of Agricultural Sciences | Plant type related protein, and coding gene and application thereof |
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Application publication date: 20130403 |