CN104745623A - Plant constitutive overexpression vector and preparation method of branched high-yield rape - Google Patents
Plant constitutive overexpression vector and preparation method of branched high-yield rape Download PDFInfo
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Abstract
The invention provides a plant constitutive overexpression vector and a preparation method of branched high-yield rape. The plant constitutive overexpression vector of the DHHC type zinc finger protein gene OsDHHC1 of rice is constructed and used for transforming cabbage type rape; and a transgenic plant with increased branch number is obtained through resistance screening, RT-PCR identification and the like. According to analysis, the yield of the transgenic plant is obviously increased over a wild plant. The result shows that multi-branch high-yield transgenic rape is cultivated through genetic transformation.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of plant composing type Overexpression vector, branch and the preparation method of high yield rape.
Background technology
Rape (Brassica napus L.) is not still only second to the second largest food oils plant of soybean in the world and is one of main source of vegetables oil and vegetable-protein simultaneously yet, accounts for 40% ~ 45% of China's oil crops ultimate production.Rape is not single species, it comprises many kinds in Brassica genus, according to the rapeseed plant morphological specificity of China's cultivation, genetic relationship, in conjunction with economical character, culture and utility feature etc. can be divided into three major types type: i.e. turnip type rape (Brassica campestris), mustard type rape (Brassica juncea) and swede type rape (Brassica napus L.).Swede type rape (Brassica napus L., AACC2n=38) be aggregate species of being evolved by Natural double after natural species hybridization by rape (AA2n=20) and wild cabbage (Brassica Olercea, CC 2n=18) and being come.
DHHC (Asp-His-His-Cys) type Zinc finger domain is a kind of Zinc finger domain being rich in halfcystine high conservative, often appears at a kind of structural motif in DBP.Belong to class C
2h
2type zinc refers to, its sequence signature is: C-X
2-C-X
9-HC-X
2-C-X
4-DHHC-X
5-C-X
4-N-X
3(C represents halfcystine to-F; H represents Histidine; X represents arbitrary amino acid).
At present, mainly concentrate on yeast and Mammals to the research of DHHC type Zinc finger domain, the DHHC type zinc finger protein research in plant is relatively less.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of plant composing type Overexpression vector, transgene rape, be intended to pass through obtained plant composing type Overexpression vector and build the new rape variety that a kind of branches and output all increases.
The present invention is achieved in that a kind of plant composing type Overexpression vector, the fusion gene that this expression vector drives OsDHHC1 gene and gus gene to be formed by 35S promoter, i.e. 35S::OsDHHC1-GUS; Wherein, described OsDHHC1 gene is another montage mode of the Os02g0819100 gene of encoding D HHC type zinc finger protein in paddy rice, and its base sequence is as shown in SEQ ID NO:1.
Invention further provides the construction process of above-mentioned plant composing type Overexpression vector, comprise the following steps:
(1) take rice cDNA as template, carry out pcr amplification by primer; Wherein, described primer is:
OsDHHC1 gene primer-F:
5’-CATGCATGCCATGGTGCTGGCACATATATCTCTTATGTC-3’，
OsDHHC1 gene primer-R:
5’-GGAAGATCTGGAAGATCTGTTGTTTGTGATCTGGAACTCAGTG-3’；
(2) pcr amplification product in step (1) is cloned in pUCm-T carrier, after order-checking qualification, with NcoI and BglII double digestion pUCm-T carrier and pCAMBIA1301 carrier, reclaims small segment and large fragment respectively, then connect;
(3) connection product conversion bacillus coli DH 5 alpha step (2) obtained, obtains positive colony through antibiotic-screening; Extract the plasmid of positive colony, obtain plant composing type Overexpression vector.
Preferably, in step (3), the method for described conversion is agrobcterium-mediated transformation.
Preferably, in step (3), the method of described conversion is by the electroporated Agrobacterium GV3101 of described plant composing type Overexpression vector, obtains positive colony, then carry out Agrobacterium-mediated genetic transformation through 100 μ g/ml Rifampins and the screening of 50 μ g/ml kantlex.
Invention further provides a kind of branch and the preparation method of high yield rape, comprise the following steps:
(1) swede type rape plant is transformed by above-mentioned plant composing type Overexpression vector;
(2) obtain obtaining branch and high yield rape by resistance screening and RT-PCR qualification.
Preferably, in step (1), described conversion adopts agriculture bacillus mediated transgenic method.
Preferably, described step (2) specifically comprises:
Carry out hygromycin resistance screening to T1 for seedling, the resistance seedling of acquisition carries out individual plant sowing;
For seedling, hygromycin resistance screening is proceeded to T2, obtains homozygous transgenic strain;
Adopt RT-PCR method to identify the homozygous transgenic strain obtained, obtain the transgenic homozygote strain that goal gene OsDHHC1 expression amount obviously increases.
For overcoming the shortcoming and defect of prior art, the invention provides a kind of plant composing type Overexpression vector, branch and the preparation method of high yield rape, utilize the DHHC type zinc finger protein gene OsDHHC1 of paddy rice, build the plant constitutive expression carrier of this gene, and transformed swede type rape, by resistance screening, RT-PCR qualification waits the transfer-gen plant obtaining branches and increase.Analyze and find, the output increase more obvious than wild-type of transfer-gen plant, namely the present invention has cultivated a multi-branched and the new rape variety of high yield by genetic transformation.
Accompanying drawing explanation
Fig. 1 is the structural representation of the T-DNA expression cassette of Ti class plasmid pCAMBIA1301 carrier of the present invention;
Fig. 2 is the structural representation that the present invention contains the T-DNA expression cassette of the Ti class plasmid pCAMBIA1301 carrier of OsDHHC1 gene; Wherein, 35S:CaMV 35S promoter; OsDHHC1: the gene of encoding D HHC type zinc finger protein in paddy rice; HygR: Hygromycin resistance marker's gene; GUS: beta-glucosiduronatase gene; LB and RB: the left margin and the right margin that are respectively T-DNA; MCS: multiple clone site; NcoI and BglII: by gene constructed for OsDHHC1 restriction enzyme site used on pCAMBIA1301 carrier;
Fig. 3 is branch and the form comparison diagram of high yield rape and wild-type rape in the present invention; Wherein, WT is wild-type to A, and 335S::OsDHHC1-GUS-1,35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3 are three different strains of transgene rape;
Fig. 4 is branch and the branches comparison diagram of high yield rape and wild-type in the present invention; Wherein, WT is wild-type, and 335S::OsDHHC1-GUS-1,35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3 are transgene rape; PCAMBIA1301 is the transfer-gen plant of pCAMBIA1301 zero load;
Fig. 5 is branch and the GUS Histochemical localization detected result figure of high yield rape and wild-type rape in the present invention; Wherein, WT is wild-type; 335S::OsDHHC1-GUS is branch and high yield rape;
Fig. 6 is branch and the detected result figure of Hyg gene in high yield rape in the present invention; Hyg gene is hygromycin selectable marker gene;
Fig. 7 is branch and the Semiquatitative RT-PCR assay qualification result figure of high yield rape in the present invention;
Fig. 8 is branch and the detection of the OsDHHC1-GUS gene of high yield rape in the present invention;
Fig. 9 is branch of the present invention and the Yield compari@figure of high yield rape and wild-type; Wherein, WT is wild-type, and 35S::OsDHHC1-GUS-1,35S:::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3 are transgene rape; PCAMBIA1301 is the transfer-gen plant of pCAMBIA1301 zero load.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1 branch and the preparation of high yield rape
1, the structure of OsDHHC1 gene clone and 35S::OsDHHC1-GUS expression vector
Paddy rice DHHC type zinc finger protein gene OsDHHC1 is the gene of contriver's new clone, and be another the montage mode of Os02g819100 in NCBI, its base sequence is as shown in SEQ ID NO:1.Adopt primer5 software design PCR primer, primer is as follows:
OsDHHC1 gene primer-F:
5’-CATG
CATGCCATGGTGCTGGCACATATATCTCTTATGTC-3’，
OsDHHC1 gene primer-R:
5’-GGA
AGATCTGGAAGATCTGTTGTTTGTGATCTGGAACTCAGTG-3’，
Above-mentioned two primer dashed part are NcoI and the BglII restriction enzyme site introduced.With the cDNA of rape for template, utilize above-mentioned primer, carry out pcr amplification.First the PCR primer obtained is cloned in pUCm-T carrier (being purchased from the raw Symbiont Engineering Co., Ltd in Shanghai), after order-checking qualification, with NcoI and BglII double digestion pUCm-T carrier and pCAMBIA1301 carrier (Fig. 1), reclaim small segment and large fragment respectively, then connect.Connect product conversion bacillus coli DH 5 alpha, obtain positive colony through microbiotic (50 μ g/ml kantlex) screening.Extract the plasmid of positive colony, obtain 35S::OsDHHC1-GUS expression vector (Fig. 2).
2, the conversion of rape
According to BIO-RAD electroporated instrument specification sheets by electroporated for 35S::OsDHHC1-GUS expression vector Agrobacterium GV3101, obtain positive colony through microbiotic (100 μ g/ml Rifampins and 50 μ g/ml kantlex) screening.The mono-clonal of the picking positive puts (containing 100 μ g/ml Rifampins and 50 μ g/ml kantlex) in 20ml LB liquid nutrient medium, and 28 DEG C shake cultivation 24 hours.Bacterium liquid is pressed the dilution proportion of 1: 100, get 200 μ l dilute the coating of bacterium liquid fresh containing antibiotic YEB solid plate, cultivate 2 ~ 3 days for 28 DEG C, with the conversion fluid of 150mL (containing the 1/2MS liquid nutrient medium of 5% sucrose, 0.05%Silwet-77,0.4% Syringylethanone, 0.002 ‰ 6-BA, PH5.8) Agrobacterium in culture dish is eluted, be configured to the Agrobacterium-mediated Transformation bacterium liquid of OD600 ≈ 0.8.Loaded by transformed bacteria liquid in plastics bag, then grab into a branch of by removing the brassica napus inflorescence of fruit pod with open flower, stretch into plastics bag gently, allow transformed bacteria liquid fully soak inflorescence, the time is 30 seconds.Inflorescence after immersion is immediately with the wrapping of sheepskin paper bag.Repeat as stated above after 2 ~ 3 days to transform once, same inflorescence total immersion bubble conversion 3 times.After completing last 7 days of transforming, extract the sheepskin paper bag on inflorescence.After seed maturity, results are dried, and obtain T0 for transgenic seed.
3, branch and the screening of high yield rapeseed plants
By branch and high yield Semen Brassicae campestris sowing in soil, spray Totomycin at Seedling Stage by 1: 200 (V/V), sprayed once every 2 ~ 3 days, continuous spraying 4 ~ 5 times.When the major branch of the rape of hygromycin and part side shoot budding and have several little the flowers are in blossom put time, with the wrapping of sheepskin paper bag, to avoid hybridization.After seed maturity, individual plant sowing is carried out to it, obtains T1 for seed, be stored in after drying-20 DEG C for subsequent use.To T2 for seedling continuation hygromycin selection, the strain that Resistant segregation no longer occurs is homozygous transgenic strain.The form of the transgene rape embodiment of the present invention obtained and wild-type rape contrasts, as shown in Figure 3.Choose three different branches and high yield rape strain 335S::OsDHHC1-GUS-1,35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3, by above three different branches and high yield rape strain plant and wild-type rape branches compare, result is as shown in Figure 4.
Embodiment 2GUS Histochemical localization detects
1, the preparation of GUS staining fluid
The preparation of GUS staining fluid used operates with reference to the formula in the Ph D dissertation of Deng Ke duty and Wang Qiming etc.
2, rape sheet dyeing process
Carry out operation with reference to the method in the Ph D dissertation of Deng Ke duty and Wang Qiming etc. and improved.First, get the seedling of the transformed plant of the gus reporter gene of the OsDHHC1 promoters driven of having cultivated 40d, be cut into the fragment that 2cm is long, be placed in 1.5ml centrifuge tube.Add GUS staining fluid submergence seedling again, vacuum infiltration (80kPa) 3h, then be placed in 37 DEG C of insulation 16 ~ 24h.Then use 70% ethanol rinse 3 times, finally take out under complete seedling is placed in Stereo microscope and observe coloration result.As shown in Figure 5, wherein, WT is wild-type to coloration result; 335S::OsDHHC1-GUS is branch and high yield rape.As can be seen from Figure 5 can see the existence of obvious sapphirine material in 335S::OsDHHC1-GUS plant, and not have in wild-type, the transgene rape plant obtaining 335S::OsDHHC1-GUS is described.
Embodiment 3 Hyg gene test
Choose three different branches and high yield rape strain 335S::OsDHHC1-GUS-1,35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3, and with wild-type rape strain WT for contrast, extract the DNA of the blade of every strain by CTAB method and carry out PCR detection with the primer of amplification Totomycin fragment (Hyg):
Hyg-F：5’-CTTCTGCGGGCGATTTGT-3’，
Hyg-R：5’-GCCGTGGTTGGC TGTATG-3’。
PCR response procedures: denaturation 94 DEG C of 3min; Sex change 94 DEG C of 30s, anneal 58 DEG C of 30s, extends 72 DEG C of 30s, 35 circulations; Rear extension 5min.
Electrophoresis detection is carried out to PCR result, detected result as shown in Figure 6, as can be seen from the figure, Hyg gene has been amplified from hygromycin resistance plant, explanation obtains containing Hyg gene transgenic plant, and 35S::osDHHC1-GUS and Hyg gene is structured on same carrier, so, obtain the transgene rape plant of 335S::OaDHHC1-GUS.
Embodiment 4 Semiquatitative RT-PCR assay is identified
In order to do RT-PCR qualification further to acquisition homozygous transgenic strain, choose three different branches and high yield rape strain 335S::OsDHHC1-GUS-1,35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3, first by homozygote branch and high yield Semen Brassicae campestris be seeded in basin dress compost in, cultivate under long-day conditions after 30 days, get blade, immediately with after liquid nitrogen flash freezer, RNA easy Mini Kit (An Biao company) is adopted to extract total serum IgE.After illustrating that use DNase (Promega, USA) removes the DNA in total serum IgE according to reagent operation, M-MLV Transcription System (Promega, USA) is adopted to synthesize cDNA.
When detecting the expression level of OsDHHC1 gene, the cDNA template of synthesis is diluted 10 times, the cDNA template that 1 μ l dilutes is added in 20 μ lPCR reaction systems, 25mM magnesium ion 1.2 μ l, 2.5 μMs of forward primer 1.5 μ l, 2.5 μMs of reverse primer 1.5 μ l, Taq archaeal dna polymerase 1 μ, then adds aseptic ddH
2o to 20 μ l.PCR response procedures is: 95 DEG C of denaturation 5min; Then 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 30s; Cycle number is 24.The PCR primer of rape house-keeping gene BnACTIN is as endogenous control, and the sequence accession number of this gene in NCBI website is AF111812.The RT-PCR reaction of each test at least in triplicate.PCR primer sequence is as follows:
OsDHHC1-F：5’-GACCCGCAGCGGCAGGGATTAAA-3’，
OsDHHC1-R：5’-TTTGTAGTTTGCATAGCCCACGCAG-3’。
BnACTIN-F：5’-TCCCTCAGCACTTTCCAACAG-3’，
BnACTIN-R：5’-AAGGACCAGAGCATCATCACAAG-3’；
After reaction terminates, get 16 μ l PCR reaction solutions, the sepharose of 1.5% carries out electrophoresis, and result as shown in Figure 7.As can be seen from the figure OsDHHC1 gene has a large amount to express in 335S::OsDHHC1-GUS-1,35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3 tri-transfer-gen plants, and for the expression of OsDHHC1 gene being detected in wild-type, the transgene rape plant obtaining 335S::OsDHHC1-GUS is described, OsDHHC1 gene a large amount in transfer-gen plant is expressed.In order to verify whether goal gene OSDHHC1 is expressed in transfer-gen plant body further, the primer devising the fusion gene of OSDHHC1 and GUS two that can increase for a pair is verified.Primer sequence is: OSDHHC1+GUS-F:5 '-ATGGCGCGGAGGAGGGGAA--3 ', the cDNA template that 1 μ l dilutes is added in OSDHHC1+GUS-R:5 '-ACCTCTCTTTAGGCATTGGTTTCGAAGC-3 ' .CR reaction system, 25mM magnesium ion 1.2 μ l, 2.5 μM forward primer 1.5 μ l, 2.5 μM reverse primer 1.5 μ l, Taq archaeal dna polymerase 1 μ, then adds aseptic ddH
2o to 20 μ l.PCR response procedures is: 95 DEG C of denaturation 5min; Then 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 30s; Cycle number is 30.Result as shown in Figure 8, transgene rape 35S::OsDHHC1-GUS-1, OSDHHC1+GUS gene can be amplified in 35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3, and in wild-type rape, have no the amplification of OSDHHC1+GUS gene, show that OsDHHC1 gene obtains expression in transgene rape.
Embodiment 5 determination of yield
Wild-type and transgenic seedlings are seeded in irrigable soil.This block ground is divided into four parts, and every part is five fritters, and being used for respectively planting containing 35S::OsDHHC1-GUS-1,35S::OsDHHC1-GUS-2 and 35S::OsDHHC1-GUS-3pCAMBIA1301 is that the T4 of pCAMBIA1301 is for transgene rape; .Seedling thickness of sowing is 30 strains/fritter, and the length and width distance between seedling is respectively 0.5 meter and 0.24 meter.Time seed maturity, picked by hand, dries, and threshing also weighs in the balance heavily.From in the beginning of October, 2010 to 2013, once, this experiment is repeated altogether three times to annual kind.Collection data are made chart, as shown in Figure 9, the individual plant fruit folder number increase more obvious than wild-type of 35S::OsDHHC1-GUS transfer-gen plant can be found out from Fig. 9 A, the increase more obvious than wild-type of the single plant yield of 35S::OsDHHC1-GUS transfer-gen plant can be found out from Fig. 9 B, the increase more obvious than wild-type of the cell production of 35S::OsDHHC1-GUS transfer-gen plant can be found out from Fig. 9 C.
Compared to the shortcoming and defect of prior art, the present invention has following beneficial effect: the present invention adopts agriculture bacillus mediated transgenic method that OsDHHC1 is imported vegetable cell, the output of rape is regulated by regulating plant branch, cultivate the new rape variety of a multi-branched high yield, adopt the Measures compare of statistical study to measure transgenic homozygote plant and WT lines, new rape variety of the present invention all has larger lifting in branches and output.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. a kind of plant composing type Overexpression vector, is characterized in that, the fusion gene that this expression vector drives OsDHHC1 gene and gus gene to be formed by 35S promoter, i.e. 35S::OsDHHC1-GUS; Wherein, described OsDHHC1 gene is another montage mode of the Os02g0819100 gene of encoding D HHC type zinc finger protein in paddy rice, and its base sequence is as shown in SEQ ID NO:1.
2. the construction process of plant composing type Overexpression vector according to claim 1, is characterized in that, comprise the following steps:
(1) take rice cDNA as template, carry out pcr amplification by primer; Wherein, described primer is:
OsDHHC1 gene primer-F:
5’-CATGCATGCCATGGTGCTGGCACATATATCTCTTATGTC-3’，
OsDHHC1 gene primer-R:
5’-GGAAGATCTGGAAGATCTGTTGTTTGTGATCTGGAACTCAGTG-3’；
(2) pcr amplification product in step (1) is cloned in pUCm-T carrier, after order-checking qualification, with NcoI and BglII double digestion pUCm-T carrier and pCAMBIA1301 carrier, reclaims small segment and large fragment respectively, then connect;
(3) connection product conversion bacillus coli DH 5 alpha step (2) obtained, obtains positive colony through antibiotic-screening; Extract the plasmid of positive colony, obtain plant composing type Overexpression vector.
3. the construction process of plant composing type Overexpression vector as claimed in claim 2, it is characterized in that, in step (3), the method for described conversion is agrobcterium-mediated transformation.
4. the construction process of plant composing type Overexpression vector as claimed in claim 3, it is characterized in that, in step (3), the method of described conversion is by the electroporated Agrobacterium GV3101 of described plant composing type Overexpression vector, obtain positive colony through 100 μ g/ml Rifampins and the screening of 50 μ g/ml kantlex, then carry out Agrobacterium-mediated genetic transformation.
5. branch and a preparation method for high yield rape, is characterized in that, comprise the following steps:
(1) swede type rape plant is transformed by plant composing type Overexpression vector described in claim 1;
(2) obtain obtaining branch and high yield rape by resistance screening and RT-PCR qualification.
6. branch as claimed in claim 5 and the preparation method of high yield rape, it is characterized in that, in step (1), described conversion adopts agriculture bacillus mediated transgenic method.
7. branch as claimed in claim 6 and the preparation method of high yield rape, it is characterized in that, described step (2) specifically comprises:
Carry out hygromycin resistance screening to T1 for seedling, the resistance seedling of acquisition carries out individual plant sowing;
For seedling, hygromycin resistance screening is proceeded to T2, obtains homozygous transgenic strain;
Adopt RT-PCR method to identify the homozygous transgenic strain obtained, obtain the transgenic homozygote strain that goal gene OsDHHC1 expression amount obviously increases.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255940A (en) * | 2015-11-27 | 2016-01-20 | 中南林业科技大学 | Plant composition type oleic acid expression vector and construction method and oilseed rape conversion method thereof |
| CN108374019A (en) * | 2018-01-30 | 2018-08-07 | 西安泽睿环境科技有限公司 | Turn the initiative and application of the heavy metal super-enriched transgenic engineering rape of Nipponbare rice Os Nramp5 genes |
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| CN102229661A (en) * | 2011-06-03 | 2011-11-02 | 湖南大学 | DHHC-type zinc finger protein gene for controlling rice tillering and application of DHHC-type zinc finger protein gene |
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2014
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102229661A (en) * | 2011-06-03 | 2011-11-02 | 湖南大学 | DHHC-type zinc finger protein gene for controlling rice tillering and application of DHHC-type zinc finger protein gene |
Non-Patent Citations (1)
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| 周波: "DHHC型锌指蛋白基因OsDHHC1在水稻株型构建中的功能分析", 《中国博士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255940A (en) * | 2015-11-27 | 2016-01-20 | 中南林业科技大学 | Plant composition type oleic acid expression vector and construction method and oilseed rape conversion method thereof |
| CN105255940B (en) * | 2015-11-27 | 2018-06-19 | 中南林业科技大学 | A kind of method of plant composing type oleic acid expression vector and its construction method and conversion rape |
| CN108374019A (en) * | 2018-01-30 | 2018-08-07 | 西安泽睿环境科技有限公司 | Turn the initiative and application of the heavy metal super-enriched transgenic engineering rape of Nipponbare rice Os Nramp5 genes |
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Application publication date: 20150701 |