CN109112141A - Influence the clone of the molecular chaperone protein OsATX1 of copper transhipment and distribution and application in rice body - Google Patents
Influence the clone of the molecular chaperone protein OsATX1 of copper transhipment and distribution and application in rice body Download PDFInfo
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Abstract
The invention belongs to field of plant genetic project technology.More particularly to clone and the application for influencing copper transhipment and the molecular chaperone protein OsATX1 distributed in rice body.The present invention includes rice copper transhipment subbase because of the separation clone of the DNA fragmentation of OsATX1 and functional verification.OsATX1 gene encodes a kind of copper ion chaperone gene, and the sequence of the protein of genetic fragment coding is as shown in SEQ ID NO:3.The segment and its exogenous regulatory sequences are directly transferred to rice, the transgenosis of overexpression OsATX1, rice significantly increases transport of the copper from root to overground part and it is shifted into tender tissue and seed.The present invention is expected by improving the copper concentration in rice grain, and then guarantees the nutritional quality of rice.
Description
Technical field
The present invention relates to field of plant genetic project technology.Influence what copper in rice body was transported and distributed more particularly to one
The clone of molecular chaperone protein OsATX1 and application.The present invention include the separation of rice copper ion chaperone OsATX1, clone,
Functional verification and application.OsATX1 is copper ion chaperone important in rice, and overexpression OsATX1 gene leads to rice
Copper content in seed significantly rises.Therefore, it is expected to by planting the rice material of overexpression OsATX1, increase in rice
Copper content, and then guarantee the nutritional quality of crops.
Background technique
Copper (Cu) is one of microelement necessary to biology growing is developed, during the growth and development of organism
It plays a significant role.The shortage of copper can be detrimental to health, and cause immune deficiency (Collins and Klevay, 2011).?
In plant, copper as a kind of various vital movements of confactor wide participation, such as: the redox in respiratory metabolism is anti-
Answer, photosynthetic electron transfer process, cell wall rebuild, oxidative stress reaction and ethylene protection etc. (Maksymiec, 1997；
Pilon et al.,2006；Yruela,2009).When lacking copper, the characters such as plant performance is to grow thickly, and top gradually bleaches are led to
Often bleach since blade tip, can be reduced when serious plant setting percentage (Burkhead et al., 2009；Huang et al.,
2016).However, excessive copper can also generate great toxicity to plant, such as generate highly reactive form of oxygen (ROS) and cellular damage etc.
(Leng et al.,2015).Therefore, the absorption and distribution of copper must be strictly controlled in plant body.
Rice (Oryza sativa L.) is one of most important cereal crops in the whole world.It is continuous with living standard
It improves, requirement of the people to rice yield and Nutrition Quality of Rice also increasingly increases.The absorption and distribution of copper ion be in rice
Influence a key factor of rice yield and Nutrition Quality of Rice.Since copper ion is to plant integration peptide, metallothionein etc.
With high-affinity, the amount of free copper ion may be limited in each cell less than an ion (Rae et in cytoplasm
al.,1999).It is rapidly absorbed into the copper ion of rice root cells, a part is isolated in vacuole, and another part is made in transpiration
With under the traction with respiration, be transported to overground part and be assigned in different organ and tissue (Yamaji and Ma,
2014；Sasaki et al.,2016).
But people do not know to what the molecular mechanism of rice absorbing and distribution copper ion understood at present.Therefore, pass through
Transgene method parses copper ion in the transhipment and distribution mechanism of plant, while guaranteeing the nutritional quality of crops, has
Important meaning.
The present invention is genetic background material, discovery overexpression coding using 11 (non-transgenic rice) are spent in rice varieties
The gene OsATX1 of copper ion chaperone can significantly increase transport of the copper from root to overground part and its to tender tissue and seed
The transfer of grain.By improving the copper concentration in rice grain, and then it ensure that the nutritional quality of crops, this is the quality of rice
Breeding provides new thinking.
Summary of the invention
The purpose of the present invention is being to overcome the deficiencies of existing technologies, copper transhipment and distribution in a kind of influence rice are provided
Copper ion chaperone gene and its application.This gene is named as OsATX1.Overexpression OsATX1 in rice, discovery
It is 26.73mg/kg DW that the copper concentration in 11 plant roots is spent in vegetative growth of rice plants phase (4 weeks seedling ages), wild type.Excess table
It is 18.4mg/kg DW up to the copper concentration in positive plant root, reduces 31.16% than WT lines (non-transgenic rice).
The copper concentration of WT lines overground part is 9.99mg/kg DW；The copper concentration of overexpression positive plant overground part is 15mg/
Kg DW increases 50.15% than wild type (non-transgenic rice).It can be seen that overexpression OsATX1 significantly increases copper
From root to the transport of overground part.Maturity period, the copper concentration in overexpression positive plant root are also significantly lower than wild type, and on the ground
Copper concentration in portion tenderer tissue and seed is significantly higher than WT lines.Wherein, the copper concentration in WT lines seed
For 11.72mg/kg DW.Copper concentration in overexpression positive plant seed is 2.55 times of wild type (non-transgenic rice),
Be conducive to the accumulation of copper in rice.
Technical scheme is as follows:
The present invention carries out transgenosis overexpression and tests using the DNA fragmentation comprising OsATX1 gene as the gene of application
The gene is transferred in rice and spends 11 by card, and overexpression positive plant shows copper accumulation in seed and dramatically increases.
Copper ion chaperone is responsible for the transhipment and distribution of copper ion in plant body, has to the balance of copper ion in plant body
Significance.Applicant utilizes one section of cDNA sequence of the copper ion chaperone Gene A tATX1 of arabidopsis, in the website NCBI
(https://www.ncbi.nlm.nih.gov/) on carry out BLAST analysis, obtain Serial No.AP003875.3One section volume
The DNA sequence dna of the gene OsATX1 of code copper ion chaperone.The nucleotides sequence of OsATX1 full-length genome be classified as 1956bp (see
Sequence table SEQ ID NO:1), encode the amino acid sequence of 81 protein (see sequence table SEQ ID NO:3).The gene includes
3 exons and 2 intrones.The present invention is by PCR method, to spend 11 cDNA to amplify in rice varieties as template
The full length coding region of OsATX1 gene (see SEQ ID NO:2)；It, will using Gateway system (Gong et al., 2013)
OsATX1 gene is connected on purpose carrier (pJC034).Agrobacterium-mediated genetic transformation is carried out again, in rice transformation kind
11 are spent, OsATX1 overexpression plant is obtained.In transgenosis T2For being found in plant, OsATX1 overexpression positive plant seed
In copper accumulation be significantly higher than WT lines (non-transgenic rice).
The present invention has the advantages that
The present invention has separated a kind of DNA fragmentation comprising OsATX1 gene and has identified its function, which influences rice body
It is spent in 11 (non-transgenic rice) in the transhipment and distribution kind of interior copper, by rice overexpression coding copper ion companion
After the gene OsATX1 of albumen, it is found that the copper concentration in overexpression positive plant root is substantially less than wild type, and overground part compared with
Tender tissue copper concentration content is significantly higher than WT lines.After tested, the copper concentration in WT lines seed is
11.72mg/kg DW, the copper concentration in overexpression positive plant seed are 2.55 times of WT lines, are conducive in rice
The accumulation of copper.
The present invention gene OsATX1 that overexpression one influence rice copper is transported and distributed in rice for the first time.For rice
It cultivates fine quality and provides new thinking,.
The genetic fragment that the present invention clones can be the research of the Nutrition and Metabolism of the cereals section crops such as rice and other crops
Support is provided.
Detailed description of the invention
SEQ ID NO:1 is full length nucleotide sequence (the wherein base 1-9 of OsATX1 gene；828-903 and 1796-
1956 sequence is the code area of the genetic fragment) sequence length be 1956bp.
SEQ ID NO:2 is the CDS sequence of OsATX1 gene, and sequence length 246bp encodes the amino of 81 protein
Acid sequence.SEQ ID NO:3 is the protein sequence of OsATX1 gene coding.
Fig. 1: general technical route map of the invention.
The structure of Fig. 2: OsATX1 gene.Description of symbols: " ATG " and " TAA " is translation initiation password and end respectively
Only password.Long arrow between " ATG " and " TAA " indicates the nucleotide area of OsATX1 gene.Wherein, orange is identified as coding
Area.Green arrow indicates 5 and 3 non-translational regions (untranslated region, UTR).
Fig. 3: with the expression of OsATX1 gene after quantitative RT-PCR technology analysis copper stress (lacking copper or excess copper) processing
Variation.Description of symbols: the A figure in Fig. 3 is to lack OsATX1 gene after copper (the miscellaneous luxuriant and rich with fragrance disulfonic acid disodium salt of 2 μm of ol) processing 7d
Expression variation；B figure in Fig. 3 is OsATX1 gene after various concentration copper (0.2,2,10,20 and 50 μm of ol) Stress treatment 7d
Expression variation；C figure in Fig. 3 is the expression variation of 20 μm of ol copper Stress treatment different time points OsATX1 genes.Standard deviation base
It is repeated in three biology, each sample is the aggregate sample of 3 plants of materials.* P < 0.05 * and (the t inspection of P < 0.01 * * are respectively represented with * *
It tests).
Fig. 4: being the structural schematic diagram of genetic transformation carrier pM999 of the invention.
Fig. 5: fusion protein OsATX1-GFP is located in the cytoplasm and nucleus of paddy rice green protoplast cell.Attached drawing
Description of symbols: using 35S:OsATX1-GFP and 35S:AtATX1-mCherry carrier cotransformation in paddy rice green protoplast
In.AtATX1-mCherry is a label of cytoplasm and nucleus.A figure in attached drawing 5 shows 35S:OsATX1-GFP
In cytoplasm and nucleus；B figure in Fig. 5, display control 35S:AtATX1-mCherry are located at cytoplasm and nucleus.Attached drawing
C figure in 5, is the light field cell under the same visual field with the A figure in Fig. 4.D figure in Fig. 5 melts for the A figure in Fig. 5 with B figure
Cell under conjunction state.Scale is 10 μm.
Fig. 6: the structural schematic diagram of genetic transformation overexpression vector pJC034.
Fig. 7: T2For the OsATX1 gene expression amount in genetic transformation plant.Control is that 11 (heredity are spent during rice varieties are
The receptor of conversion).Overexpression plant is OX-1, OX-2 and OX-3.Standard deviation is based on three biology and repeats, and each sample is
The aggregate sample of 3 plants of materials.* P < 0.05 * and P < 0.01 * * (t inspection) are respectively represented with * *.
Fig. 8: T2For the content of the intracorporal copper in genetic transformation plant.Description of symbols: in the A figure and Fig. 8 in Fig. 8
B figure be 4 weeks seedling age phase plant above ground portion's copper content testings of vegetative growth of rice plants phase as a result, wherein the A figure in Fig. 8 is 4 weeks
The content of seedling age rice plant overground part copper；B figure in Fig. 8 is the content of the copper in 4 weeks seedling age rice plant roots；C in Fig. 8
Figure is the content of copper in rice maturity seed.Check variety is to spend 11 in rice；Overexpression plant be OX-1, OX-2 and
OX-3.Standard deviation is based on three biology and repeats, and each sample is the aggregate sample of 3 plants of materials.* P < 0.05 * is respectively represented with * *
With P < 0.01 * * (t inspection).
Specific embodiment
Following embodiment further defines the present invention, and describes separation OsATX1 gene, genetic transformation and copper
The measuring method of content and the expression pattern of the gene in rice.According to description below and these implementation examples, this field
Technical staff can determine essential characteristic of the invention, and without departing from the spirit and scope of the invention, can be right
The present invention makes various changes and modifications, so that it is applicable in various uses and condition.
The sequence and structural analysis of embodiment 1:OsATX1 gene
The prediction of OsATX1 gene structure:
Applicant of the present invention is used using one section of cDNA sequence of the copper ion chaperone Gene A tATX1 of arabidopsis
BLAST method retrieval RiboaptDB GenBank (http://www.ncbi.nlm.nih.gov), find the cDNA sequence
With one from rice varieties OryzasativaLcv.Nipponbare be located at No. 8 chromosomes of rice, long 126112bp sequence (GenBank number of registration:AP003875.3) in one section of (the 16242nd to 18197 place) very high homology.Rice genome database TIGR (http:// rice.plantbiology.msu.edu/) in, the rice sequences of this segment and AtATX1 DNA homolog are noted as a base
Cause, number is LOC_Os08g10480.This unnamed gene is OsATX1 gene (Antioxidant by applicant
protein1).OsATX1 full-length genome sequence is 1956bp, encodes 81 amino acid.The gene includes 3 exons and 2
A introne (see Fig. 2, nucleotide Xu sequence and code area are as shown in SEQ ID NO:2).
1, separation, clone's OsATX1 gene from rice varieties " in spend 11 "
It is middle that spend 11 (ZH11) be a rice varieties commonly used in the art.Applicant is according to OsATX1 gene in japonica rice
Position and structure (rice genome database TIGR) in " OryzasativaLcv.Nipponbare " genome, with the genome sequence of OsATX1 gene two sides
It is classified as template, devises two PCR primers i.e.: OsATX1-oxF (5 '-AAAAAGCAGGCTTAATGGCTGAGACTGTTGTGCTC-3 ') (underscore represents the site attB1) and OsATX1-oxR (5 '-AGAAAGCTGGGTATTCCACTGGGAAATTGTTACATC-3 ') (underscore represents the site attB2), it therefrom spends in 11 and expands
It obtains comprising OsATX1 gene, the DNA fragmentation that length is 330bp, it includes complete OsATX1 gene order.
2, the structural analysis of OsATX1 gene
In order to obtain OsATX1 full length gene cDNA sequence, 11 total serum IgE is spent in applicant's extraction rice varieties.Specific step
Suddenly are as follows: take 3~5g total serum IgE DNaseI (Invitrogen company of the U.S.) processing 15min to remove contaminating genomic DNA, so
Afterwards referring to the method for the report such as Zhou (2002), use oligo (dT)15Oligomer primers and the M-MLV reverse transcriptase (U.S.
Promega company) reverse transcription is carried out into cDNA.Then PCR primer OsATX1-F and OsATX1-R are utilized, using total cDNA as mould
Plate amplifies the full-length cDNA of OsATX1 gene.Sequencing analysis is carried out to PCR product, obtains the cDNA sequence of OsATX1 gene.
The genome sequence and cDNA sequence of comparative analysis OsATX1 gene determine OsATX1 gene by 1956 nucleotide
Composition only includes 3 exons, and 2 containing sub (Fig. 2).The cDNA sequence of OsATX1 gene is made of 246 nucleotide (to be located at
At the 1-9bp of sequence table SEQ ID NO:1,828-903bp, 1796-1956bp), 81 amino acid are encoded altogether.
3, the coded product analysis of OsATX1 gene
With the method analysis of BLASTP (Altschul etc., 1997) report, the copper ion of OsATX1 protein and arabidopsis
The amino acid sequence similarity of chaperone AtATX1 is 87%.
Embodiment 2: the expression analysis of OsATX1 gene after copper Stress treatment
In order to verify the regulation whether OsATX1 gene participates in the transhipment of rice Copper uptakie, the present invention uses quantitative PCR first
(Quantitative real-time PCR, qRT-PCR) technology analyzes the reaction of OsATX1 gene pairs copper Stress treatment.This
Invention uses hydroponic method rice cultivation.Nutrient solution culture nutrient solution uses standard recipe.In standard liquid nutrient culture solution
Mantoquita (copper sulphate) content is 0.12 μm of ol.In the experiment of scarce copper Stress treatment, the miscellaneous phenanthrene of copper ion chelator is added in culture solution
Disulfonic acid disodium salt (BCS), miscellaneous phenanthrene disulfonic acid disodium salt concentration for the treatment of is 2 μm of ol.In the experiment of copper excess Stress treatment, culture
The concentration for the treatment of of copper is 20 μm of ol in liquid.11 Solution culture methods to 4 leaf phases will be spent to start to carry out copper Stress treatment in rice varieties.
It fetches water rice seedling (shoot) respectively in different time points after copper Stress treatment and root, extracted total RNA is simultaneously inverted
Record.Specific steps: take 3~5g total serum IgE DNaseI (Invitrogen company of the U.S.) processing 15min to remove genomic DNA
Pollution carries out reverse transcription using 15 oligomer primers of oligo (dT) and M-MLV reverse transcriptase (Promega company of the U.S.).Using reality
When quantitative PCR analysis kitGreen PCR Master Mix (purchased from precious bioengineering Dalian Co., Ltd), root
It is operated according to the operation instructions of kit, in ABI 7500Real-Time PCR system instrument (U.S. Applied
Biosystems company) on carry out real-time quantitative PCR reaction.Quantify gene expression (Livak and using relative quantitation method
Schmittgen,2001).It is measured with the expression quantity of rice ubiquitin (ubiquitin, ubi) gene and is uniformed sample RNA and contained
Amount.OsATX1 gene specific PCR primer in qRT-PCR analysis is OsATX1-qF (5 '-TTACACCAGATGCCGTTCTTC-
3 ') and OsATX1-qR (5 '-AGCAGCAGTAGCTTCAACAG-3 '), Maize Ubiquitin gene PCR primer are ubi-F (5 '-
AACCAGCTGAGGCCCAAGA-3 ') and ubi-R (5 '-ACGATTGATTTAACCAGTCCATGA-3 ').Quantitative RT PCR analysis
It has been shown that, under the processing of high concentration copper, OsATX1 gene is by inducing expression.This prompt OsATX1 gene may participate in Copper uptakie
The regulation of transhipment, is as a result shown in Fig. 3.
Embodiment 3:OsATX1 gene encoding production Subcellular Localization
1, the building of OsATX1 subcellular localization transient expression vector
Agrobacterium-mediated genetic transformation carrier used in the present invention is pM999 (structure is shown in Fig. 4).It carries CaMV35S
Promoter and green fluorescence protein gene (GFP) label.Using overall length OsATX1 gene cloning as template, expanded using round pcr
The code area segment DNA of OsATX1 gene.PCR primer is OsATX1-scF (5 '-CGGAATTCATGGCTGAGACTGTTGTGCTC-
3 ') (underscore represents EcoR1 digestion with restriction enzyme site) and OsATX1-scF (5 '-
GGGGTACCTTAAGATGAAGCAGCAGTAGCTTC-3 ') (underscore represents KpnI digestion with restriction enzyme site).It will
PCR product is connect with T-A cloning vector pGEM-T (being purchased from U.S. Promega company), and the carrier connected is carried out electrotransformation
(electric converter is eppendorf Products, and the present embodiment applied voltage is 1800V, and concrete operations refer to the use of the instrument
Specification) enter Escherichia coli, by digestion screening positive clone, then through sequence verification, whether there is or not base mutations.With restricted interior
The positive colony and pM999 carrier of code area of enzyme cutting EcoR1 and the KpnI digestion with OsATX1 gene.It is purified with digestion
The good carrier of DNA fragmentation and digestion comprising the gene coding region OsATX1 does connection reaction.It is screened by EcoR1 and KpnI digestion
The positive colony (Fig. 4) of exogenous sequences forward direction insertion.
2, the separation of paddy rice green protoplast, conversion and Subcellular Localization
11 seeds are spent to germinate in root media according to conventional Plant Tissue Breeding and genetic transforming method by middle, 28
DEG C illumination cultivation 10-15d.The separation and conversion of paddy rice green protoplast mainly carry out on the basis of the method that forefathers report
Optimization (Xie and Yang, 2013).
Reagent and instrument:
(1) plasmid of QIAGEN plasmid Midi Kit (Cat no.12143) purifying.
Prepare following mother liquor:
(1) 0.8mol Mannitiol (Sigma M1902, FW:182.17): 7.287g adds sterilizing ddH2O, constant volume arrive
50mL is dissolved in 55 DEG C of water-baths.
(2) 0.6mol Mannitiol (Sigma M1902, FW:182.17): 2.186g adds sterilizing ddH2O, constant volume arrive
20mL is dissolved in 55 DEG C of water-baths.Or it is directly diluted with 0.8mol Mannitiol.
(3) 0.2mol KCl (Sigma P5405, FW:74.55): 0.746g adds sterilizing ddH2O, constant volume to 50mL.
(4)1mol CaCl2·2H2O (Sigma C7902, FW:147.01): 7.35g adds sterilizing ddH2O, constant volume arrive
50mL。
(5)0.5mol MgCl2·6H2O (Sigma M2393, FW:203.3): 5.083g adds sterilizing ddH2O, constant volume arrive
50mL。
(6) 1.54mol NaCl (Sigma S6191, FW:58.44): 4.5g adds sterilizing ddH2O, constant volume to 50mL.
(7) 0.1mol MES-KOH, pH5.7:MES hydrate (Sigma M8250, FW:195.24): 0.195g, which adds, to go out
Bacterium ddH2O ,~150 μ L KOH (Sigma P5958) are adjusted to pH 5.7, and KOH is about 2mol, constant volume to 10mL.
Other reagents and instrument:
The present invention uses following instrument and equipments: Cellulose RS (Yakult Honsha)；Macerozyme R10
(Yakult Honsha)；BSA(SIGMA B4287)；β-Mercaptoethanol(SIGMA M3148)；PEG4000(SIGMA
81240)；Fetal Bovine Serum(gibco by life technologies,10099-133)；Adjustable acceleration-deceleration
Basket-hanging refrigerated centrifuge；2mL round bottom centrifuge tube (Eppendorf 0030123.344) and 50mL round bottom centrifuge tube
(Beckman 357006)。
2, operating procedure:
Enzymolysis liquid is prepared, ready-to-use:
The preparation method of 1 enzymolysis liquid of table
Concrete operation step:
(1) prepare enzymolysis liquid, take out 15 10-15 days seedling, in being cut on filter paper less than 0.5mm segment, be transferred in time
In 0.6mol Mannitol.
(2) after all seedling cut, 10min is balanced in 0.6mol Mannitol.Enzymolysis liquid is down to room temperature, is added
Subsequent reagent.Mannitol solution is sucked, room temperature enzymolysis liquid is added.It is placed in 30min in vacuum tank, is placed in room temperature, 50rpm enzymatic hydrolysis
3.5h。
(3) prepare PEG-CaCl2·2H2O solution, prepares 1mL, and ingredient is as shown in table 2.
2 PEG-CaCl of table2·2H2The ingredient of O solution
(4) when enzymatic hydrolysis is nearly completed, W5 and MMG solution is prepared, ingredient is shown in Table 3 and table 4.
The ingredient of 3 W5 solution of table
The ingredient of 4 MMG solution of table
(5) after the completion of digesting, after 10mL W5 is added, protoplast, release are discharged with 80rpm speed on horizontal shaker
10min。
(6) 100g, centrifugal filtration liquid 7min, acceleration-deceleration are set as slow at room temperature.Slowly Aspirate supernatant, not by institute
There is supernatant to suck.
(7) about 3mL W5 suspension protoplast is added, shakes gently centrifuge tube to shake scattered protoplast.It is primary to draw 10 μ L
Plastid suspension microscopy, adjustment protoplast concentration to 0.5-1x107/ mL sets Protoplast suspension in dark place, room temperature preservation
60min.Prepare the plasmid of conversion.
(8) in centrifuge, 100g is centrifuged 7min at room temperature, discards supernatant.Protoplast is suspended in MMG solution, and
Concentration is adjusted to 0.5-1x107/mL。
(9) 5 μ g plasmids are diluted to 10 μ L, 100 μ L Protoplast suspensions is added.After jog mixes, 110 μ L are added
PEG-CaCl2Solution flicks mixing.28 DEG C, dark in convert 10-15min.
(10) 440 μ L W5 solution are added, turn upside down to stop converting.
(11) in centrifuge, 100g is centrifuged 7min at room temperature, during which, handles 24 hole cell culture with 5% calf serum
Plate.It discards supernatant, 400 μ L WI solution is added, protoplast is resuspended, and be transferred into tissue culture plate.It is pre- in culture plate
400 μ L WI solution are first added.
(11) incubated at room temperature 12-16h collects protoplast.
The ingredient of 6 WI solution of table
(12) 12-16h is cultivated at room temperature, collects protoplast.
(13) expression of GFP is observed under laser confocal microscope.The result shows that OsATX1-GFP and arabidopsis
AtATX1-mCherry common location is in cytoplasm and nucleus (Fig. 5).
Embodiment 4: overexpression OsATX1 gene verifies its function in the transhipment of rice Copper uptakie
1. the building of overexpression genetic transformation carrier
Used carrier of the present invention is that (carrier of this experimental reconstruction, can be used for the overexpression of gene to pJC034, and Vector map is shown in
Fig. 6).The carrier carries the heredity with the mediated by agriculture bacillus of Maize Ubiquitin gene promoter of composing type and overexpression feature
Conversion carrier.Applicant is mixed by the PCR product of OsATX1 gene and donor vehicle pDONR207 (being purchased from Invitrogen company)
It closes, BP recombination mixed enzyme is added and carries out recombining reaction, can convert and obtain the entry vector with target gene.Then it is obtaining
To entry vector clone in, target gene is recombinated again on destination carrier pJC034 using LR enzyme (Gong et al.,
2013)。
2. genetic transformation and T0For the analysis of genetic transformation plant
By Agrobacterium, (EHA105 provides by the Australian laboratory CAMBIA, referring to New Agrobacterium
Helper plasmids for gene transfer to plants, 1993, Transgenic Res 2:208-218) it is situated between
Lead with rice transformation system, the plasmid correctly cloned is imported into rice varieties and is spent in 11, by preculture, infect,
Co-culture, screening has the callus of hygromycin resistance, is broken up again, is taken root, hardening and transplanting and other steps, obtain turning base
Because of plant.Rice (japonica rice subspecies) genetic conversion system that Agrobacterium (EHA105) mediates is mainly in the side that Hiei et al. is reported
It is optimized on the basis of method (Livak and Schmittgen, 2001).The present invention obtains 80 plants of independent transformation plant altogether.
Using Real-time PCR Analysis detection gene in the intracorporal expression quantity of plant.By obtained overexpression plant into
Row breeding, obtains T1And T2For transgenic plant.The results show that compared with spending 11 in control wild type (non-transgenic rice), sun
The expression quantity of the property intracorporal OsATX1 gene of transformed plant is increased substantially (Fig. 7).
3. the analysis of copper content in overexpression plant
Whether can enhance the ability to Copper uptakie and accumulation for verifying genetic transformation plant, the present invention is in T0Representative reaches
Measure the T of the 3 genetic transformation plant (number OsATX1.OX-1, OsATX1.OX-2 and OsATX1.OX-3) significantly improved2Generation
Transgenic positive family has carried out the analysis of copper content.In different growing (4 weeks seedling ages, heading stage and maturity period) to each group
It knits to be sampled and be analyzed with copper assay.Copper content testing method mainly optimizes in reported method
(Yang et al.,2013；Yang et al.,2014).
The results show that the copper concentration in the tenderer tissue and seed in the plant above ground portion of overexpression OsATX1 gene is all aobvious
It writes and improves (referring to Fig. 8).11 (non-transgenic rice) plant above ground are spent in vegetative growth of rice plants phase (4 weeks seedling ages), wild type
The copper concentration in portion is 9.99mg/kg DW, and the copper concentration in overexpression positive transgenic plant above ground portion is 15mg/kg DW, than
The copper concentration in 11 plant above ground portions is spent to increase 50.15% (the A figure in Fig. 8) in wild type.And the copper in WT lines root
Concentration is 26.73mg/kg DW.Copper concentration in overexpression positive transgenic plant root is 18.4mg/kg DW, compares wild type
Reduce 31.16% (the B figure in Fig. 8).These are the results show that overexpression OsATX1 gene can promote rice that will inhale in root
The copper ion of receipts is transported to overground part.And the copper concentration in the maturity period, overexpression positive transgenic plant root is also significant low
In wild type, and the copper concentration in the tenderer tissue and seed of overground part is then significantly higher than in wild type and spends 11 plant.Wherein, wild
It is 11.72mg/kg DW that the copper concentration in 11 plant seeds is spent in raw type, the copper in overexpression positive transgenic plant seed
Concentration is that 2.55 times (the C figures in Fig. 8) of 11 plant are spent in wild type, which is conducive to the accumulation of copper in rice.
Annex: related reagent and its preparation method:
65%HNO3: SIGMA-ALDRICH, USA, CAS:30709；
Deionized water: Millipore 18.2M Ω ultrapure water；
Outer titer: Agilent Environmental Calibration Standard, USA, CAS:5183-
4688；
Internal standard liquid: Agilent ICP-MS Internal Std Mix, USA, CAS:5188-6525；
Tune liquid: Agilent Tuning Solution for ICP-MS 7500cs, USA, CAS:5185-5959；
Bush branch and leaf Standard Reference Materials for Determination: the People's Republic of China (PRC), GBW07602 (GSV-1).
Instrument:
Microwave dissolver: CEM MARS 6, USA；
Icp ms (ICP-MS): Agilent 7700series ICP-MS, USA；
Micro-wave digestion and copper assay operating procedure:
(1) sample is placed on and dries 3d in 80 DEG C of baking ovens, until constant weight；Then sample is ground into carefully with tissue pulverizer
Powder.
(2) claim sample: weighing the plant tissue powder sample that 0.2g (being accurate to 0.0001g) dries to constant weight, pour into microwave and disappear
Liner bottom of the tube is solved, is careful not to be sticked to nozzle.
(3) it clears up: 10mL 65%HNO is added3, elastic slice is put, lid is covered tightly, is sequentially placed into resolution pipe support in order, is put
It is put into 6 microwave cavity of microwave dissolver MARS after full, fastens chamber door, run after recalling corresponding resolution program confirmation.
(4) it catches up with acid: being finished to the operation of micro-wave digestion program, when temperature is down to 80 DEG C or less, sample is taken out, in draught cupboard
It twists the lid off 160 DEG C and catches up with sour 40min.
(4) constant volume: when catching up with sour instrument temperature to be down to room temperature, by sample constant volume to 50mL, after placing clarification after mixing
Upper machine measures (ICP-MS).
Bibliography
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homeostasis.New Phytol 182:799-816；
2.Collins JF,Klevay LM(2011)Copper.Adv Nutr 2:520-522；
3.Gong L,Chen W,Gao Y,Liu X,Zhang H,Xu C,Yu S,Zhang Q,Luo J(2013)
Genetic analysis of the metabolome exemplified using a rice population.Proc
Natl Acad Sci USA 110:20320-20325；
4.Huang XY,Deng F,Yamaji N,Pinson SR,Fujii-Kashino M,Danku J,Douglas
A,Guerinot ML,Salt DE,Ma JF(2016)A heavy metal P-type ATPase OsHMA4prevents
copper accumulation in rice grain.Nat Commun 7:12138；
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Integr Genomics 15:673-684；
6.Livak KJ,Schmittgen TD(2001)Analysis of relative gene expression
data using real-time quantitative PCR and the 2(-Delta Delta C(T))
Method.Methods 25:402-408；
7.Maksymiec W(1997)Effect of copper on cellular processes in higher
plants.Photosynthetica 34:321-342；
8.Pilon M,Abdel-Ghany SE,Cohu CM,Gogolin KA,Ye H(2006)Copper cofactor
delivery in plant cells.Curr Opin Plant Biol 9:256-263；
9.Rae TD,Schmidt PJ,Pufahl RA,Culotta VC,O'Halloran TV(1999)
Undetectable intracellular free copper:the requirement of a copper chaperone
for superoxide dismutase.Science 284:805-808
10.Sasaki A,Yamaji N,Ma JF(2016)Transporters involved in mineral
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11.Xie K,Yang Y(2013)RNA-guided genome editing in plants using a
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distribution in graminaceous plants.Trends Plant Sci 19:556-563；
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Is a Vascular Bundles-Specific Manganese Transporter That Is Responsible for
Manganese Distribution in Rice.PLoS One 8；
14.Yang M,Zhang Y,Zhang L,Hu J,Zhang X,Lu K,Dong H,Wang D,Zhao FJ,
Huang CF,et al(2014)OsNRAMP5 contributes to manganese translocation and
distribution in rice shoots.J Exp Bot 65:4849-4861；
15.Yruela I(2009)Copper in plants:acquisition,transport and
interactions.Funct Plant Biol 39:409-430；
16.Gong L,Chen W,Gao Y,Liu X,Zhang H,Xu C,Yu S,Zhang Q,Luo J(2013)
Genetic analysis of the metabolome exemplified using a rice population.Proc
Natl Acad Sci USA 110:20320-20325。
Sequence table
<110>Hua Zhong Agriculture University
<120>clone of the molecular chaperone protein OsATX1 of copper transhipment and distribution and application in rice body are influenced
<141> 2018-06-10
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1956
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> gene
<222> (1)..(1956)
<400> 1
atggctgagg tacggccccg cgacagaact ccttgtttat tcgtttggtg ttaggggcgc 60
ggtcccttcc tgcggtcctg gagagagaag agagagagag agagagagag agagagagag 120
agagagagag agagagagag gagagggttg atcgataggg gtagatgggg aggccaagag 180
aggaggattc ctcttttttt tttttggcgt cgagttgttt gaatatgggg gaagaagcgt 240
gttcttttag cttgcgtgtg gggttaatta gggatctgat tatgatctga tgattggccg 300
gcgcgctgat cgatcagctt gcttgttctt gcgcgtgctt gtcttggctt tccttcatcg 360
tgcttgcagg ccagggtatg acttggggat gatgttttgg gcgaaatctt gctaggaggg 420
cggtatacct ttcagagttt cagtagttca aacaccaact gatggacaag ttttagaact 480
tcatggtggg ggtcgaaact agcttgatga ttttgatttg tgtacgttct tgttgaaacg 540
agtacagatt gcggagtagg cccgattgga tagttgttgt acctacctta aatgagccat 600
cttgttggtt atggtctgtg agttcaatga tagagcatga ttgtccccat ctgattatta 660
ctagttccct tccagaatga agcataaatt catttaattg ttgtcggtta cagtaaatct 720
tgattgggaa ttatgatgta ttccgagtac catgtggcgt gtgcgaagat gtgattgagg 780
acgaatgagg acttagctga tccttgctat ttttgtctat tgtgcagact gttgtgctca 840
gggttgggat gtcctgtgaa ggttgtgttg gagctgttaa gcgggtactg ggaaagatgc 900
aaggttagtt tagctctgtc ctcagcgttc tgtttgttga gaactttttc ctttttgcga 960
gcaattttta caatgtaacg cgtgcaatgc aacattggac ttaacttatg ccaatgctga 1020
atctgaacca atcctggtat atctctgcaa ttggtatctc ctctttaaaa attcccttaa 1080
ctgtatgctt ttgacgaaca ctggataatt ttgattgggt gaaatcttgt gcaatttcta 1140
gtttcaaatt gtacctgcgt ctgatttcta ggcataaaga tattgttttg taatattgct 1200
ttcgattgta ttttggactc atactttggc aattatgtac ctttctgtca ctaccatgac 1260
actcaccact tgtgtaagtg gcaatgtcca tagacaaaag gaaattaagg tatccggagt 1320
gcagacgcaa acaagttagg caacaacttt agtattcaaa tgagaattta tagctgtatt 1380
tgcttgtttt gctggtatta taaataaaat tttgagctct gttgttaatg attgctattt 1440
gaaattatcc acaactatag agtatagtgg gcaaacatat aatttcgaat ttctctcctg 1500
catgctattt ataaattcaa ttaattttta atcaaattga aagaatggtt ggtaaaaagg 1560
aaaactacat gtgcttcatc tttggcacat tttggtccaa ggcagttgct tgaataatgc 1620
taaggctatc tttcaaaccc aacctctgca atcagcccta atgacttcca atattctgca 1680
ttagatcatg gaagcacaaa tgattattta tacttgcata tgtattcttg taagtgtcac 1740
tgcaatattt agcgcatcga tctaaacatc tgagttggac ttgtggttgc ttcaggagtg 1800
gagtcctttg acgtagacat caaggagcag aaggtcactg tcaagggaaa tgttacacca 1860
gatgccgttc ttcagaccgt ttcaaagacg ggcaagaaga cctcgttctg ggatgctgag 1920
cctgcacctg ttgaagctac tgctgcttca tcttaa 1956
<210> 2
<211> 246
<212> DNA
<213>rice (Oryza sativa)
<220>
<221> gene
<222> (1)..(246)
<220>
<221> CDS
<222> (1)..(246)
<400> 2
atg gct gag act gtt gtg ctc agg gtt ggg atg tcc tgt gaa ggt tgt 48
Met Ala Glu Thr Val Val Leu Arg Val Gly Met Ser Cys Glu Gly Cys
1 5 10 15
gtt gga gct gtt aag cgg gta ctg gga aag atg caa gga gtg gag tcc 96
Val Gly Ala Val Lys Arg Val Leu Gly Lys Met Gln Gly Val Glu Ser
20 25 30
ttt gac gta gac atc aag gag cag aag gtc act gtc aag gga aat gtt 144
Phe Asp Val Asp Ile Lys Glu Gln Lys Val Thr Val Lys Gly Asn Val
35 40 45
aca cca gat gcc gtt ctt cag acc gtt tca aag acg ggc aag aag acc 192
Thr Pro Asp Ala Val Leu Gln Thr Val Ser Lys Thr Gly Lys Lys Thr
50 55 60
tcg ttc tgg gat gct gag cct gca cct gtt gaa gct act gct gct tca 240
Ser Phe Trp Asp Ala Glu Pro Ala Pro Val Glu Ala Thr Ala Ala Ser
65 70 75 80
tct taa 246
Ser
<210> 3
<211> 81
<212> PRT
<213>rice (Oryza sativa)
<400> 3
Met Ala Glu Thr Val Val Leu Arg Val Gly Met Ser Cys Glu Gly Cys
1 5 10 15
Val Gly Ala Val Lys Arg Val Leu Gly Lys Met Gln Gly Val Glu Ser
20 25 30
Phe Asp Val Asp Ile Lys Glu Gln Lys Val Thr Val Lys Gly Asn Val
35 40 45
Thr Pro Asp Ala Val Leu Gln Thr Val Ser Lys Thr Gly Lys Lys Thr
50 55 60
Ser Phe Trp Asp Ala Glu Pro Ala Pro Val Glu Ala Thr Ala Ala Ser
65 70 75 80
Ser
Claims (2)
- Application of the 1.OsATX1 genetic fragment in enhancing rice body in copper transhipment and distribution, which is characterized in that OsATX1 gene The nucleotide sequence of segment is as shown in sequence table SEQ ID NO:1.
- Application of the 2.OsATX1 genetic fragment in enhancing rice body in copper transhipment and distribution, which is characterized in that OsATX1 gene The protein sequence of fragment coding is as shown in sequence table SEQ ID NO:3.
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