CN108707622A - The method for improving China pink conversion ratio based on Tobacco rattle virus silencing system - Google Patents
The method for improving China pink conversion ratio based on Tobacco rattle virus silencing system Download PDFInfo
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Abstract
The invention belongs to horticulture and flower field of plant genetic, and in particular to the method for improving China pink conversion ratio based on Tobacco rattle virus silencing system.The silencing system of the present invention contains SEQ ID NO：PDS gene orders shown in 1 design special primer according to China pink PDS genetic fragments, amplify the cDNA segments of 305bp in China pink by PCR, the segment of acquisition is connected to TRV2On carrier, structure obtains recombinant expression carrier TRV2- DcPDS infects China pink using agriculture bacillus mediated mechanical damage method, and utilizes recombinant expression carrier TRV2- DcPDS converts China pink by transient transformation methods, and silence occurs for induction China pink target gene, to identify the gene function of Caryophyllales.The present invention can be used for the Rapid identification and transgeneic procedure of China pink gene function.
Description
Technical field
The invention belongs to horticulture and flower gene engineering technology fields, and in particular to one kind being based on Tobacco rattle virus silence body
The method that China pink conversion ratio improves in system, the present invention can be used for improving the work(of China pink conversion ratio and Rapid identification China pink endogenous gene
Energy.
Background technology
China pink is Caryophyllaceae Carnation herbaceos perennial, originates in China and East Asia Region.China pink cultivation history is long,
With higher ornamental value, horticultural use and economic value, it can be used for flower bed, flower border, flower stand, potting, also can be used as cut-flower etc.
It uses.China pink can also sulfur dioxide absorption and chloride, be excellent material for air purification；China pink is also research 5 radixes flower hair
The important Model of Scientific Research plant educated.Improve China pink ornamental value, resistance, blooming period prolonging and medical value etc. be in research and production urgently
Problem to be solved.Therefore, China pink related gene function is studied, the merit of China pink itself can be improved, increases ornamental plant
Using type, achieve the purpose that economizing type beautification, greening.But at present lack to control these character gene functions carry out quickly,
The system of efficient verification.Virus induced gene silencing technology (virus-induced gene silence, VIGS) has succeeded
The fast verification of gene function is carried out applied to various plants, although forefathers have relevant report to China pink VIGS systems, is answered
The induced efficiency converted with China pink after the system is poor, and only 20%, and changing effect is slow, just occurs within 20 days or so after infecting white
Change phenotype.And the system that the present invention is built uses new varieties and new method, induced efficiency to be up to 64.7%, while changing effect
Soon, albefaction phenotype will occur within about 7 days or so after infecting, substantially increase the efficiency of conversion, saved the time.This will be big
It is big to promote the verification of horticulture and flower plant gene function and analysis efficiency.
Invention content
It is an object of the invention to overcome defect of the existing technology, provide a kind of based on Tobacco rattle virus silence
The method that system improves China pink conversion ratio, the present invention can obtain the gene silencing character of Tobacco rattle virus induction, Jin Eryou
Effect is quickly and efficiently identified for China pink gene function.
To achieve the above object, the present invention is adopted the following technical scheme that：
Applicant clones to obtain a kind of endogenous DcPDS genetic fragments of China pink, the nucleotide sequence such as SEQ ID of the segment
NO：Shown in 1.
Nucleotide sequence containing above-mentioned DcPDS genetic fragments can be used as recombinant expression carrier, transgenic cell line or
The foreign gene of transgenic engineered bacteria.
The structure of plant expression vector, above-mentioned recombinant expression carrier are based on a kind of virus induction of Tobacco rattle virus
Gene silencing vector will contain such as sequence table SEQ ID NO：DcPDS genetic fragments shown in 1 are inserted into TRV2The BamH of carrier
Between I and Xho I sites, plant recombination expression vector TRV is obtained2-DcPDS。
It can be by genetic engineering bacterium by above-mentioned plant recombination expression vector TRV2- DcPDS transformed competence colibacillus Agrobacteriums
Obtain genetically modified plants.
Above-mentioned DcPDS genetic fragments can be applied in the Gene Silencing carrier of structure Tobacco rattle virus.
Carry above-mentioned DcPDS genetic fragments, the conversion carrier of recombinant expression carrier and genetic engineering bacterium can be in Rapid identification
It is applied in China pink gene function.
A kind of method of Rapid identification China pink gene function is applicant provided, main technological route includes：It will contain interior
The TRV of source target gene fragment2Viral silence expression vector plasmid is induced by mechanical damage method instantaneous conversion China pink in China pink
Silence occurs for source target gene, so as to quick and precisely identify the function of the endogenous target gene.
Specifically, applicant provide a kind of method of Rapid identification China pink gene function, the method further include as
Lower step：
(1) structure plant expression vector TRV2-DcPDS：Using the cDNA of China pink as template, with forward primer DcPDS-F (sequences
Row such as SEQ ID NO：Shown in 2) and reverse primer DcPDS-R (sequence such as SEQ ID NO：Shown in 3) it is that primer combination carries out PCR
Amplified reaction amplifies the PDS genetic fragments of 305bp sizes, and gained segment is connected to PMD18-T carriers (Fig. 6 is purchased from
Precious bioengineering Dalian Co., Ltd) on, obtain transfer vector plasmid PMD18-T-DcPDS (Fig. 8).By the carrier of successful connection
The heat-shock transformed bacillus coli DH 5 alpha competence of plasmid PMD18-T-DcPDS, in the LB containing 100 μ g/ml ampicillins (Apm)
It cultivates, is detected by positive single bacterium colony, sequence verification in culture dish, obtain the vector plasmid (Fig. 8) of PMD18-T-DcPDS.It will
PMD18-T-DcPDS vector plasmids obtain PDS endonuclease bamhis with BamH I and Sal I double digestions, with T4 ligases and BamH I
With the expression vector TRV of Xho I double digestions linearisation2(Fig. 7) connect, heat-shock transformed bacillus coli DH 5 alpha competence, containing
It is cultivated in the LB culture dishes of 100 μ g/ml kanamycins (Kan), positive single bacterium colony detection, sequence verification.By the plant of above-mentioned structure
Object expression vector TRV2- DcPDS (Fig. 9) electrotransformation Agrobacterium competence GV3101, the detection of picking single bacterium colony is positive, chooses positive
Clone shakes bacterium and preserves, for converting China pink blade.TRV is utilized simultaneously2Unloaded electrotransformation Agrobacterium competence GV3101, as
Convert the negative control of China pink blade.
(2) it prepares and infects buffer solution：Contain plant from picking on the LB tablets for being added to 100 μ g/ml of kanamycins (Kan)
Expression vector TRV2-DcPDS、TRV2The positive Agrobacterium GV3101 progress monoclonals of unloaded control vector, TRV1 carriers, respectively
It is incubated overnight in the fluid nutrient medium for being added to 100 μ g/ml gentamicins (Gen) and 100 μ g/ml kanamycins (Kan), so
Bacterium solution is transferred to respectively afterwards and is added to 100 μ g/ml gentamicins (Gen), 100 μ g/ml kanamycins (Kan), 0.5M/L 2-
It is incubated overnight again in (N- morpholines) ethanesulfonic acid monohydrate (MES) and 100mM acetosyringones (AS) fluid nutrient medium, then
Centrifugation goes supernatant to collect each thalline, each thalline is resuspended and is adjusted OD with buffer solution is infected600To 2.0, by TVR1Respectively with TRV2-
DcPDS and TRV2Unloaded control vector suspension bacteria liquid mixes in equal volume, and dark is lower to stand 3h, is buffered for China pink Infectikon
Liquid.
1M/L MES mother liquors：MES 9.76g are weighed, is dissolved in a small amount of ultra-pure water, ultra-pure water is used in combination to be settled to 50ml,
It is filtered to the sterile centrifugation seal of tube using the sterilised membrane filter in 0.2 μm of aperture in superclean bench, 4 DEG C save backup.
100mM AS mother liquors：It weighs AS 196.2mg, AS is dissolved in a small amount of DMSO (dimethyl sub-maple) in draught cupboard
In, ultra-pure water is settled to 10ml, mixing, is filtered to sterile centrifugation tube using the sterilised membrane filter in 0.2 μm of aperture in superclean bench
Sealing, -20 DEG C save backup.
2M/L MgCl2Mother liquor：Weigh MgCl220.33g or MgCl26H2O 30.942g add a small amount of ultra-pure water
Dissolving, is used in combination ultra-pure water to be settled to 50ml；120 DEG C of high pressure steam sterilization 20min are sub-packed in sterile centrifugation tube in superclean bench
In, 4 DEG C save backup.
Infect buffer (500ml, now with the current)：5ml 1M/L MES, 10ml 100mM AS, 2.5ml 2M/L
MgCl2, sterile water is settled to 500ml.
(3) buffer solution is infected using mechanical damage method (that is, with clean dry sanding paper polishing blade by prepared by step (2)
Front is removed after wound applies 1min with the cotton for speckling with bacterium solution) conversion China pink seedling leaves.
(4) it is cultivated after the conversion of China pink seedling：China pink seedling after conversion is transferred to illumination box (condition of culture：16h light
According to/8h dark；Cultivation temperature is 26 DEG C/24 DEG C；Relative humidity (RH) is 65%；Intensity of illumination is 4500LUX), black is used in combination
Polybag covers China pink seedling, raises afterwards for 24 hours, continues to be placed in illumination box and cultivate.
(5) observation of China pink plant phenotype is converted：It is in albefaction phenotype when observing China pink plant leaf after infecting 7~8 days,
Photograph to record image.
(6) the RT-PCR detections of the endogenous target gene DcPDS of China pink：Choose TRV1/TRV2The albefaction leaf that-DcPDS infects
Piece, TRV1/TRV2The China pink blade that zero load is infected, and the China pink blade of normal plant that does not infect, extract stone according to a conventional method
The leaf of bamboo piece RNA, reverse transcription cDNA, RT-PCR primer (the forward primer DcPDS-RT-F of design amplification DcPDS genetic fragments
(primer sequence is shown in SEQ ID NO：4) (primer sequence is shown in SEQ ID NO with reverse primer DcPDS-RT-R：5), with TRV1/TRV2
The empty carrier China pink blade infected and the normal China pink plant leaf not infected are control, detect the endogenous purpose DcPDS bases of China pink
The expression of cause.
Beneficial effects of the present invention：
The present invention contains the TRV of endogenous target gene2Viral silence expression vector plasmid, by blade mechanism damage method wink
When convert blade, make the endogenous target gene of China pink that silence occur.The present invention builds to obtain China pink TRV-VIGS silencing endogenous genes
System, at low cost, efficient, effect is fast, can obtain Tobacco rattle virus induction gene silencing character, and then solve have
Effect demonstrate,proves the problem of China pink gene function.In addition, with forefathers' report compared to it is of the invention have the advantages that it is following prominent：
(1) survival rate of plant height is infected, survival rate can reach 94.12%.
(2) it is high to infect plant detection efficiency, infect efficiency is up to 64.7%.
(3) greatly shorten transformation time, albefaction phenotype can occur within 7~8 days or so after infecting.
(4) operating method of the present invention is easy.
Description of the drawings
Sequence table SEQ ID NO:1 is China pink PDS gene orders.
Sequence table SEQ ID NO:2 be forward primer DcPDS-F sequences.
Sequence table SEQ ID NO:3 be reverse primer DcPDS-R sequences.
Sequence table SEQ ID NO:4 be forward primer DcPDS-RT-F sequences.
Sequence table SEQ ID NO:5 be reverse primer DcPDS-RT-R sequences.
Fig. 1：General technical flow chart of the present invention.
Fig. 2：China pink kind " satellite " scarlet series infects the electrophoresis pattern of the TVR viral diagnosis of plant.Reference numeral is said
It is bright：Swimming lane 4,5,6,10 and No. 11 samples are to infect successful China pink plant.
Fig. 3：Infect the scarlet series VIGS interference configuration figure of plant " satellite " China pink.Reference sign：Label
TRV:PDS is the plant that PDS infects, and TRV is unloaded adjoining tree, and Control is positive control.
Fig. 4：The scarlet serial genes silencing system (VIGS) of China pink kind " satellite " interferes phenotype.Reference sign：Figure
Piece is followed successively by：RV:PDS-1,TRV:PDS-2,TRV:PDS-3 indicates different strains, their phenotype is to enhance successively.Its
In：TRV is unloaded compares；Control is positive control.
Fig. 5：Expression analysis after the scarlet Series P DS gene interferences of China pink kind " satellite ".Reference sign：TRV:
PDS-1,TRV:PDS-2,TRV:PDS-3 is different strain after interference, and phenotype is to enhance successively, TRV be it is unloaded compare,
Control is positive control.
Fig. 6：PMD18-T vector plasmid collection of illustrative plates.
Fig. 7：pTRV2Vector plasmid collection of illustrative plates.
Fig. 8：PMD18-T-DcPDS vector plasmid collection of illustrative plates.
Fig. 9：pTRV2- DcPDS vector plasmid collection of illustrative plates.
Specific implementation mode
In the examples below, unless specifically indicated, involved carrier and experiment material are those skilled in the art
Known.
Embodiment 1
(1) clone of DcPDS genetic fragments
Fresh China pink blade 0.5g is taken, is said with reference to the Trizol RNA extracts kits of precious bioengineering Dalian Co., Ltd
The operating method of bright book extracts blade total serum IgE, is inverted with precious bioengineering Dalian Co., Ltd M-MLV reverse transcription reagent box
Record obtains the cDNA of China pink blade, according to the sequence information of the gene in China pink library, with 5 Software for Design of Primer as
The lower Specific primer pair expands DcPDS genetic fragments, and it is as described below that primer combines particular sequence；
Forward primer DcPDS-F：(sequence is the same as SEQ ID NO by 5 '-CCATTGAAGGTGGTGTGTGTAGA-3 '：2)
Reverse primer DcPDS-R：(sequence is the same as SEQ ID NO by 5 '-TTGGGGTAAGCCCCAAAGA-3 '：3)
Using the cDNA of China pink blade as template, PCR reactions are carried out.In 20 μ L reaction systems：0.2ul Taq enzymes, 2.0ul
Buffer, 1.0ul cDNA templates, 1.0ul DcPDS-F primers, 1.0ul DcPDS-R primers and 14.4ul dd H2O.It will be each
After reagent mixing, centrifugation sky gets rid of 3~5s；Response procedures：95 DEG C of pre-degeneration 3min；Amplification 35 cycle, 95 DEG C denaturation 40s, 59
DEG C annealing 30s, 72 DEG C extension 1min；72 DEG C of extension 10min；4 DEG C keep the temperature to 10min.
(2) plant expression vector TRV2The structure of-DcPDS
Using the PCR product of DcPDS-F, DcPDS-R primer amplification with gel reclaims kit (model AXYGEEN, USA)
Recycling connects target gene fragment DcPDS with PMD18-T carriers, establishes 20ul linked systems：10ul Solution1,1ul
Vector PMD-18T, 9ul DcPDS genetic fragments.Mixing, concussion, sky get rid of 3~5s of centrifugal enrichment.16 DEG C of constant temperature stand 8h,
It can be obtained recombinant vector PMD18-T-DcPDS connection liquid.By the heat-shock transformed large intestines of carrier PMD18-T-DcPDS of successful connection
Bacillus DH5 α competence is cultivated in the LB culture dishes containing 100 μ g/ml ampicillins (Apm), positive single bacterium colony detection,
Sequence verification obtains the vector plasmid of PMD18-T-DcPDS.PMD18-T-DcPDS vector plasmids are used into BamH I and Sal I
It carries out double digestion and obtains PDS endonuclease bamhis, with T4 ligases (purchased from precious bioengineering Dalian Co., Ltd) and BamH I and Xho
The expression vector TRV of I double digestions linearisation2Connection, heat-shock transformed bacillus coli DH 5 alpha competence, containing 100 μ g/ml cards that
It is cultivated in the LB culture dishes of mycin (Kan), is finally positive single bacterium colony detection and sequence verification.
By the plant expression vector TRV of above-mentioned structure2- DcPDS electrotransformation Agrobacterium competence GV3101, picking single bacterium colony
Detection is positive, chooses positive colony and shakes bacterium and preserve, for converting China pink blade.With by TRV2Empty carrier electrotransformation Agrobacterium sense
By state GV3101, the negative control as conversion China pink blade.
(3) TRV that Agrobacterium GV3101 is mediated2- DcPDS instantaneous conversion China pink blades
Contain plant expression vector TRV from picking on the LB tablets for being added to 100 μ g/ml of kanamycins (Kan)2-
DcPDS、TRV2Unloaded control vector, TRV1The positive Agrobacterium GV3101 monoclonals of carrier are being added to 100 μ g/ml celebratings respectively
Carry out being incubated overnight in the 10ml fluid nutrient mediums of big mycin (Gen) and 100 μ g/ml kanamycins (Kan) 12h (28 DEG C,
200rpm), then bacterium solution is transferred to respectively be added to 100 μ g/ml gentamicins (Gen), 100 μ g/ml kanamycins (Kan),
In the 50ml fluid nutrient mediums of 0.5M/L 2- (N- morpholines) ethanesulfonic acid monohydrate (MES) and 100mM acetosyringones (AS) again
Secondary to be incubated overnight 12h (28 DEG C, 200rpm), then 5000rpm, 25 DEG C, centrifuges 15min, removes each thalline of supernatant collection, use
Buffer solution is infected each thalline is resuspended and adjusts OD600To 2.0, by TVR1Respectively with TRV2- DcPDS and TRV2Unloaded control vector is outstanding
Floating bacterium solution mixes in equal volume, and dark is lower to stand 3h, is used for China pink Infectikon liquid.
(4) buffer (500ml, now with the current) is infected：5ml 1M/L MES, 10ml 100mM AS, 2.5ml
2M/L MgCl2, sterile water is settled to 500ml..
(5) after planting 4 weeks China pink seedling, when there is 6 pairs of blades opened completely, with least significant end 1 to blade and centre 1
Blade is used as mechanical damage method and infects object.Liquid is infected using mechanical damage method (i.e. with clean dry sanding paper polishing China pink
Face of blade removes after wound applies 1min with the cotton for speckling with bacterium solution), by converting China pink seedling leaves, turned
China pink seedling after change.
(6) plant expression vector TRV2Culture after-DcPDS instantaneous conversion China pink blades and Phenotypic Observation
China pink seedling after conversion is transferred to illumination box (condition of culture：Illumination 16h/8h；26 DEG C/24 DEG C of temperature；RH
65%；Intensity of illumination 4500LUX), it is used in combination black plastic bag to cover culture, raises black plastic bag afterwards for 24 hours, will continue to be placed in
It is cultivated in illumination box.After infecting 7~8 days, TRV2The plant that-DcPDS infects shows that apparent albefaction table occurs in newborn blade
Type, and TRV2It is identical as normal plant phenotype (see Fig. 3, Fig. 4) without albefaction phenotype to infect plant.
5, the expression quantity of DcPDS genes is detected using RT-PCR
Take TRV1/TRV2Albefaction blade, the TRV that-DcPDS infects1/TRV2The blade and do not infect normal that zero load is infected
Plant leaf extracts blade total serum IgE, and reverse transcription is cDNA, designs DcDcPDS gene RT-PCR primer (forward primers
DcPDS-RT-F, sequence are shown in SEQ ID NO：4), reverse primer DcPDS-RT-R, sequence are shown in SEQ ID NO：5), with TRV1/
TRV2Blade that zero load is infected and the normal China pink plant leaf not infected are control, the endogenous target gene DcPDS of detection China pink
Expression (result is shown in Fig. 5).If endogenous gene expression is low (after being silenced), there is albefaction phenotype in blade.According to table
Judge the function (preventing blade photooxidation function) of DcPDS genes with the relevance of albefaction leaf morphology up to horizontal reduce.
Detailed process of the present invention is as follows：
(1) acquisition of plant is infected：Using after planting 4 weeks China pink seedling, there are 6 pairs of blades opened completely, with most
Low side 1 handles blade to blade and intermediate 1 pair of blade mechanical damage method, infects object as Agrobacterium.
(2) plant expression vector is built：Using China pink cDNA as template, 305bpPDS genetic fragments are gone out by PCR amplification, and
The segment is connected on PMD18-T carriers, you can obtain transfer vector plasmid PMD18-T-DcPDS.
(3) PMD18-T-DcPDS vector plasmids are carried out with BamH I and Sal I after double digestion obtains PDS endonuclease bamhis
With the expression vector TRV of T4 ligases and the linearisation of BamH I and Xho I double digestions2Connection.
(4) by the TRV obtained by above-mentioned (3) step2- DcPDS transfer vector plasmid electrotransformation Agrobacterium competence GV3101
And carry out the PCR detections of positive colony.Simultaneously with TRV2Empty carrier electrotransformation Agrobacterium competence GV3101, as conversion China pink
The negative control of blade.
(5) TRV will be contained at room temperature2The Agrobacterium of-DcPDS recombinant plasmids is infecting buffer solution (MES+AS+MgCl2)
Dark is lower to stand 3h, for infecting China pink blade.
(6) the buffer solution conventional mechanical damage method that infects of above-mentioned preparation (such as is polished China pink with clean dry sanding paper
Face of blade removes after wound applies 1min with the cotton for speckling with bacterium solution), the China pink seedling after being converted.
(7) it is cultivated after the conversion of China pink seedling：China pink seedling after conversion is transferred to illumination box (condition of culture：Illumination
16h/8h；26 DEG C/24 DEG C of temperature；RH 65%；Intensity of illumination 4500LUX), be used in combination black plastic bag cover culture, for 24 hours after
It raises, continues to be placed in illumination box and cultivate.
(8) transformant Phenotypic Observation：The China pink plant leaf for infecting observation conversion in 7~8 days is albefaction phenotype, is photographed to record
Image.
(9) the RT-PCR detections of endogenous target gene DcPDS：Choose TRV1/TRV2The albefaction phyllostachys nuda leaf that-DcPDS infects
Piece, TRV1/TRV2The blade of blade and the normal China pink plant that do not infect that zero load is infected extracts blade RNA according to a conventional method,
And reverse transcription is cDNA, RT-PCR primer (forward primer DcPDS-RT-F (the SEQ ID of design amplification DcPDS genetic fragments
NO.：4), reverse primer DcPDS-RT-R (SEQ ID NO：5)), with TRV1/TRV2The blade and do not infect just that zero load is infected
Normal plant leaf is control, the expression of the endogenous target gene DcPDS of detection China pink.
In conclusion the present invention constructs silence recombinant expression carrier TRV2- DcPDS, and pass through agriculture bacillus mediated leaf
Piece mechanical damage method carries out instantaneous conversion to China pink, to realize the silence of China pink endogenous gene.
Sequence table
<110>Hua Zhong Agriculture University
<120>The method for improving China pink conversion ratio based on Tobacco rattle virus silencing system
<141> 2018-04-03
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 305
<212> DNA
<213>Phytoene dehydrogenase (Crocus sativus)
<220>
<221> gene
<222> (1)..(305)
<400> 1
ccattgaagg tggtgtgtgt agattatcca agacctgagc ttgataacac agtgaattac 60
ttggaggcag cttacttgtc ttcaactttt cggaattcgc ctcgtcctca gaagccatta 120
gatgttgtta ttgctggagc tggtttggca ggcctttcca ctgcaaagta tttagcggat 180
gcgggtcaca gacccctatt gttggaaggg agagatgttt taggaggaaa gattgctgca 240
tggaaagatg aggatgggga ctggtacgag acggggctac atatattctt tggggcttac 300
ccgaa 305
<210> 2
<211> 23
<212> DNA
<213>Phytoene dehydrogenase (Crocus sativus)
<220>
<221> primer_bind
<222> (1)..(23)
<400> 2
ccattgaagg tggtgtgtgt aga 23
<210> 3
<211> 19
<212> DNA
<213>Phytoene dehydrogenase (Crocus sativus)
<220>
<221> primer_bind
<222> (1)..(19)
<400> 3
ttggggtaag ccccaaaga 19
<210> 4
<211> 21
<212> DNA
<213>Phytoene dehydrogenase (Crocus sativus)
<220>
<221> primer_bind
<222> (1)..(21)
<400> 4
cttggaggca gcttacttgt c 21
<210> 5
<211> 20
<212> DNA
<213>Phytoene dehydrogenase (Crocus sativus)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 5
ttcgggtaag ccccaaagaa 20
Claims (6)
1. such as SEQ ID NO：DcPDS genetic fragments shown in 1 are carried in the Gene Silencing of structure Tobacco rattle virus
Application in body.
2. a kind of recombinant expression carrier of conversion China pink, it is characterised in that：By SEQ ID NO：DcPDS genetic fragments shown in 1
It is inserted into TRV2The recombinant expression carrier TRV obtained between BamH I and the Xho I sites of carrier2-DcPDS；
Wherein：
TRV2The collection of illustrative plates of plasmid is as shown in Figure 7.
3. application of the recombinant expression carrier in Rapid identification China pink gene function described in claim 2.
4. a kind of method of Rapid identification China pink gene function, it is characterised in that：Utilize the TRV containing endogenous target gene fragment2
Silence occurs for viral silence expression vector plasmid instantaneous conversion China pink, the endogenous target gene DcPDS of induction China pink, to which identification should
The function of endogenous target gene；
Wherein：
TRV2Viral silence expression vector recombinant expression carrier TRV as claimed in claim 22- DcPDS passes through mechanical damage method
By the recombinant expression carrier TRV described in claim 22- DcPDS instantaneous conversion China pinks, induction such as SEQ ID NO：Shown in 1
Silence occurs for the endogenous purpose Phytoene dehydrogenase gene DcPDS of China pink, identifies the endogenous target gene DcPDS's
Function.
5. the method for Rapid identification China pink gene function as claimed in claim 4, feature further include following steps：
(1) construction of expression vector TRV2-DcPDS：Using the cDNA of China pink as template, with SEQ ID NO：Forward primer described in 2
DcPDS-F, SEQ ID NO：Reverse primer DcPDS-R described in 3 is that primer pair carries out PCR, and amplification obtains the PDS bases of 305bp
Because of segment, the PDS genetic fragments of gained are connected on carrier PMD18-T, obtain intermediate carrier plasmid PMD18-T-DcPDS；
(2) by the heat-shock transformed bacillus coli DH 5 alpha competence of the PMD18-T-DcPDS carriers of successful connection, containing 100 μ g/ml
It is cultivated in the LB culture dishes of ampicillin, carries out positive single bacterium colony detection and sequence verification, obtain the load of PMD18-T-DcPDS
Constitution grain；PMD18-T-DcPDS vector plasmids are subjected to double digestion with BamH I and Sal I and obtain PDS endonuclease bamhis, then use T4
The expression vector TRV of ligase and the linearisation of BamH I and Xho I double digestions2Connection, heat-shock transformed bacillus coli DH 5 alpha impression
State is cultivated in the LB culture dishes containing 100 μ g/ml kanamycins, carries out positive single bacterium colony detection and sequence verification；Use plant
Expression vector TRV2- DcPDS electrotransformation Agrobacterium competence GV3101, the detection of picking single bacterium colony is positive, chooses positive colony and shakes
Bacterium simultaneously preserves, for converting China pink blade；Use TRV2Unloaded electrotransformation Agrobacterium competence GV3101, as conversion China pink blade
Negative control；
(3) it prepares and infects buffer solution：From picking expression vector TRV on the LB tablets for being added to 100 μ g/ml of kanamycins2-
DcPDS, TRV2Unloaded control vector, TRV1The positive Agrobacterium GV3101 monoclonals of carrier, respectively added with 100 μ g/ml celebratings
It is incubated overnight in the fluid nutrient medium of big mycin and 100 μ g/ml kanamycins, it is big mould that bacterium solution is transferred to 100 μ g/ml celebratings respectively
Element, 100 μ g/ml kanamycins, the liquid of 0.5M/L2- (N- morpholines) ethanesulfonic acid monohydrate and 100mM acetosyringones (AS)
It is incubated overnight again in culture medium, is then centrifuged for each thalline of supernatant collection, each thalline is resuspended with buffer solution is infected, adjusts
OD600Value is to 2.0, by TVR1Respectively with TRV2- DcPDS and TRV2Unloaded control vector suspension bacteria liquid is dark to mix in equal volume
Lower standing 3h infects buffer solution for China pink blade,
Wherein：
1M/L 2- (N- morpholines) ethanesulfonic acid monohydrate mother liquor：MES 9.76g are weighed, is dissolved in a small amount of ultra-pure water, is used in combination
Ultra-pure water is settled to 50ml, is filtered to the sterile centrifugation seal of tube, in 4 using the sterilised membrane filter in 0.2 μm of aperture in superclean bench
It is saved backup at DEG C；
100mM acetosyringone mother liquors：AS 196.2mg are weighed, AS is dissolved in a small amount of DMSO (dimethyl in draught cupboard
Sub- maple) in, it is settled to 10ml with ultra-pure water, mixing is filtered with the sterilised membrane filter in 0.2 μm of aperture to sterile in superclean bench
The seal of tube is centrifuged, -20 DEG C save backup；
2M/L MgCl2Mother liquor：Weigh MgCl220.33g or MgCl26H2O 30.942g add a small amount of ultrapure water dissolution,
It is used in combination ultra-pure water to be settled to 50ml；120 DEG C of high pressure steam sterilization 20min, are sub-packed in sterile centrifugation tube in superclean bench,
It is saved backup at 4 DEG C；
Infect buffer：5ml 1M/L (N- morpholines) ethanesulfonic acid monohydrate, 10ml 100mM acetosyringones, 2.5ml
2M/L MgCl2, 500ml is settled to sterile water；
(4) buffer solution that infects prepared by step (2) is converted into China pink seedling leaves, the stone after being converted using mechanical damage method
Bamboo seedling；
(5) it is cultivated after China pink conversion：China pink seedling after conversion is transferred in illumination box and is cultivated, condition of culture：16h illumination/
8h is dark；Cultivation temperature is 26 DEG C/24 DEG C；Relative humidity is 65%；Intensity of illumination is 4500LUX, is covered with black plastic bag
China pink seedling is simultaneously raised in for 24 hours afterwards, is continued to be placed in illumination box and be cultivated；
(6) Phenotypic Observation of China pink transformed plant：Infect whether 7~8 days observation China pink transformed plant blades albefaction phenotype occur,
It photographs to record；
(7) RT-PCR of endogenous target gene DcPDS is detected：Choose TRV1/TRV2Albefaction China pink blade that-DcPDS infects,
TRV1/TRV2The zero load China pink blade infected and the normal China pink plant leaf not infected extract RNA, and reverse transcription is cDNA,
DcPDS gene RT-PCR primers are designed, with TRV1/TRV2The zero load China pink blade infected and the normal China pink Plant Leaf not infected
Piece is control, detects the expression of endogenous target gene DcPDS.
6. the method for Rapid identification China pink gene function as described in claim 4 or 5, which is characterized in that the DcPDS bases
Because RT-PCR primer includes such as SEQ ID NO：Forward primer DcPDS-RT-F shown in 4, such as SEQ ID NO：It is reversed shown in 5
Primer DcPDS-RT-R.
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