CN105039360B - A kind of method for improving anthocyanidin content - Google Patents
A kind of method for improving anthocyanidin content Download PDFInfo
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Abstract
The present invention relates to the method that citrus phytoene synthase gene is used to improve strawberry anthocyanidin content and improve strawberry anthocyanidin content using the citrus phytoene synthase gene, comprise the following steps：Build citrus phytoene synthase gene expression plasmid；Optimize Transformation of Strawberry system；Citrus phytoene synthase gene expression plasmid is transferred in strawberry using agrobacterium-mediated transformation, obtains the strawberry of expression citrus phytoene synthase gene；The strawberry for being transferred to citrus phytoene synthase gene is subjected to molecular biology identification, detects the content of anthocyanidin in the strawberry of expression citrus phytoene synthase gene.The content of anthocyanidin in strawberry can be significantly improved using citrus phytoene synthase gene and above-mentioned method.
Description
Technical field
The present invention relates to genetic engineering field, more particularly to one kind to utilize citrus phytoene synthase gene
(CitPSY) method of strawberry anthocyanidin content is improved.
Background technology
Strawberry (Fragaria anannassa Duch.) belongs to rose family Fragaria herbaceos perennial.Strawberry full genome
Smaller, the Genome Size about 240Mbp of diploid of group.It is less demanding to growing environment, it can be planted in various regions.With it is other
Fruit tree is compared, and the growth period of strawberry is shorter, can obtain fruit in a short time.The new way of genetic engineering's teacher Strwberry Breeding
One of, its regeneration and genetic conversion system etc. are more ripe.In addition strawberry is vegetative propagation, and target gene is transferred in plant and expressed
Afterwards, it can be preserved and be expanded numerous by tissue cultures, division propagation etc. vegetative manner.Therefore, strawberry is the children such as citrus
One of good vegetable material of related gene functional verification in the fruit tree of phase length.
The pigment mainly contained in strawberry is anthocyanidin.Anthocyanidin is a kind of water colo(u)r, and its basic framework is 2- benzene
Base chromene cation (2-phenylbenzo-pyryalium), (Zhang Ning, 2008) as shown in Equation 1.Dissociate in nature
Anthocyanidin it is unstable, often form glucosides with one or more glucose, arabinose, galactolipin, rhamnose etc. and be housed in liquid
In bubble, glucosides makes anthocyanidin more stable, but not influences the color of anthocyanidin, and the color of anthocyanidin is by R1 and R2 positions
Different substituents determine (formula 1).Common cyanine is known as six kinds in plant：Delphinidin (Dp), cyanidin
(Cy), pelargonidin (Pg), malvidin (Mv), paeonidin (Pn) and morning glory pigment (Pt).
In formula 1, R1=R2It is pelargonidin (Pelargonidin) during=H；R1=H, R2It is cornflowerblue during=OH
Plain (Cyanidin)；R1=R2It is delphinidin (Delphinidin) during=OH；R1=H, R2=OCH3When, it is paeonidin
(Peonidin)；R1=OH, R2=OCH3When, it is morning glory pigment (Petunidin)；R1=R2=OCH3When, it is high mallow pattern
Plain (Malvidin).
Anthocyanidin has good healthcare function, such as：Strengthen eyesight, anti-eye strain；Delay cranial nerve aging, change
Kind sleep；There is therapeutic action to the microangiopathy as caused by diabetes；Strengthen cardio-pulmonary function；Prevent senile dementia, pre- anti-cancer
Disease；Beautifying face and moistering lotion, remove free radical.
Phytoene synthetase (PSY) is the core enzyme in Carotenoid Metabolism, and most important rate-limiting enzyme.
According to existing achievement in research, the effect of phytoene synthetase is to be catalyzed Mang ox base Mang ox base Jiao's phosphorus of 2 molecules
Sour (GGPP), so as to generate colourless phytoene.Because the phytoene synthetase speed limit important to carotenoid
Effect, therefore, presently mainly changes the accumulation of carotenoid using PSY genes by technique for gene engineering.At present from
PSY genes (Palaisa K A, 2003) have been isolated in the various plants such as corn, arabidopsis, satsuma orange, capsicum.
Anthocyanidin is mainly contained in strawberry, and comprises only a small amount of carotenoid.Substantial amounts of class Hu trailing plants is then accumulated in citrus
Bu Su, this shows plant preferred accumulation a certain kind pigment, and plant, which accumulates any pigment, to be determined by plant evolution.Flower
Blue or green element is water colo(u)r, and carotenoid is fat-soluble pigment, and both have significant difference, anabolism on molecular structure
Approach is also entirely different.Davison etc. (2002) research discoveries, the overexpression HYD genes in arabidopsis, carotenoid
Content increase, the anthocyanidin of accumulation are reduced；Cao Hongbo (2012) has found a large amount of of carotenoid when studying apple callus
Accumulation can cause the synthesis of anthocyanidin to reduce, and the gene expression amount of regulation and control anthocyanidin synthesis is lowered.
The content of the invention
In view of the problems of prior art, the present invention provides a kind of citrus phytoene synthase gene
CitPSY new application, and improve strawberry anthocyanidin content using citrus phytoene synthase gene CitPSY
Method, containing for anthocyanidin in strawberry fruit can be significantly improved by expressing citrus phytoene synthase gene in strawberry
Amount.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
The invention provides citrus phytoene synthase gene CitPSY new application, i.e., by citrus octahydro tomato
Red pigment synthase gene CitPSY is used to be transferred in strawberry to improve the purposes of strawberry anthocyanidin content.
The details of the citrus phytoene synthase gene CitPSY can be compiled in the inquiry of NCBI websites
Number it is Gene ID:102578001.Can also be referring to bibliography：Zhang Jiancheng Cara Cara carotenoid synthase genes
(CsPSY, CsLCYb) functional analysis and influence [Ph.D. Dissertation] of CrtB transgenosis to herxheimer-liked reaction are military
The Chinese：Hua Zhong Agriculture University, 2009.Cara Cara carotenoid synthase gene (CsPSY) in above-mentioned document is the present invention's
Entitled citrus phytoene synthase gene (CitPSY) in description.
A kind of method for improving strawberry anthocyanidin content, comprises the following steps：
1) citrus phytoene synthase gene CitPSY expression plasmids are built；
2) the citrus phytoene synthase gene CitPSY for being built step 1) using agrobacterium-mediated transformation is expressed
Plasmid is transferred in strawberry, obtains the strawberry for turning citrus phytoene synthase gene CitPSY；
3) strawberry for turning citrus phytoene synthase gene CitPSY is subjected to molecular biology identification；
4) anthocyanidin content in detection expression citrus phytoene synthase gene CitPSY strawberry；
5) turn anthocyanidin Metabolism-Related Genes Expression amount in citrus phytoene synthase gene CitPSY strawberries to examine
Survey.
Beneficial effects of the present invention：The present invention can be significantly improved in strawberry by expressing CitPSY genes in strawberry
The content of anthocyanidin.
The method of the present invention for improving strawberry anthocyanidin content is CitPSY to be carried in the form of plasmid and by one
Rise to be transferred in strawberry and expressed, those skilled in the art can select appropriate expression vector and be optimized accordingly, real
Apply and the preferable technical scheme of effect is merely provided in example, but be not limited solely to the technical scheme in embodiment.In addition, except matter
Beyond the form of grain, in other way, such as CitPSY can also be incorporated on the chromosome of strawberry so as to improve grass
The content of anthocyanidin in the certain kind of berries.
On the basis of above-mentioned technical proposal, the present invention can also do following improvement.
Further, the step 2) method for obtaining the strawberry for turning citrus phytoene synthase gene includes following
Step：
21) strawberry in vitro cuttings material is prepared；
22) the strawberry explant of inoculation is prepared；
23) leaf disk method Agrobacterium-mediated transformation is utilized.
Further, step 23) is described carries out conversion using leaf disk method Agrobacterium and specifically includes following steps：
A) Agrobacterium containing CitPSY expression plasmids is rule and cultivated；
B) strawberry explant is obtained into leaf dish in solid explant regeneration culture medium light culture；
C) take step a) single bacterium colony to be inoculated in the fluid nutrient medium containing antibiotic to be cultivated；
D) take the bacterium solution of culture in step c) to be inoculated into the fluid nutrient medium for not containing antibiotic, expand culture；
E) the bacterium solution centrifugation that step d) is cultivated is taken, supernatant is abandoned, is resuspended；
F) the leaf dish aseptic condition low suspension cultivated step b) is standby；
G) liquid by step f) suspensions is filtered out, and the bacterium solution that step e) is resuspended is added into leaf dish, is shaken, is filtered out bacterium
Liquid, after the unnecessary bacterium solution of blade is blotted, it is connected on the explant regeneration culture medium of solid and is co-cultured, the co-cultivation is dark
Culture, postponement screening is carried out afterwards, the postponement screening is also light culture；
H) blade postponed after screening is connected to screening and culturing medium；
I) carry out being transferred to illumination cultivation after light culture to blade grows callus, screen resistant budses；
J) resistant budses obtained after screening and culturing are cut, are connected in the plant subculture medium containing antibiotic and cultivate,
After sprout grows and more vanelets occurs, root induction in the root media containing antibiotic is moved to；
Above-mentioned steps c), j) described in antibiotic resistance and citrus phytoene synthase gene expression plasmid
Entrained resistant gene resistance is consistent.
Further, in step g), the explant regeneration culture medium is based on MS culture mediums, and addition is final concentration of
The clever and final concentration of 0.2mg/L of 1.5mg/L disleave indolebutyric acid.The co-cultivation time is 3d, postpones the time of screening
For 7-14d.
Further, in step h), the screening and culturing medium adds final concentration of 1.5mg/L's based on MS culture mediums
Clever, the final concentration of 0.2mg/L of disleave indolebutyric acid, final concentration of 250mg/L cephalosporin and final concentration of 10mg/L
Kanamycins.
Further, in step j), the plant subculture medium containing antibiotic is based on MS culture mediums, and addition is eventually
Concentration be 0.2mg/L 6-benzyladenine, final concentration of 0.02mg/L indolebutyric acid, final concentration of 0.2mg/L it is red mould
Plain, final concentration of 10mg/L kanamycins and final concentration of 250mg/L cephalosporin.
Further, in step j), the root media containing antibiotic for after twice of the dilution of MS culture mediums simultaneously
Add the cephalo of final concentration of 0.2mg/L indolebutyric acid, final concentration of 10mg/L kanamycins and final concentration of 250mg/L
Mycin.
It is using the above-mentioned further beneficial effect of scheme：Suitable turn is have selected in method for transformation of the present invention
Change condition and the concentration using reagent, have higher regeneration frequency and higher transformation frequency, have been successfully prepared turn
CitPSY strawberry, and find false positive rate than relatively low in follow-up incubation.
The fluid nutrient medium containing antibiotic in above-mentioned steps c) refers to be closed suitable for culture containing citrus phytoene
, can be according to entrained by containing citrus phytoene synthase gene expression plasmid into the Agrobacterium of enzyme gene expression plasmid
Resistant gene select suitable antibiotic and concentration, the fluid nutrient medium containing antibiotic used in this implementation be containing
Kan concentration is 50mg/L LB fluid nutrient mediums.
Brief description of the drawings
Fig. 1 is plasmid pBI121-Cit-PSY expression vector T-DNA areas schematic diagram of the present invention, wherein：LB represents T-
DNA left margins；RB represents T-DNA right margins；Npt II represent neomycin phosphotransferase II genes；
Fig. 2 includes Fig. 2A and Fig. 2 B, and Fig. 2A is the process signal of the method for raising strawberry anthocyanidin content of the present invention
Figure；Fig. 2 B are Agrobacterium tumefaciens mediated Strawberry Leaves genetic transformation process schematic of the present invention；
Fig. 3 is that transgenic positive strain PCR of the present invention expands CitPSY genetic results, wherein：M is 1kb DNA
ladder；Q is CitPSY plasmids；C compares for non-transgenic strawberry；Band 1-14 is resistance strawberry；
Fig. 4 is to determine anthocyanidin chromatogram in different strawberry strains, including Fig. 4 A to 4F using HPLC；Wherein, ordinate
For content (AU), abscissa is time (unit is minute)；Fig. 4 A to 4F are to utilize HPLC measure sweet tea Charlie, gold and jade, red respectively
Face, a chapter Ji, PMV strains, the anthocyanidin chromatogram of 4-1 strains；
Fig. 5 is anthocyanin accumulation feature comparative result in strawberry fruit, including Fig. 5 A and Fig. 5 B, wherein：A, b is represented in P<
Significant difference be present in 0.05 level；Ordinate is content, and unit is μ g/g；Abscissa is the kind of strawberry；Fig. 5 A are difference
The comparision contents of the fish pelargonium -3-O- glucosides of strawberry strain；Fig. 5 B are the corn flower -3-O- glucose of different strawberry strains
The comparision contents of glycosides；
Fig. 6 is expression characteristic result figure of the anthocyanidin synthesis related gene between different strawberry cultivars, including Fig. 6 A are to scheming
6J；Wherein, ordinate is content, and unit is μ g/g；Abscissa is the kind of strawberry, wherein, 4-1 represents 4-1 strains, TCL generations
Table sweet tea Charlie, HY represent beauty, and ZJ represents a chapter Ji, and JY represents gold and jade；Fig. 6 A to 6J be respectively respectively for CHS, CHI, F3H,
Expression characteristic result figure between DFR, ANS, UFGT, MYB1, MYB10, bHLH3, bHLH33 different strawberry cultivars.
Embodiment
The principle and feature of the present invention are described below in conjunction with accompanying drawing, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
First, vegetable material：
Strawberry cultivars ' all-star ' (F.ananassa Duch. ' Allstar '), domestic Main Cultivars, are drawn by the U.S.
Enter, have the characteristics that high yield, storage endurance, resistance against diseases are strong；The strawberry cultivars fruits such as sweet tea Charlie, gold and jade, beauty, a chapter Ji, are purchased from
Crop Institute, Hunan Academy of Agricultural Sciences's base greenhouse.
Detoxification test tube plantlet：From Agricultural University Of Shenyang Zhang Zhihong teacher laboratory, a large amount of aseptic seedlings are obtained by expansion is numerous.
Using blade as explant material in embodiment.
2nd, bacterial material：
Coli strain DH5 α, purchased from Wuhan Ding Guo Bioisystech Co., Ltd；Agrobacterium tumefaciems
(Agrobacterium tumefaciens) bacterial strain EHA105 (may be referred to document：Jiancheng Zhang,Nengguo
Tao,Qiang Xu,Wenjing Zhou,Hongbo Cao,Juan Xu,Xiuxin Deng.Functional
characterization of Citrus PSY gene in Hongkong kumquat(Fortunella hindsii
Swingle).Plant Cell Rep(2009)28:1737-1746), by the gardening plant biology Ministry of Education of Hua Zhong Agriculture University
Key lab preserves, can be with for the public to obtain.
3rd, main biochemical reagent：
Kanamycins (Kan):Purchased from Ding Guo biotech firms, 50mg/mL mother liquor, 0.22 μm of filter membrane are configured to sterilized water
Filtration sterilization, preserved at -20 DEG C.
Cephalosporin (cefotaxime, Cef):Purchased from Sigma companies, 400mg/mL mother liquor is configured to sterilized water,
0.22 μm of membrane filtration sterilizing, is preserved at -20 DEG C.
Disleave spirit (TDZ):Purchased from Wuhan Ding Guo Bioisystech Co., Ltd, after a small amount of 2mol/L KOH dissolvings, then use
Sterilized water is configured to 20mg/mL mother liquor, is preserved at -4 DEG C.
Other main agents are mainly purchased from Sigma companies, Takaya companies.
4th, culture medium involved in the present embodiment：
1st, for cultivating the culture medium of Agrobacterium tumefaciems
LB fluid nutrient mediums：Contain 10g/L pancreases albumen, 5g/L yeast extracts, 10g/L NaCl；
LB solid mediums：7g/L agar is added on the basis of LB fluid nutrient mediums.
2nd, plant fast breeding culture medium：Final concentration of 0.5mg/L BA, final concentration of is added on the basis of MS culture mediums
0.02mg/L IBA and final concentration of 0.2mg/L GA；
3rd, plant subculture medium：Final concentration of 0.2mg/L BA, final concentration of is added on the basis of MS culture mediums
0.02mg/L IBA and final concentration of 0.2mg/L GA；
4th, explant regeneration culture medium：Final concentration of 1.5mg/L TDZ and final concentration of is added on the basis of MS culture mediums
0.2mg/L IBA；
5th, screening and culturing medium is postponed：Final concentration of 1.5mg/L TDZ, final concentration of is added on the basis of MS culture mediums
0.2mg/L IBA and final concentration of 250mg/L Cef；
6th, screening and culturing medium：Final concentration of 1.5mg/L TDZ, final concentration of 0.2mg/L are added on the basis of MS culture mediums
IBA, final concentration of 250mg/L Cef and final concentration of 10mg/L Kan；
7th, root media：Final concentration of 0.2mg/L IBA is added after twice of the dilution of MS culture mediums simultaneously.
Above-mentioned all hormone concentration units are mg/L, and the medium pH based on MS is adjusted to 5.85, and sterilize standby
With.
On solid medium liquid medium within component base, 7g/L agar is added.
The specific composition of MS culture mediums and compound method referring to《Plant Tissue Breeding experiment instruction》(Gong Yifu, 2011).
IBA refers to indolebutyric acid (Indole-3-Butytric acid), and BA refers to 6-benzyladenine (6-
Benzyladenine), GA refers to gibberellin (Gibberellin), is purchased from Wuhan Ding Guo Bioisystech Co., Ltd.
The primer is by Primer 5 and Primer Express Software for Design in experimental example.
If not particularly pointing out, reagent of the present invention or method can refer to《Molecular Cloning:A Laboratory guide》(second
Version), those skilled in the art can realize technical scheme described in the invention.
Embodiment 1
Build citrus phytoene synthase gene CitPSY expression plasmids.
CitPSY amplification and the method detailed content of structure CitPSY expression plasmids are shown in document：Zhang Jiancheng red meat navels
Orange carotenoid synthase gene (CsPSY, CsLCYb) functional analysis is with CrtB transgenosis to herxheimer-liked reaction
Influence [Ph.D. Dissertation] Wuhan：Hua Zhong Agriculture University, 2009.The a length of 1540bp of its cDNA of the CitPSY of clone, comprising
1308bp opening code-reading frame, 436 amino acid are encoded, there is the non-translational region that 49bp grows at 5 ' ends, and 3 ' ends include 160bp non-volume
Code sequence；The molecular weight and isoelectric point of prediction are respectively 49.5kDa and 5.86.
The title of Cara Cara carotenoid synthase gene (CsPSY) in the description of the invention in above-mentioned document
For citrus phytoene synthase gene (CitPSY).
The entitled pBI121- of the expression vector pBI-CsPSY of structure in above-mentioned document in the description of the invention
Cit-PSY.Plasmid pBI121-Cit-PSY expression vector T-DNA areas schematic diagram is as shown in Figure 1.
Plasmid pBI121-Cit-PSY is transformed into Agrobacterium tumefaciens strain EHA105, obtains bacterial strain EHA105/
pBI121-Cit-PSY.Specific method for transformation may be referred to above-mentioned document.
Embodiment 2
Agrobacterium tumefaciens mediated Strawberry Leaves genetic transformation, detailed process are as shown in Figure 2.
1st, the preparation of strawberry in vitro cuttings material
In vitro cuttings are seeded in into quick breeding solid medium, and (addition is dense eventually on the basis of plant fast breeding culture medium
Spend the agar of sucrose and final concentration of 7g/L for 30g/L) on Multiplying culture, single-plant propagation, each triangular flask is inoculated with 4 explants
Body, it is placed on the culture of illumination cultivation room.Condition of culture：25 DEG C, intensity of illumination 2000lx, photoperiod 16h/d of temperature.Through 2-3
Individual month, a large amount of in vitro cuttings are obtained, it is standby as experiment material.
2nd, it is inoculated with explant
Take and the consistent blade of 30-40d or so, growth site growing way is grown on step 1 culture medium, cut into 4mm × 4mm's
Block leaf dish, the explant as inoculation in experiment.All explant adaxial and its surfaces contact with culture medium in culture dish, are connect per ware
35 explants of kind, each processing are repeated 3 times.
3rd, Agrobacterium leaf disk method Transformation Program
(1) by EHA105/pBI121-Cit-PSY bacterium solutions in LB (Kan contained concentration is 50mg/L) solid medium
Upper line, 28 DEG C of culture at least 2d.
(2) blade (i.e. strawberry explant) is cut into 4mm × 4mm or so bulks, it is dark in solid explant regeneration culture medium
2d is cultivated, obtains leaf dish.
(3) above-mentioned bacterium single bacterium is taken to fall within 50mL LB fluid nutrient mediums (Kan contained concentration is 50mg/L), 200r/
Min, 28 DEG C, concussion 16h, OD600 values are about 0.8-1.0.
(4) above-mentioned bacterium solution 1mL is taken in 50mL LB fluid nutrient mediums, and 200r/min, 28 DEG C, concussion 10h, OD600 values are about
For 0.3-0.4.
(5) 5000r/min normal temperature centrifugation 5min, abandons supernatant, adds same volume MS fluid nutrient mediums (being not added with hormone) and suspends.
(6) leaf dish of preculture suspends standby in the MS fluid nutrient mediums of the sterile sucrose containing 30g/L.
(7) above-mentioned MS fluid nutrient mediums are filtered out, adds the bacterium solution being resuspended in step (5), shaken 5-7min, filter out bacterium
Liquid, the unnecessary bacterium solution of blade is blotted, be connected to solid explant regeneration culture medium, co-culture 3d (darkroom), trained using screening is postponed
Foster base carries out postponing screening 7-14d, and this process is light culture.
(8) postpone the blade after screening and be connected to screening and culturing medium.
(9) selection culture：Light culture to blade is transferred to illumination cultivation after growing callus, selects the time of culture altogether
For 35-45d, primary screening culture medium is changed per every about 20d or so, the time by callus to generation resistant budses is 15-
20d。
(10) propagation of resistant budses is with taking root：The resistant budses obtained after screening and culturing (length is more than 1cm) are cut, are connected on
Simultaneously containing 10mg/L Kan, 250mg/L Cef plant subculture medium in cultivate, when sprout grows to 2-3cm, and go out
After existing more vanelets, move to containing 10mg/L Kan, 250mg/L Cef root media in root induction, it is anti-from obtaining
Property bud to the time to send out roots is about 30d.
While embodiment 2 are carried out, inventor has also carried out comparative example 2-1 to 2-8 work simultaneously, so as to test
The selection of design parameter has the advantage that in card technical solutions according to the invention and technical scheme.Contrast experiment's example it is specific
Content is as follows.
Comparative example 2-1
In above-mentioned steps (7), adjust solid explant regeneration culture medium in TDZ concentration be respectively 1.0mg/L,
The TDZ of the various concentrations such as 1.5mg/L, 2.0mg/L, 2.5mg/L and 3.0mg/L, adventitious shoot regeneration situation is counted after cultivating 50d,
Per 20d subcultures once.Remaining step is the same as embodiment 2.
Comparative example 2-2
In step (9), Strawberry Leaves are inoculated on regeneration culture medium, are placed in darkroom culture, the dark treatment time is respectively：
7d, 14d, 21d, 28d and 35d.Remaining step is the same as embodiment 2.
Comparative example 2-3
In step (8), by blade inoculation on the screening and culturing medium of different Kan concentration, observation is not per 20d subcultures once
Normal bud regenerates situation, selects the minimum Kan concentration for making leaf regeneration callus but being unable to regenerated adventitious bud, sets at following concentration
Reason：0mg/L, 10mg/L, 20mg/L, 30mg/L and 40mg/L.Remaining step is the same as embodiment 2.
Comparative example 2-4
In step (8), leaf dish is infected with OD600=0.4 Agrobacterium bacterium solution, aseptic filter paper, which blots to be seeded in after bacterium solution, to be added
On the screening and culturing medium for adding different concentration of Ce f, Agrobacterium growth inhibitory effect is observed.Following concentration is set to handle：0mg/L、
100mg/L, 250mg/L, 400mg/L and 550mg/L.And count Agrobacterium growth time and growing state.Remaining step is the same as real
Apply example 2.
Comparative example 2-5
The Agrobacterium tumefaciems after activation is suctioned out into 1mL in step (4), added in the LB culture mediums newly prepared, shake respectively to
OD600 be 0.1,0.3,0.5,0.7, after centrifugation, with isometric MS liquid regenerations culture medium be resuspended, infect respectively 1min, 5min,
10min, 3d is co-cultured, kanamycin-resistant callus tissue growing state is observed after 25d.Remaining step is the same as embodiment 2.
Comparative example 2-6
Blade is placed in preculture 0d, 1d, 2d, 3d, 4d on explant regeneration culture medium in step (2), after use OD600=
0.5 bacterium solution infects 5min, co-cultures 3d, kanamycin-resistant callus tissue growing state is observed after 25d.Remaining step is the same as embodiment 2.
Comparative example 2-7
In step (7), by blade preculture 2d, infect 5min with OD600=0.5 bacterium solution afterwards, co-culture 2d, 3d,
4d, continue to cultivate after sloughing Agrobacterium tumefaciems, observe kanamycin-resistant callus tissue growing state.Remaining step is the same as embodiment 2.
Comparative example 2-8
In step (7), after co-culturing 3d, the blade infected is inoculated in the solid explant regeneration containing Kan and Cef respectively
Screened immediately in culture medium, in the postponement screening and culturing medium for comprise only Cef postpone screening, respectively postpone screening 2d, 7d,
The growth of statistical observation kanamycin-resistant callus tissue and budding situation after 14d, 30d.Remaining step is the same as embodiment 2.
The strawberry for being transferred to CitPSY obtained by the above method, and molecular biology identification is carried out, qualification result is sun
The Strain Designation of property is 4-1 strains.
It is ' complete bright that the plasmid pBI121 for the empty carrier for not containing CitPSY is transferred to strawberry cultivars according to the method described above simultaneously
Star ', it is named as PMV strains.
The 4-1 strains, the plant of PMV strains are preserved to country of Hua Zhong Agriculture University citrus breeding center.
For 4-1 strains compared with PMV strains, the blades of 4-1 strains shows that light green color, blade are relatively thin, fruit type is smaller, color
It is more bright-coloured, in peony.PMV strain blades are dark green, blade is thicker, stem is more sturdy.
Sample pretreatment
Collection 4-1 strains, PMV strains, sweet tea Charlie, gold and jade, beauty, the fruit (being complete red phase fruit) of a chapter Ji, with clear
Water cleans up fruit, removes base of fruit, and fruit is divided into three parts of repetitions, is frozen with liquid nitrogen and is sealed later at -80 DEG C
In refrigerator, for molecular biology identification and the measure of various metabolites.
The molecular biology identification of the Transgenic Strawberry of embodiment 3
1st, strawberry DNA is extracted：Using CTAB a small amount of method methods extraction leaf DNAs, specific steps see (Yunjiang Cheng,
Wenwu Guo,Hualin Yi,Xiaomin Pang,Xiuxin Deng.An efficient protocol for
genomic DNA extraction from Citrus species.Plant Molecular Biology Reporter,
2003,21:177a-177g)。
2nd, identified using PCR
Embodiment 2 is obtained into 14 plants of resistance seedlings after Kan is screened.Using CitPSY special primers (fwd5 '-
CGGGATCCATGTCTGTTACA-3 ', rev5 '-GGGGTACCTTAAGCCTTACT-3 ') (Zhang Jiancheng Cara Cara carotenoids
Plain synthase gene (CsPSY, CsLCYb) functional analysis and influence [doctor of the crtB transgenosis to herxheimer-liked reaction
Academic dissertation] Wuhan：Hua Zhong Agriculture University, 2009), the blade of resistance seedling is extracted into DNA, enters performing PCR amplification.Positive control
For Agrobacterium plasmid, negative control is the strawberry DNA of non-transgenosis.As a result it is as shown in Figure 3, it can be seen that after PCR amplifications, 3 strains
It is to amplify a 1300bp or so band, reporter gene nptII (fwd5 '-GTGCCCTGAATGAACTGC-3 ', rev5 '-
GCCTTGAGCCTGGCGAAC-3 ') (Zhang Jiancheng Cara Cara carotenoid synthase gene (CsPSY, CsLCYb) function point
Analysis and influence [Ph.D. Dissertation] Wuhan of the crtB transgenosis to herxheimer-liked reaction：Hua Zhong Agriculture University,
2009) corresponding band is also amplified.
Second Year, positive strain obtained above and unloaded strain (PMV strains) are transferred in experiment greenhouse, allow it to give birth to
Long result, qualification result are named as 4-1 strains for positive strain.
Pcr amplification reaction liquid is prepared according to the reaction system in table 1.
The pcr amplification reaction liquid of table 1
According to the condition operation PCR reactions in table 2.
The PCR of table 2 reacts operation program
| Total denaturation | Denaturation | Renaturation | Extension | Period |
| 94 DEG C, 7min | 94 DEG C, 1min | 55 DEG C, 50s | 72 DEG C, 2min | 35 |
Comparative example 3-1
Respectively using PMV strains, sweet tea Charlie, gold and jade, beauty, a chapter Ji fruit (be complete red phase fruit) as compareing, she enters
Row comparative example.Specific operation process is the same as embodiment 3.
The sweet tea Charlie, gold and jade, beauty, a chapter Ji are big purchased from Crop Institute, Hunan Academy of Agricultural Sciences base
Canopy.
Embodiment 4
Anthocyanidin content is determined using HPLC, specific steps are referred to Bordonaba JG, Terry
LA.Biochemical profiling and chemometric analysis of seventeen UK-grown black
currant cultivars.Agricul tural and Food Chemistry,2008,56(16):Described in 7422-7430
Method.
1) by sample liquid nitrogen grinding, 1g samples are accurately weighed into 10mL centrifuge tubes.
2) 5mL extract solution is added into centrifuge tube, and (extract solution is the mixed liquor of hydrochloric acid and methanol solution, wherein salt
The volume fraction of acid is 1%) vortex 2min.
3) test tube after vortex is put into ultrasonic 40min in Ultrasound Instrument.
4) 5000g centrifugations 10min after ultrasound, 0.22 μm of filter membrane is crossed, prepares loading.
Wherein, chromatographic column is 4.6mm × 250mm, 5 μm (Sigma, USA)；Mobile phase A is that water (is containing mass fraction
1% methanol), Mobile phase B is methanol, flow velocity 1mL/min.
Specific elution requirement is referring to table 3.
The HPLC of table 3 determines the elution requirement of anthocyanidin
| Time | Elution requirement |
| 0-10min | 10%-20%B |
| 10-15min | 20%-30%B |
| 15-50min | 30%-50%B |
| 50-60min | 50%-60%B |
| 60-68min | 60%-10%B |
| 68-70min | 10%B |
Select fish pelargonium -3-O- glucosides (The nature network, 97%), C-3-G
(Shanghai Hui Cheng biologies, 98%) is used as standard items, is diluted to 512,256,128,64,32,16,8,4,2,1 μ g/ml respectively.Do
Go out standard curve, draw quantitative equation and R values.
Comparative example 4
Respectively using PMV strains, sweet tea Charlie, gold and jade, beauty, a chapter Ji fruit (be complete red phase fruit) as compareing, she enters
Row comparative example.Specific operation process is the same as embodiment 4.
Embodiment 5 is analyzed with anthocyanidin related gene expression
1.RNA reverse transcriptions are into single-stranded cDNA：With reference to RevertAid M-MuLV KIT method.Made with 1-1.5 μ g mRNA
For template carry out reverse transcription, after the completion of cDNA add 80 μ L ddH20 dilution；
2. according to document has been reported, Primer 5 and Primer Express Software for Design real-time fluorescence quantitative PCRs are utilized
Primer, wherein CHS fwd5 '-CCGACTACTACTTTCGTATCACCA-3 ', rev5 '-ACTACCACCATGTCTTGTCTTGC-
3’；CHI fwd5’-TTTTCAATGGCTTTCGCTTCTG-3’,rev5’-GTGACAATGATACTACCGCTGACG-3’；F3H
fwd5’-GTGCGCCACCGTGACTACTC-3’,rev5’-ATGCCTTTGTCAATGCCTCC-3’；DFR fwd5’-
GGGTGGTGTTTACATCTTCGG-3’,rev5’-CTGCTTGCTCGGCTAGAGTTT-3’；ANS fwd5’-
ATCGTCATGCACATAGGCGACACC-3’,rev5’-CCTTGGGCGGCTCACAGAAAA-3’；UFGT fwd5’-
ATCGTGGCTTGACAAACAGAA-3’,rev5’-TGACCACAAGAATGGAACCCTA-3’；MYB1fwd5’-
ATGAGGAAGCCCTGCTGCGA-3 ', rev5 '-AACGACGCAACCCTGCAGCC-3 ' (it may be referred to document：
Salvatierra A,Pimentel P,Moya-león MA,Herrera R.Increased accumulation of
anthocyanins in Fragariachiloensis fruits by transient suppression of
FcMYB1gene.Phytochemistry,2013,90:25-36.)；MYB10fwd5’-TCAAATCAGGCTTAAACAGA-3’,
rev5’-TTAAAGACCACCTGTTTCCT-3’；bHLH3fwd5’-ACCGAGTAGTAGCAGACTCCGTGGTAT-3’,
rev5’-CCATCTGCCCATATTAACATCCCTTG-3’；bHLH33fwd5’-AATCCATGAGAGGGTGCCTGAGAAT-3’,
Rev5 '-CAGCACCCCTTGTTGAGTTGTTGA-3 ' (may be referred to document Espley RV, Hellens RP, Putterill
J,Stevenson DE,Kutty-Amma S,Allan AC.Red colouration in apple fruit is due to
the activity of the MYB transcription factor,MdMYB10.Plant Journal,2007,49:
414-427.).Fluorescence real-time quantitative PCR is in ABI7500Real Time System (PEApplied Biosystems；
Foster City, CA, USA) on carry out, method and response procedures bibliography：The dark willows of Liu Qing ' ' the sweet orange red mutation bodily form
Molecular mechanism research [Ph.D. Dissertation] the Wuhan that shape is formed：Hua Zhong Agriculture University, 2008..By target gene and internal reference base
Because of GADPH specific primers (fwd5 '-TCTTTGATGCCAAGGCTGGA-3 ', rev5 '-TCACACGGGAACTGTAACCC-
3 ') andGREEN Master Mix are well mixed, and are added in the reaction tube added with template in advance, and reaction system is
10 μ L, detailed component part table 4-1.Real-time fluorescence quantitative PCR response procedures are to be shown in Table 4-2.
Table 4-1 quantitative fluorescent PCR reaction systems
Table 4-2 real-time fluorescence quantitative PCR response procedures
Experimental result：
Statistical method on experimental result：Total explant number of explant regeneration rate=regeneration callus number/inoculation ×
100%；Total explant number × 100% of explant number/inoculation of adventitious shoot regeneration rate=regenerated adventitious bud；Average regeneration bud number
Explant number × 100% of=regenerated adventitious bud sum/regeneration bud.
In embodiment 2, preculture 2d leaf dish progress Agrobacterium (OD600=0.4) is infected, after co-culturing 3d, transfer
To postponing on screening and culturing medium, start callus occur after 7d, color is turned white.Leaf dish is connected to screening after 2 weeks (being abbreviated as w)
In culture medium, the later stage carries out illumination cultivation, callus raised growth, and most of callus presses close to the gradual browning in culture medium position,
Callus in growth gradually shows light green, small part browning.The explant that the callus of browning is depended on gradually becomes
It is black to withered.The kanamycin-resistant callus tissue of continued growth, after normal growth, adventitious bud is differentiated on a small quantity.After adventitious bud gradually extends, by it
Cut, be transferred to containing 10mg/L Kan, in 250mg/L Cef strawberry subculture medium, treat resistant budses further
After growing up, screening pressure Kan concentration is improved to 20mg/L.The resistant budses later stage elongation, propagation growth course in, Partial Conversion bud by
Gradually albefaction is dead, bud of as escaping, also occurs half white half green phenomenon, as chimera.Remaining resistant budses, keep green
Color, growth are normal.
When resistant budses grow to 2-3cm, it is placed in containing 10mg/L Kan, enters in 250mg/L Cef root media
The induction of row root.All 19 plants of resistant budses in root media is seeded to, do not take root always by preceding 8w.By subculture for several times, subtract
Antibiotic concentration in few culture medium, finally has 14 plants to take root.
First, the specific experiment result and contrast experiment's example on Agrobacterium tumefaciens mediated Strawberry Leaves genetic transformation part
Comparing result is as follows.
1st, with comparative example 2-1 comparing result
For ' all-star ' blade, with MS+TDZ+IBA (0.2mg/L) for explant regeneration culture medium, tables of data in table 5
Bright, with the increase of TDZ concentration, callus regeneration rate and adventitious shoot regeneration level all increase.Technical solution of the present invention
The middle TDZ concentration used can both ensure with higher adventitious shoot regeneration rate for 1.5mg/L and avoid regeneration well
Bud vitrification phenomenon.Because TDZ is expensive, take low concentration TDZ be also beneficial to it is cost-effective.
The various concentrations TDZ of table 5 is to the induction of ' all-star ' blade callus and the influence of adventitious shoot regeneration
2nd, with comparative example 2-2 comparing result
As can be seen from Table 6, the present invention is carrying out light culture of the Transformation of Strawberry experiment using 21-30d, compared to other
Light culture time, both having shortened the time and can of Callus formation enough makes the callus after illumination cultivation gradually become
It is green.All-star blade is positioned under illumination and cultivated again after light culture, therewith the increase of light culture time, callus regeneration rate,
Adventitious bud bud ratio gradually steps up.Light culture 14d, callus regeneration rate and adventitious shoot regeneration rate all reach highest, with light culture
Time increases, and regeneration rate and bud ratio have declined.It is observed that blade light culture can shorten callus shape in experiment
Into time, the callus originally formed is turned white, and after illumination cultivation, the gradual greening of callus, indefinite budding takes place afterwards.Infect
Experiment, screening test show that when being grown on the culture medium containing antibiotic, Callus formation, adventitious bud occur all blade
Postpone.
The different light culture times of table 6 are to the induction of ' all-star ' blade callus and the influence of adventitious shoot regeneration
3rd, with comparative example 2-3 comparing result
' all-star ' leaf dish is inoculated into the explant regeneration culture medium containing different Kan concentration, resistance is observed after 30d
Callus regenerates situation, and adventitious bud budding and growing state are observed after 50d.Inductions of the Kan to strawberry leaf dish callus and indefinite
The differentiation of bud has significant inhibitory action.
Show in table 7, compared to other concentration, present invention selection 10mg/L Kan can not only reach the purpose of screening but also avoid
Influences of the Kan of excessive concentrations to adventitious shoot regeneration.
Because as the raising of Kan concentration, callus regeneration frequency, the differentiation rate and survival rate of adventitious bud are all notable
Reduce.When Kan concentration is more than 20mg/L, callus can still induce generation, but adventitious bud can not regenerate, bigger concentration
Kan the rapid browning of blade can be caused dead.When Kan concentration is 10mg/L, although can also induce adventitious bud, later stage culture
In, the gradual albefaction of adventitious bud is dead.It is undifferentiated to be cured after squamous subculture 50d in the screening and culturing medium containing various concentrations Kan
It is dead after the gradual yellow of explant blade of injured tissue, albefaction.On additional Kan screening and culturing medium, it is observed that explant
Body Callus of Leaf, which produces, relatively compares late 5-10d, and adventitious bud budding Time transfer receiver shines late 30-45d.
Influence of the kanamycins of table 7 to blade adventitious shoot regeneration
4th, with comparative example 2-4 comparing result
Selection to bacteriostatic agent Cef concentration, it is desirable to it can either effectively suppress the raised growth of Agrobacterium, and it is thin to explant
The influence of intracellular growth is minimum.
As can be seen from Table 8, postponed using 250mg/L Cef in the present invention and being cultivated on screening and culturing medium, compared
Other concentration, optimal fungistatic effect can be reached and make antiseptic influence to diminish on blade.When Cef concentration is more than 400mg/L
When, it can effectively suppress the growth and breeding of Agrobacterium.When Cef concentration is less than 100mg/L, the growth of Agrobacterium hardly by
To suppression.When Cef concentration is 250mg/L, although Agrobacterium has also grown, the speed of growth is slower.Therefore, in this experiment first
1-2 rinsing is carried out to the blade of co-cultivation using the Cef liquid explant regeneration culture mediums containing low concentration, then by blade
Liquid explant regeneration culture medium, which blots, to be followed by being cultivated on containing 250mg/L Cef postponement screening and culturing mediums, can reach
Optimal fungistatic effect, and make antiseptic influence to diminish on blade.
The various concentrations cephalosporin of table 8 influences on Agrobacterium inhibition and on leaf growth
5th, with comparative example 2-5 comparing result
As can be seen from Table 9, when the present invention is infected explant and infected using the Agrobacterium tumefaciems that OD600 is 0.5
Between be 5-7min, compared to other infection conditions, there is higher kanamycin-resistant callus tissue regeneration rate.Agrobacterium tumefaciems concentration relationship to conversion
The height of rate, too low bacterial concentration are unfavorable for attachment of the Agrobacterium tumefaciems on explant, and too high bacterial concentration is unfavorable for
Agrobacterium tumefaciems is removed after co-cultivation, and explant is injured larger.Select raising of the suitable bacterial concentration to conversion ratio
It is critically important.When OD600 values are in 0.3-0.5, gained kanamycin-resistant callus tissue regeneration rate highest, and shadow is grown to explant after co-culturing
Ring little.When OD600 is 0.1, explant blade conversion ratio is relatively low, when the later stage is gradually dead, OD600 is 0.7, explant by
Agrobacterium, which is infected, to have a great influence, and is postponing the quick Necrosis of screening stage.Experiment is infected from OD600 ≈ 0.4 for Agrobacterium
Optimum concentration.
Time of infection is equally an important factor for influenceing explant transformation efficiency.Time of infection is short, and Agrobacterium is not inoculated into
Wound face, the long Agrobacterium of time of infection, which excessively injures explant, causes otch browning dead.
The influence of the bacterial concentration of table 9 and time of infection to conversion ratio
6th, with comparative example 2-6 and 2-7 comparing result
As can be seen from Table 10, the present invention use pre-incubation time for 2d and co-cultivation the time be 3d, compared to others
Incubation time, callus can be made comparatively fast to be formed and there was only a small amount of blade browning.It is most to convert preceding carry out on plant explant
The research of preculture shows that explantation tissue's metabolism can be improved by carrying out preculture, promote cell division, reduce incubation time, profit
Culture medium nutrition is absorbed after explant infects, so as to improve conversion ratio.As can be seen from Table 10, preculture 2d, conversion effect
Rate is all higher than other incubation times.Because generally for the blade of preculture, incision cell is hurt gradual reparation,
Under the induction of regeneration culture medium, the vigorous division of cell, more competent cell is formed, now Agrobacterium tumefaciems is infected more
Sensitivity, the insertion beneficial to T-DNA are integrated, and improve conversion ratio.When pre-incubation time is more than 4d, paddle cutout heals substantially, profit
Reduced in the small molecule phenols material yield of conversion, cell is no longer sensitive, and so as to which rate of rotation reduces, the later stage causes blade in crown gall
Agrobacterium is infected and gradually death under antibiotic-screening dual-pressure.
The principle of Agrobacterium tumefaciens transformation is that T-DNA is transferred in plant cell, and is incorporated into explant genome.
It can not immediately be converted after Agrobacterium attachment, at least 16h ability induced tumors must be grown at incisional wound position and insert T-DNA.
The appropriate co-cultivation time, transformation efficiency is influenceed very crucial.It can be seen that, present invention selection co-cultures 2-3d from table 10,
Compared to other incubation times, there is higher kanamycin-resistant callus tissue regeneration rate；And when co-culturing 4d, because Agrobacterium tumefaciems grew
Amount, the quick browning of explant can be caused dead, conversion ratio decline.If co-cultivation can give birth to more than 4d, explant body cell because of Agrobacterium
Grow and can not regenerate, kanamycin-resistant callus tissue certainly will be cannot get.
The influence of the pre-incubation time of table 10 and co-cultivation time to leaf regeneration and transformation efficiency
7th, with comparative example 2-8 comparing result
As can be seen from Table 11, the present invention carries out the screening of resistant budses using the selection mode for postponing 7-14d screenings, compares
Others postpone screening time, have higher callus regeneration rate and leaf growth good.
As can be seen from Table 11, it is stepped up Kan screenings pressure or delay screening is advantageous to the raising of conversion ratio, reason can
It can be after co-cultivation, explant progressively restoration ecosystem vigor first on screening and culturing medium is postponed, be screened again afterwards,
Environment can preferably be adapted to and grown.
Table 11 postpones influence of the screening time to leaf regeneration and transformation efficiency
| Postpone screening (d) | Callus regeneration rate (%) | Leaf growth situation |
| 0 | 0 | Blade wound browning is serious, most of dead after 5d |
| 2 | 8.7 | Most of blade wound browning is serious, can form callus on a small quantity |
| 7 | 47.6 | 5-7d starts callus occur, well-grown |
| 14 | 48.6 | 5-7d starts callus occur, well-grown |
2nd, HPLC determines strawberry anthocyanidin result
Anthocyanidin in HPLC measure sweet teas Charlie, gold and jade, beauty, a chapter Ji and PMV strains, elution produce three peaks, respectively
For peak 1, peak 2, peak 3；Anthocyanidin in HPLC measure 4-1 strains, equally produces three peaks during elution, but the 3rd peak and peak
3 appearance time is significantly different, is named as peak 4 (such as Fig. 4).
The standard items of fish pelargonium -3-O- glucosides and C-3-G are diluted to respectively 512 μ g/mL,
256μg/mL、128μg/mL、64μg/mL、32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL.Do bid
Directrix curve, the quantitative calculation formula drawn.According to the standard of fish pelargonium -3-O- glucosides and C-3-G
Curve can learn that peak 1 is C-3-G, and peak 2 is fish pelargonium -3-O- glucosides.From this Fig. 5 A and Fig. 5 B
It can be seen that mainly accumulate fish pelargonium -3-O glucosides in all strawberry cultivars；Fish pelargonium -3-O- grapes in 4-1 strains
The content of glucosides and C-3-G is extremely significantly above other kinds；Be not detected by sweet tea Charlie corn flower-
3-O- glucosides.
3rd, gene expression analysis result
At present, the metabolic pathway of anthocyanidin is substantially clear, each structural gene of control anthocyanidin synthesis by gram
Grand, anthocyanidin content is mainly by gene expression regulation (Espley RV, Hellens RP, Putterill J, Stevenson
DE,Kutty-Amma S,Allan AC.Red colouration in apple fruit is due to the
activity of the MYB transcription factor,MdMYB10.Plant Journal,2007,49:414-
427).The reason for annotate anthocyanidin height accumulation in 4-1 strains, inventor also measured were and strawberry anthocyanidin is metabolized dependency structure
The expression characterization of gene and transcription factor between different cultivars.
From fig. 6, it can be seen that with PMV strains (zero load) to compare, the expression quantity of most detection genes in 4-1 strain fruits
Higher than PMV strains.Wherein raise trend most notably chalcone synthase genes (CHS), 8.5 times of the gene upregulation.Secondly
It is enzyme, namely chalcone isomerase gene (CHI), 6.1 times has been raised in 4-1 strains.In addition, flavanones -3- hydroxylases in 4-1 strains
Gene (F3H), flavanonol-4-reductase gene (DFR), anthocyanin synthase gene (ANS), flavonoids -3-O- Portugals
Glucosyl transferase gene (UFGT) also has 2-4 times of up-regulation trend.The MYB1 gene related to anthocyanidin, MYB10 genes,
BHLH3 genes, bHLH33 gene expression amounts measure in, MYB10 genes, bHLH3 gene upregulations are obvious in 4-1 strains, up-regulation
3-4 times.For MYB1 genes and bHLH33 genes, the variation tendency unobvious of 4-1 strains.
How to make Agrobacterium tumefaciens mediated Strawberry Leaves genetic transformation that there is higher conversion ratio and citrus octahydro tomato
Red pigment synthase gene CitPSY stable expression in strawberry is that the key content of technical scheme of the present invention is also difficult point.
The process of genetic transformation is considerably complicated, audient's multifactor impact, as Agrobacterium tumefaciems thalline vigor, institute's band plasmid cause
Characteristic of disease, concentration, time of infection, co-cultivation time, class of antibiotic, screening pressure concentration, recipient cell vigor, recipient cell DNA
Complexity, regeneration condition of culture, explant regeneration period etc., these factor reciprocations, so as to influence transformation efficiency.Crown gall
The concentration and time of infection of Agrobacterium bacterium solution, it is necessary to which disclosure satisfy that can obtain higher regeneration frequency and higher conversion frequency
Rate, the bacterium solution time of infection of low concentration is long or high concentration infects the short period and can not all obtain good changing effect.Kan
It is antibiotic the most frequently used in Genetic Transformation in Higher Plants, it is successfully to screen and obtain the key of resistant budses to select suitable Kan concentration.
Kan has bad influence for plant tissue, and excessive concentration can cause violent toxic action to transformed cells, can cause explant
Body quick death；Concentration is low, and screening is not thorough, can obtain many false positive seedling or chimera seedling.Cef is conventional agriculture bar
Bacteria inhibitor, to be recipient cell normal development, to suppress Agrobacterium continued growth after co-cultivation.Cef is similarly for explant
The toxic effect of body material, Callus formation and adventitious shoot regeneration can be suppressed, while can make to be infected leaf development deformity.This
In invention, when Kan concentration reaches 10mg/L, the callus regeneration rate of blade has become extremely low, almost completely inhibits adventitious bud.
The blade of inoculation and the adventitious bud of regeneration are gradually shoaled dead to albefaction by green.When Kan concentration is higher than 20mg/L, leaf dish obtains
Regeneration is almost totally constrained.Cef concentration can preferably suppress the breeding of Agrobacterium tumefaciems, then high concentration in 250mg/L
It is easily the growth deformity of blade.In the present invention, the Cef liquid regeneration cultures of the also same concentrations of the leaf dish after co-cultivation
Base rinses 1-2 times, can have effective suppression Agrobacterium, reduce the browning of callus and the teratogenesis of blade well.This hair
The bright selection for Kan concentration and conventional dosage difference are very big.Kan is very bright to the adventitious shoot regeneration inhibitory action of Strawberry Leaves
It is aobvious, but the strawberry of different genotype is different to its susceptibility, and the technology of the present invention can not be directly used according to existing dosage
In scheme.In the present invention, early stage presses using 10mg/L Kan as screening, and after Elongation of adventitious bud, screening pressure is progressively increased to 15mg/
L.In root media, Kan is taken root with strong inhibitory action, after Elongation of adventitious bud growth, inoculation to transformed plant
In the root media containing 10mg/L Kan, just taken root after 10w, afterwards normal growth per 3w subcultures once.
It is optimal to postpone changing effect after 2w (i.e. 2 weeks) screenings in the present invention, and non-immediate screening.After leaf dish co-cultures
It is connected to immediately on screening and culturing medium, wound browning is serious, and most of leaf dish yellow or albefaction, regeneration rate substantially reduce.Speculate
Reason may be：Leaf dish starts to expand, be upturned or crimp in edge in culture, if being inoculated in the sieve containing Kan immediately
Screening and culturing on culture medium is selected, is influenceed by antibiotic in the middle part of leaf dish and produces yellow or albefaction, cause auxin and nutrition supplying
Should be insufficient, the cell converted on the edge of leaf dish is unable to differentiation and bud formation.In addition, single transformed cells may not bear Kan choosings
Select pressure and dead, postpone 10d screenings, form a large amount of cell masses around it, the ability to bear that increase screening is pressed, but such case
Under, non-transformed cell and transformed cells grow simultaneously, and false positive and chimera occur being inevitable.In the present invention, obtain
The slowly albefaction in later stage subculture of false positive plant, occur half albefaction in chimera plant, half keeps the state of green,
Also die off afterwards.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (9)
1. citrus phytoene synthase gene is used to be transferred in strawberry to improve the purposes of strawberry anthocyanidin content.
A kind of 2. method for improving strawberry anthocyanidin content, it is characterised in that comprise the following steps：
1) citrus phytoene synthase gene expression plasmid is built；
2) the citrus phytoene synthase gene expression plasmid that step 1) is built is transferred to grass using agrobacterium-mediated transformation
In the certain kind of berries, the strawberry for turning citrus phytoene synthase gene is obtained；
3) strawberry for turning citrus phytoene synthase gene is subjected to molecular biology identification；
4) content of anthocyanidin in the strawberry of expression citrus phytoene synthase gene is detected；
5) anthocyanidin Metabolism-Related Genes Expression amount in the strawberry of citrus phytoene synthase gene is transferred to detect.
A kind of 3. method for improving strawberry anthocyanidin content according to claim 2, it is characterised in that the step 2) acquisition
The method for turning the strawberry of citrus phytoene synthase gene comprises the following steps：
21) strawberry in vitro cuttings material is prepared；
22) the strawberry explant of inoculation is prepared；
23) leaf disk method Agrobacterium-mediated transformation is utilized.
A kind of 4. method for improving strawberry anthocyanidin content according to claim 3, it is characterised in that the step 23) profit
Conversion, which is carried out, with leaf disk method Agrobacterium specifically includes following steps：
A) Agrobacterium containing citrus phytoene synthase gene expression plasmid is rule and cultivated；
B) strawberry explant is obtained into leaf dish in solid explant regeneration culture medium light culture；
C) take step a) single bacterium colony to be inoculated in the fluid nutrient medium containing antibiotic to be cultivated；
D) take the bacterium solution of culture in step c) to be inoculated into the fluid nutrient medium for not containing antibiotic, expand culture；
E) the bacterium solution centrifugation that step d) is cultivated is taken, supernatant is abandoned, is resuspended；
F) leaf dish cultivated step b) is standby in aseptic condition low suspension；
G) liquid for being used to suspend in step f) is filtered out, the bacterium solution that step e) is resuspended is added into leaf dish, shaken, filter out bacterium
Liquid, after the unnecessary bacterium solution of blade is blotted, it is connected on the explant regeneration culture medium of solid and is co-cultured, the co-cultivation is dark
Culture, postponement screening is carried out afterwards, the postponement screening is also light culture；
H) blade postponed after screening is connected to screening and culturing medium；
I) carry out being transferred to illumination cultivation after light culture to blade grows callus, screen resistant budses；
J) resistant budses obtained after screening and culturing are cut, is connected in the plant subculture medium containing antibiotic and cultivates, work as bud
After body grows and vanelets occurs, root induction in the root media containing antibiotic is moved to；
Above-mentioned steps c), j) described in antibiotic resistance and citrus phytoene synthase gene expression plasmid taken
The resistant gene resistance of band is consistent.
5. a kind of method for improving strawberry anthocyanidin content according to claim 4, it is characterised in that described in step g)
Explant regeneration culture medium be added based on MS culture mediums final concentration of 1.5mg/L disleave it is clever and final concentration of
0.2mg/L indolebutyric acid.
A kind of 6. method for improving strawberry anthocyanidin content according to claim 4, it is characterised in that in step g), training altogether
It is 3d to support the time, and the time for postponing screening is 7-14d.
7. a kind of method for improving strawberry anthocyanidin content according to claim 4, it is characterised in that described in step h)
Screening and culturing medium is the Yin for clever, the final concentration of 0.2mg/L of disleave that final concentration of 1.5mg/L is added based on MS culture mediums
The kanamycins of diindyl butyric acid, final concentration of 250mg/L cephalosporin and final concentration of 10mg/L.
8. according to a kind of any one of claim 4 to 7 method for improving strawberry anthocyanidin content, it is characterised in that step
J) in, the plant subculture medium containing antibiotic is the 6- that final concentration of 0.2mg/L is added based on MS culture mediums
Benzyladenine, final concentration of 0.02mg/L indolebutyric acid, final concentration of 0.2mg/L gibberellin, final concentration of 10mg/L
Kanamycins and final concentration of 250mg/L cephalosporin.
9. a kind of method for improving strawberry anthocyanidin content according to claim 8, it is characterised in that described in step j)
Root media containing antibiotic is the indoles fourth for adding final concentration of 0.2mg/L after MS culture mediums are diluted twice simultaneously
Sour, final concentration of 10mg/L kanamycins and final concentration of 250mg/L cephalosporin.
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| CN102660561A (en) * | 2012-05-25 | 2012-09-12 | 西南大学 | Three key genes for anthocyanin biosynthesis from mulberry and application of three key genes |
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| "澳洲青苹"苹果套袋处理后果实着色相关基因的克隆及表达分析;张小军;《中国博士学位论文全文数据库农业科技辑》;20140515(第05期);摘要，第4页第1段，第14页倒数第1段 * |
| Nutrient Limitation is the Main Regulatory Factor for Carotenoid Accumulation and for Psy and Pds Steady State Transcript Levels in Dunaliella salina (Chlorophyta) Exposed to High Light and Salt Stress;Sacha Nicole Coesel et al.;《Mar Biotechnol》;20080501;第10卷;第602-611页 * |
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