CN116042692A - Genetic transformation method of hemerocallis and application thereof - Google Patents
Genetic transformation method of hemerocallis and application thereof Download PDFInfo
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8205—Agrobacterium mediated transformation
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Abstract
The invention provides a genetic transformation method of hemerocallis, which comprises the following steps: infection of a daylily transformed receptor with an agrobacterium-based infection solution, the agrobacterium comprising a gene of interest and a gene encoding an ANS, the agrobacterium being capable of inserting the gene of interest and the gene encoding an ANS into the daylily transformed receptor and co-expressing the gene of interest and the gene encoding an ANS. According to the method, the pansy ANS gene is used for infecting the hemerocalli for the first time, so that the pansy ANS gene with the sequence shown as SEQ ID No.1 can be proved to be capable of effectively improving the success rate of genetic transformation of the hemerocalli, the average positive rate can reach 65% through detection under a fluorescence microscope, and the construction of a hemerocalli genetic transformation system is realized; the antibiotic concentration is gradually reduced in the screening culture process of the hemerocalli of the hemerocallis, so that the browning rate and albino rate of the hemerocallis can be effectively reduced, the genetic transformation system of the hemerocallis is more optimized, and the success rate of genetic transformation of the hemerocallis is further improved.
Description
Technical Field
The application relates to the field of molecular breeding, in particular to a genetic transformation method of hemerocallis and application thereof.
Background
The hemerocallis are perennial root herbs of hemerocallis in lily, have unique flowers and various colors, are easy to cultivate and reproduce, can be used for arranging various flower beds, road isolation belts and forestation grass slopes, are also good ground cover plants, and have good landscaping effects. Research on hemerocallis in China mainly focuses on aspects of hybridization, ploidy breeding, introduction cultivation, variety screening evaluation and the like. Research on hemerocallis at home and abroad mainly focuses on resistance genes, ploidy breeding, genetic characteristics and the like. The flower color of hemerocallis is single, so that ornamental research of flowers and leaves is also a factor which needs to be considered seriously for variety breeding. However, the conventional breeding technology has the problems of great difficulty and long time in breeding rare flower colors or leaf color varieties. Therefore, there is a need to establish a high-efficiency and feasible genetic transformation system for hemerocallis.
The ANS gene is an important anthocyanin synthase and an important regulatory gene in the downstream pathway of anthocyanin synthesis, and can catalyze colorless flavonols to form brick-red geranium pigment, red cyanidin and blue-violet delphinium pigment. The ornamental value of the leaf color of many varieties is not inferior to that of the flower color, and especially for the plants with shorter flowering period such as hemerocallis, the ornamental value of the flower color can be expanded, and the commodity value of the hemerocallis can be improved. There is no prior art currently relating ANS genes to genetic transformation of Hemerocallis.
Disclosure of Invention
The invention aims to provide a high-efficiency and feasible genetic transformation system for hemerocallis, which utilizes agrobacterium to insert target genes and coding genes of pansy ANS (VwANS) into hemerocallis transformation receptors, and enables the target genes and the coding genes of ANS to be co-expressed, so that the genetic transformation efficiency of the hemerocallis is improved.
In one aspect, the present application provides a genetic transformation method of hemerocallis, comprising the steps of:
infection of a daylily transformed receptor with an agrobacterium-based infection solution, the agrobacterium comprising a gene of interest and a gene encoding an ANS, the agrobacterium being capable of inserting the gene of interest and the gene encoding an ANS into the daylily transformed receptor and co-expressing the gene of interest and the gene encoding an ANS.
Further, the coding gene of the ANS is derived from pansy, arabidopsis thaliana, papaya, kumquat, sweet orange or apple; preferably, the gene encoding the ANS is from pansy; more preferably, the coding gene of the ANS comprises the sequence shown as SEQ ID No.1 and/or a sequence having at least more than 90% sequence identity with the sequence of SEQ ID No. 1.
The skilled artisan can design primer sequences for obtaining the coding gene for ANS according to conventional techniques.
In a preferred embodiment, the pansy is used as an RNA template, and the primers shown in SEQ ID No.2-3 are used for PCR amplification reaction to obtain the ANS coding gene shown in SEQ ID No. 1.
VwANS-F(SEQ ID No.2)：GCTCTAGAGCCAGTTGGGCTGGGCTTAGAGVwANS-R(SEQ ID No.3)：GCTCGAGCGGGACCATGTTGTGGAGGAT
Further, the agrobacterium is agrobacterium tumefaciens or agrobacterium rhizogenes; preferably, the agrobacterium is agrobacterium tumefaciens; more preferably, the agrobacterium tumefaciens is selected from one or more of EHA105, EHA101, LBA4404, GV3101, AGL 1; more preferably, the agrobacterium tumefaciens is LBA4404.
Further, the agrobacterium contains an expression vector; preferably, the expression vector is selected from one or more of PBI121, pCAMBIA1300, pCAMBIA1301 and pCAMBIA 3300; more preferably, the expression vector is PBI121;
preferably, the gene of interest and the gene encoding the ANS are located on an expression vector.
Preferably, the expression vector is PBI121 with a fluorescent site, more preferably, the fluorescent site is a GEP fluorescent marker gene.
In a preferred embodiment, the GEP fluorescent gene is used as a target gene, a PBI121-GEP-ANS recombinant vector is constructed, and the recombinant vector is transferred into agrobacterium LBA4404 to infect hemerocalli by utilizing the characteristics of agrobacterium.
It will be appreciated by those skilled in the art that the genetic transformation methods described herein may be used to select a desired gene according to the circumstances, and that the selection of the desired gene does not affect the specific effects of the genetic transformation methods described herein.
Likewise, other fluorescent genes may be selected by those skilled in the art as fluorescent markers.
The fluorescent marker in the application is used for assisting an experimenter to rapidly screen and determine plants or calli successfully transferred into an expression vector. Among them, a person skilled in the art can obtain a known fluorescent marker gene by purchase or synthesis, and design and synthesize its corresponding primer based on the fluorescent marker gene. The fluorescent marker gene may be one or more selected from GEP, GFP, YFP, CFP, BFP, as long as the effect of screening by fluorescent markers can be achieved, and is not particularly limited herein.
Further, the agrobacteria invasion solution OD 600 The value of (1) is 0.5-1.0, and the infection time is 10-20min; preferably, the Agrobacterium invasion solution OD 600 The value of (2) is 0.5, and the infection time is 15min;
preferably, the agrobacterium infection solution also comprises Acetosyringone (AS), and the concentration of the acetosyringone is 100-200 mu mol/L; more preferably, the acetosyringone concentration is 150. Mu. Mol/L.
Further, the genetic transformation method further comprises the step of performing dark culture on the infected hemerocallis transformation receptor; preferably, the dark culture conditions include: the culture temperature is 22-30 ℃, and the dark culture time is 1-2d; more preferably, the dark culture conditions include: the culture temperature is 24-28 ℃, and the dark culture time is 2d.
In a preferred embodiment, the infection of the calli of hemerocalli comprises the following steps:
the prepared (PBI 121-GEP-ANS) Agrobacterium LBA4404 invader solution was introduced into a 100mL conical flask with OD as the invader solution concentration 600 Infection of callus was performed at an as concentration of 150 (. Mu.mol/L) for 15min, =0.5. Then transferring to MS+6-BA0.6mg/L, and dark culturing for 2d at 24-28 ℃.
Further, the hemerocallis transformation receptor comprises hemerocallis root, stem, leaf, flower and/or callus; preferably, the callus is selected.
In the application, the callus obtained by taking different parts of hemerocallis as explants in the hemerocallis transformation process is found to have no obvious difference, so the source part of the callus is not particularly limited.
Further, after infection of the callus, screening and culturing in a screening culture medium, wherein the screening culture medium comprises antibiotics; preferably, the antibiotic comprises one or more of kanamycin (Kan), carbenicillin (Car) and thiabendazole (Cef); preferably, the antibiotics include kanamycin and thiamycin; more preferably, the antibiotics include 50-200mg/L kanamycin and 100-200mg/L thiamycin; more preferably, the antibiotics include 50mg/L kanamycin and 200mg/L thiamycin;
more preferably, the concentration of antibiotic in the screening medium is gradually reduced to 0mg/l during the culture process.
Specifically, during the first stage of culture, the concentration of the antibiotic in the screening medium is as follows: kan 50mg/L and Cef200mg/L;
during the second stage of culture, the antibiotic concentration of the screening medium is as follows: kan 25mg/L and Cef200mg/L;
in the third stage of culture, the antibiotic concentration of the screening culture medium is as follows: kan 0mg/L and Cef 150mg/L;
in the fourth stage of culture, the antibiotic concentration of the screening culture medium is as follows: kan 0mg/L and Cef 100mg/L;
in the fifth stage of culture, the antibiotic concentration of the screening culture medium is as follows: kan 0mg/L and Cef 50mg/L;
in the sixth stage of culture, the antibiotic concentration of the screening culture medium is as follows: kan 0mg/L and Cef 25mg/L;
in the seventh stage of culture, the antibiotic concentration of the screening culture medium is as follows: kan 0mg/L and Cef 0mg/L.
The incubation time of each stage can be adjusted by the person skilled in the art according to the actual situation, for example 10 days in the present application.
Preferably, the screening medium further comprises MS+6-BA0.6 mg/L+30g/L sucrose+6 g/L agar.
In a preferred embodiment, a method for improving genetic transformation efficiency of hemerocallis using pansy ANS (VwANS) gene is provided, comprising the steps of:
1) 2 times of subculturing by using healthy hemerocalli, selecting sterile hemerocalli without adventitious buds, and preculturing for 2d in MS+6-BA0.6mg/L preculture medium;
2) Genetic expression of pansy ANS in hemerocalli of hemerocalli is carried out, specifically, the agrobacterium LBA4404 invasion dye solution containing PBI121-GEP-ANS recombinant vector is used for infecting the hemerocalli of hemerocalli in step 1), and the infection solution concentration is OD 600 =0.5, AS concentration 150 μmol/L, infection time 15min, transfer to MS+6-BA0.6mg/L co-culture medium after infection, dark culture at 24-28deg.C for 2d;
3) Performing degerming on infected hemerocalli, transferring into MS+6-BA0.6mg/L+Kan50mg/L+Cef200mg/L screening culture medium after degerming, performing screening culture to obtain transgenic callus with Kan and Cef resistance, gradually reducing antibiotic concentration in the culture process, and transferring into MS+6-BA0.6mg/L+IAA0.1mg/L induction culture medium without antibiotics after 60 d;
4) And carrying out fluorescence microscope positive detection, phenotype observation and PCR positive identification on the transgenic callus.
The genetic transformation system of the hemerocallis is established and optimized for the pansy ANS gene for the first time, a theoretical basis and a test basis are provided for the molecular breeding work of the hemerocallis, and the genetic system process of the hemerocallis is effectively promoted.
In another aspect, the present application also provides a product for genetic transformation of daylily with agrobacterium, the product comprising: an agent capable of inserting a target gene and a gene encoding an ANS into a Hemerocallis transformation receptor and coexpression of the target gene and the gene encoding an ANS, the agent comprising Agrobacterium including the target gene and the gene encoding an ANS, using the genetic transformation method as described above.
On the other hand, the application also provides an application of the genetic transformation method in preparing hemerocallis.
The invention has the following beneficial effects:
1. according to the method, the pansy ANS gene is used for infecting the hemerocallis for the first time, so that the pansy ANS gene with the sequence shown as SEQ ID No.1 can effectively improve the success rate of genetic transformation of the hemerocallis, the average positive rate can reach 65%, the final PCR detection positive rate is 41%, and the construction of a high-efficiency hemerocallis genetic transformation system is realized;
2. in the application, the antibiotic concentration is gradually reduced to 0mg/l in the screening culture process of the hemerocallis, so that the browning rate and whitening rate of the hemerocallis can be effectively reduced, the minimum can be within 2%, the genetic transformation system of the hemerocallis is more optimized, and the success rate of genetic transformation of the hemerocallis is further improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 is a diagram of the nucleotide sequence and amino acid sequence of pansy and their correspondence;
FIG. 2 is an electrophoretogram of recombinant PBI121-GEP-ANS vector, wherein M: marker DL2000,1: a PCR product;
FIG. 3 is a plot of a screening media optimization test line, wherein the abscissa represents incubation time (d) and the ordinate represents percentage;
FIG. 4 is a fluorescent map of transgenic plant identification, wherein A: effect of wild hemerocalli on natural light, B: effect of wild type hemerocalli under fluorescence microscope, C: effect of transgenic hemerocalli under natural light, D: effect of transgenic hemerocalli under fluorescence microscope;
FIG. 5 is an electrophoretogram of transgenic plant identification, wherein M: marker DL2000,1-2: transgenic plant DNA.
Detailed Description
In order to more clearly illustrate the general concepts of the present application, the following detailed description is made by way of example with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
In the following embodiments, unless specified otherwise, the reagents or apparatus used are conventional products available commercially without reference to the manufacturer.
The media and their formulations referred to in this application are shown in tables 1 and 2:
TABLE 1
Note that: the additional reagent can be adjusted according to the actual requirement
TABLE 2
The preparation of the agrobacterium competent cells can be selected by a person skilled in the art or can be obtained by purchasing, and the preparation method of the agrobacterium competent cells comprises the following steps:
the first step: picking newly activated LBA4404 colonies from the LB plate, inoculating the newly activated LBA4404 colonies into 10mL of LB liquid medium, and shake culturing overnight at 28 ℃;
and a second step of: inoculating 1mL of bacterial liquid into 100mL of LB liquid culture medium, and shake culturing overnight at 28 ℃ by a shaking table until A600 is equal to 0.6;
and a third step of: placing the culture on ice for 10min;
fourth step: transferring the culture into a 50mL centrifuge tube, and centrifuging at 4000rpm for 10min at 4 ℃;
fifth step: the supernatant was carefully removed and the cells were treated with 30mL of cold 0.1M CaCl 2 Suspending and precipitating, ice-bath for 20min, and centrifuging at 4000rpm and 4deg.C for 10min;
sixth step: the supernatant was carefully removed and the cells were treated with 2mL of cold 0.1M CaCl 2 The pellet was suspended and ice-bathed for 10min, and the suspension was dispensed into 1.5mL centrifuge tubes at 200. Mu.L per tube. Finally, the agrobacteria competent cells are stored at-80 ℃ and are preferably prepared on site so as to ensure the activity of the competent cells.
EXAMPLE 1 construction of recombinant PBI121-GEP-ANS
1. Cloning of pansy ANS
Taking pansy as an RNA template, carrying out PCR amplification reaction by using a primer shown as SEQ ID No.2-3, wherein the PCR reaction program is that the reaction is finished in 35 cycles of pre-denaturation at 95 ℃ for 5min, pre-denaturation at 94 ℃ for 1min, pre-denaturation at 58 ℃ for 1min and pre-denaturation at 72 ℃ for 90s, and the reaction is finished at 72 ℃ for 5 min.
The agarose gel electrophoresis is used for recovering the gene sequence with the length of about 1000bp, the specific pansy ANS gene sequence is shown as SEQ ID No.1, the specific nucleotide sequence, the amino acid sequence and the corresponding relation thereof are shown as figure 1, escherichia coli DH5 alpha is transformed after being connected with pMD18-T, LB+100mg/L ampicillin flat-plate culture is carried out, the culture condition is 37 ℃ overnight, white single colony is selected and inoculated in LB liquid culture medium for culture, the culture condition is 37 ℃ overnight, plasmid containing target ANS gene is selected and sequenced and verified, the specific verification result is shown as figure 2, and the pMD18-T recombinant plasmid connected with the pansy ANS gene sequence can be successfully obtained from figure 2.
The amplification primer (containing the cleavage site) sequence is as follows:
VwANS-F(SEQ ID No.2)：GCTCTAGAGCCAGTTGGGCTGGGCTTAGAG
VwANS-R(SEQ ID No.3)：GCTCGAGCGGGACCATGTTGTGGAGGAT
2. construction of PBI121-GEP-ANS plant expression vector
The recombinant plasmid (containing the target gene fragment ANS with correct sequencing result) and the PBI121-GEP are respectively digested with XbaI and BamHI, 2 plasmids are connected, escherichia coli DH5 alpha is transformed, LB+100mg/L kanamycin flat plate culture is carried out, the culture condition is 37 ℃ overnight, white single colony is selected and inoculated in LB liquid culture medium for culture, and the culture condition is 37 ℃ overnight, thus obtaining the recombinant plasmid PBI121-GEP-ANS.
3. Preparation of Agrobacterium PBI121-GEP-ANS suspension (Agrobacterium invasion solution)
The recombinant plasmid extracted after the enzyme digestion reaction is transformed with agrobacterium, and the transformation method is as follows:
the first step: agro-competent cells (LBA 4404) were removed from-80℃and thawed on ice and immediately added to 1-5. Mu.L plasmid DNA and centrifuge tubes;
and a second step of: placing the centrifuge tube on ice for 30min;
and a third step of: placing the centrifuge tube into liquid nitrogen for cooling for 1min;
fourth step: placing the centrifuge tube into a water bath kettle for thawing for 5min at 37 ℃;
fifth step: adding 800 mu L of LB liquid medium without antibiotics into the centrifuge tube, and shake culturing for 2-4h at 28 ℃;
sixth step: centrifuging at 4000rpm for 5min, collecting thallus, suspending and precipitating with 100 μl of LB liquid medium, sucking out excessive liquid, uniformly coating the bacterial liquid on LB+100mg/L kanamycin solid medium, and standing at 28deg.C for 2-3d.
Picking single colony for containingLB liquid culture medium with 100mg/L kanamycin resistance is subjected to shake culture in a constant temperature shaking table at 28 ℃ for 2d until the bacterial liquid OD 600 The value is between 0.5 and 1.0, and the agrobacterium infection solution is obtained and used for infecting the calli of hemerocalli of hemerocallis.
Example 2 method for transforming Hemerocallis calli Using ANS Gene
A method for transforming hemerocalli using an ANS gene, comprising the steps of:
1) Agrobacterium liquid activation
Picking single colony of agrobacterium PBI121-GEP-ANS in example 1, performing LB liquid culture medium containing kanamycin resistance of 100mg/L, shake culturing at constant temperature of 28deg.C for 2d, and culturing until the bacterial liquid OD 600 The value is between 0.5 and 1.0, and is used for infecting calli of hemerocalli of hemerocallis.
2) Pre-culture
Laboratory-cultured, sterile, day lily callus without adventitious buds was selected and precultured for 2d at 24℃to 28℃in MS+6-BA0.6mg/L preculture medium.
3) Co-cultivation
Since no complete genetic system for hemerocalli exists at present, in order to find a proper infection method, the OD (optical density) of infection liquid is designed 600 Four factors including value, infection time, AS concentration and culture condition are designed, four-factor three-level orthogonal tests are designed, and each treatment is repeated for 3 times, so that an optimal infection scheme is obtained. Specific orthogonal test designs are shown in table 3, and specific results are shown in table 4:
TABLE 3 orthogonal test design
Table 4 statistics of test data
Note that: k1 and K2 are average values, R1 represents the extreme value of mortality, and R2 represents the extreme value of positive rate.
As can be seen from the results of table 4, the comparison of the magnitudes of the combined R values (factor difference values) can be seen from three factors: for R1 (mortality, R values ranging from small to large), factor four culture conditions are the optimal factor, followed by factor three AS concentrations, and then by factor one OD 600 The value is finally factor two infection time; for R2 (positive rate, R values are arranged from large to small), factor-OD 600 The value is the optimal factor, the infection time of the second factor and the concentration of the third AS, and the culture condition of the fourth factor.
Specifically combining the optimal levels of the factors, the death rate and the positive rate are considered (low K1 death rate and high K2 positive rate), and the factor is OD 600 The first level is optimally OD 600 =0.5, the second level of factor two infection time was optimally 15min, the second level of factor three AS concentration was optimally 150 μmol/L, and the first level of factor four culture conditions was optimally dark culture 2d.
In summary, the optimal infection scheme in the genetic transformation method of hemerocallis is as follows: the concentration of the agrobacterium infection liquid is OD 600 =0.5, as concentration 150 μmol/L, infection time 15min, dark incubation 2d.
And (3) carrying out infection on the hemerocalli of hemerocallis according to the optimal infection scheme, wherein the method specifically comprises the following steps of:
the prepared agrobacterium infection solution is introduced into a 100mL conical flask, and the concentration of the infection solution is OD 600 Infection of callus was performed at an as concentration of 150 (. Mu.mol/L) for 15min, =0.5. Then transferring to MS+6-BA0.6mg/L co-culture medium, and dark culturing at 26+ -2deg.C for 2d.
EXAMPLE 3 cultivation of Hemerocallis fulva callus
The infected calli of hemerocalli obtained in example 2 were subjected to subsequent cultivation, specifically comprising two steps of degerming and screening cultivation.
1、Degerming：
Firstly, soaking the callus in sterilized water containing 200mg/mLCar for 20min, and pouring out the sterilized water; soaking the callus in sterilized water containing 150mg/mLCar, washing for 15min, and pouring out the sterilized water; soaking the callus in sterilized water containing 100mg/mLCar, washing for 10min, and pouring out sterilized water; finally, the callus is soaked in sterilized water containing 50mg/mLCar for 5min, the sterilized water is poured out, and the moisture on the surface of the callus is sucked by sterilized filter paper.
The day lily calli were subjected to the above degerming treatment every 10 d.
2. Screening and culturing:
in the process of culturing transgenic hemerocalli, whitening and browning phenomena occur to different degrees, and the literature proves that the phenomena are related to the concentration and the type of antibiotics, so that the orthogonal test of the type and the concentration of the antibiotics is carried out on the hemerocalli cultured by the same infection scheme, and the optimal combination and concentration of antibiotics which are most suitable for the growth of the hemerocalli are selected, and the primary positive rate detection is carried out by a fluorescence microscope. Each treatment was repeated 3 times, the specific orthogonal test design is shown in table 5 and the results analysis is shown in table 6:
TABLE 5 screening of Medium antibiotics 3 factor 3 horizontal test design
TABLE 6 screening test data statistics for culture medium antibiotics
Note that: k1 and K2 are average values, R1 represents the extreme value of mortality, and R2 represents the extreme value of positive rate.
From the range analysis table, it is known that: from the comparison of the magnitudes of the combined R values (factor difference values), it is known that the optimal factor is the concentration of Cef, followed by the concentrations of Kan and Car for R1 (mortality, R values ranging from small to large), and R2 (positivity, R values ranging from large to small).
Specifically, by combining the optimal levels of the factors, the mortality and the positive rate are considered (the death rate of K1 is low, the positive rate of K2 is high), the second level of the factor I Kan is optimal to be 50mg/L, the first level of the factor II Car is optimal to be 0mg/L, and the third level of the factor Cef is optimal to be 200mg/L.
All antibiotic combination tests were orthogonal tests performed under the same conditions as the optimal infection combination, and from the results of the orthogonal tests, the optimal antibiotic combination was Kan50mg/L+Cef200mg/L.
In summary, the method for culturing the infected hemerocalli comprises the following steps: after the degerming is completed, the strain is transferred into a screening culture medium of MS+6-BA0.6 mg/L+Kan50mg/L+Cef200mg/L for bacteriostasis culture.
Example 4 screening Medium optimization test
In this application, it is found that, on the one hand, the addition of antibiotics to the screening medium in example 3 can well inhibit the growth of agrobacterium, but the too high antibiotic concentration has a certain degree of inhibition effect on plant growth, in order to improve browning and whitening of the calli of hemerocalli after infection of agrobacterium, and to better optimize the genetic transformation system of hemerocalli, the optimal infection concentration and time of the calli of hemerocalli infected with agrobacterium are set, the optimal antibiotic concentration is used as a control, and the experiment of reducing the antibiotic concentration of the medium is carried out along with the increase of the cultivation time of the calli of hemerocalli, and each group of calli infected with hemerocalli is repeated for 100 times. The test results are shown in Table 7 and FIG. 3.
TABLE 7
Fig. 3 is a line graph made from the data of table 7, wherein:
average positive rate (%) of fluorescence detection: y= -0.1521x+66.256, r 2 ＝0.2727
Average browning rate (%) of day lily calli: y= -5.6568x+40.417, r 2 ＝0.9953
Average albino rate (%) of day lily calli: y= -2.1361x+15.99, r 2 ＝0.9814
According to the test results, the antibiotic concentration in the culture medium is gradually reduced along with the increase of the culture time, and the average positive rate of fluorescence detection is basically stabilized at about 65%, which shows that the reduction of the antibiotic concentration does not influence the positive rate, and especially when the concentrations of antibiotics Kan and Cef are reduced to 0, the browning rate of the calli is reduced to 2.01%, the whitening rate is reduced to 1.57%, so that the genetic transformation system of hemerocallis can be better optimized.
Example 5 pansy ANS Gene Positive Rate versus other Gene test
Under the condition that the orthogonal test factors and the levels are consistent with those of the embodiment 4, the result shows that the average positive rate of the Hemerocallis calluses is 32.11 percent (specific data are shown in the following table 8) when the Hemerocallis calluses are infected by using the F3'5' H genes of petunia, and the average positive rate of the Hemerocallis calluses is basically consistent with that of other Hemerocallis genetic transformation tests, and the average positive rate of the Hemerocallis subjected to the pansy ANS is 65 percent, which indicates that the pansy ANS genes can improve the positive rate of the calluses.
TABLE 8
EXAMPLE 6 transgenic plant acquisition
The transgenic calli obtained in example 4 were observed under a fluorescence microscope, as shown in FIG. 4, and the transgenic calli showed a green fluorescence effect under the microscope, whereas the wild type did not.
EXAMPLE 7 rooting culture of transgenic plants
The adventitious buds of the transgenic hemerocalli obtained in example 4 are cut and placed in a rooting culture medium of 1/2MS+NAA 0.5mg/L to induce rooting for 30d, and domesticated and transplanted to a big pot, so that 200 positive plants can be obtained.
The DNA of the transgenic hemerocallis plant is extracted, the PCR detection is carried out on the positive hemerocallis by utilizing the specific primer SEQ ID No.2-3 of the gene, as shown in the result of figure 5, the gene is successfully transferred into the hemerocallis, the target band is about 1000bp and is consistent with the size of the target gene, the obtained hemerocallis is proved to be the transgenic hemerocallis with pansy ANS gene, and the number of positive seedlings is 82 in the experiment.
The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.
Claims (10)
1. A genetic transformation method of hemerocallis, which is characterized by comprising the following steps:
infection of a daylily transformed receptor with an agrobacterium-based infection solution, the agrobacterium comprising a gene of interest and a gene encoding an ANS, the agrobacterium being capable of inserting the gene of interest and the gene encoding an ANS into the daylily transformed receptor and co-expressing the gene of interest and the gene encoding an ANS.
2. The genetic transformation method according to claim 1, wherein the ANS encoding gene is from pansy, arabidopsis, papaya, kumquat, orange or apple; preferably, the gene encoding the ANS is from pansy; more preferably, the coding gene of the ANS comprises the sequence shown as SEQ ID No.1 and/or a sequence having at least more than 90% sequence identity with the sequence of SEQ ID No. 1.
3. The genetic transformation method according to claim 1, wherein the agrobacterium is agrobacterium tumefaciens or agrobacterium rhizogenes; preferably, the agrobacterium is agrobacterium tumefaciens; more preferably, the agrobacterium tumefaciens is selected from one or more of EHA105, EHA101, LBA4404, GV3101, AGL 1; more preferably, the agrobacterium tumefaciens is LBA4404.
4. The genetic transformation method according to claim 3, wherein the agrobacterium contains an expression vector; preferably, the expression vector is selected from one or more of PBI121, pCAMBIA1300, pCAMBIA1301 and pCAMBIA 3300; more preferably, the expression vector is PBI121;
preferably, the gene of interest and the gene encoding the ANS are located on an expression vector.
5. The genetic transformation method according to claim 1, wherein the agrobacterium infection solution OD 600 The value of (1) is 0.5-1.0, and the infection time is 10-20min; preferably, the Agrobacterium invasion solution OD 600 The value of (2) is 0.5, and the infection time is 15min;
preferably, the agrobacterium infection solution also comprises acetosyringone, and the concentration of the acetosyringone is 100-200 mu mol/L; more preferably, the acetosyringone concentration is 150. Mu. Mol/L.
6. The genetic transformation method according to claim 1, further comprising the step of dark culturing the infected hemerocallis transformed recipient; preferably, the dark culture conditions include: the culture temperature is 22-30 ℃, and the dark culture time is 1-2d; more preferably, the dark culture conditions include: the culture temperature is 24-28 ℃, and the dark culture time is 2d.
7. The genetic transformation method according to claim 1, wherein the hemerocallis transformation receptor comprises hemerocallis root, stem, leaf, flower and/or callus; preferably, the callus is selected.
8. The genetic transformation method according to claim 7, wherein the callus is subjected to a screening culture in a screening medium after infection is completed, the screening medium comprising an antibiotic; preferably, the antibiotics comprise one or more of kanamycin, carbenicillin and thiabendazole; preferably, the antibiotics include kanamycin and thiamycin; more preferably, the antibiotics include 50-200mg/L kanamycin and 100-200mg/L thiamycin; more preferably, the antibiotics include 50mg/L kanamycin and 200mg/L thiamycin;
more preferably, the concentration of antibiotic in the screening medium is gradually reduced to 0mg/l during the culture process.
9. A product of genetic transformation of hemerocallis using agrobacterium, comprising: an agent capable of inserting a gene of interest and a gene encoding an ANS into a hemerocallis transformation receptor and coexpression of the gene of interest and the gene encoding an ANS using the genetic transformation method according to claim 1, the agent comprising agrobacterium comprising the gene of interest and the gene encoding an ANS.
10. Use of the genetic transformation method according to any one of claims 1-8 for the preparation of hemerocallis.
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