CN115992169A - Efficient lily quilt sheet transient expression method and application - Google Patents
Efficient lily quilt sheet transient expression method and application Download PDFInfo
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Abstract
The invention discloses a high-efficiency lily quilt sheet transient expression method and application, and relates to the technical field of plant genetic engineering. The influence of three factors of agrobacterium strain type, acetosyringone (AS) concentration and suction air pressure on the transient expression efficiency of GUS genes is determined through an orthogonal test, and the method is used for carrying out the function verification of three genes, namely LoWRKY33, loSAG5 and LoSAG10, of Super1300 vector with GFP tag protein, so that the protein coded by the target gene can be smoothly expressed in lily quilt pieces. The method greatly shortens the time for verifying the gene functions in the lily, and lays a good foundation for screening and researching the functions of important genes in the lily flower senescence-opening process.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a high-efficiency lily quilt sheet transient expression method and application.
Background
Lily (Lilium spp.) is a bulb flower of Lily genus of family Liliaceae, and perennial bulb herb is mainly distributed in temperate regions of northern hemisphere such as east Asia, europe, and North America. China is the natural distribution center of the world Lily plant, which has about 110-115 species, and China has 55 species and 18 varieties (Li Jiewen, etc., 2019). Lily is an important cut flower type in the world, and in the production of fresh cut flowers in China, the planting scale of lily is continuously enlarged, and the annual increase is more than 20%. However, the cut lily flowers cannot be stored and transported, the economic loss caused by the storage and fresh-keeping links reaches as much as 40% (Wu Zhongjun, etc. 2013), and in addition, the cut lily flower vase has a short service life, which severely restricts the ornamental quality. The screening and identification of key genes in the aging process of lily flowers, and further the delay of the aging of lily flowers by molecular breeding or preservation technology have important theoretical value and potential application value.
To study gene function, previous attempts have developed methods such as agrobacterium-mediated genetic transformation, virus-induced gene silencing and transient overexpression. Agrobacterium-mediated genetic transformation can be further subdivided into inflorescence dipping, leaf disc, somatic embryo transformation, apical meristem transformation, and the like, depending on the plant material transformed. The inflorescence dipping method is suitable for model plant Arabidopsis thaliana, the infection process can be completed only by soaking inflorescences in bacterial liquid carrying target genes, and then the transgenic positive seedlings are obtained by carrying out resistance screening on seeds (Zhang et al, 2006). The leaf disc method is widely used for transformation of plants such as chrysanthemum (Hu et al, 2018) and tobacco (Wang et al, 2019). In recent years, embryo transformation has also been successful in flowers such as China rose (Zhang et al, 2021), rose (Shen et al, 2019) and lily (Yan et al, 2020). In addition, the apical meristem of plants can directly form shoots without undergoing dedifferentiation and redifferentiation processes (Nguyen et al, 2016), and methods of transformation using the apical meristem have been applied in some plants with difficult regeneration, such as cotton (Rajasekaran et al, 2019) and wheat (Hamada et al, 2017). Agrobacterium-mediated genetic transformation can obtain stably expressed transgenic plants, but has good regeneration capability and higher transformation efficiency depending on the transformed plant material, and the process is quite long.
To rapidly identify gene function, the former developed a method of virus-induced gene silencing (virus-induced gene silencing, VIGS) based on tobacco brittle virus (tobacco rattle virus, TRV) for silencing of genes of interest (Liuet al, 2002). The TRV virus is derived from tobacco, so that the TRV virus has higher infection efficiency on the solanaceae plants such as tobacco, tomatoes, peppers and the like, and has good effect on the function research of important genes in apple fruits, china rose and carnation flowers. In lily, the function of the LoPIP2 gene in lily anther dehiscence (Tong et al, 2013) and LsGRP1 gene in lily leaf infection fungal disease process was also studied by VIGS technology (Lin et al, 2020). There are also methods of transient overexpression corresponding to transient silencing. This method finds application in China rose and chrysanthemum flowers (Luo et al, 2021a; wang et al, 2021;Yasmin and Debener,2010), whereas in lily, wu et al also tried transient overexpression of the LlHsfA3A and LlHsfA3B genes in lily sheets (Wu et al, 2018), which requires tiling lily sheets on MS medium for culture, requires high requirements for aseptic operation conditions, and the culture time is only 2 days, and no phenotypic change can be observed.
The former research shows that the lily has long growth period and difficult genetic transformation, so that developing a fast and efficient transient transformation system to screen and identify key genes affecting lily flower opening and aging has important theoretical and application values.
Disclosure of Invention
The invention aims to solve the problems and provide a high-efficiency lily quilt sheet transient expression method. The optimal transformation conditions were determined by orthogonal experiments as follows: the instantaneous expression efficiency of petals is highest and reaches 94.3% when the agrobacterium strain is GV3101, AS concentration is 100 mu mol/L and suction air pressure is 0.7 standard atmospheric pressure.
Another object of the present invention is to perform functional verification of lily senescence-associated genes LoWRKY33, loSAG5 and LoSAG10 using the above-described transient expression method. First, super1300 vector with GFP tag protein was transformed into lily quilt pieces, and GFP fluorescence showed that the transient expression system was viable. Secondly, the system is used for carrying out functional verification of three genes, namely LoWRKY33, loSAG5, loSAG10 and the like, which shows that the aging of lily flowers can be obviously promoted after the LoWRKY33 and the LoSAG10 are over-expressed.
In order to achieve the above purpose, the technical scheme of the invention is as follows: an efficient lily quilt sheet transient expression method comprises the following steps:
s1, taking oriental lily 'Siberia' as a material, and converting a binary expression vector PBI121 carrying a GUS reporter gene into lily sheets through mediation of agrobacterium tumefaciens and negative pressure suction;
s2, after infection, washing out agrobacterium attached to the lily quilt pieces by using deionized water;
s3, spreading the washed lily quilts in a culture dish filled with two layers of wet filter paper, performing dark culture for 3 days at the temperature of 23 ℃, transferring to the illumination condition of 23 ℃ for culture, recording for 0 days, photographing every 2 days, recording phenotype changes, and preliminarily determining gene functions through physiological index measurement and gene expression level detection, so as to establish an efficient transient expression system in the lily quilts.
The invention also provides application of the high-efficiency lily quilt piece transient expression method, and application of the lily quilt piece transient expression method in LoWRKY33 gene function research.
Further, the LoWRKY33 gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
The invention also provides application of the high-efficiency lily quilt piece transient expression method, and application of the lily quilt piece transient expression method in LoSAG5 gene function research.
Further, the sequence of the LoSAG5 gene is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
The invention also provides application of the high-efficiency lily quilt piece transient expression method, and application of the lily quilt piece transient expression method in LoSAG10 gene function research.
Further, the sequence of the LoSAG10 gene is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6.
Compared with the prior art, the beneficial effect of this scheme:
the lily quilt piece transient expression method is simple and convenient to operate, low in cost and high in conversion efficiency, the transient conversion efficiency is up to 94.3%, and the functions of a plurality of genes in the lily senescence process are achieved by utilizing the method, so that the lily senescence is obviously promoted after LoWRKY33 and LoSAG10 are over-expressed, and important gene resources are provided for research of the flower senescence mechanism and molecular breeding of anti-aging varieties.
Drawings
FIG. 1 is a technical flow chart of a method according to an embodiment of the invention;
FIG. 2 shows GUS staining of 16 treated festooned discs in an example of the invention;
FIG. 3 is a graph of GFP fluorescence observations of the infected and uninfected lily quilt discs in the examples of the invention;
FIG. 4 is a construction map of LoWRKY33 overexpression vector in the examples of the invention;
FIG. 5 illustrates the transient overexpression of LoWRKY33 in the perianth of the lilium oriental hybrid;
FIG. 6 is an analysis chart of the expression of salicylic acid synthesis genes in LoWRKY33 overexpression samples in the examples of the present invention;
FIG. 7 is a chart showing GFP fluorescence observation in Super1300 empty transient over-expression lily quilts in an embodiment of the present invention;
FIG. 8 is a graph showing GFP fluorescence in a sheet of lily by transient over-expression of LoSAG5 in an embodiment of the invention;
FIG. 9 is a graph showing GFP fluorescence in a sheet of lily by transient over-expression of LoSAG10 in an embodiment of the invention;
FIG. 10 phenotype of LoSAG5 and LoSAG10 overexpressing lily pieces in the examples of the invention;
FIG. 11 is a graph showing ion permeability and MDA content of a LoSAG5 and LoSAG10 overexpressing lily quilt sheet in an embodiment of the present invention;
FIG. 12 overexpression of LoSAG5 and LoSAG10 of gene expression levels in lilium in examples of the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the illustrated embodiments are merely exemplary of some, but not all, of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
Example 1:
transient expression of GUS reporter gene in lily quilt pieces
1) Preparation of strains, vectors, culture Medium and drugs
Agrobacterium strains EHA105, GV3101, LAB4405 and AGL1 were purchased from Wohan LvRi Biotech Co.
Expression vector PBI121 was self-contained by the laboratory.
The culture medium used for the agrobacterium culture is LB (1L): 5g of Yeast Extract (Yeast Extract), 10g of peptone (Tryptone) and 10g of sodium chloride (NaCl), adjusting the pH to 7.2-7.4 after the Yeast Extract, the peptone (Tryptone) and the sodium chloride are fully dissolved, fixing the volume to 1L, and sterilizing at the high temperature of 121 ℃ for 15min. For solid LB culture medium, agar 15g/L should be added after regulating pH, then volume is fixed to 1L, high temperature sterilization is carried out, when cooling to about 55 ℃, filter sterilized Kanamycin (Kanamycin, kan) is added into an ultra clean workbench, kan final concentration is 50mg/L, after uniform mixing, the mixture is poured into sterilized flat plates, and about 30mL of culture medium is poured into each plate.
1mol/L2- (N-morpholino) ethanesulfonic acid [2- (N-morpholinic) ethanesulfonic acid, MES ] mother liquor: 19.52g of MES powder was dissolved in 100mL of distilled water, sterilized by suction filtration through a sterilized 0.22 μm filter, sub-packaged in 10mL sterile centrifuge tubes and stored at 4 ℃.
0.05mol/LAS: 1g of AS powder was dissolved in 100mL of dimethyl sulfoxide (DMSO), and the solution was sufficiently dissolved, without sterilization, and stored at 4 ℃.
GUS histochemical staining solution (100 mL): 500. Mu. L X-Gluc solution (100. Mu.g/. Mu.L), 75.5mL of 0.1mol/L phosphate buffer (pH=7.0), 1mL of 5mmol/L potassium ferricyanide, 1mL of 5mmol/L potassium ferrocyanide, 2mL of 0.5mol/L EDTA, 20mL of methanol, 100. Mu.LTriton X-100. Wherein, the preparation of X-Gluc solution comprises the following steps: 20-Gluc was dissolved in 200. Mu.LN, N-dimethylformamide to give an X-Gluc solution having a final concentration of 100. Mu.g/. Mu.L.
2) Transformation of Agrobacterium
Sterilizing and ventilating the ultra-clean workbench, taking out the stored agrobacterium from the temperature of minus 80 ℃, thawing, adding 5 mu LPBI121 plasmid into the agrobacterium, gently mixing, and carrying out ice bath for 30min;
the bacterial liquid mixed with the plasmids is placed into liquid nitrogen to be frozen for 3min, then placed into warm water with the temperature of 37 ℃ to be bathed for 5min, and then the pipe is rapidly inserted into ice for 5min;
600 mu L of LB culture solution is added into an ultra-clean workbench, and a shaking table at 28 ℃ is put for culturing for 4 hours at a rotating speed of 200 rpm;
taking out the bacterial liquid, centrifuging for 6min at the normal temperature with the rotation speed of 5000rpm, removing 600 mu L of supernatant in an ultra-clean workbench, and uniformly stirring and mixing the residual 100 mu L of bacterial liquid by using a pipetting gun;
burning and sterilizing the coating agent for coating the plate with alcohol in an ultra-clean workbench, and using after cooling;
sucking 50-100 mu L of fungus liquid drop on LB culture medium containing Kan antibiotics, and coating the fungus liquid with a coater;
after the bacterial liquid is dried, sealing the flat plate by using a sealing film, and placing the flat plate in a 28 ℃ incubator for inversion culture for 2 days.
3) Treatment of lily cut-flower material
Fresh oriental lily 'Siberia' cut flowers are purchased from flowers and trees in the southern lake of Wuhan, hubei province, transported to a laboratory within half an hour, stem segments of about 10cm of the basal parts of the flowers are subtracted from tap water, leaves of 15cm or less of the basal parts of the flowers are removed, and the basal parts of the flowers are soaked in deionized water for later use.
Before infection, a festoon sheet of flowers in a full bloom stage is taken, a puncher is used for punching out a circular sheet with the diameter of 12mm on the festoon sheet, and the circular sheet is soaked in deionized water for standby.
4) Exploration of transformation conditions
Three factors four-level orthogonal experiments were designed by optimizing three factors that may affect transformation efficiency, agrobacterium strain type, AS concentration, and suction air pressure (table 1).
Table 1 three-factor four-level orthometric table
5) Culturing and transforming process of agrobacterium
Culturing agrobacterium:
agrobacterium monoclonal was picked from the plates with toothpicks, inoculated into fresh LB medium (+50 mg/LKan), cultured overnight in a shaker at 28℃and detected as positive by PCR. The check primers used were:
detection of up 5'-AGGCTTTACACTTTATGCTTCC-3' by PBI121
PBI121 detection Low 5'-GCCCGGCTTTCTTGTAACGCGCTTT-3'
Agrobacterium that detected positive was selected, 200. Mu.L of the broth was added to 200mL of fresh LB medium (+50 mg/L Kan+10mmol/L MES+20. Mu. Mol/L AS) and incubated overnight in a shaker at 28 ℃.
Taking out the bacterial liquid, centrifuging at 5000rpm and 8 ℃ for 6min, removing the supernatant, re-suspending bacterial blocks with the infection liquid, and regulating the bacterial liquid concentration to OD by using a spectrophotometer 600nm =0.8, and left to stand in dark for 4 h.
Preparing additional invasion dye liquor:
500mL of an counterstain was prepared, which contained magnesium chloride (final concentration 10 mM), MES (final concentration 10 mM) and AS. The amount of AS was added according to the concentration gradient in the orthogonal test. After the drug is sufficiently dissolved, the pH is adjusted to 5.6.
According to the orthogonal test design, soaking lily slices in bacterial solutions of different treatments, placing bottles filled with the bacterial solutions in an air suction disc, reducing the air pressure in the air suction disc to 0.4-0.7 atmosphere (atm) by using a vacuum pump, and then slowly recovering to normal air pressure;
cleaning the lily quilt wafer 3 times by tap water after suction and 2 times by deionized water;
spreading two pieces of qualitative filter paper in a plastic culture dish with the diameter of 15cm, adding a proper amount of deionized water, and keeping the filter paper moist;
spreading the lily quilt discs on filter paper, and placing 35-40 discs on each disc;
the dishes were incubated at 23℃in the dark for 3 days.
6) GUS staining and data statistics
Taking out the lily quilt discs after being subjected to dark culture for 3 days after being subjected to agrobacterium infection;
adding GUS histochemical staining solution until the petals are immersed;
culturing overnight at 37 ℃;
taking out the overnight dyed lily quilt wafer, adding 70% alcohol for rinsing, photographing according to the dyeing condition of the lily quilt wafer, and counting the dyeing condition. GUS staining of 16 treated perianth discs is shown in FIG. 2.
To quantify the stained condition of the floral disc, the following 4 grades were assigned according to the size of GUS stained area:
TABLE 2GUS staining series partitioning
Based on the number of quantization steps, the number of weighting steps for each process is calculated to determine the optimal expression condition for transient expression, and the result is shown in Table 3.
TABLE 3GUS staining results and weighted ranking results
From the classification weighted results, when the agrobacterium strain is GV3101, the AS concentration is 100 μmol/L, and the suction air pressure is 0.7atm, the gene transient expression efficiency is highest, 4 out of 70 total flower and plant discs are not stained with GUS, 36 discs are 1% -25%, 18 discs are 26% -50%, 12 discs are 51% -100%, the weighted progression = (4×0+36×1+18×2+12×3)/70=1.54, and the expression efficiency = (36+18+12)/70 is 94.3%.
Example 2:
transient expression of GFP reporter gene in lily sheet
Sterilizing and ventilating the ultra-clean bench, taking out the stored agrobacterium GV3101 competence from the temperature of-80 ℃, thawing, and transferring 5 mu L of Super1300 plasmid into agrobacterium; the following primers were used to detect agrobacterium positivity.
Superup：5'-GGATAAATAGCCTTGCTTCC-3'
GFPlow：5'-GAACTTGTGGCCGTTTACG-3'
Agrobacteria carrying Super1300 plasmid were transferred into lily discs by vacuum aspiration, and during infection, AS concentration was 100. Mu.M and aspiration pressure was 0.7 atm.
After infection with Agrobacterium, the cells were dark-cultured at 23℃for 3 days, and GFP fluorescence was observed under a split microscope. GFP fluorescence was randomly observed for 24 post-infection lily and 4 uninfected floral discs, indicating that 21 of the 24 post-infection floral discs exhibited varying degrees of GFP fluorescence with an infection efficiency of 87.5% and 4 uninfected discs were not fluorescent (fig. 3).
Example 3:
instantaneous expression of LoWRKY33 gene in lily quilt sheet
1) Cloning of LoWRKY33 Gene
Based on the sequencing result of a second generation and third generation transcriptome in the early lily opening process (Luoetal, 2021 b) of the applicant, a WRKY gene LoWRKY33 which presents a higher expression level in the lily senescence process is screened, the sequence number is i1_LQ_ly14_c 15136/f1p0/1297, and the length is 1300bp. The full length sequence of the LoWRKY33 gene was obtained with reference to the previously published lily second generation transcriptome (Dual., 2017; shi et al.,2018; villacota-Martin et al., 2015). The following full-length primers were designed using PrimerPremier5 software, and the full length of LoWRKY33 gene was amplified from the floral plate cDNA of 'Siberia' lilium orientale.
LoWRKY33ORF up:5’-ATGACTTCATCCTCAGGAAGCAT-3’
LoWRKY33ORF low:5’-TCAGCAAGGCATGGAGTCG-3’
The PCR reaction system is shown in Table 4.
TABLE 4PCR reaction System
The PCR amplification conditions are shown in Table 5.
TABLE 5PCR amplification conditions
The amplified PCR product was separated into target bands by agarose gel electrophoresis, and the target bands were recovered using a full-size gold gel recovery kit (cat# EG 101-01), methods of which were referred to in the kit instructions. And (3) connecting the amplified PCR product into a pCloneEZ vector (product number: C5852-50), screening positive clones, and sequencing to obtain the full-length sequence of the LoWRKY33 gene, wherein the nucleotide sequence is shown as SEQ ID NO.1, the length is 1653bp, 550 amino acids are encoded, and the encoded amino acid sequence is shown as sequence SEQ ID NO. 2.
2) Construction of LoWRKY33 Gene overexpression vector
By performing cleavage sites on the LoWRKY33 full-length sequence, combining with the cleavage sites available in the Super1300 vector multiple cloning site (Multiple cloning site, MCS) region, two cleavage sites, salI and KpnI, were selected for vector construction.
First, loWRKY33 full-length primer with enzyme cutting site is designed:
LoWRKY33ORFup(+SalI)：
5'-TATGTCGACATGACTTCATCCTCAGGAAGC-3'
LoWRKY33ORFlow(+KpnI)：
5'-TATGGTACCGCAAGGCATGGAGTCGAG-3'
amplifying the full length LoWRKY33 with the cleavage site from the pCloneEZ plasmid containing the corrected full length LoWRKY33 sequence with the primer with the cleavage site;
separating the target strip by agarose gel electrophoresis, and recovering the target strip by using a gel recovery kit;
LoWRKY33 recovered product and Super1300 was digested overnight empty with restriction enzymes SalI and KpnI, and the target bands were isolated and recovered, and the digestion system is shown in Table 6.
TABLE 6 enzyme digestion system
Ligation of LoWRKY33 and Super1300 vector cleavage products was performed using Thermo T4 DNA ligase (cat# EL 0014), the ligation system is shown in Table 7;
table 7 connection system
After overnight ligation at 16 ℃, the ligation product was transformed into E.coli competent, spread on LB+kanamycin (Kanamycin, kan) plates, and cultured in an incubator at 37℃for 15-16 hours in an inverted manner;
and (3) selecting positive bacteria from a bacterial plate, shaking the bacteria, and carrying out PCR positive detection and sequencing verification to obtain the Super1300-LoWRKY33 vector (figure 4) which is successfully constructed.
3) Super1300-LoWRKY33 converted lily quilt sheet
The Super1300 and Super1300-LoWRKY33 plasmids are transformed into agrobacterium GV3101 to be competent by a freeze thawing method, and are coated on a flat plate of LB+Kan, and are inversely cultured for 48 hours in a 28 ℃ incubator;
a single colony was picked and shaken for a single colony, positive detection was performed using LoWRKY33 full-length primer with cleavage site, and then secondary shaking and bacterial collection were performed by referring to the method in example 2, and lily sheet was infected.
As shown in FIG. 5A, aging of LoWRKY33 overexpressing treated group festooned sheets was significantly accelerated after 4 days of infestation compared to control group. Ion permeability was measured 4 days after infection of the petals in both groups and was found to be significantly higher in the over-expressed group than in the control group (fig. 5B). The over-expression after 4 days of infection and the expression quantity of LoWRKY33 in the control group are detected by using real-time quantitative PCR, and the expression level of the LoWRKY33 gene in the LoWRKY33 over-expression treatment sample is found to be extremely higher than that of the control group (P <0.01, figure 5C).
The applicant has found in earlier studies that the content of salicylic acid increases sharply during the senescence process of lily flowers, and in order to solve whether the synthesis of salicylic acid is affected after LoWRKY33 is over-expressed, the expression of salicylic acid synthesis genes in LoWRKY33 over-expressed samples is further detected by fluorescence quantitative PCR. The results showed that the expression of the salicylic acid synthesis key genes PBS3 and ICS in the over-expressed samples was significantly higher than in the control group (fig. 6). This result suggests that LoWRKY33 may promote senescence of lily by promoting salicylic acid synthesis.
Example 4: transient expression of the LoSAG5 and LoSAG10 genes in lilium brownii sheets
1) Cloning of the LoSAG5 and LoSAG10 genes
Based on transcriptome sequencing of the early lily flowers in the subject group at different stages of opening (stages S1-S4), 11 SAG genes were screened, whose expression levels were all drastically increased at the late stage of flower senescence (Luoetal., 2021 b), and the gene function notes of the database of SwissProt et al showed that SAG1, SAG2 and SAG7 were senescence-specific cysteine protease genes, SAG3, SAG10 and SAG11 were nuclease genes, and SAG4, SAG5, SAG6, SAG8 and SAG9 were thiol protease genes (Table 8).
TABLE 8 11 SAG genes obtained by transcriptome sequencing
Based on the analysis of the expression patterns of 11 SAG genes in lily flowers in the process of opening and in different organs such as roots, stems, leaves, flowers and the like, and after abscisic acid and salicylic acid treatment, the preliminary assumption is that LoSAG5 and LoSAG10 can play a certain role in lily flowers in the process of aging. The full length primers for LoSAG5 and LoSAG10 were designed using Primer Premier5 software, the sequences of the primers were as follows:
LoSAG5 ORF up:5’-ATGGAGAACAATTCCACATCCTC-3’
LoSAG5 ORF low:5’-CTAGAGTTCATCTTTTTCTGGATTTG-3’
LoSAG10 ORF up:5’-ATGTATTCATTTTCAACCATCTCCG-3’
LoSAG10ORF low:5’-TCAGGTTGGTGGCGGTATTTC-3’
cloning procedure of the full length of the LoSAG5 and LoSAG10 genes reference was made to cloning of the LoWRKY33 gene in example 3. Obtaining the full-length sequence of the LoSAG5 gene, wherein the nucleotide sequence is shown as SEQ ID NO.3, the length is 1071bp, 356 amino acids are encoded, and the encoded amino acid sequence is shown as sequence SEQ ID NO. 4; the full-length sequence of the LoSAG10 gene is obtained, the nucleotide sequence of the LoSAG10 gene is shown as SEQ ID NO.5, the length of the LoSAG10 gene is 921bp, 306 amino acids are encoded, and the encoded amino acid sequence of the LoSAG10 gene is shown as sequence SEQ ID NO. 6.
2) Construction of LoSAG5 and LoSAG10 Gene overexpression vectors
Referring to the procedure for Super-LoWRKY33 vector construction in example 3, the full length primers of LoSAG5 and LoSAG10 with cleavage sites were designed:
LoSAG5ORF up(+SalI)：5’-TATGTCGACATGGAGAACAATTCCACATCCTC-3’
LoSAG5ORF low(+KpnI)：5’-TATGGTACCGAGTTCATCTTTTTCTGGATTTG-3’
LoSAG10ORF up(+SalI)：5’-TATGTCGACATGTATTCATTTTCAACCATCTCCG-3’
LoSAG10ORF low(+KpnI)：5’-TATGGTACCGGTTGGTGGCGGTATTTC-3’
the coding regions of LoSAG5 and LoSAG10 were constructed into the over-expression vector Super1300 by restriction and ligation, with reference to the procedure of Super-LoWRKY33 vector construction in example 3.
3) Super1300-LoSAG5/LoSAG10 conversion lily quilt sheet
The transient overexpression test of Super-LoWRKY33 in reference example 3, vacuum aspiration infestation via mediation of Agrobacterium GV3101, was performed for transient overexpression of Super1300-LoSAG5 and LoSAG10 in lily sheets.
After agrobacterium infection, culturing in dark at 23 ℃ for 3 days, randomly selecting 12 lily cover discs from Super1300 idle, loSAG5 and LoSAG10 over-expression samples for GFP fluorescence observation, and judging the expression condition of a target gene. The results showed that significant fluorescence appeared in all three groups of lily quilted samples, indicating that both the LoSAG5 and LoSAG10 were expressed to different extents in lily quilted sheets (FIG. 7).
After 3 days of dark culture at 23 ℃, phenotypic observation of the transiently overexpressed lily pieces of LoSAG5 and LoSAG10 was started, recorded as 0 days, and no apparent phenotype of the overexpressed lily pieces of LoSAG5 and LoSAG10 was observed compared with the Super1300 no-load control. After 2 days, the phenotype change of the festooned sheet of the LoSAG5 overexpression system is not obvious, and the aging degree of the festooned sheet of the LoSAG10 overexpression system is obviously accelerated (figure 8).
The aging degree of the lily quilt sheet after over-expression of the LoSAG5 and the LoSAG10 is reflected by the ion permeability and the MDA content. As shown in fig. 9, after 2 days, the ion permeability of the losg 10 overexpressed lily sheet was much higher than that of the losg 5 overexpressed sample and the Super1300 empty control sample. The MDA content measurement result shows that: the MDA content of the festive film of the SAG10 over-expression system is obviously higher than that of the Super1300 empty control group, the MDA value of the festive film is about 7 times of that of the Super empty control group, the MDA value of the SAG5 over-expression system is slightly lower than that of the Super empty control group, and the damage degree of the festive film system of the over-expression system is lower (figure 9).
To verify the variation of the expression levels of the LoSAG5 and LoSAG10 genes in the lily sheet transiently overexpressed samples, RNA extraction and fluorescent quantitative PCR experiments were performed on samples of 0 and 2 days. The results show that at day 0, the expression level of the LoSAG5 in the over-expression sample is slightly higher than that of the Super1300 control; the expression level of the LoSAG5 in the over-expressed sample is significantly higher than that of the control at 2 days. For LoSAG10, the expression level of the gene in the over-expressed sample was 3.12.+ -. 0.51 times in the Super1300 control sample at day 0, and the expression level in the over-expressed sample was 12.35.+ -. 1.38 times in the Super1300 control group at day 2 (FIG. 10).
The above specific embodiments are provided for illustrative purposes only and are not intended to limit the invention, and modifications, no inventive contribution, will be made to the embodiments by those skilled in the art after having read the present specification, as long as they are within the scope of the patent statutes.
Claims (7)
1. A high-efficiency lily quilt sheet transient expression method is characterized in that: the method comprises the following steps:
s1, taking oriental lily 'Siberia' as a material, and converting a binary expression vector PBI121 carrying a GUS reporter gene into lily sheets through mediation of agrobacterium tumefaciens and negative pressure suction;
s2, after infection, washing out agrobacterium attached to the lily quilt pieces by using deionized water;
s3, spreading the washed lily quilts in a culture dish filled with two layers of wet filter paper, performing dark culture for 3 days at the temperature of 23 ℃, transferring to the illumination condition of 23 ℃ for culture, recording for 0 days, photographing every 2 days, recording phenotype changes, and preliminarily determining gene functions through physiological index measurement and gene expression level detection, so as to establish an efficient transient expression system in the lily quilts.
2. The application of the high-efficiency lily quilt sheet transient expression method as claimed in claim 1, which is characterized in that: the transient expression method in the lily quilt piece is applied to LoWRKY33 gene function research.
3. The application of the high-efficiency lily quilt sheet transient expression method as claimed in claim 2, which is characterized in that: the LoWRKY33 gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
4. The application of the high-efficiency lily quilt sheet transient expression method as claimed in claim 1, which is characterized in that: the application of the instant expression method in the lily quilt piece in the functional research of the LoSAG5 gene.
5. The application of the high-efficiency lily quilt sheet transient expression method as claimed in claim 4, which is characterized in that: the sequence of the LoSAG5 gene is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
6. The application of the high-efficiency lily quilt sheet transient expression method as claimed in claim 1, which is characterized in that: the application of the instant expression method in the lily quilt piece in the functional research of the LoSAG10 gene.
7. The application of the high-efficiency lily quilt sheet transient expression method as claimed in claim 6, which is characterized in that: the sequence of the LoSAG10 gene is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6.
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