CN116751791A - Application of PvPSK3 gene in improving genetic transformation efficiency of gramineous plants - Google Patents
Application of PvPSK3 gene in improving genetic transformation efficiency of gramineous plants Download PDFInfo
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Abstract
The application relates to a PvPSK3 gene and application thereof in improving the genetic transformation efficiency of gramineous plants, belonging to the technical field of biology, wherein the application comprises application of a recombinant vector, an expression cassette or recombinant bacteria containing the PvPSK3 gene in improving the genetic transformation efficiency of gramineous plants. According to the application, the PvPSK3 gene is cloned from switchgrass and integrated on the chromosome of the gramineous plant through a plant expression vector in a genetic engineering way, and the PvPSK3 gene is found for the first time, so that the genetic transformation efficiency of the gramineous plant can be remarkably improved. Through research on the gene, the gene over-expression can improve the callus acquisition rate and regeneration efficiency of the transformed calli or young embryo positive calli of switchgrass, sorghum and corn, shorten the time of forming green buds by calli differentiation, and have no adverse effect on the subsequent growth and development of transgenic plants.
Description
Technical Field
The application belongs to the field of biotechnology. More particularly, it relates to the use of a PvPSK3 gene (pavir.3KG025900) for improving the genetic transformation efficiency of gramineous plants.
Background
Switchgrass (panicumvirginum), sorghum (sorghum) and corn (Zeamays) crops are important energy grasses, food crops and model plants, and have important values in green energy, animal industry development, food safety and plant foundation research. In the research of genetic improvement of gramineous plants, genetic transformation is an indispensable means, and plays an important role in the processes of genome editing, character precise improvement, plant molecular design breeding, gene function research and the like. The genetic transformation technology of switchgrass, sorghum and corn is mature, but the problems of serious genotype limitation, low transformation efficiency and the like still exist, so that only few varieties/genotype genetic transformation systems are successfully established at present.
Phytosulfokine (PSK) is a sulfonated pentapeptide growth factor, is commonly found in monocotyledonous and dicotyledonous plants, and plays an important role in promoting proliferation and differentiation of plant cells. However, in the past PSK was applied exogenously in culture to regulate cell proliferation and differentiation, but exogenously applied has an effect on endogenous responses, resulting in PSK having different effects on different tissues of different varieties/genotypes, not having universality, poor reproducibility, and currently not available in the market for this compound. The gene is not used as an auxiliary transformation molecular tool in plant tissue culture rapid propagation and genetic transformation, so the novel genetic transformation auxiliary molecular tool capable of promoting callus formation and regeneration simultaneously is constructed, is used for genome editing and genetic improvement of grasses such as switchgrass, sorghum, corn and the like, greatly improves the genetic transformation efficiency of the grasses, promotes the development of transgenic breeding technology and molecular precise design breeding technology, and promotes the cultivation process of new varieties with high yield, high quality and strong stress resistance.
Disclosure of Invention
Aiming at the aim of improving the genetic transformation efficiency of the gramineous plants, the application provides the application of the PvPSK3 gene in improving the genetic transformation efficiency of the gramineous plants, wherein the switchgrass PvPSK3 gene can improve the positive callus acquisition rate, the callus regeneration efficiency and shorten the time for callus differentiation into green buds so as to improve the genetic transformation efficiency.
The application is realized by the following technical scheme:
in a first aspect, the application provides an application of a PvPSK3 gene in improving the genetic transformation efficiency of gramineous plants, wherein the PvPSK3 gene can improve the positive callus acquisition rate and regeneration efficiency of switchgrass, sorghum and corn, shorten the green bud formation time of callus differentiation, and the nucleotide sequence of the PvPSK3 gene is shown as SEQ ID NO. 1.
The second aspect of the present application provides the use of a recombinant expression vector for the PvPSK3 gene, a genetically engineered bacterium, a transgenic cell line, or a protein encoded by the PvPSK3 gene in 1) or 2) as follows:
1) Improving the yield and regeneration efficiency of positive calli of switchgrass, sorghum and corn, and shortening the time for positive calli to differentiate and form green buds;
2) And (3) raising the gramineous plant varieties such as switchgrass, sorghum, corn and the like, wherein the yield of positive callus is improved or the regeneration efficiency of the callus is improved or the time for forming green buds by callus differentiation is shortened.
In a third aspect, the present application provides a method for increasing the yield or regeneration efficiency of switchgrass, sorghum and maize positive calli or for shortening the time for callus differentiation to form green shoots comprising the steps of transforming a plant with a nucleotide sequence as set forth in any one of the following a) -d) and allowing expression of said nucleotide sequence in said plant;
a) A DNA fragment shown in SEQ ID NO. 1;
b) A DNA fragment encoding the amino acid sequence shown in SEQ ID NO. 2;
c) A DNA fragment which has 75% or more identity with the DNA fragment defined in a) or b) and which encodes a protein functionally equivalent to the protein shown in SEQ ID NO. 2;
d) A cDNA fragment or a DNA fragment which hybridizes under stringent conditions to the DNA fragment of a) or b) and codes for the protein shown in SEQ ID NO. 2.
In a fourth aspect, the present application provides a method for breeding transgenic switchgrass, sorghum or maize with increased positive callus availability or increased callus regeneration or reduced callus differentiation time to form green shoots, by introducing a nucleotide sequence as set forth in any one of the following a) -d) into switchgrass, sorghum or maize to obtain a transgenic plant with increased genetic transformation efficiency;
a) A DNA fragment shown in SEQ ID NO. 1;
b) A DNA fragment encoding the amino acid sequence shown in SEQ ID NO. 2;
c) A DNA fragment which has 75% or more identity with the DNA fragment defined in a) or b) and which encodes a protein functionally equivalent to the protein shown in SEQ ID NO. 2;
d) A cDNA fragment or a DNA fragment which hybridizes under stringent conditions to the DNA fragment of a) or b) and codes for the protein shown in SEQ ID NO. 2.
In the above method, the method for introducing the nucleotide sequence into switchgrass, sorghum and corn comprises: polyethylene glycol method, agrobacterium mediated method or gene gun bombardment method.
In the above method, the improvement in genetic transformation efficiency of switchgrass, sorghum, and corn is at least one of the following 1) -5):
1) In the transformation process, in the infected switchgrass calli, the proliferation speed of positive cells is improved, so that the obtaining efficiency of the positive calli is improved;
2) In the transformation process, the formation efficiency of the embryogenic callus of the young embryo of the infected sorghum or corn is improved, and the obtaining efficiency of the positive callus is further improved;
3) In the transformation process, the time for forming green buds by callus differentiation is longer than that of a control group during the positive callus regeneration induction process of the transgenic switchgrass, sorghum and corn;
4) In the transformation process, the positive callus regeneration induction process of the transgenic switchgrass, sorghum and corn has higher callus regeneration efficiency than that of the control group.
Compared with the prior art, the application has the beneficial effects that: according to the application, a novel PvPSK3 gene is cloned from switchgrass, the function of the gene for improving genetic transformation efficiency is studied in gramineous plants for the first time, an over-expression vector is constructed by utilizing a DNA sequence of CDS full-length fragments containing the gene, agrobacterium is introduced, and in the process of converting the switchgrass calli by using an agrobacterium-mediated method, compared with pANIC6B empty vector control, over-expression of PvPSK3 can promote positive calli to obtain efficiency, positive calli differentiation is remarkably advanced, more green buds are differentiated, and regeneration efficiency is improved. Meanwhile, the agrobacterium-mediated method is used for transforming the PvPSK3 over-expression vector into the sorghum and maize immature embryo, so that the immature embryo can be promoted to form embryogenic callus, the obtaining efficiency of positive callus is further improved, the regeneration efficiency of positive callus is improved, and the time for forming green buds by differentiating positive callus is shortened. Therefore, the gene is over-expressed and applied to gramineous backbone germplasm with difficult genetic transformation, which is helpful to improve the genetic transformation efficiency, and further improve the yield, quality and stress resistance of the gramineous backbone germplasm, and has important economic value and social benefit.
Drawings
Fig. 1: pvPSK 3_panc6b versus control pANIC6B empty vector transformed switchgrass A3 (genetically inefficient genotype) callus selection culture process map;
a is the new callus state after 4 weeks of pANIC6B empty vector transformed switchgrass A3 callus screening;
b is pANIC6B empty vector transformed switchgrass A3 positive callus rate (GUS chromogenic rate);
c is the newly generated callus state after 4 weeks of screening the calli of the switchgrass A3 transformed by the PvPSK3_pANIC6B;
d is the PvPSK3_pANIC6B transformed switchgrass A3 positive callus rate (GUS chromogenic rate);
e is PvPSK3_pANIC6B and pANIC6B empty vector comparison positive callus acquisition rate data statistical analysis after A3 callus transformation.
Fig. 2: comparative plot of PvPSK3_cub versus control CUB empty vector transformed sorghum Tx430 immature embryo callus induction process; A-D is the callus induction culture process of the young embryo of the sorghum Tx430 transformed by the CUB empty vector; E-H is a callus induction culture process of converting PvPSK3_CUB into sorghum Tx430 immature embryos, wherein A, E is a mature callus state formed by each young embryo, B, F is an embryogenic state of each mature callus, C, G is a positive callus rate (eGFP luminescence callus rate) obtained by each young embryo, D, H is a positive callus rate (eGFP luminescence callus rate) in one of the calli; i is statistical analysis of PvPSK3_CUB and control CUB empty vector transformed sorghum Tx430 young embryo positive callus acquisition rate data.
Fig. 3: a comparative map of PvPSK3_panic6b versus control panic6b empty vector transformed switchgrass A3 callus regeneration culture process; A-C is the resistance callus regeneration induction culture process obtained by converting pANIC6B empty vector into switchgrass A3, and D-F is the resistance callus regeneration induction culture process obtained by converting PvPSK3_pANIC6B into switchgrass A3; G. h is the statistical analysis result of the days of induction and callus regeneration efficiency of the green bud formation by the differentiation of the resistant callus obtained by transforming the PvPSK3_panic6b with the control panic6b vector into A3; i is the detection result of the GUS sequence of T0 generation positive transgenic plants obtained by converting switchgrass A3 by PvPSK3_pANIC6B, numbers of the transgenic plants indicated by 1-22, NC indicates a wild genome negative control, PC indicates an expression vector plasmid positive control, and M indicates Marker indicating bands.
Fig. 4: a comparison chart of positive callus regeneration culture processes obtained by transforming seed sorghum Tx430 immature embryos with PvPSK3_CUB and a control CUB empty vector; A-B is a positive callus regeneration induction culture process obtained by converting sorghum Tx430 with CUB empty vector; C-D is a positive callus regeneration induction culture process obtained by converting PvPSK3_CUB into sorghum Tx 430; E. f is the statistical analysis result of the induction days of green bud formation and the callus regeneration efficiency in the positive callus regeneration culture process obtained by transforming seed sorghum Tx430 immature embryos by using PvPSK3_CUB and a control CUB empty vector; FIG. G shows the result of detecting the eGFP sequence of T0-generation positive transgenic plants obtained by converting PvPSK3_CUB into seed sorghum Tx430, numbers of the transgenic plants indicated by 1-22, NC indicating a wild type genome negative control, PC indicating an expression vector plasmid positive control, and M indicating Marker indicating bands.
Fig. 5: a comparison chart of positive callus regeneration culture processes obtained by transforming maize 18-599R young embryo with PvPSK3_CUB and a control CUB empty vector; A-B is a positive callus regeneration induction culture process obtained by transforming maize 18-599R immature embryo with CUB empty vector; C-D is a positive callus regeneration induction culture process obtained by transforming 18-599R young embryo of corn by PvPSK3_CUB; E. f is the statistical analysis result of the number of days of green bud formation induction and the callus regeneration efficiency in the positive callus regeneration culture process obtained by transforming maize 18-599R immature embryos with PvPSK3_CUB and a control CUB empty vector; FIG. G shows the result of detecting the eGFP sequence of T0-generation positive transgenic plants obtained by transforming corn 18-599R with PvPSK3_CUB, wherein the numbers of the transgenic plants are 1-22, NC represents a wild genome negative control, PC represents an expression vector plasmid positive control, and M represents Marker indicating bands.
Detailed Description
The application is further illustrated in the following drawings and specific examples, which are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
When the genetic transformation system of the switchgrass is established, callus formed by the induction of seeds, young ears and stems is a common explant. Genotype is a major factor affecting the callus transformation efficiency of switchgrass, and most of the calli of switchgrass cause genetic transformation difficulties due to low positive callus acquisition rate and low callus regeneration rate after transformation. At present, researches on improving the positive callus acquisition rate and the callus regeneration efficiency after the callus transformation of the switchgrass are mainly focused on a culture medium, a transformation mode and a callus material, and genes capable of improving the callus proliferation rate and the callus regeneration efficiency of the switchgrass are rarely reported.
Based on the above, the application carries out intensive research on the gene capable of regulating and controlling the proliferation rate and the regeneration efficiency of the switchgrass, and the research discovers that the gene pavir.3KG025900 has very low expression level in the mutant material with the loss of the regeneration capacity of the switchgrass, and has higher expression level in the material with high regeneration rate of the switchgrass. Through phylogenetic tree analysis of protein sequences, the gene codes for pentapeptide (DYIYT) of plant sulfopeptide and is closer to the Phytosulfofonepre pre 3 (AtPSK 3) gene in arabidopsis thaliana, so that the inventor names the gene as PvPSK3, the nucleotide sequence of the gene is shown as SEDIDNO.1, the full length of the coding sequence of the gene is 369bp, the amino acid sequence of the gene is shown as SEDIDNO.2, and the coding sequence of the gene is 122 amino acids.
The application obtains the PvPSK3 gene from the cDNA of the switchgrass callus by amplifying, constructs an over-expression vector by using a DNA sequence containing the CDS full-length fragment of the PvPSK3 gene, then introduces the over-expression vector into agrobacterium, infects the switchgrass callus by using an agrobacterium-mediated method, and then carries out screening culture and regeneration induction culture until a transgenic positive strain is obtained. The inventor finds that the overexpression of PvPSK3 can promote the acquisition of positive callus of transformed callus, improve the regeneration rate of positive callus, shorten the time for forming green buds by differentiation of positive callus, improve the acquisition rate of positive callus and the regeneration capacity of callus, be an important way for improving the genetic transformation efficiency of switchgrass, can be used for assisting the efficient transformation of other genes, and generate important economic value and social benefit. Meanwhile, the PvPSK3 gene is excessively expressed in young embryos of sorghum and corn, and the gene is found to promote embryogenic callus formation of the young embryos, is consistent with the action expressed in switchgrass transformation, and finally, the genetic transformation efficiency of the sorghum and the corn is obviously improved.
Based on the above-found PvPSK3 gene, the scope of the present application also includes DNA fragments homologous to the above-mentioned gene, and DNA fragments functionally equivalent to the encoded protein shown in SEQ ID NO. 2. The expression "functionally equivalent to the protein shown in SEQ ID No. 2" as used herein means that the protein encoded by the target DNA fragment is identical or similar to the protein shown in SEQ ID No.2 of the present application in terms of biological function, physiological and biochemical characteristics, etc. The application discovers that the typical biological function of the protein shown in SEQ ID NO.2 is to promote the improvement of the transformation efficiency of the immature embryo of the switchgrass callus, the sorghum and the corn, and the protein shown in SEQ ID NO.2 can be used for improving the genetic transformation efficiency of the switchgrass, the sorghum and the corn by up-regulating the expression quantity and/or the activity of the protein.
These DNA fragments homologous to the PvPSK3 gene include alleles, homologous genes, mutant genes and derivative genes corresponding to the nucleotide sequence of the present application (SEQ id No. 1); the protein encoded by the polypeptide is similar to the protein shown in SEQ ID NO.2 of the present application, or the substitution, deletion or insertion phenomenon of one, a plurality or dozens of amino acids exists, which belongs to the content of the present application.
From the application point of view, the DNA fragment of the PvPSK3 gene or the homologous gene thereof is likely to create a transgenic plant line with improved positive callus acquisition efficiency and callus regeneration capacity after being introduced into switchgrass, sorghum or corn plants. The present application is not limited to plasmid vectors for carrying out transformation of plant cells, as long as they are capable of expressing a load-bearing gene in plant cells. For those skilled in the art, various means may be used to introduce the plasmid vector into plant cells, such as polyethylene glycol (PEG) method, electroporation (electric corporation) method, agrobacterium-mediated method, gene gun bombardment method, etc., and to develop transformed cells into transgenic plants. In the plant field, various transgenic techniques tend to be mature and are widely used. These and other similar methods are applicable in the field of the present application.
In one embodiment of the application, the gene is obtained by amplification from switchgrass callus cDNA, comprising the following steps:
1) Extracting RNA of the switchgrass callus and reversely transcribing the RNA into cDNA;
2) Cloning of the PvPSK3 gene.
PCR amplification was performed using switchgrass callus cDNA as template, using the following primer pairs:
an upstream primer: 5'-AGCAGGCTTTGACTTTATGAGGCGTTGCGGCGGTCTCT-3', as shown in SEQ ID NO. 3;
a downstream primer: 5'-TGGGTCTAGAGACTTTCATGGCTTGCCCTTGTGCT-3', as shown in SEQ ID NO. 4;
note that the italic base sequence is the homologous recombination linker sequence used for pGWC vector ligation.
The PCR amplification system was 1. Mu.L of the upstream primer (10 pmol/. Mu.L), 1. Mu.L of the downstream primer (10 pmol/. Mu.L), 10. Mu.L of 2 XPhantaMix, 1. Mu.L of cDNA template, and deionized water was added to make up the total volume to 20. Mu.L.
The amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 20s, and circulation 34 times; extending at 72℃for 5min.
The PCR product was subjected to agarose gel electrophoresis, the target band was subjected to gel recovery, 2. Mu.L of the gel recovered product was ligated with pGWC linear vector (Ahd 1 restriction enzyme) and the procedure was performed according to the In-fusion HDCloning kit product instruction provided by Taraka Co. Then, the ligation product was transformed into E.coli DH5a, and cultured overnight on LB solid medium containing chloramphenicol (50 mg/L). Single colonies were picked and cultured overnight in LB liquid medium containing chloramphenicol (50 mg/L). Plasmid DNA was extracted by an alkaline method, and the sequence was determined. The sequence of the amplified product is shown as SEQ ID NO.1 after sequencing analysis, which shows that the PvPSK3 gene is connected to pGWC vector, and the cloning vector construction is completed. Then, the cloning vector and pANIC6B empty expression vector were ligated by using LRMix, and the ligation product was transformed into E.coli DH5a, and cultured overnight on LB solid medium containing kanamycin (50 mg/L). Single colonies were picked and cultured overnight in LB liquid medium containing kanamycin (50 mg/L). Plasmid DNA was extracted by an alkaline method, and the sequence was determined. Sequencing analysis shows that the sequence of the amplified product is shown as SEQ ID NO.1, and the amplified product shows that PvPSK3 is connected to pANIC6B vector, and the construction of the overexpression vector PvPSK3_pANIC6B is completed. The inventors further transformed agrobacterium with the obtained PvPSK 3_panc6b overexpression vector.
Finally, the inventor utilizes an agrobacterium-mediated method to transform a callus line A3 with low transformation efficiency of switchgrass (the positive callus obtaining efficiency is less than 10 percent and the callus regeneration efficiency is less than 40 percent), and discovers that PvPSK3 can promote infected callus to obtain positive callus in the transformation process (figure 1), and the days of green bud formation by callus differentiation are obviously shortened and the callus regeneration efficiency is obviously improved in the positive callus regeneration induction process (figure 3), thereby overcoming the dependence of genetic transformation of switchgrass on genotype. Therefore, the gene is developed into a genetic transformation molecule auxiliary tool which is used for transforming difficult backbone germplasm, so that the genetic transformation efficiency of backbone germplasm can be improved, and the working process of molecular design breeding is promoted.
In another embodiment of the application, positive callus availability and callus regeneration capacity conditions after transformation of seed sorghum Tx430 immature embryos by PvPSK3_cub were observed as follows:
PCR amplification was performed using switchgrass callus cDNA as template, using the following primer pairs:
an upstream primer: 5'-CATCAGTCGCTGAGGATGAGGCGTTGCGGCGGTCTCT-3', as shown in SEQ ID NO. 5;
a downstream primer: 5'-CACCATCACCTCAGCTCATGGCTTGCCCTTGTGCT-3', as shown in SEQ ID NO. 6;
note that the italic base sequence is a homologous recombination linker sequence used for CUB vector ligation.
The PCR amplification system was 1. Mu.L of the upstream primer (10 pmol/. Mu.L), 1. Mu.L of the downstream primer (10 pmol/. Mu.L), 10. Mu.L of 2 XPhanta Mix, 1. Mu.L of cDNA template, and deionized water was added to make up the total volume to 20. Mu.L.
The amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 20s, and circulation 34 times; extending at 72℃for 5min.
The PCR product was subjected to agarose gel electrophoresis, the target band was subjected to gel recovery, 2. Mu.L of the gel recovered product was ligated with CUB linear vector (BbvCI enzyme digestion), and the procedure was performed according to the In-Fusion HD Cloning kits product instruction provided by Taraka Co. The ligation product was then transformed into E.coli DH5a and cultured overnight on LB solid medium containing kanamycin (50 mg/L). Single colonies were picked and cultured overnight in LB liquid medium containing kanamycin (50 mg/L). Plasmid DNA was extracted by an alkaline method, and the sequence was determined. The sequence of the amplified product is shown as SEQ ID NO.1 after sequencing analysis, which shows that the PvPSK3 gene is connected to the CUB vector, and the construction of the overexpression vector PvPSK3_CUB is completed. The inventors further transformed Agrobacterium using the obtained PvPSK3_CUB overexpression vector.
Taking sorghum ears about 12-14 days after pollination, placing the sorghum ears into a sterile 200ml culture bottle, sterilizing and disinfecting (20 min of 75% alcohol and 10min of 20% Naclo solution) in an ultra-clean workbench, stripping glume, stripping sorghum young embryos, infecting the sorghum young embryos by using agrobacterium, and carrying out tissue culture. The inventors observed the induction condition of the callus in the tissue culture process, found that the immature embryo was subjected to the induction culture for 4 weeks to form mature callus (A and E in FIG. 2), wherein the immature embryo of CUB empty vector was transformed, and the formed resistant callus was mostly milky soft callus with fluffy structure (A and B in FIG. 2), and the positive rate was low (C, D and I in FIG. 2). In contrast, the immature embryos transformed with PvPSK3_CUB produced resistant calli which were mostly pale yellow, tightly structured, hard calli (E, F in FIG. 2) with high positive rates (G, H and I in FIG. 2). Positive mature callus (eGFP-luminescent callus) is transferred to a regeneration induction medium for light culture, and the time for forming the green buds of the callus, the regeneration rate of the green buds and the positive seedling rate are counted. The calli transformed with the PvPSK3_cub vector were found to differentiate green shoots earlier, and the callus regeneration efficiency and positive shoot rate were significantly improved relative to the calli of the control group transformed with the CUB empty vector (fig. 4). The inventors identified T0 generation transgenic plants by designing eGFP primers, resulting in transgenic positive plants (G in fig. 4).
In yet another embodiment of the present application, pvPSK3_CUB transformed maize 18-599R post-immature embryo positive callus acquisition rate and callus regeneration capacity were found to be significantly improved relative to the CUB empty vector control group. The method comprises the following specific steps:
the inventors infects young embryos of maize 18-599R about 12-14 days after pollination with Agrobacterium harboring the PvPSK3_CUB vector and perform tissue culture. The inventors observed the induction condition of the callus in the tissue culture process, found that positive callus formed by the immature embryo of the overexpression PvPSK3_CUB has better embryology, compact callus structure and light yellow color, and the positive callus is transferred to a regeneration induction culture medium for illumination culture, and the formation time, regeneration rate and positive seedling rate of the callus are counted (figure 5). The callus formation time (E in fig. 5), callus regeneration rate (D, F in fig. 5) and positive shoot ratio (G in fig. 5) were found to be significantly advanced relative to the empty vector control, which was transformed with PvPSK 3_cub. The inventors identified T0 generation transgenic plants by designing eGFP primers, resulting in transgenic positive plants (G in fig. 5). The result shows that the PvPSK3 has the capability of promoting the formation of maize immature embryo embryogenic callus, and improves the regeneration capability of the callus, thereby improving the genetic transformation efficiency. The development of the PvPSK3 into a genetic transformation molecule auxiliary tool is beneficial to overcoming the bottleneck of difficult transformation of maize backbone inbred lines, accelerating the molecular design breeding process and promoting the cultivation of good varieties.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present application are all conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers.
Example 1: construction of PvPSK3 Gene clone and plant overexpression vector
1. Extraction of total RNA from switchgrass calli:
the RNA extraction kit used in this experiment was E.Z.N.A.plant RNAkit provided by Omega company, USA, and the specific experimental procedure was as follows:
1) Sample treatment: weighing about 0.1g of switchgrass callus material, fully grinding in liquid nitrogen, transferring the ground powder into a 2mL centrifuge tube, immediately adding 500ul RBBuffer, fully vibrating and uniformly mixing to fully crack a tissue sample, and standing at room temperature for 5min;
2) Transferring the liquid to a gDNA filtering column, removing genome DNA, and centrifuging at 12,000rpm/min for 5min at room temperature;
3) Transferring the supernatant to a new 1.5ml centrifuge tube, adding 0.5X volume of absolute ethyl alcohol, and uniformly mixing for 20s;
4) Transferring the liquid to a HiBindRNAMiniColumbn column, and centrifuging at 10,000rpm/min at room temperature for 1min;
5) 400ul RWFWAshBuffer is added into the column, and the mixture is centrifuged for 30s at room temperature at 10,000rpm/min, and the waste liquid in the collecting pipe is poured out;
6) Adding 500ul RNAWashBufferII to the column, centrifuging at 10,000rpm/min at room temperature for 30s, and pouring out the waste liquid in the collecting pipe;
7) Repeating the step (6) once, centrifuging the empty column at the room temperature of 14,000rpm/min for 2min, and drying the column;
8) Transferring the column to a new 1.5ml centrifuge tube, adding 50-100ul DEPC water to dissolve RNA;
9) Centrifuging at room temperature for 2min at 14,000rpm/min, and freezing the obtained RNA at-80deg.C for use.
Synthesis of first strand cDNA
The first strand cDNA synthesis kit used in this experiment was completed by EasScript One-Step gDNARemoval and cDNA Synthesis SuperMix supplied by the full gold Bio Inc. The specific experimental operation steps are as follows:
and taking out the template RNA from the refrigerator at the temperature of minus 80 ℃, placing the template RNA on ice for thawing, taking out the kit in advance, thawing at room temperature, and rapidly placing the kit on ice after thawing. Preparing a reaction solution of a first strand cDNA synthesis and gDNA removal system, wherein the preparation method is shown in table 1:
table 1: first strand cDNA synthesis and gDNA removal reaction system
2) Adding the above components according to the above table, thoroughly mixing, centrifuging briefly, placing in a PCR instrument, and incubating at 42deg.C for 30min;
3) Incubation at 85℃for 5s, inactivationRT/RI Enzyme and gDNA remote were then stored in a-20deg.C freezer for further experimental manipulation.
Cloning of PvPSK3 Gene
PCR amplification was performed using reverse transcribed cDNA as template, using the following primer pairs:
an upstream primer: 5'-ATGAGGCGTTGCGGCGGTCTCT-3', as shown in SEQ ID NO. 7;
a downstream primer: 5'-TCATGGCTTGCCCTTGTGCT-3', as shown in SEQ ID NO. 8;
the PCR amplification system was 1. Mu.L of the upstream primer (10 pmol/. Mu.L), 1. Mu.L of the downstream primer (10 pmol/. Mu.L), 10. Mu.L of 2 XPhanta Mix, 1. Mu.L of cDNA template, and deionized water was added to make up the total volume to 20. Mu.L.
The amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 20s, and circulation 34 times; extending at 72℃for 5min.
4. Construction of plant expression vector PvPSK3_pANIC6B
1) Amplifying the PvPSK3 gene by using the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4 and taking cDNA of the switchgrass callus as a template;
2) Performing agarose gel electrophoresis detection on the PCR product, performing gel recovery on the target strip, connecting 2 mu L of gel recovery product with pGWC linear vector (obtained by Ahd1 enzyme digestion), and performing the operation steps according to In-Fusion HD Cloning kits product specifications provided by Taraka company;
3) The ligation product was transformed into E.coli DH5a and cultured overnight on LB solid medium containing chloramphenicol (50 mg/L);
4) Single colonies were picked and cultured overnight in LB liquid medium containing chloramphenicol (50 mg/L);
5) Plasmid DNA was extracted by an alkaline method, and the sequence was determined. Sequencing analysis shows that the sequence of the amplified product is shown as SEQ ID NO.1, which shows that the PvPSK3 gene is connected to the pGWC vector, and shows that the construction of the cloning vector is completed;
6) Ligating the cloning vector with pANIC6B empty expression vector by LR Mix, transforming the ligation product into E.coli DH5a, and culturing overnight on LB solid medium containing kanamycin (50 mg/L);
7) Single colonies were picked and cultured overnight in LB liquid medium containing kanamycin (50 mg/L);
8) Plasmid DNA was extracted by an alkaline method, and the sequence was determined. Sequencing analysis shows that the sequence of the amplified product is shown as SEQ ID NO.1, which shows that PvPSK3 is connected to pANIC6B vector, and shows that the construction of the overexpression vector PvPSK3_pANIC6B is completed;
9) The correct expression vector PvPSK 3_panc6b for sequencing analysis was transformed into agrobacterium EHA105 and the agrobacterium strain available for transformation was obtained.
5. Construction of plant expression vector PvPSK3_CUB
1) Amplifying the PvPSK3 gene by using the primer pair shown in SEQ ID NO.5 and SEQ ID NO.6 and taking cDNA of the switchgrass callus as a template;
2) Performing agarose gel electrophoresis detection on the PCR product, performing gel recovery on the target strip, connecting 2 mu L of gel recovery product with a CUB linear carrier (BbvCI enzyme digestion), and performing the operation steps according to In-Fusion HD Cloning kits product specifications provided by Taraka company;
3) The ligation product was transformed into E.coli DH5a and cultured overnight on LB solid medium containing kanamycin (50 mg/L);
4) Single colonies were picked and cultured overnight in LB liquid medium containing kanamycin (50 mg/L);
5) Plasmid DNA was extracted by an alkaline method, and the sequence was determined. Sequencing analysis shows that the sequence of the amplified product is shown as SEQ ID NO.1, which shows that the PvPSK3 gene is connected to the CUB vector, and the construction of the overexpression vector PvPSK3_CUB is finished;
6) The correct expression vector PvPSK3_cub for sequencing analysis was transformed into agrobacterium EHA105 and agrobacterium strains were obtained for transformation.
Example 2: agrobacterium-mediated transformation of switchgrass calli and acquisition of positive plants
The genetic transformation of the switchgrass adopts an agrobacterium-mediated callus genetic transformation method, and the agrobacterium infection steps are as follows:
1) Streaking an EHA105 engineering bacterial solution stored at the temperature of minus 80 ℃ and carrying recombinant plasmids on a bacterial culture medium to activate the bacterial strain, selecting a small quantity of shaking bacteria for positive bacterial colonies, adding 100 mu L of activated bacterial solution into 50mL of YEP liquid culture medium containing 50 mg.L-1 kanamycin and 50 mg.L-1 rifampicin, and carrying out shaking culture at the temperature of 28 ℃ and 180rpm for overnight;
2) When the OD600 of the bacterial liquid measured by a spectrophotometer is 0.3-0.4, AS is added until the working concentration is 100 mu M;
3) Shaking culture at 28 deg.C and 180rpm until OD600 is 0.6-0.8, 18 deg.C, 4,500g, centrifuging for 10min, and collecting thallus;
4) Re-suspending and precipitating by using SM3 infection liquid, transferring the re-suspension to a tissue culture bottle, adding the SM3 infection liquid until the bacterial liquid concentration OD600 is 0.2-0.3, and adding AS until the working concentration is 100 mu M;
5) Taking 5-8 dishes of calli grown on SM5 culture medium for 10-15 days, adding into the heavy suspension, and gently shaking and mixing. Placing the tissue culture bottle filled with the callus in a vacuum dryer, and vacuumizing for 10min; ultrasonic treatment at 15 ℃ for 5min; vacuumizing for 10min;
6) Pouring the heavy suspension as clean as possible, airing the callus on sterile filter paper for 30min, transferring the callus to new sterile filter paper, and co-culturing for 3-4 days under dark condition;
7) Transferring the calli to switchgrass selection medium, dark culturing for about 3 weeks (depending on the growth of resistant calli);
8) Transferring the resistant callus onto the switchgrass differentiation medium, and replacing the new differentiation medium every 3 weeks until seedlings grow out;
9) Transferring the differentiated seedlings to a rooting culture medium until the tissue culture seedlings root, transplanting the tissue culture seedlings into nutrient soil, and growing in a greenhouse;
10 Identification of transgenic positive plants mainly comprises genomic DNA extraction and PCR identification of GUS sequences of target genes or marker genes, GUS staining and qRT-PCR identification of the expression level of the target genes in the transgenic plants. Wherein, GUS detects the upstream primer: 5'-GGAGAAGATTCGGACGTTTG AG-3', as shown in SEQ ID NO. 9; GUS detection downstream primer: 5'-GTGATGGTGATGGTGA TGGCTA-3' as shown in SEQ ID NO. 10.
TABLE 2 Medium formulation during infection and cultivation in experiments
Example 3: agrobacterium-mediated transformation of young sorghum and maize embryos and acquisition of positive plants
Genetic transformation of sorghum and corn adopts an agrobacterium-mediated young embryo genetic transformation method, and agrobacterium infection steps are as follows:
1) Streaking an EHA105 engineering bacterial solution stored at the temperature of minus 80 ℃ and carrying recombinant plasmids on a bacterial culture medium to activate the bacterial strain, selecting a small quantity of shaking bacteria of positive bacterial colonies, adding 100 mu L of activated bacterial solution into 50mL of YEP liquid culture medium containing 50mg/L kanamycin and 50mg/L rifampicin, and carrying out shake culture at the temperature of 28 ℃ and 180rpm for overnight; when the OD600 of the bacterial liquid is 0.3-0.4, AS is added to the working concentration of 100 mu M, the bacterial liquid is continuously cultured in an oscillating way until the OD600 is 0.6-0.8, the temperature is 18 ℃, the bacterial liquid is 4,500g, the bacterial liquid is centrifuged for 10min, the bacterial liquid is collected, and the bacterial liquid is resuspended by the infection liquid added with AS (the working concentration of 100 mu M);
2) Sucking the prepared agrobacterium infection liquid into a 1.5mL centrifuge tube containing sorghum or corn young embryo, gently reversing and uniformly mixing for 1min, and immersing the young embryo into the bacterial liquid;
3) Standing for 10min, and sucking out bacterial liquid;
4) Inoculating the infected young embryo on a co-culture medium by using a sterile scalpel, and co-culturing in a dark incubator at 25 ℃;
5) After co-culturing for 2-3d, transferring the young embryo to a recovery culture medium, and culturing in dark at 28 ℃;
6) Cutting off embryo buds after recovering to culture for 3-5d, continuously recovering to culture for about 7d, expanding embryo buds, and generating primary callus;
7) Transferring the expanded primary callus to a screening culture medium, and continuing dark culture at 28 ℃ for 28d until the immature embryo forms a mature callus;
8) Transferring the mature callus into a regeneration culture medium, and culturing in an illumination incubator with the illumination intensity of 1600lx at the temperature of 25 ℃ until the callus is differentiated to bud;
9) Transferring the green buds obtained by regenerating the callus into a rooting culture medium until the green buds grow out of more than 3 root systems with the length of more than 5cm, transplanting the root systems into nutrient soil, and placing the root systems in a greenhouse for growth culture;
10 Identification of transgenic positive plants mainly comprises genomic DNA extraction and PCR identification of the sequence of interest or marker gene eGFP sequence. Wherein, eGFP detects the upstream primer: 5'-ATGGTGAGCAAGG GCGAGGA-3', as shown in SEQ ID NO. 11; eGFP detection downstream primer: 5'-TTACTTGTAC AGCTCGTCCATGCC-3', as shown in SEQ ID NO. 12.
TABLE 3 Medium formulation during infection and cultivation in experiments
Example 4: conversion data statistical analysis
1. Effect of conversion of PvPSK3 on switchgrass A3 positive callus acquisition efficiency, callus green bud formation time, and callus regeneration efficiency
For the reliability of experimental data, we repeatedly transformed switchgrass A3 calli 3 times and counted 3 times, and the results showed that transforming PvPSK3 significantly improved positive callus acquisition efficiency (E in fig. 1), shortened callus green bud formation time (G in fig. 3), and improved callus regeneration efficiency (H in fig. 3). In E in fig. 1, positive callus availability = GUS chromogenic callus number/inoculation callus number x 100%; in H in fig. 3, the callus regeneration efficiency=the number of green shoots/the number of inoculated calli×100%.
2. Effect of transformation of PvPSK3 on efficiency of sorghum Tx430 positive callus acquisition, callus green bud formation time and callus regeneration efficiency
For the reliability of experimental data, we repeatedly transformed sorghum Tx430 young embryos 3 times and counted 3 times, and the results show that transforming PvPSK3 can significantly improve the efficiency of obtaining positive calli of sorghum young embryos (I in fig. 2), shorten the time of forming calli green buds (E in fig. 4), and significantly improve the efficiency of calli regeneration (F in fig. 4). In fig. 2I, positive callus availability = eGFP number of luminescent callus/number of chick embryos inoculated x 100%; in F in fig. 4, callus regeneration efficiency = number of regenerated green shoots/number of inoculated young embryos x 100%.
3. Effect of transformed PvPSK3 on maize 18-599R callus bud formation time and callus regeneration efficiency
For the reliability of experimental data, we repeatedly transformed maize 18-599R young embryos 3 times and counted 3 times, and the results show that transforming PvPSK3 can significantly shorten the callus regeneration shoot induction time (fig. 5E) and improve the callus regeneration efficiency (fig. 5F). In fig. 5F: callus regeneration efficiency = number of regenerated green shoots/number of inoculated young embryos x 100%.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
- The application of the PvPSK3 gene in improving the genetic transformation efficiency of gramineous plants is characterized in that the PvPSK3 gene can improve the positive callus acquisition rate and regeneration efficiency of switchgrass, sorghum and corn, shorten the time for callus differentiation and form green buds, and the nucleotide sequence of the PvPSK3 gene is shown as SEQ ID NO. 1.
- 2. Use of a recombinant expression vector comprising a PvPSK3 gene in 1) or 2) as follows: the method is characterized in that 1) the method improves the yield and regeneration efficiency of positive calli of switchgrass, sorghum and corn, and shortens the time for forming green buds by callus differentiation;and 2) in the cultivation of gramineous plant varieties including switchgrass, sorghum and corn, the positive callus acquisition rate and regeneration efficiency are improved, and the time for forming green buds by callus differentiation is shortened.
- 3. The application of the genetically engineered bacteria containing the PvPSK3 gene in the following 1) or 2) is characterized in that 1) the time for forming green buds by callus differentiation is shortened for improving the positive callus acquisition rate and regeneration efficiency of switchgrass, sorghum and corn;and 2) in the cultivation of gramineous plant varieties including switchgrass, sorghum and corn, the positive callus acquisition rate and regeneration efficiency are improved, and the time for forming green buds by callus differentiation is shortened.
- 4. Use of a transgenic cell line comprising a PvPSK3 gene in 1) or 2) wherein 1) is to increase the positive callus availability and regeneration efficiency of switchgrass, sorghum and maize and to reduce the time for callus differentiation to green shoots;and 2) in the cultivation of gramineous plant varieties including switchgrass, sorghum and corn, the positive callus acquisition rate and regeneration efficiency are improved, and the time for forming green buds by callus differentiation is shortened.
- 5. The application of the protein coded by the PvPSK3 gene in the following 1) or 2) is characterized in that 1) the positive callus acquisition rate and regeneration efficiency of switchgrass, sorghum and corn are improved, and the time for forming green buds by callus differentiation is shortened;and 2) in the cultivation of gramineous plant varieties including switchgrass, sorghum and corn, the positive callus acquisition rate and regeneration efficiency are improved, and the time for forming green buds by callus differentiation is shortened.
- 6. The use according to any one of claims 2 to 5, wherein the method comprises the steps of transforming a plant with a nucleotide sequence according to any one of the following a) to d) and allowing expression of the nucleotide sequence in the plant;a) A DNA fragment shown in SEQ ID NO. 1;b) A DNA fragment encoding the amino acid sequence shown in SEQ ID NO. 2;c) A DNA fragment which has 75% or more identity with the DNA fragment defined in a) or b) and which encodes a protein functionally equivalent to the protein shown in SEQ ID NO. 2;d) A cDNA fragment or a DNA fragment which hybridizes under stringent conditions to the DNA fragment of a) or b) and codes for the protein shown in SEQ ID NO. 2.
- 7. The use according to any one of claims 2-5, characterized in that the nucleotide sequence of any one of a) to d) is introduced into switchgrass, sorghum or maize to obtain transgenic plants with improved genetic transformation efficiency;a) A DNA fragment shown in SEQ ID NO. 1;b) A DNA fragment encoding the amino acid sequence shown in SEQ ID NO. 2;c) A DNA fragment which has 75% or more identity with the DNA fragment defined in a) or b) and which encodes a protein functionally equivalent to the protein shown in SEQ ID NO. 2;d) A cDNA fragment or a DNA fragment which hybridizes under stringent conditions to the DNA fragment of a) or b) and codes for the protein shown in SEQ ID NO. 2.
- 8. The method of claim 6, wherein said introducing said nucleotide sequence into switchgrass, sorghum, and corn comprises: polyethylene glycol method, agrobacterium mediated method or gene gun bombardment method.
- 9. The method of claim 8, wherein in the method the increase in genetic transformation efficiency of switchgrass, sorghum, and corn is at least one of the following 1) -4):1) In the transformation process, in the infected switchgrass calli, the proliferation speed of positive cells is improved, so that the obtaining efficiency of the positive calli is improved;2) In the transformation process, the formation efficiency of the embryogenic callus of the young embryo of the infected sorghum or corn is improved, and the obtaining efficiency of the positive callus is further improved;3) In the transformation process, the time for forming green buds by callus differentiation is longer than that of a control group during the positive callus regeneration induction process of the transgenic switchgrass, sorghum and corn;4) In the transformation process, the positive callus regeneration induction process of the transgenic switchgrass, sorghum and corn has higher callus regeneration efficiency than that of the control group.
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