CN102936279A - Plant somatic embryogenesis gap-associated protein GhSERK2 as well as encoding gene and application thereof - Google Patents

Abstract

The invention discloses plant somatic embryogenesis-related protein GhSERK2 and its coding gene and application. Experiments have proved that, in the transgenic Arabidopsis line expressing the gene GhSERK2 shown in Sequence 1 of the Sequence List, the number of shoots of each hypocotyl after induction and differentiation is significantly higher than that of the wild-type and empty vector controls; In the transgenic tobacco line with the gene GhSERK2, the number of buds of each explant leaf piece induced and differentiated was significantly higher than that of the wild type and the empty vector control; indicating that the GhSERK2 protein and its encoding gene can regulate the somatic embryogenesis of the target plant Ability. The present invention clarifies the network regulation and genetic mechanism in the cotton somatic cell culture process, improves the plant regeneration rate and genetic transformation rate of cotton, overcomes the genotype limitation of cotton somatic cell culture, and selects excellent genotypes purposefully to obtain regenerated plants. It is of great significance to obtain new and excellent cotton varieties quickly and efficiently.

Description

Plant somatic embryogenesis-related protein GhSERK2 and its encoding gene and application

technical field

The invention relates to a plant somatic embryogenesis-related protein GhSERK2 and its coding gene and application.

Background technique

Cotton is one of the most important economic crops in the world, providing important raw materials for the textile industry and oil industry. At the same time, cotton is the most successful crop that combines modern biotechnology with traditional breeding and applies transgenic technology, and cotton tissue culture technology is an important platform for the realization of transgenic technology.

Cotton tissue culture mainly includes organogenesis pathway and embryoid body pathway. Plant organogenesis in vitro refers to the process of differentiation of tissues or cell mass (callus) under culture conditions to form adventitious roots, adventitious shoots and other organs; embryoid is the somatic embryo Or somatic embryo, which is an embryo-like structure produced by somatic cells under specific conditions in certain plants using specific parts as explants outside the plant. Its development process is similar to that of the zygotic embryo, passing through several stages such as proembryo, globular embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledon embryo; in the earliest stage of its occurrence, it has two polarities, that is, the root end (radicle) and stem end (germ) and has no direct connection to the maternal cells or vascular bundles of the explant, unlike organogenesis. Thus, the embryoid body starts out as a rudimentary form of a complete plant, which can obtain nutrition from the explant or callus through the root tip or stalk-like structure.

Cotton tissue culture technology includes tissue culture systems such as somatic cell culture, anther culture, shoot tip culture, ovule and embryo culture, and protoplast culture, among which cotton somatic cell culture is one of the most mature technologies. Through the method of cotton somatic cell culture, the method of genetically transforming the hypocotyl, petiole, embryogenic callus, etc. as receptors to obtain transgenic cotton has been widely used.

Since Price and Smith first reported in 1979 that somatic embryos were obtained from the cell suspension culture of Gossypium klotzschianum wild cotton, it has been reported in upland cotton (Gossypium hirsutum L.), sea island cotton (Gossypium barbadense L. ), Gossypium arboreum L., Gossypium herbaceum L., Gossypium klotzschianum Anderss, Gossypium aridum, Gossypium davidsonii, Raymond's Gossypium raimondii, Gossypium thurberi, Gossypium gossypioides, Gossypiumtomentosum, Gossypium nelsonii, Gossypium australe, Gossypium gossypioides Gossypium stocksii) and other 14 species have obtained embryoid bodies or regenerated plants, especially in upland cotton cultivars, many varieties of regeneration systems have been obtained.

At present, most of the cotton varieties that have established a regeneration system through the somatic cell culture process have been eliminated from production, and the offspring of transgenic plants obtained by genetic transformation of cotton using these varieties have poor agronomic traits, which increases the difficulty of screening good varieties later.

Contents of the invention

The object of the invention is a plant somatic embryogenesis-related protein GhSERK2 and its coding gene and application.

The somatic embryogenesis-related protein provided by the present invention is derived from upland cotton (Gossypium hirsutum L.), and the name is GhSERK2, which is the protein of a) or b) as follows:

a) A protein composed of the amino acid sequence shown in Sequence 2 of the Sequence Listing;

b) A protein derived from (a) that is related to plant somatic embryogenesis by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence of Sequence Listing 2;

The amino acid sequence shown in Sequence 2 of the Sequence Listing consists of 620 amino acid residues.

In order to facilitate the purification of the protein in (a) above, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 2 of the Sequence Listing can be attached with the tags shown in Table 1.

Table 1 Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The gene encoding the protein in (b) above can be deleted by deleting one or several codons of amino acid residues in the DNA sequence shown in the 148th to 2010th positions of Sequence Listing Sequence 1, and/or adding one or several A missense mutation of 1 base pair, and/or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

The gene encoding the protein also belongs to the protection scope of the present invention.

The gene encoding the protein is any one of the following 1)-4) genes:

1) Its nucleotide sequence is the DNA molecule shown from No. 148 to No. 2010 in Sequence 1 of the Sequence Listing;

2) Its nucleotide sequence is the DNA molecule shown in Sequence 1 of the Sequence Listing;

3) At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of the DNA sequence defined in 1) or 2) %, DNA molecules that are at least 98% or at least 99% homologous and encode said protein;

4) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) or 3) under stringent conditions and encodes the protein.

Sequence Listing Sequence 1 is composed of 2054 deoxynucleotides, wherein the open reading frame from the 148th to the 2010th position of Sequence Listing Sequence 1 encodes the protein shown in Sequence Listing Sequence 2.

The stringent conditions can be described as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na ₃ PO ₄ and 1 mM EDTA, at 50°C, 2×SSC, 0.1% SDS Rinse in medium; can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1mM EDTA, rinse in 50°C, 1×SSC, 0.1% SDS; can also be: 50°C , hybridized in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1mM EDTA, rinsed at 50°C, 0.5×SSC, 0.1% SDS; also: 50°C, in 7% SDS, 0.5M Na ₃ Hybridize in a mixed solution of PO ₄ and 1mM EDTA, rinse at 50°C, 0.1×SSC, 0.1% SDS; also: 50°C, in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1mM EDTA hybridization at 65°C, rinsed in 0.1×SSC, 0.1% SDS; alternatively: in a solution of 6×SSC, 0.5% SDS, hybridization at 65°C, and then with 2×SSC, 0.1% SDS and 1 ×SSC and 0.1% SDS were used to wash the membrane once.

Recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses containing the genes also belong to the protection scope of the present invention.

An existing plant expression vector can be used to construct a recombinant expression vector containing the gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyA signal can direct polyA to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of protein gene) has similar functions. When using the gene to construct a recombinant plant expression vector, any enhanced promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, maize ubiquitin promoter ( Ubiquitin), constitutive promoters or tissue-specific expression promoters (such as seed-specific expression promoters), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector , can also use enhancers, including translation enhancers or transcription enhancers, these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the same reading frame as the coding sequence to ensure that the entire sequence correct translation of . The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to metharexate, the EPSPS gene that confers resistance to glyphosate) or the marker gene for resistance to chemical agents (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene.

The recombinant vector containing the gene can be a recombinant expression vector obtained by inserting the gene into the vector pBI121, specifically, it can be obtained by inserting the DNA molecule shown in Sequence 1 of the sequence listing between the Xba Ⅰ and Sac Ⅰ restriction sites of the vector pBI121 recombinant expression vectors.

The invention protects the application of the protein or the gene in regulating the somatic embryogenesis of the target plant.

Another object of the present invention is to provide a method for cultivating transgenic plants, which is to introduce the gene into the target plant to obtain a transgenic plant whose somatic embryogenesis ability is higher than that of the target plant; the introduction is through the recombination carried by the carrier.

The present invention also provides a method for improving the somatic embryogenesis ability of the target plant, including the step of expressing the protein or its coding gene in the target plant.

The somatic embryogenesis ability is embodied in the embodiments of the present invention as the number or ability of each explant to obtain green shoots through dedifferentiation and differentiation.

In the above application and method, the target plant may be a monocotyledon or a dicotyledon; specifically, the dicotyledon may be Arabidopsis or tobacco.

Experiments have proved that, in the transgenic Arabidopsis line expressing the gene GhSERK2 shown in Sequence 1 of the Sequence List, the number of shoots of each hypocotyl after induction and differentiation is significantly higher than that of the wild-type and empty vector controls; In the transgenic tobacco line with the gene GhSERK2, the number of buds of each explant leaf piece induced and differentiated was significantly higher than that of the wild type and the empty vector control; indicating that the GhSERK2 protein and its encoding gene can regulate the somatic embryogenesis of the target plant Ability. The present invention clarifies the network regulation and genetic mechanism in the cotton somatic cell culture process, improves the plant regeneration rate and genetic transformation rate of cotton, overcomes the genotype limitation of cotton somatic cell culture, and selects excellent genotypes purposefully to obtain regenerated plants. It is of great significance to obtain new and excellent cotton varieties quickly and efficiently.

Description of drawings

Figure 1 is the electrophoresis diagram of PCR amplification of GhSERK2 gene. Wherein, M is the molecular weight standard, and 1 is the product of the target gene amplified by PCR.

Fig. 2 is the double restriction electrophoresis diagram of the recombinant plasmid pBI121-GhSERK2. Wherein, M is the molecular weight standard, and 1 is the product of pBI121-GhSERK2 double-digested by XbaI and SacI.

Fig. 3 is the PCR detection result of the transgenic Arabidopsis T ₃ generation strain. Among them, M is the molecular weight standard D2000DNAladder, "+" is the plasmid pBI121-GhSERK2 positive control; "-" is the wild-type Arabidopsis negative control; "1~5" are the transgenic Arabidopsis T ₃ generation plants.

Figure 4 is the observation of the seedling stage phenotype of transgenic Arabidopsis T ₃ generation plants. "W" is the wild-type Arabidopsis negative control; "T" is the T ₃ generation plant of transgenic Arabidopsis.

Figure 5 shows the expression results of the gene GhSERK2 detected by semi-quantitative RT-PCR. Among them, WT is the negative control of wild-type Arabidopsis; "SK1-7~SK3-5" respectively represent the transgenic Arabidopsis T ₃ generation lines.

Figure 6 shows the callus induced by the hypocotyls of transgenic Arabidopsis T ₃ generation plants (left picture) and differentiated buds from the callus after subculture (right picture). The length of the scale bar in the figure is 1cm.

Fig. 7 is the PCR detection result of the transgenic tobacco T ₃ generation strain. Among them, M is the molecular weight standard D15000DNAladder, "+" is the positive control of plasmid pBI121-GhSERK2; "-" is the negative control of wild-type Nicotiana Shanxi; "1~5" are the plants of transgenic tobacco T ₃ generation lines.

Fig. 8 is the observation of the phenotype of transgenic tobacco T ₃ generation plants at seedling stage. Among them, "W" is the negative control of wild-type Tobacco Shanxi; "T" is the _T3 transgenic tobacco line.

Fig. 9 is a semi-quantitative RT-PCR detection gene GhSERK2 expression in _T3 transgenic tobacco lines. Among them, WT is the negative control of wild-type Tobacco Shanxi; "SK1-3~SK3-9" are the homozygous transgenic tobacco lines of _T3 generation respectively.

Figure 10 shows the callus induced by leaves of transgenic tobacco T ₃ generation plants (left picture) and the shoots differentiated from the callus after subculture (right picture). The length of the scale in the figure is 1cm.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the cloning of cotton GhSERK2 gene

Young leaves of upland cotton (Gossypium hirsutum L.) cultivar Coker201 (Wu Xiuming, Liu Chuanliang, Zhang Chaojun, Li Fuguang. Research progress on somatic embryogenesis of cotton. Bulletin of Botany, 2008,25(4):469-475) As the material, extract the total RNA, use PrimeScipt RT reagent Kit With Gdna Eraser (Prefect RealTime) (TaKaRa) to digest the genomic DNA in the total RNA, and reverse transcribe to obtain cDNA; design primers L and R, and use the cDNA as a template for PCR Amplify.

The primer sequences for the above-mentioned PCR amplification are as follows:

Primer L: 5'-TATTTGCTCGTTGATTCGG-3';

Primer R: 5'-TGTTGGAGAAGACCCTTTG-3'.

The above reaction system for PCR amplification: cDNA template 10ng, 10×PCR Buffer 2.5μl, 10mmol/L dNTPMixture 2μl, 2μmol/L upstream and downstream primers 0.5μl each, Taq DNA polymerase 1.25U, ddH ₂ O to make up to the total Volume 25 μl.

The reaction program of the above PCR amplification: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 30s, annealing at 53-57°C for 30s, extension at 72°C for 30s-3min, 30 cycles; extension at 72°C for 10 minutes.

The obtained PCR amplification product was subjected to 1% agarose gel electrophoresis, and a fragment of about 2 kb was recovered, connected to pMD18-T Simple Vector (purchased from Bao Biological Engineering Co., Ltd., catalog number D103A) to transform Escherichia coli DH5α strain, using Positive clones were screened by blue-white screening method, and the positive clones were sequenced. The sequencing results showed that the recombinant vector pMD18-T-GhSERK2 was obtained, that is, the vector pMD18-T Simple Vector was inserted with a 2054bp DNA fragment (named GhSERK2 gene) shown in Sequence 1 of the Sequence Listing, from the 148th to 2010th position of the Sequence 1 of the Sequence Listing It is an open reading frame with a length of 1863bp, encoding a protein GhSERK2 consisting of 620 amino acid residues as shown in Sequence 2 of the Sequence Listing, eg.

Embodiment 2, the construction of recombinant plant expression vector

1. Acquisition of the target gene GhSERK2

Using pMD18-T-GhSERK2 as a template and primers L2 and R2 as specific primers, PCR amplification was carried out, and the PCR amplification products were detected by 1% agarose gel electrophoresis. A PCR product of about 2kb was recovered and purified (Fig. 1).

Primer L2: 5′–GC TCTAGA GCTATTTGCTCGTTGATTCGG–3′;

Primer R2: 5'-C GAGCTC GTGTTGGAGAAGACCCTTTG-3'.

2. Construction of recombinant plant expression vector

① Use restriction endonucleases Xba Ⅰ and Sac Ⅰ to digest the purified PCR product in step 1, and recover the digested product;

② Digest the plant expression vector pBI121 (China Plasmid Vector Strain Cell Strain Gene Collection Center, product number; Biovector008-32) with restriction endonucleases XbaⅠ and SacⅠ, and recover the vector skeleton;

③ connecting the digested product of step ① to the carrier backbone of step ②;

④ Heat-shock transformed the ligation product of step ③ into Escherichia coli DH5α strain, cultured overnight at 37°C, picked positive clones for sequencing; the sequencing results showed that the recombinant plasmid pBI121-GhSERK2 was obtained, which was the Xba Ⅰ and Sac in the vector pBI121. A 2054bp DNA fragment shown in Sequence 1 of the Sequence Listing was inserted between the Ⅰ restriction sites, and it was verified to be correct by XbaI and SacI double digestion (Figure 2).

Embodiment 3, cotton GhSERK2 gene transformation Arabidopsis

1. Construction of recombinant Agrobacterium

The recombinant plant expression vector pBI121-GhSERK2 prepared in Example 2 was used to transform the competent cells of Agrobacterium EHA105 (purchased from China Plasmid Vector Strain Cell Line Gene Collection Center, product number Biovector008) with liquid nitrogen quick-freezing-dissolving method, and stored at 28°C in Select and culture in YEB solid culture containing 50 μg/ml kanamycin sulfate and 50 μg/ml rifampicin; pick a single colony for shaking, and extract the plasmid DNA, carry out enzyme digestion verification on the recombinant plasmid, and identify the correct The recombinant Agrobacterium containing the recombinant plant expression vector pBI121-GhSERK2 is named EHA105/pBI121-GhSERK2.

2. Obtaining transgenic Arabidopsis

1) Preparation of Agrobacterium infection solution

Take the single colony of recombinant Agrobacterium EHA105/pBI121-GhSERK2 from step 1 and inoculate it in 5ml YEB liquid medium (containing 50μg/ml kanamycin sulfate and 50μg/ml rifampicin), shake the bacteria overnight, and spin the bottle the next day Into 500ml YEB liquid medium, culture at 28°C until the OD ₆₀₀ is 1.6~2; centrifuge to collect the bacteria, and use MS solution (MS basic medium, add 5% sucrose, 40μl Silwet L-77) to suspend until the OD ₆₀₀ is 0.8 ~1. Obtain the Agrobacterium infection solution.

2) Preparation of target plant Arabidopsis

The Colombian wild-type Arabidopsis Col-0 (Arabidopsis thaliana) (purchased from The Arabidopsis Information Resource, website: http://www.arabidopsis.org/index.jsp, accession number: Species Variant: 90) seeds were planted in nutrient soil : In the mixed soil of vermiculite=1:2, it can be transformed after it has bolted moss and grown more flower buds and entered the flowering period.

3) Conversion

Cut off the flowering and pollinated flower organs of Arabidopsis thaliana in step 2), and put the whole plant upside down together with the flowerpot in a container containing 500ml of Agrobacterium infection solution prepared in step 1) for soaking and transformation; Arabidopsis thaliana The plants were placed sideways in a tray and covered with a black plastic cloth. One day later, the plastic cloth was uncovered, the flowerpot was placed upright, and normal light cultivation was carried out to collect mature _T1 generation seeds.

4) Screening of resistant seedlings

After sterilizing the _T1 generation seeds, spread them on MS solid medium (containing 50 μg/ml kanamycin sulfate), vernalize at 4°C for 2-3 days, cultivate them at 21-23°C, and select them after about 7 days For resistant plants, move them into the mixed soil of nutrient soil: vermiculite = 1:2, and harvest the T ₂ generation seeds per plant after 45-60 days. Plant and screen T ₂ generation seeds in the same way, transplant 4 T ₂ generation strains with a segregation ratio of resistance of 3:1, and harvest T ₃ generation seeds on each individual plant in the T ₂ generation strains , randomly selected 10 seeds of T ₃ generation strains and carried out resistance screening in the same way, and obtained 5 homozygous transgenic Arabidopsis lines that no longer produced resistance segregation in T ₃ generation.

At the same time, the empty vector pBI121 was transformed into wild-type Arabidopsis thaliana according to the above steps 1 and 2 to obtain an empty vector-transferred control Arabidopsis line that no longer produces resistance in the T ₃ generation.

T ₁ generation represents the seeds produced by the self-crossing of the transformed contemporary plants and the plants grown from it; T ₂ generation represents the seeds produced by the self-crossing of the T ₁ generation and the plants grown from it; T ₃ generation represents the self-growth of the T ₂ generation The seed produced by intercourse and the plant that grows from it.

3. PCR identification of transgenic Arabidopsis plants

Genomic DNA was extracted from homozygous transgenic Arabidopsis thaliana plants and leaves of wild-type Arabidopsis thaliana that no longer produced resistance in the T ₃ generation obtained in step 2, and used primers KanF1:5'-CACTGAAGCGGGAAGGGACT-3' and KanR1:5 '-CGATACCGTAAAGCACGAGGAA-3'PCR amplified the kanamycin resistance marker gene, and the predicted product size was 500bp. As a result, all the homozygous transgenic lines were positive, and the wild type was negative. The results are shown in Figure 5. The same method was used to detect the empty vector control Arabidopsis thaliana line plants that no longer produce resistance in the T ₃ generation, and the results were all positive.

4. Phenotype identification of transgenic Arabidopsis plants

The seeds of the T ₃ generation homozygous GhSERK2 gene Arabidopsis strain identified as positive by the PCR in the above step 3, the positive T ₃ generation homozygous transfer of empty vector control Arabidopsis strain seeds and wild type Arabidopsis seeds were killed. After bacteria, they were spread on MS medium and cultured in the dark for 7 days to obtain germinated sterile hypocotyls, and the hypocotyl fragments were cut and placed in induction medium (B5 medium + 5mg/L2, 4-D + 0.5mg /L KT) at 21°C, with a photoperiod of 16 hours of light/8 hours of darkness per day, and a light intensity of 2000LUX for 12-15 days to obtain callus, and then put the callus on fresh induction medium After two subcultures (15 days each), the callus with a diameter of 10 mm (the left picture in Figure 6) was placed on the differentiation medium (MS medium + 5 mg/L KT + 0.1 mg/L IAA) on Differentiation culture was carried out under the conditions of 21°C, photoperiod of 16 hours of light/8 hours of darkness per day, and light intensity of 2000LUX. After 30 days, the number of buds was counted (the number of buds refers to the number of differentiated buds on the callus, as shown on the right in Figure 6 shown in the figure).

B5 medium formula: KNO ₃ 2.5g/L, CaCl ₂ 2H ₂ O 0.15g/L, MgSO ₄ 7H ₂ O 0.25g/L, (NH4) ₂ SO ₄ 0.134g/L, KI 0.75mg/L , H ₃ BO ₄ 3.0mg/L, MnSO ₄ 4H ₂ O 10mg/L, ZnSO ₄ 7H ₂ O 2.0mg/L, Na ₂ MoO ₄ 2H ₂ O 0.25mg/L, CoCl ₂ 6H ₂ O 0.025mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, Na ₂ -EDTA 37.3mg/L, FeSO ₄ 7H ₂ O 27.8mg/L, Inositol 100mg/L, Niacin 1.0mg/L, Hydrochloric acid Pyridoxine 1.0mg/L, ammonium sulfate hydrochloride 10mg/L, sucrose 20g/L, agar 7g/L, pH 5.5;

MS medium formula: KNO ₃ 1.9g/L, NH ₄ NO ₃ 1.65g/L, KH ₂ PO ₄ 0.17g/L, MgSO ₄ 7H ₂ O 0.37g/L, CaCl ₂ .2H ₂ O 0.44g/L L, KI 0.83mg/L, H ₃ BO ₄ 6.2mg/L, MnSO ₄ 4H ₂ O 22.3mg/L+ZnSO ₄ 7H ₂ O 8.6mg/L+Na ₂ MoO ₄ 2H ₂ O 0.25mg/L L, CoCl ₂ 6H ₂ O 0.025mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, Na ₂ -EDTA 37.3mg/L, FeSO ₄ 7H2O 27.8mg/L, Inositol 100mg/L, Glycine 2mg /L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, sucrose 30g/L, agar 7g/L, pH 5.6;

The experiment was repeated 3 times, and 6 hypocotyl materials were taken for each repetition of each strain for induction culture, and the average number of germinations obtained by the above-mentioned induction and differentiation culture, that is, somatic embryogenesis, of each repeated hypocotyl material of each strain was counted. , and the results are shown in Table 2.

Table 2. The number of shoots induced by hypocotyl differentiation of seeds of transgenic Arabidopsis lines with GhSERK2

Note: WT means the untransgenic wild-type Arabidopsis control; SK1-7, SK1-9, SK2-3, SK2-7 and SK3-5 are the five Arabidopsis lines homozygously transfected with the GhSERK2 gene in the T ₃ generation . There was no significant difference between the results of the empty vector control Arabidopsis line and the wild-type Arabidopsis; * indicates a significant difference at P﹤0.05 compared with the wild-type Arabidopsis.

Further observe the seedling growth phenotype of the seeds of _T3 generation homozygous GhSERK2 gene Arabidopsis strains, such as root length, leaf color and size, the results are shown in Figure 4, compared with the control wild-type Arabidopsis , there was no significant difference in root length, leaf color and size of transgenic Arabidopsis lines, which indicated that the expression of GhSERK2 gene had no adverse effect on the growth and development of recipient materials.

The above results show that the GhSERK2 protein and its coding gene can regulate the plant somatic embryogenesis ability and plant regeneration ability (each differentiated bud can further develop into a plant after being stripped, so the number of buds in Arabidopsis represents the number of subsequent regenerated plants.)

5. Semi-quantitative RT-PCR detection

Take the positive T ₃ generation homozygous GhSERK2 gene transgenic Arabidopsis plant, the positive T ₃ generation homozygous empty vector control Arabidopsis plant and wild type Arabidopsis plant leaves identified by PCR in the above step 3, RNA was extracted by CTAB-LiCl method (Xie Chuansheng et al., Comparison of Different Extraction Methods of Arabidopsis and Tobacco Young Seed RNA. China Agricultural Science Bulletin, 2009, 25(23):78-81), cDNA was synthesized by reverse transcription, and The Arabidopsis Atactin gene was used as an internal reference, and the expression of the target gene GhSERK2 in recipient plants was identified by semi-quantitative RT-PCR. The results are shown in Figure 5.

The primers for detection of Atactin gene expression are as follows:

AtactinF1:5'-CCATGAAACCACCTATAACTCC-3';

AtactinR1:5'-TACTCTGCCTTTGCGATCCAC-3';

The primers for detecting the expression of GhSERK2 gene are as follows:

Semi-GhSERKF1:5'-CGGCGATTGATTTCCTTTTGT-3'; (corresponding to the 166th-186th position of sequence 1 in the sequence listing)

Semi-GhSERKR1:5'-GTAGTCGGGATGTCTCCAGTT-3'. (corresponding to No. 540-560 of Sequence 1 of the Sequence Listing)

The results showed that the expression of the target gene GhSERK2 could be detected in the transgenic Arabidopsis line material, but the expression could not be detected in the wild type Arabidopsis plant, and the results of the empty vector control Arabidopsis line plant were the same as those of the wild type Arabidopsis plants had the same results.

Embodiment 4, cotton GhSERK2 gene transformation tobacco

1. Obtaining genetically modified tobacco

Shanxi Tobacco (Zhang Junfang, Yue Yuesen, Zhang Jiajia. Agrobacterium-mediated SOS1 Gene Transformed Tobacco and Preliminary Detection of Salt Resistance of Transformed Plants. Chinese Science and Technology Papers Online Excellent Papers, 2010, 3(19): 2027-2033. Publicly available (obtained from China Agricultural University) seeds were sterilized with 20% H ₂ O ₂ for 15 minutes, rinsed with sterile water for 5 times, and the treated seeds were spread on MS medium to germinate sterile seedlings. Get the young leaves of sterile seedlings, remove the veins, cut to ^1cm size, place in the Agrobacterium infection solution obtained in Step 2 of Example 3 and soak for 15min, suck off the excess suspension on the surface of the leaves with sterile filter paper, and place Leaves were co-cultured on differentiation medium (MS medium + 3mg/L6-BA + 0.2mg/L NAA + plant gel 2.7g/L + glucose 30g/L) at 19°C for 48h, then rinsed with sterile water ( benzyl penicillin 200mg/L) and washed several times, the excess water on the filter paper was blotted and inoculated into the tobacco differentiation screening medium (MS medium+3mg/L6-BA+0.2mg/L NAA+plant gel 2.7g/L+glucose 30g/ L+carbenicillin 200mg/L+kanamycin sulfate 100mg/L) for differentiation and screening to obtain regenerated tobacco plants, which were transplanted and planted to harvest _T1 generation seeds.

2. Screening of resistant seedlings

After sterilizing the _T1 generation seeds, spread them on MS solid medium (containing 50 μg/ml kanamycin sulfate), vernalize them at 4°C for 2-3 days, and cultivate them at 21-23°C for about 7 days. Select resistant plants and move them into the mixed soil of nutrient soil: vermiculite = 1:2, and harvest the T ₂ generation seeds per plant after 45-60 days. Plant and screen T ₂ generation seeds in the same way, transplant 4 T ₂ generation strains with a segregation ratio of resistance of 3:1, and harvest T ₃ generation seeds on each individual plant in the T ₂ generation strains , 12 seeds of T3 _generation lines were randomly selected for resistance screening in the same way, and 5 homozygous transgenic tobacco lines that no longer produced resistance segregation in T3 _generation were obtained.

At the same time, the empty vector pBI121 was transformed into wild-type Nicotiana Shanxi according to the above steps 1 and 2 to obtain a _T3 generation homozygous empty vector-transformed control tobacco line.

T ₁ generation represents the seeds produced by the self-crossing of the transformed contemporary plants and the plants grown from it; T ₂ generation represents the seeds produced by the self-crossing of the T ₁ generation and the plants grown from it; T ₃ generation represents the self-growth of the T ₂ generation The seed produced by intercourse and the plant that grows from it.

3. PCR identification of transgenic tobacco plants

Genomic DNA was extracted from the homozygous transgenic tobacco line plants and wild-type Shanxi tobacco leaves that were no longer resistant to isolation of the T ₃ generation obtained in step 2, and primers KanF1:5'-CACTGAAGCGGGAAGGGACT-3' and KanR1:5'- CGATACCGTAAAGCACGAGGAA-3'PCR amplified the kanamycin resistance marker gene, and the predicted product size was 500bp. As a result, all the homozygous transgenic tobacco plants were positive, and the wild-type Nicotiana Shanxi was negative. The results are shown in Figure 7. The same method was used to detect the empty carrier control tobacco line plants that no longer produce resistance isolation in the _T3 generation, and the results were all positive.

4. Phenotypic identification of transgenic tobacco plants

The positive T ₃ generation homozygous GhSERK2 gene tobacco strain seeds identified as positive by PCR in the above step 3, the positive T ₃ generation homozygous transfer empty vector control tobacco strain seeds and wild type Shanxi tobacco seeds were sterilized and spread respectively Cultured on MS medium for 30 days at 22-23°C and 2000LX light conditions to obtain aseptic seedlings containing 2-3 true leaves, and averaged the second leaf from the bottom to the top of each aseptic seedling. Cut into 4 pieces (each piece is an explant), place tobacco callus differentiation medium (MS medium (the formula is the same as the MS medium formula in Example 3, but does not contain agar)+3mg/L 6-BA+0.2mg/L NAA+plant gel 2.7g/L+glucose 30g/L) at 22~23℃, photoperiod of 16 hours of light/8 hours of darkness, and light intensity of 2000LUX for 2030 days Callus, the callus was placed on the fresh above-mentioned tobacco callus induction differentiation medium and subcultured once (20 days each time), and the callus with a diameter of 10mm (the left picture in Figure 10) was placed Carry out differentiation culture on the above-mentioned tobacco induction differentiation medium at 22-23°C, photoperiod of 16 hours of light/8 hours of darkness per day, and light intensity of 2000LUX, count the number of buds after 30 days (the number of buds refers to the number of calli The number of shoots that differentiated above, right panel in Fig. 10).

The experiment was repeated 3 times, and each strain was repeated to get 3 sterile seedlings, and the average number of buds obtained by each explant of each strain in each repetition was obtained through the above-mentioned induction and differentiation culture, that is, somatic embryogenesis, and the result as shown in Table 3.

Table 3. The number of induced differentiation shoots per explant of transgenic GhSERK2 tobacco plants

Note: WT means the non-transgenic wild-type Shanxi tobacco control; SK1-3, SK2-5, SK2-6, SK2-11 and SK3-9 are five _T3 generation homozygous GhSERK2 gene transgenic tobacco lines. There was no significant difference between the results of the empty vector control tobacco line and the wild-type Nicotiana Shanxi; * indicates a significant difference at P﹤0.05 compared with the results of the wild-type Nicotiana Shanxi.

Further observe the seedling growth phenotypes of the _T3 generation homozygous GhSERK2 gene tobacco line plants, such as plant height, leaf color and size, the results are shown in Figure 8, compared with the control wild-type Shanxi tobacco, the transgenic There were no significant differences in plant height, leaf color and size among tobacco lines, which indicated that the expression of GhSERK2 gene had no adverse effect on the growth and development of recipient materials.

The above results indicate that the GhSERK2 protein and its coding gene can regulate the somatic embryogenesis ability and plant regeneration ability of the target plant.

6. Semi-quantitative RT-PCR detection

Take the positive T ₃ generation homozygous GhSERK2 gene tobacco strain plants, positive T ₃ generation homozygous transfer empty vector control tobacco strain plants and wild-type Shanxi tobacco plant leaves identified as positive by PCR in the above step 3, and use CTAB-LiCl Method (Xie Chuansheng et al. Comparison of different extraction methods for Arabidopsis and tobacco young seed RNA. China Agricultural Science Bulletin, 2009, 25(23): 78-81) to extract RNA, synthesize cDNA by reverse transcription, and use tobacco NtTac9 gene as As an internal reference, semi-quantitative RT-PCR identified the expression of the target gene GhSERK2 in recipient plants, and the results are shown in FIG. 9 .

The primers for detecting NtTac9 gene expression are as follows:

NtTac9F1:5'-CCCTCCCACATGCTATTCT-3';

NtTac9AtactinR1:5'-AGAGCCTCCAATCCAGACA-3';

The primers for detecting the expression of GhSERK2 gene are as follows:

Semi-GhSERKF1:5'-CGGCGATTGATTTCCTTTTGT-3'; (corresponding to the 166th-186th position of sequence 1 in the sequence listing)

Semi-GhSERKR1:5'-GTAGTCGGGATGTCTCCAGTT-3'. (corresponding to No. 540-560 of Sequence 1 of the Sequence Listing)

The results showed that the expression of the target gene GhSERK2 could be detected in the transgenic tobacco line material, but could not be detected in the wild-type Shanxi tobacco plants, and the results of the empty vector control tobacco line plants were the same as those of the wild-type tobacco plants .

Claims (10)

1. protein, be following a) or b) protein:

A) protein that is formed by the aminoacid sequence shown in the sequence table sequence 2;

B) with the aminoacid sequence of sequence table sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and occur relevant by (a) derivative protein with plant somatocyte embryo.

2. the encoding gene of the described protein of claim 1;

Described gene specifically can be following 1)-4) in the gene any one:

1) its nucleotide sequence is the dna molecular shown in the 148th to the 2010th in the sequence table sequence 1;

2) its nucleotide sequence is the dna molecular shown in the sequence table sequence 1;

3) with 1) or 2) dna sequence dna that limits has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least 99% homology and a described protein DNA molecule of coding claim 1;

4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization and the described protein DNA molecule of coding claim 1 that limit.

3. the recombinant vectors, expression cassette, transgenic cell line, recombinant bacterium or the recombinant virus that contain the described gene of claim 2.

4. recombinant vectors according to claim 3 is characterized in that: the recombinant expression vector that described recombinant vectors obtains for Xba I and Sac I with the described gene insertion vector PBI121 of claim 2.

5. the described protein of claim 1 or the described gene of claim 2 application in regulation and control purpose plant somatocyte embryo occurs.

6. a method of cultivating transgenic plant is that the described gene of claim 2 is imported in the purpose plant, obtains the transgenic plant that Somatic embryogenetic ability is higher than described purpose plant.

7. method according to claim 6 is characterized in that: described importing realizes by the described recombinant vectors of claim 4.

8. a method that improves purpose plant somatocyte embryo generating ability is included in the step of expressing the described protein of claim 1 or its encoding gene in the described purpose plant.

9. arbitrary described application or method according to claim 5-8 is characterized in that: described purpose plant is monocotyledons or dicotyledons.

10. application according to claim 9 or method, it is characterized in that: described dicotyledons is Arabidopis thaliana or tobacco.

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