A method for improving plant tissue culture differentiation efficiency by transferring NtZFP8 gene

Abstract

The present invention discloses a method for improving the differentiation efficiency of plant tissue culture by transferring the NtZFP8 gene. The present invention provides a gene fragment, the nucleotide sequence of which is shown in SEQ ID NO.1. The present invention also discloses a recombinant vector and a recombinant bacterium comprising the aforementioned gene fragment and their uses. The present invention significantly improves the regeneration ability of plant explants and callus tissues by transferring the NtZFP8 gene fragment into plants, reduces the cycle of obtaining regenerated seedlings, and obtains regenerated plants with utilization value. The method can significantly improve the regeneration ability of plant explants and callus tissues, and has potential application value and economic benefits.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a method for improving the differentiation efficiency of plant tissue culture by transferring NtZFP8 gene. Background Art

[0002] Plant tissue culture technology is the embodiment of cell totipotency. With the in-depth research on the mechanism of somatic embryogenesis and the rapid development of related fields, plant tissue culture technology has not only been widely used in the rapid propagation of forestry, crops and horticultural crops, but also plays an important role in the breeding of genetically engineered varieties. Plant tissue culture is mainly carried out through two pathways: one is direct induction of explants; the other is indirect induction, that is, inducing embryonic callus through somatic cells, and then differentiating and sprouting callus to obtain regenerated plants. Differentiation efficiency is an important indicator to measure the efficiency of plant tissue culture technology.

[0003] The differentiation of explants and callus is a complex biological process that requires some specific physical and chemical factors to initiate and maintain, such as the genotype of the recipient plant, the type and physiological state of the explant, the composition of the culture medium, and the composition of plant hormones. For example, studies in rice have found that different genotypes of rice have different callus differentiation abilities, and japonica rice is significantly better than indica rice (Lin YJ, Zhang Q. Optimising the tissue culture conditions for high efficiency transformation of indica rice [J]. Plant Cell Reports, 2005, 23 (8): 540-547.), and it also affects the transgenic efficiency of indica rice callus (Tie W, Zhou F, Wang L, et al. Reasons for lower transformation efficiency in indica rice using Agrobacterium tumefaciens-mediated transformation: lessons from transformation assays and genome-wide expression profiling [J]. Plant Molecular Biology, 2012, 78 (1): 1-18.). The problem of explant and callus differentiation efficiency increases the cycle of plant tissue culture factory-scale breeding and increases production costs.

[0004] Previous researchers have improved differentiation capacity by adjusting hormone ratios, changing culture medium components, and improving culture environment (Zhu Keming, Tao Huimin, Xu Shuo, et al. Research Progress on Rice Callus Differentiation and Regeneration [J]. Seed, 2016(11):55-59.). With the development of molecular biology technology, improving regeneration capacity by transforming receptor plants through plant bioengineering has become a new research direction. Summary of the invention

[0005] The purpose of the present invention is to provide a new gene and improve the differentiation efficiency of plant tissue culture by transgenic means.

[0006] The present invention first provides a gene fragment, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0007] The present invention also provides a recombinant vector, which comprises the nucleotide sequence shown in SEQ ID NO. 1. Preferably, the recombinant vector is a recombinant pCAMBIA1301 vector.

[0008] The present invention also provides a recombinant bacterium, which comprises the aforementioned recombinant vector. Preferably, the recombinant bacterium is a recombinant Agrobacterium, and more preferably is a recombinant Agrobacterium EHA105.

[0009] The present invention also provides a protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The present invention also provides the use of the aforementioned gene fragment, recombinant vector, recombinant bacteria and protein in improving the differentiation efficiency of plant explants and / or callus tissues.

[0011] Wherein, the plant is tobacco.

[0012] The present invention also provides a method for improving the differentiation efficiency of plant tissue culture, which comprises taking the aforementioned gene fragments, recombinant vectors, and recombinant bacteria, and transferring them into plants to obtain plants or seeds that stably express the aforementioned proteins.

[0013] Wherein, the plant is tobacco.

[0014] The present invention provides a method for improving the regeneration rate of plant tissue culture by NtZFP8 transfection. The NtZFP8 gene fragment of the present invention of tobacco is used to obtain a stably inherited transgenic material, and verification experiments show that the NtZFP8 gene can play an important role in the differentiation of plant explants and callus tissues, and the regenerated buds obtained by differentiation can develop into plants. Therefore, the gene and recombinant plasmid can be used in variety breeding and tissue culture industrialization, serving application and production through a new approach of plant genetic engineering.

[0015] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0016] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the electrophoresis diagram of PCR amplification of NtZFP8 gene, wherein lane M is a molecular weight marker (AL2000 DNA Marker, purchased from Adele Company), and lanes 1-2 are the amplification results of NtZFP8.

[0018] Figure 2 The electrophoresis diagram is the PCR of Agrobacterium colony transformed with NtZFP8 gene, wherein lane M is the molecular weight marker (AL2000 DNA Marker, purchased from Adelaide Company), and lanes 1-6 are the PCR amplification results of Agrobacterium bacterial solution.

[0019] Figure 3 Identification results of NtZFP8 gene expression level, where 1 is the wild-type comparison, and 2 and 3 are the overexpression experimental groups.

[0020] Figure 4 The diagrams show the callus differentiation of wild-type and NtZFP8-overexpressing tobacco, where A is the wild-type comparison and B is the overexpression experimental group. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described are carried out with reference to the specific methods listed in the Molecular Biology Experiment Manual (Ma Wenli, 2011), or are carried out according to the kits and product instructions.

[0022] Example 1 Isolation of the NtZFP8 gene and construction of transgenic materials of the present invention

[0023] This example mainly describes the methods of obtaining the NtZFP8 gene, constructing vectors and preparing transgenic materials, which are briefly introduced as follows: The tobacco background material used in the experiment is Guiyan No. 6, which is preserved in the Plant Development Physiology Laboratory of the School of Life Sciences of Sichuan University.

[0024] (1) Construction of gene fragments and vectors

[0025] Weigh 0.5 g of fresh tobacco leaves and extract tobacco RNA using PrimeScript TM cDNA was synthesized using RT reagent Kit (RR037Q), primers (NtZFP8-F: 5'-ATGGACAAAACAGACAGAGAAAC-3'; NtZFP8-R: 5'-TCACAAGTGTAGATCTAAACTCACAT-3') were designed, and PCR amplification was performed to obtain a fragment of the target band size (the results are shown in Figure 1 The PCR product was purified using a gel recovery kit (Takara, 805A), connected to a T vector (Transgene Biotech, CT101-01), and transformed into competent E. coli DH5α using the heat shock method. The transformants were screened on LB plates containing kanamycin, and single clones were picked and sent to Chengdu Qingke Zixi Biotechnology Co., Ltd. for sequencing.

[0026] The nucleotide sequence of the amplified target fragment NtZFP8 gene (SEQ ID NO.1) is:

[0027] ATGGACAAAACAGACAGAGAAACTCGTGATTTCATGAACGTGGAGTCCTTCTCAGCTACCCTTTATTCGACCAGCACCAGCTAATAAAGAAAAAGCCATTAGGCTTTTTGGCAAAGAATTTGGTACTGCTGGTGACTCCATGGCTGCAACAGAATCCACTGAAACAAACCCTAGCCAAGAAGAAATCAAA GACCATATTAGCAACACAGTGAGAGCAGTCGAAAATTCGAGTGCCATTATTGTTGCAGGAATTTCCCGACTTCACAAGCACTAGGAGGACATCAAAATGCTCATAAGAGAGAACGCCAGAATGCTAAAAGAGCGCATCTTCAATCTGCAATGGTACACGGTGCTTTAACAGAGGCAAATATTTATGGAATAA TGAATTACCATCGTCTTGGTAGTGCGCCAACAGCAGCTTATCATCACTACGCAACCAACAATATTAACAGTGCTACTAGGTTTTATGGAAGCCATCACCATGTTAATAGTACCCCTTATAATTCTCATCAAACTCCTATAAATGGAAGTCCTTAGCTTTATGGAGGATTCCTGCAGCAACTAGTGTTCA TCATACTTCTTCGTCTTCGTCTAATTTTGGCCGTGAACGATCCATGAATATGCAGCCATTGCCAGTATTTGCTGCTAATAATGAGGATTTCAAGCCTTCTCCAATCATCAATAGCTCAAGTTCTCAAAGAGGGTTCGGATATGAAACAAAGGCCGGAATTAAAGATCATGTGAGTTTAGATCTACACTTGTGA

[0028] The NtZFP8 gene described in the aforementioned nucleotide sequence can be obtained by adopting the above method, or can be directly synthesized.

[0029] The plasmid containing the NtZFP8 gene was extracted, double-digested, and connected to the pCAMBIA1301 vector with T4-DNAligase (RT406). After the connection was incubated at 16°C overnight, the recombinant was transferred into the competent E. coli cells to obtain transformants. The plasmid of the transformant was extracted and transformed into the competent Agrobacterium EHA105. A single colony was picked for colony PCR. The colony PCR results (results are shown in Figure 2 The amplified product with the same size as the NtZFP8 gene appeared.

[0030] (2) Preparation of transgenic plants

[0031] The recombinant Agrobacterium strain was cultured until the bacterial liquid concentration reached OD600=0.6.

[0032] Immerse the tobacco leaf disc in the bacterial solution for 10 minutes. Place it in MURASHIGE SKOOG (MS) basal medium (purchased from PhytoTech, USA) containing 2.0 mg / L 6-benzylaminoadenine and 0.5 mg / L naphthaleneacetic acid, and culture it in the dark for 2 days; in order to remove excess Agrobacterium, transfer the tobacco leaf disc to MS medium containing 2.0 mg / L 6-benzylaminoadenine, 0.5 mg / L naphthaleneacetic acid and 300 mg / L cephalosporin.

[0033] After two weeks of growth of tobacco leaf discs on MS medium containing 6-benzylaminoadenine 2.0mg / L, naphthaleneacetic acid 0.5mg / L, kanamycin 100mg / L and cephalosporin 300mg / L, it was observed that the leaf discs were swollen, the color became lighter, and callus appeared on the edge of the leaves. After two weeks of treatment, the leaf discs were transferred to MS medium containing 6-benzylaminoadenine 2.0mg / L, naphthaleneacetic acid 0.5mg / L, kanamycin 50mg / L and cephalosporin 300mg / L for screening culture. After 3 weeks of culture, a large number of clustered buds were produced on the callus on the edge of the leaves. When the clustered buds grow to about 2cm, they are moved to MS medium containing 100mg / L ampicillin to induce rooting. After two weeks, the seedlings are trained and transplanted into flower pots for growth after three days.

[0034] The tobacco seeds harvested after the above Agrobacterium infection were collected and screened on MS medium containing 100 mg / L ampicillin. The seedlings that could grow normally were selected and planted in soil. After maturity, the seeds were collected and screened on MS medium containing 100 mg / L kanamycin.

[0035] Positive seeds were taken for molecular identification and the expression of NtZFP8 gene was determined. Figure 3 As shown, it is proved that the present invention has indeed obtained a homozygous body with stable expression of the target gene NtZFP8 gene.

[0036] The amino acid sequence of the protein expressed by the NtZFP8 gene (SEQ ID NO.2) is:

[0037] MDKTDRETRDFMNVESFSQLPFIRPAPANKEKAIRLFGKEFGTAGDSMAATESTETNPSQEEIKDHISNNSESSRKFECHYCCRNFPTSQALGGHQNAHKRERQNAKRAHLQSAMVHGALTEANIYGI MNYHRLGSAPTAAYHHHYATNNINSATRFYGSHHHVNSTPYNSHQTPINGSPLALWRIPAATSVHHTSSSSSNFGRERSMNMQPLPVFAANNEDFKPSPIINSSSSQRGFGYETKAGIKDHVSLDLHL

[0038] The beneficial effects of the present invention are described below by way of test examples:

[0039] Experimental Example 1 Ability of NtZFP8 gene to directly improve differentiation efficiency of plant explants

[0040] 1. Test methods

[0041] Wild-type tobacco seeds and NtZFP8 overexpressing tobacco seeds (seeds containing NtZFP8 gene prepared in Example 1) were sterilized in sodium hypochlorite (1%) for 4 minutes, dried on sterilized filter paper, and placed on a plate containing MS solid medium for germination. After 7 days, they were transferred to a culture bottle containing MS solid medium for culture, and after 15 days, they were used for explant differentiation test.

[0042] The third leaf of the wild-type and NtZFP8-overexpressing tobacco sterile seedlings was taken respectively, and leaves of the same size were obtained using a 1 cm borer. The leaves were placed on a plate containing MS solid culture medium for differentiation induction, and the differentiation efficiency was observed after 20 days.

[0043] 2. Test results

[0044] The results showed that after the induction of wild-type tobacco leaf discs, buds appeared but could not grow and no regenerated buds with utilization value could be obtained; after the induction of NtZFP8-overexpressing tobacco leaf discs, buds began to appear after 7 days, and the buds could gradually grow to more than 3 cm, and regenerated buds with utilization value were obtained.

[0045] The test results show that after the NtZFP8 gene of the present invention is transferred into tobacco, the differentiation ability of tobacco explants can be improved.

[0046] Experimental Example 2 Ability of NtZFP8 gene to improve plant callus differentiation efficiency

[0047] The leaf disks were obtained according to the method described in Experimental Example 1 and cultured on MS medium containing 2.0 mg / L 6-benzylaminoadenine and 0.5 mg / L naphthaleneacetic acid. After 10 days, the callus differentiation and the number of buds were counted; after 15 days, the number of regenerated seedlings was counted.

[0048] The results show that if Figure 4 As shown, the positive tobacco leaf callus was produced early, with more bud-like protrusions on the surface, and the number of regenerated shoots obtained was significantly higher than that of wild-type tobacco.

[0049] The test results show that after the NtZFP8 gene of the present invention is transferred into tobacco, the differentiation ability of tobacco callus can be improved.

[0050] In summary, the present invention prepares the NtZFP8 gene and recombinant bacteria, and transferring them into plants can effectively improve the differentiation efficiency of plant explants and callus tissues, thereby improving the efficiency of plant tissue culture technology, and has good application prospects. SEQUENCE LISTING <110> Sichuan University <120> A method for improving plant tissue culture differentiation efficiency by transferring NtZFP8 gene <130> GY007-17P1102 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 771 <212> DNA <213> Nucleotide sequence of NtZFP8 gene <400> 1 atggacaaaa cagacagaga aactcgtgat ttcatgaacg tggagtcctt ctctcagcta 60 ccctttattc gaccagcacc agctaataaa gaaaaagcca ttaggctttt tggcaaagaa 120 tttggtactg ctggtgactc catggctgca acagaatcca ctgaaacaaa ccctagccaa 180 gaagaaatca aagaccatat tagcaacaac agtgagagca gtcgaaaatt cgagtgccat 240 tattgttgca ggaatttccc gacttcacaa gcactaggag gacatcaaaa tgctcataag 300 agagaacgcc agaatgctaa aagagcgcat cttcaatctg caatggtaca cggtgcttta 360 acagaggcaa atatttatgg aataatgaat taccatcgtc ttggtagtgc gccaacagca 420 gcttatcatc atcactacgc aaccaacaat attaacagtg ctactaggtt ttatggaagc 480 catcaccatg ttaatagtac cccttataat tctcatcaaa ctcctataaa tggaagtcct 540 ttagcttat ggaggattcc tgcagcaact agtgttcatc atacttcttc gtcttcgtct 600 aatttggcc gtgaacgatc catgaatatg cagccattgc cagtatttgc tgctaataat 660 gaggattca agccttctcc aatcatcaat agctcaagtt ctcaaagagg gttcggatat 720 gaaacaaagg ccggaattaa agatcatgtg agtttagatc tacacttgtg a 771 <210> 2 <211> 256 <212> PRT <213> NtZFP8 is the most commonly used source <400> 2 Met Asp Lys Thr Asp Arg Glu Thr Arg Asp Phe Met Asn Val Glu Ser 1 5 10 15 Phe Ser Gln Leu Pro Phe Ile Arg Pro Ala Pro Ala Asn Lys Glu Lys 20 25 30 Ala Ile Arg Leu Phe Gly Lys Glu Phe Gly Thr Ala Gly Asp Ser Met 35 40 45 Ala Ala Thr Glu Ser Thr Glu Thr Asn Pro Ser Gln Glu Glu Ile Lys 50 55 60 Asp His Ile Ser Asn Asn Ser Glu Ser Ser Arg Lys Phe Glu Cys His 65 70 75 80 Tyr Cys Cys Arg Asn Phe Pro Thr Ser Gln Ala Leu Gly Gly His Gln 85 90 95 Asn Ala His Lys Arg Glu Arg Gln Asn Ala Lys Arg Ala His Leu Gln 100 105 110 Ser Ala Met Val His Gly Ala Leu Thr Glu Ala Asn Ile Tyr Gly Ile 115 120 125 Met Asn Tyr His Arg Leu Gly Ser Ala Pro Thr Ala Ala Tyr His His 130 135 140 His Tyr Ala Thr Asn Asn Ile Asn Ser Ala Thr Arg Phe Tyr Gly Ser 145 150 155 160 His His His Val Asn Ser Thr Pro Tyr Asn Ser His Gln Thr Pro Ile 165 170 175 Asn Gly Ser Pro Leu Ala Leu Trp Arg Ile Pro Ala Ala Thr Ser Val 180 185 190 His His Thr Ser Ser Ser Ser Ser Asn Phe Gly Arg Glu Arg Ser Met 195 200 205 Asn Met Gln Pro Leu Pro Val Phe Ala Ala Asn Asn Glu Asp Phe Lys 210 215 220 Pro Ser Pro Ile Ile Asn Ser Ser Ser Ser Gln Arg Gly Phe Gly Tyr 225 230 235 240 Glu Thr Lys Ala Gly Ile Lys Asp His Val Ser Leu Asp Leu His Leu 245 250 255 <210> 3 <211> 23 <212> DNA <213> NtZFP8-F <400> 3 atggacaaaa cagacagaga aac 23 <210> 4 <211> 26 <212> DNA <213> NtZFP8-R <400> 4 tcacaagtgt agatctaaac tcacat 26

Claims

1. Use of overexpressing the gene shown in SEQ ID NO.1 or the protein shown in SEQ ID NO.2 in accelerating the production of callus in tobacco tissue culture; the application is to improve the differentiation efficiency of tobacco callus.

2. A method for accelerating callus production in plant tissue culture, characterized in that : The gene fragment shown in SEQ ID NO.1, a recombinant vector containing the gene fragment or a recombinant bacterium containing the gene fragment is transferred into tobacco to obtain plants or seeds stably expressing the protein shown in SEQ ID NO.2; the protein shown in SEQ ID NO.2 is overexpressed in tobacco plants or seeds, and the differentiation efficiency of tobacco callus is improved.

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