CN117987427A - Deep-sowing-resistant gene ZmSRO e and application thereof - Google Patents
Deep-sowing-resistant gene ZmSRO e and application thereof Download PDFInfo
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of biological gene engineering, and particularly relates to a deep-sowing-resistant gene ZmSRO e and application thereof. Specifically, the invention separates a deep-sowing-resistant gene from corn, which is named ZmSRO e, and experiments prove that the gene is related to the elongation of the hypocotyl in the corn, the hypocotyl of a plant (such as common corn) transformed with the gene is obviously elongated, the deep-sowing-resistant capability is obviously improved, theoretical basis and practical guarantee are provided for cultivating the deep-sowing-resistant plant, and the invention has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biological gene engineering, and particularly relates to a deep-sowing-resistant gene ZmSRO e and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Most corns are planted in arid and semiarid regions, seeds in the regions need to be sown into deeper soil, and the deep soil moisture can be effectively utilized to meet the moisture requirement of seed germination, so that the adverse effect of surface soil drought in the sowing period is reduced. For common corn varieties, increasing the sowing depth can lead to the decrease of the emergence rate, and the establishment of seedlings and the final yield are affected. The corn variety with deep sowing resistance has longer mesocotyl and stronger soil-jacking capability during deep sowing, has high emergence rate, can absorb moisture from deeper soil, and is beneficial to drought resistance of corn. Identification of deep-sowing-resistant key genes and cultivation of deep-sowing-resistant varieties are of great significance to maize production in arid and semiarid regions
The new character is transferred into the high biomass plant by using the transgenic improved plant technology, so that the novel variety of the high-efficiency transgenic plant is developed and used for improving the drought resistance of crops in arid and semiarid areas, and the technology has wide application prospect.
Research on deep sowing resistance of plants is developed by utilizing related technologies of molecular genetics. Candidate genes related to the deep-sowing resistance are identified, and allelic mutation of the genes is found, so that the deep-sowing resistance of plants is weakened. However, at present, the genetic analysis of the deep-seeding resistance of most crops is mainly focused on the discovery of deep-seeding resistance candidate sites, and the functions and specific molecular mechanisms of deep-seeding resistance genes are rarely reported, so that the enhancement is needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a deep-sowing-resistant gene ZmSRO e and application thereof. Specifically, the invention separates a deep-sowing-resistant gene from corn, which is named ZmSRO e, and experiments prove that the gene is related to the elongation of the hypocotyl in the corn, the hypocotyl of a plant transformed with the gene is obviously elongated, and the deep-sowing-resistant capability is obviously improved. Based on the above results, the present invention has been completed.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
In a first aspect of the present invention, there is provided a deep-cast-resistant gene designated ZmSRO e, said deep-cast-resistant gene having any one of the nucleotide sequences (a 1) to (a 4):
(a1) A nucleotide sequence shown as SEQ ID NO. 1;
(a2) A nucleotide sequence which encodes a protein of the same amino acid sequence as the nucleotide sequence of (a 1) but differs in sequence due to the degeneracy of the genetic code;
(a3) A nucleotide sequence which has more than or equal to 90% identity with the nucleotide sequence shown in (a 1) or (a 2) and codes for the same functional protein;
(a4) A nucleotide sequence complementary to any one of (a 1) to (a 3).
In a second aspect of the invention, a deep-seeding-resistant protein is provided, wherein the protein is obtained by encoding the deep-seeding-resistant gene.
In a third aspect of the present invention, it is also within the scope of the present invention to provide a recombinant expression vector, transgenic cell line, host bacterium or transgenic plant comprising the above-described deep-broadcast-resistant gene.
In a fourth aspect, the invention provides the use of the aforementioned deep-broadcast-resistant gene, deep-broadcast-resistant protein, recombinant expression vector containing the aforementioned deep-broadcast-resistant gene, transgenic cell line, host bacterium or transgenic plant in any one or more of the following:
(b1) Regulating and controlling the length of hypocotyls in plants;
(b2) Cultivating deep-sowing-resistant plants.
In a fifth aspect of the invention, there is provided a method of growing a deep-plant tolerant plant, the method comprising: the deep-sowing resistant gene is expressed in a target plant.
The beneficial technical effects of one or more of the technical schemes are as follows:
according to the technical scheme, the existing plant genetic engineering technology is utilized for the first time, the related gene ZmSRO e of the maize mesocotyl elongation is obtained through cloning, the gene is excessively expressed in the maize through a method mediated by agrobacterium tumefaciens, and comparison analysis proves that the mesocotyl of the transgenic plant is obviously elongated, the deep-sowing resistance is obviously improved, theoretical basis and practical guarantee are provided for cultivating the deep-sowing-resistant plant, and the method has a wide application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows the analysis of the expression level of ZmSRO e in example 1 according to the present invention after the dark and light treatment.
FIG. 2 is an identification of ZmSRO e transgenic maize at the gDNA and RNA level in example 4 of the present invention.
Wherein: (a) ZmSRO e transgenic maize is tested at the gDNA level; (b) ZmSRO e transgenic maize was tested at the RNA level.
FIG. 3 shows the phenotype of ZmSRO e transgenic maize in example 4 of the present invention under deep sowing.
Wherein: (a) the phenotype of ZmSRO e transgenic maize at 5cm deep sowing; (b) ZmSRO e phenotype of transgenic maize at 10cm deep sowing; (c) ZmSRO1e transgenic maize was deep-sown with 5cm hypocotyl phenotype; (d) ZmSRO1e transgenic corn is subjected to hypocotyl phenotype under deep sowing of 10 cm; (e) ZmSRO1e transgenic corn is subjected to deep sowing under 15cm to obtain a mesocotyl phenotype; (f) Carrying out statistics on the emergence rate of ZmSRO e transgenic corns under deep sowing; (g) ZmSRO1E transgenic corn is subjected to mesocotyl length statistics under deep sowing.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
In the present invention, a "nucleic acid molecule" or "nucleic acid sequence" is a linear fragment of single or double stranded DNA or RNA that can be isolated from any source. In the context of the present invention, preferably, the nucleic acid molecule is a DNA fragment. "nucleic acid molecule" is also known as a polynucleotide molecule.
"Plant" is any plant at any stage of development, in particular a seed plant; in particular, the plant is a monocot such as sweet sorghum, maize, wheat, rice, etc., with maize being most preferred.
The phrase "substantially identical" in the context of two nucleic acid or protein sequences refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 85%, more preferably 90%, even more preferably 95% and most preferably at least 99% nucleotide or amino acid residue identity, as determined using one of the following sequence comparison algorithms or visual inspection, when compared and aligned for maximum correspondence. Preferably, substantial identity exists over a region of the sequence that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences in at least about 150 residues are substantially identical. In a particularly preferred embodiment, the sequences are substantially identical throughout the length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences have substantially identical functions.
For sequence comparison, typically, a sequence is compared to the test sequence as a reference sequence. When using the sequence comparison algorithm, the detection and reference sequences are input into a computer, the coordinates of the subsequences are designated if necessary, and the parameters of the sequence algorithm program are designated. The sequence comparison algorithm will then calculate the percent sequence identity (identity) of the test sequence relative to the reference sequence based on the selected program parameters.
"Transformed/transgenic/recombinant" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the host genome or the nucleic acid molecule may be present as an extrachromosomal molecule. Such extrachromosomal molecules may be autonomously replicating. Transformed cells, tissues, or plants are understood to include not only the end product of the transformation process but also the transgenic progeny thereof. "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, such as a bacterium or plant, that does not contain a heterologous nucleic acid molecule.
The terms "polynucleotide", "polynucleotide molecule", "polynucleotide sequence", "coding sequence", and the like as used herein include single-or double-stranded DNA and RNA molecules, and may comprise one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences comprising exons and introns, chemically synthesized DNA and RNA sequences, and sense and corresponding antisense strands.
Methods of producing and manipulating the polynucleotide molecules and oligonucleotide molecules disclosed herein are known to those of skill in the art and can be accomplished according to the described recombinant techniques.
Once the desired nucleotide sequence has been transformed into a particular plant species, it can be propagated in that species or transferred into other varieties of the same species, including in particular commercial varieties, using conventional breeding techniques.
Preferably, the nucleotide sequences of the invention are expressed in transgenic plants, thereby causing biosynthesis of the corresponding spikelet proteins in the transgenic plants. In this way, transgenic plants with improved traits can be produced. In order to express the nucleotide sequences of the present invention in transgenic plants, the nucleotide sequences of the present invention may require modification and optimization. All organisms have a specific codon usage preference, as is known in the art, which may be altered to conform to plant preferences while maintaining the amino acids encoded by the nucleotide sequences of the invention.
At the same time, the nucleotide sequence can be optimized in this way for expression in any plant. It is recognized that all or any portion of the gene sequence may be optimized or synthesized. That is, synthetic or partially optimized sequences may also be utilized.
A wide variety of transformation vectors useful for plant transformation are known to those skilled in the art of plant transformation, and the nucleic acid molecules of the invention may be used in combination with any such vector. The choice of vector will depend on the preferred transformation technique used for transformation and the target plant species. For some target species, different antibiotic or herbicide selectable markers may be preferred. Selectable markers commonly used in transformation include the nptII gene, which confers resistance to kanamycin and related antibiotics, and the like. However, it should be noted that the selection of the selectable marker is not critical to the invention.
As described above, the genetic analysis of the deep-seeding resistance of most crops is mainly focused on the development of candidate deep-seeding resistance sites, and the functions and specific molecular mechanisms of deep-seeding resistance genes are rarely reported, so that the enhancement is needed.
In view of this, in an exemplary embodiment of the present invention, there is provided a deep-cast-resistant gene ZmSRO e having any one of the nucleotide sequences (a 1) to (a 4):
(a1) A nucleotide sequence shown as SEQ ID NO. 1;
(a2) A nucleotide sequence which encodes a protein of the same amino acid sequence as the nucleotide sequence of (a 1) but differs in sequence due to the degeneracy of the genetic code;
(a3) A nucleotide sequence having greater than or equal to 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence) identity to the nucleotide sequence set forth in (a 1) or (a 2) and encoding the same functional protein;
(a4) A nucleotide sequence complementary to any one of (a 1) to (a 3).
In still another embodiment of the present invention, there is provided a deep-seeding-resistant protein obtained by encoding the above deep-seeding-resistant gene.
The deep-seeding-resistant gene ZmSRO e and protein (polypeptide) of the invention can endow transgenic plants containing the gene with deep-seeding-resistant characters. Preferably, the deep-broadcast tolerant genes ZmSRO e and proteins of the invention have the ability to improve crop plant traits. The characteristics at least comprise obvious elongation of hypocotyl of the plant and obvious improvement of deep sowing resistance.
Therefore, in yet another embodiment of the present invention, it is also within the scope of the present invention to provide a recombinant expression vector, transgenic cell line, host bacterium or transgenic plant containing the above-mentioned deep-cast-resistant gene.
In one embodiment of the present invention, the recombinant expression vector is obtained by operably linking the above-described deep-cast-resistant gene to an expression vector, which is any one or more of a viral vector, a plasmid, a phagemid, a cosmid, or an artificial chromosome; specifically, the expression vector may be a maize over-expression vector pUNTF.
The transgenic cell line may be isolated, cultured, or preferably a part of a plant.
Wherein the host bacteria can be eukaryotic bacteria or prokaryotic bacteria.
The host bacterium may be agrobacterium, more specifically agrobacterium tumefaciens (EHA 105).
The transgenic plant may be a crop, particularly a monocot crop (e.g., sweet sorghum, maize, wheat, rice, etc.), and in one embodiment of the invention, the plant is maize. The transgenic plants of the invention also relate to transgenic seeds from said plants.
In yet another embodiment of the present invention, there is provided the use of the above-described deep-sowing-resistant gene, deep-sowing-resistant protein, recombinant expression vector containing the above-described deep-sowing-resistant gene, transgenic cell line, host bacterium or transgenic plant in any one or more of the following:
(b1) Regulating and controlling the length of hypocotyls in plants;
(b2) Cultivating deep-sowing-resistant plants.
Wherein the plant may be a crop, particularly a monocot crop (e.g., sweet sorghum, maize, wheat, rice, etc.), and in one embodiment of the invention the plant is maize.
In yet another embodiment of the present invention, there is provided a method of growing a deep-plant tolerant plant, the method comprising: the deep-sowing resistant gene is expressed in a target plant.
The plant may be a crop, particularly a monocot crop (e.g., sweet sorghum, maize, wheat, rice, etc.), and in one embodiment of the invention the plant is maize.
The expression can be over-expression, and in a specific embodiment of the invention, the deep-sowing-resistant gene is over-expressed in corn by a rhizobium mediated method, so that the mesocotyl of the transgenic corn is obviously elongated, and the deep-sowing-resistant capability is obviously improved.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. In the examples described below, materials, reagents, carriers, strains and the like used, unless otherwise specified, were all obtained commercially.
Cloning of example 1, zmSRO e
1.1 Extraction of corn Total RNA
① Placing the tissue material into a ceramic mortar, quickly freezing with liquid nitrogen and grinding into powder, and filling into a RNAASE FREE 2.0ml EP tube to 1/3 volume;
② 1ml of TRIzol extract of Invitrogen company is added into each tube, the sample is mixed by intense shaking, the sample is fully cracked, and the mixture is left at room temperature for 5min;
③ Adding 0.2ml chloroform (chloroform), mixing, and standing at room temperature for 10min;
④ Centrifuging at 12000rpm at 4deg.C for 15min;
⑤ The supernatant was taken into a new 1.5ml EP tube, added with equal volume of isopropanol (about 500. Mu.l), mixed well upside down and left at room temperature for 10 minutes;
⑥ Centrifuging the sample at 12000rpm for 10min at 4deg.C, and discarding supernatant;
⑦ Adding 1ml of 75% ethanol to wash the precipitate, centrifuging at 12000rpm for 10min at 4 ℃ and collecting the precipitate;
⑧ Repeating step ⑦ once;
⑨ Removing supernatant, precipitating RNA, air drying on sterile operation table for about 7-10min, adding RNase-free water with proper volume (30-50 μl) after the water in test tube is completely dried, and dissolving thoroughly (can be stored at-80deg.C for a long time);
⑩ 1% Agrose gel electrophoresis and ultraviolet spectrophotometry to detect RNA quality and concentration.
Note that: a) RNA quality and size were detected by 1% agrose gel electrophoresis. A1% agarose gel containing EB was prepared, 1. Mu.l of RNA was extracted, 3. Mu.l of RNase-free water was added, 1. Mu.l of loading buffer was added, and immediately after rapid mixing, electrophoresis was performed on a horse for 15min at 120V, and immediately after the electrophoresis was completed, the gel was taken out from the electrophoresis tank and observed. The RNA with better quality is three bands, and the bands are clear and tidy.
B) The concentration of RNA was measured by UV spectrophotometry. The ratio of RNA at OD 260/OD280 was determined to detect the purity of RNA, and the ratio at OD 260/OD280 should be between 1.8 and 2.0, indicating that RNA purity is high and there is no contamination of DNA and protein.
1.2CDNA reverse transcription
Reverse transcription kit was used: FASTKING CDNA first Strand Synthesis kit (Degenome) (KR 116) cDNA was synthesized as follows:
① The system for removing genomic DNA was prepared as follows, and was thoroughly mixed, centrifuged briefly, and then incubated at 42℃for 3min. And then placed on ice.
5×gDNA Buffer 2μl
Total RNA 50ng-2μl
RNAase-Free ddH 2 O was made up to 10. Mu.l
② Preparing a mixed solution Mix of a reverse transcription reaction system, adding the mixed solution Mix into a reaction solution in ①, and fully and uniformly mixing. Incubate at 42℃for 15min.
③ Incubating at 95deg.C for 3min, placing on ice, and preserving at low temperature to obtain cDNA
1.3 Cloning and sequencing of open reading frames
① Primer sequence: based on the sequencing results, gene upstream and downstream primers ZmSRO e-F and ZmSRO e-R were designed to amplify the open reading frames of the genes.
ZmSRO1e-F:ATGGATTTGTCATCCGAC(SEQ ID NO.2)
ZmSRO1e-R:CATCGTCGAAGTTTCTTG(SEQ ID NO.3)
② PCR reaction system (50 μl):
③ The PCR reaction procedure was:
④ The amplified fragment was recovered, ligated with pEASY-Blunt simple vector, and transformed into E.coli Trans 1T 1, and sequencing was completed by Qingdao Optimaceae.
1.4 Gene expression analysis
① Extraction of RNA
The seed of the maize inbred line B73 is normally germinated, is cultured in nutrient soil in the dark for 10 days, is treated for 0, 1, 6 and 12 hours under light, and the hypocotyl is taken to extract RNA.
B. Reverse Transcription (RT) to generate cDNA
Reverse transcription produces cDNA, as described above.
PCR reaction and electrophoresis
1. PCR was performed using cDNA as a template. The primers are as follows
ZmSRO1e-qF：5’-GCTATTCCTCAGCTATGGGATG-3’(SEQ IDNO.4)
ZmSRO1e-qR：5’-GCGTTGTTCCTCACGAATC-3’(SEQ ID NO.5)
PCR system:
PCR procedure:
95℃5min,25～30cycles 95℃20s,57℃60s,72℃60s；72℃7min。
And determining the cycle number of PCR according to the amplification condition of the internal reference action, and adjusting the addition amount of the cDNA template.
The results are shown in FIG. 1.
Example 2 construction of plant expression vectors (Ubi promoter)
1.1 Construction of Ubi promoter plant expression vectors
The plant expression vector pUNTF is utilized, bamHI and SacI are selected to respectively carry out double enzyme digestion on pUNTF and pEASY-Blunt simple vector containing target genes, large vector fragments and small target gene fragments are respectively recovered, the large vector fragments and the small target gene fragments are connected by T 4 DNA ligase, then competent cells of escherichia coli Trans 1T 1 are transformed, and the plant expression vector with the target genes is obtained after identification.
① The plasmid pUNTF is empty and BamHI and SacI of pEASY-Blunt simple vector containing target gene are digested.
Extracting pUNTF empty vector and pEASY-blue simple plasmid by alkaline hydrolysis, and respectively taking 4 mug of enzyme digestion, wherein the enzyme digestion system is as follows:
and (3) performing enzyme digestion for 1 hour in a water bath with constant temperature of 37 ℃. After double digestion, the digested product was subjected to 1% agarose gel electrophoresis using 1×TAE as running buffer. A large fragment of the vector of pUNTF kb and a band of the gene of interest of about 1.3kb in pEASY-T1 were excised under a UV transilluminator with a clean blade, and the band was recovered.
② The molar ratio of ZmSRO e fragment to carrier fragment after recovery was 5:1 are connected for 15min at 25 ℃.
③ The ligation product was transformed into competent cells of E.coli Trans 1T 1 by heat shock, and the transformant was cultured on LB solid plates containing Kan 50. Mu.g/ml for about 16 hours at 37 ℃.
④ Identification of recombinants
A) PCR verification of plasmids
Single colonies were picked and inoculated in 5ml LB liquid medium containing Kan, shake-cultured overnight at 37℃and plasmids were extracted by alkaline denaturation, PCR amplification was performed using on-vector primers UBF (upstream primer) and UTA (downstream primer), the primers were as follows:
UBF:5’-GCCCTGCCTTCATACGCT-3’(SEQ ID NO.6)
UTA:5’-GCGGGACTCTAATCATAAA-3’(SEQ ID NO.7)
The system is as follows:
The PCR conditions were as follows:
The PCR products were identified by electrophoresis on a 1.0% agarose gel.
B) Plasmid enzyme digestion identification
The plasmid was digested with BamHI and SacI in the same manner as described above. 1.0% agarose gel electrophoresis, detecting whether the fragment with expected molecular weight is contained, and verifying the correct construction of the vector.
Example 3 preparation and transformation of Agrobacterium competence
1.1 Preparation of Agrobacterium EHA105 competence
① Single colonies of Agrobacterium tumefaciens were picked from YEP plates (containing 50. Mu.g/ml rifampicin) and inoculated at 50. Mu.g/ml
The medium was incubated overnight at 28℃at 200rpm/min in YEP liquid medium.
② 2Ml of overnight broth was inoculated into 50ml of YEP liquid medium containing the same antibiotic and cultured under the same conditions to OD 600 of 0.5.
③ The bacterial liquid is subjected to ice bath for 30min,4 ℃ and centrifugation at 5000rpm for 10min, and bacterial cells are collected.
④ The cells were resuspended in 10ml of 0.15mol/L NaCl in an ice bath, and collected by centrifugation.
⑤ Then, the solution is suspended in 1ml of 20mmol/L CaCl 2 solution precooled by ice, bacterial liquid is split into 1.5mlEppendorf tubes by 200 mu L/tube, frozen in liquid nitrogen for 1min at-70 ℃ for standby.
1.2 Freeze thawing method for transforming Agrobacterium tumefaciens EHA105
① The competent cells of Agrobacterium were thawed at room temperature, 1. Mu.g of expression vector plasmid DNA was added, and after mixing well, the ice bath was performed for 5min.
② Quick freezing with liquid nitrogen for 5min, rapidly transferring to 37deg.C, maintaining the temperature for 5min, and ice-bathing for 5min again.
③ YEP 800. Mu.l without antibiotic was added and shake cultured at 28℃for 3 hours.
④ The cells were collected by centrifugation at 7000rpm for 30s and plated on YEP plates containing 50. Mu.g/ml rifampicin, 50. Mu.g/ml Kan, and cultured in inverted dark at 28℃for 2-3 days.
1.3 PCR identification of thallus
Single colonies were picked and PCR amplified using UBF (upstream primer) and UTA (downstream primer), the primers were as follows:
UBF:5’-GCCCTGCCTTCATACGCT-3’(SEQ ID NO.8)
UTA:5’-GCGGGACTCTAATCATAAA-3’(SEQ ID NO.9)
The system is as follows:
The PCR conditions were as follows:
The PCR products were identified by electrophoresis on a 1.0% agarose gel.
Example 4 transgene functional verification-maize transformation functional verification
1.1 Maize transformation
① One day before transformation, 2ml of activated Agrobacterium EHA105 was taken and added to 200ml of YEP medium containing the corresponding antibiotic and incubated overnight to OD 600 = 1.0-1.2.
② The cells were collected by centrifugation and resuspended in the counterstain solution to OD 600 =0.8.
③ Taking young ears of a maize inbred line Z32 pollinated for 8-15 days, separating immature maize embryos into a centrifuge tube containing LS-inf culture medium, swirling for 5s at 2500rpm, and removing the culture medium; carrying out water bath heat treatment at 46 ℃ for 3min and ice water bath for 1min; 2ml LS-inf medium was added and centrifuged at 20000g for 10min at 4℃and the medium was removed.
④ 1Ml of the infested solution was added to a centrifuge tube containing the immature embryos, vortexed at 2500rpm for 30s, left at room temperature for 5min, and 0.7ml of the suspension was removed.
⑤ Transferring the immature embryo onto a solid LS-AS medium, and co-culturing for 7 days at 25 ℃ under dark conditions; transferring the embryo to LSD1.5A screening culture medium, and culturing at 25deg.C in dark for 10 days; embryos are transferred to LSD1.5B selection medium and incubated for 21 days at 25℃in the dark.
⑥ Cutting type I callus, transferring to LSD1.5B screening culture medium, culturing at 25deg.C in dark for 21 days, and amplifying type I callus to obtain transgenic callus.
⑦ The amplified type I calli were excised and transferred to LSZ medium and incubated at 25℃for 14 days under continuous light.
⑧ Regenerated shoots were transferred to LSZ medium and incubated at 25℃for 14 days under continuous light.
⑨ Planting the regenerated transgenic plant into soil for 3-4 months, and harvesting offspring seeds.
⑩ The offspring seeds are grown in the soil for 10 days after germination, leaf tissues are taken, DNA and RNA are extracted, and the transgenic plants are detected by PCR and qRT-PCR methods, and the result is shown in figure 2.
1.2 Functional verification of transgenic maize
① Uniformly mixing the sieved soil with clear water, keeping the humidity of the soil consistent, and putting the soil into a plastic flowerpot (with the length of 100cm, the width of 60cm and the height of 35 cm) to enable the soil height to reach 10cm.
② The seeds of transgenic corn with the same size as that of the control line Z31 are selected, 20 seeds of each line are uniformly sown on the surface of soil, then 20cm, 10cm and 5cm of soil are covered, and the length of hypocotyls and the emergence rate are observed and counted after 10 days, and the result is shown in figure 3.
It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A deep-cast-resistant gene ZmSRO e, characterized in that the deep-cast-resistant gene has the nucleotide sequence of any one of (a 1) - (a 4):
(a1) A nucleotide sequence shown as SEQ ID NO. 1;
(a2) A nucleotide sequence which encodes a protein of the same amino acid sequence as the nucleotide sequence of (a 1) but differs in sequence due to the degeneracy of the genetic code;
(a3) A nucleotide sequence which has more than or equal to 90% identity with the nucleotide sequence shown in (a 1) or (a 2) and codes for the same functional protein;
(a4) A nucleotide sequence complementary to any one of (a 1) to (a 3).
2. A deep-seeding-resistant protein, wherein the protein is encoded by the deep-seeding-resistant gene of claim 1.
3. A recombinant expression vector, transgenic cell line, host bacterium or transgenic plant comprising the deep-broadcast-resistant gene of claim 1.
4. The recombinant expression vector, transgenic cell line, host bacterium, or transgenic plant of claim 3, wherein the recombinant expression vector is obtained by operably linking the deep-broadcast tolerant gene of claim 1 to an expression vector that is any one or more of a viral vector, a plasmid, a phagemid, a cosmid, or an artificial chromosome; further, the expression vector is a maize over-expression vector pUNTF.
5. The recombinant expression vector, transgenic cell line, host bacterium, or transgenic plant of claim 3, wherein the host bacterium is a eukaryotic fungus or a prokaryotic fungus;
further, the host bacterium is agrobacterium, more specifically, agrobacterium tumefaciens (EHA 105).
6. The recombinant expression vector, transgenic cell line, host bacterium, or transgenic plant of claim 3, wherein the transgenic plant is a crop, particularly a monocot crop, more particularly the plant is maize.
7. Use of the deep-broadcast-resistant gene of claim 1, the deep-broadcast-resistant protein of claim 2, a recombinant expression vector comprising the deep-broadcast-resistant gene of claim 1, a transgenic cell line, a host bacterium or a transgenic plant in any one or more of the following:
(b1) Regulating and controlling the length of hypocotyls in plants;
(b2) Cultivating deep-sowing-resistant plants.
8. The use according to claim 7, wherein the plant is a crop, in particular a monocot crop, more in particular the plant is maize.
9. A method of growing a deep-plant tolerant plant, the method comprising: allowing the deep-seeding-resistant gene of claim 1 to be expressed in a target plant.
10. The method of claim 9, wherein the plant is maize;
furthermore, the expression is over-expression, and specifically, the deep-sowing-resistant gene is over-expressed in corn by a rhizobium mediated method.
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