CN115851755A - Meng nong hybrid wheatgrass tillering angle regulation factor and application thereof - Google Patents
Meng nong hybrid wheatgrass tillering angle regulation factor and application thereof Download PDFInfo
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Abstract
The invention discloses an application of a Mongolian hybrid wheatgrass tillering angle regulation factor, belonging to the technical field of plant genetic engineering. The invention uses Mongolian hybrid agropyron cristatum tillering node RNA as a material, adopts a method of ORF full-length cloning, and obtains a full-length sequence of Meng Nong hybrid agropyron cristatum tillering angle regulating factor LAZY1 or WOX gene. Constructing a plant over-expression vector pCAMBIA1300-UBI, transferring the pCAMBIA1300-UBI into receptor material rice by using an agrobacterium-mediated method, and expressing a transgenic rice plant of LAZY1 in an over-expression way to show a phenomenon that a tillering angle is increased. Therefore, the regulatory gene provided by the invention can participate in the regulation and control of the tillering angle of plants, can improve the plant type of the plants in plant genetic engineering and accelerate the breeding process of high-yield compact plant varieties.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of a Mongolian hybrid agropyron tillering angle regulation factor WOX11 gene and an LAZY1 gene.
Background
The information disclosed in this background section is only for enhancement of 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 that is already known to a person of ordinary skill in the art.
The regulation and control of the tillering angle of the plant is a complex biological process, and has important significance in the aspect of constructing the ideal plant type of the plant under the common regulation and control of environmental factors such as temperature, illumination, nutrients and non-environmental factors such as hormones, genetic genes and the like. In gramineous plants, such as cereal crops like rice, the number of tillers and the size of tillering angles have a direct relationship with the yield of the crop.
Transcription Factors (TF) are a class of trans-acting factors that regulate gene expression. Often, a single transcription factor can regulate the expression of multiple functional genes simultaneously relative to the functional gene. Therefore, it is very significant to analyze and identify the gene function of the transcription factor. Through a transgenic technology, a transcription factor transformation plant capable of simultaneously regulating and controlling a plurality of functional genes is expected to realize the rapid cultivation of a new variety with excellent comprehensive characters.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide the application of the regulating factor for the tillering angle of the Mengnong hybrid agro-iceberg.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the Meng Nong hybrid wheatgrass tillering angle regulating factor is WOX11 gene or LAZY1 gene, and the nucleotide sequence of the regulating factor is shown as SEQ _ NO.1 and SEQ _ NO. 2.
A plant over-expression vector containing the Mongolian hybrid agropyron cristatum tillering angle regulating and controlling factor.
Furthermore, the plant overexpression vector of the Meng Nong hybrid wheatgrass tillering angle regulating factor is pCAMBIA1300-UBI.
An application of the said regulating factor for tillering angle of Mongolian hybrid agropyron plant in breeding plant.
Further, the application comprises the following steps:
1) Constructing a plant over-expression vector of Meng Nong hybrid agropyron cristatum tillering angle regulating and controlling factor;
2) Transforming the constructed plant over-expression vector of the Meng Nong hybrid agropyron cristatum tillering angle regulating factor into plants or plant cells;
3) And culturing and screening to obtain transgenic plants with different tillering angles.
Furthermore, hygromycin is used as a screening marker of the transgenic plant in the vector pCAMBIA1300-UBI, or kanamycin is used for screening the transgenic plant.
Further, the plant is a gramineous plant, and further, the plant is rice.
Further, the application comprises the improvement of plant type of the plant, and further the regulation and control of the tillering angle of the plant.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses an application of a Mongolian hybrid wheatgrass tillering angle regulation factor, belonging to the technical field of plant genetic engineering. The invention takes Mongolian hybrid agropyron cristatum tillering node RNA as a material, adopts an ORF full-length cloning method to obtain the full-length sequences of Meng Nong hybrid agropyron cristatum tillering angle regulating factor WOX11 gene and LAZY1 gene, as shown in SEQ _ WOX11 and SEQ _ LAZY 1. Constructing a plant over-expression vector pCAMBIA1300-UBI, transferring the pCAMBIA1300-UBI into receptor material rice by using an agrobacterium-mediated method, and expressing a phenomenon that a transgenic rice plant of WOX11 or LAZY1 over-expresses a tillering angle is increased. Therefore, the regulatory gene provided by the invention can participate in the regulation and control of the tillering angle of the plant, can improve the plant type of the plant and accelerate the breeding process of high-yield compact plant varieties in plant genetic engineering.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a diagram of the structure of plant expression vector pCAMBIA1300-UBI;
FIG. 2 is a table diagram of tillering angles of transgenic rice overexpressing LAZY1 gene;
FIG. 3 is a phenotype diagram of tillering angle of transgenic rice over-expressing WOX11 gene.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.
The material used in the application is Mongolian hybrid wheatgrass tillering node which is collected in a greenhouse of inner Mongolian agriculture university in 2019 and 12 months.
Example 1: cloning of the full length ORF of the WOX11 Gene
Based on Meng Nong hybrid agropyron cristatum tillering node transcriptome sequencing result, the Open Reading Frame (ORF) full-length sequence of WOX11 gene is designed and amplified by specific primer.
(1) Amplification of ORF sequence: based on Meng Nong hybrid wheatgrass tillering node transcriptome sequencing result, designing a specific primer to amplify the full-length sequence of an Open Reading Frame (ORF) of a WOX11 gene, wherein the ORF specific primer comprises: WOX11 forward primer: 5'-TAGCTCGTCTGCTAGCTCG-3' (SEQ _ No. 3) and WOX11 reverse primer: 5'-CATTAATTTGACCTCGCG-3' (SEQ _ NO. 4). The RNA extraction of the tillering node of the Mongolian hybrid wheatgrass is carried out by adopting a Trizol method, and the First chain of cDNA is synthesized by a RevertAID First Strand cDNA Synthesis Kit of Fermentas company. The ORF sequence of WOX11 gene was amplified using KOD FXneo (1U/ul) enzyme, PCR reaction amplification program: 5min at 98 ℃; 10s at 98 ℃; 30s at 55 ℃;72 ℃ for 2min (35 cycles); 10min at 72 ℃. Carrying out electrophoretic separation on the PCR product by using 1% agarose gel, taking pictures and cutting gel by a gel imaging system, carrying out amplification fragment recovery by using a DNA agarose gel recovery kit, carrying out T4 cloning vector connection and transformation of escherichia coli competent cells on a fresh recovery product, and finally sending positive bacteria liquid detected by bacteria liquid PCR to a biological engineering limited company for sequencing.
(2) And (3) according to a sequencing peak diagram, carrying out sequence splicing, removing a vector sequence, then carrying out alignment on NCBI, and further determining a final result, wherein the final result is shown as SEQ _ NO. 1.
Example 2: construction and genetic transformation of WOX11 gene plant expression vector
Construction of an overexpression vector for the WOX11 gene. Specific PCR primers are used, cDNA is used as a template, PCR amplification is carried out, and the WOX11 gene open reading frame sequence is constructed into a vector pCAMBIA1300-UBI. Selecting positive clones from a screening culture plate to carry out PCR detection and sequencing verification, confirming that the construction of an overexpression vector is successful after the PCR detection and the sequencing verification, and naming the overexpression vector as pCAMBIA1300-UBI (figure 1), wherein the gene is positioned behind a promoter UBI, and the pCAMBIA1300 can be efficiently expressed in a receptor plant under the drive of the promoter UBI.
The specific method for constructing the vector comprises the following steps:
(1) amplification of a Gene fragment of interest
Designing specific primer according to target gene sequence
Using E.coli DH5 alpha (pGEM-T-easy-target gene) as a template, establishing a PCR reaction system as follows to amplify a pre-expressed target gene fragment;
Pre mixtaq 25uL
upstream primer 1uL
Downstream primer 1uL
pGEM-T-easy-target Gene 1uL
22uL of water
Total volume 50uL
The PCR reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 60 s at 72 ℃ for 35 cycles; extension at 72 ℃ for 10min.
The PCR product was electrophoresed on 1% agarose gel, and the desired fragment was recovered using E.N.Z.A DNA gel recovery kit, and cleaved with KpnI/BamHI in duplicate as follows:
10×Buffer 5uL
PCR product 35uL
5uL of water
KpnI 2.5uL
BamHI 2.5uL
Total volume 50uL
After 3h is incubated at 37 ℃, 1% agarose gel electrophoresis is carried out, and the target fragment after enzyme digestion is recovered by a DNA gel recovery kit.
(2) Enzyme digestion preparation of overexpression vectors
Selecting a fresh-cultured colony of DH5a (pCAMBIA 1300-UBI), inoculating the colony to 10 mL LB liquid medium (containing 50 ug/mL kalina), and performing shaking culture at 37 ℃ for 12 h; extracting plasmid pCAMBIA1300-UBI by using a small-amount plasmid preparation kit of E.Z.N.A.; the double digestion was carried out with KpnI/BamHI, and the reaction system is shown in tables 4-3: after incubation at 37 ℃ for 3h, the samples were subjected to 1% agarose gel electrophoresis, 100V electrophoresis 1 h. And (5) recovering the plasmid after enzyme digestion by using an E.Z.N.A.DNA gel recovery kit.
10×Buffer 5uL
pCAMBIA1300-UBI vector 35uL
5uL of water
KpnI 2.5uL
BamHI 2.5uL
Total volume 50uL
(3) Connection of enzyme-digested vector and enzyme-digested target gene
The following ligation reactions were set up, 16 ℃ overnight ligation.
10 XDNA ligation buffer 1uL
After enzyme digestion, pCAMBIA1300-UBI vector 2uL
After enzyme digestion, the target gene fragment is 6uL
T4 DNA ligase 1uL
Total volume 10uL
(4) Transformation of the ligation products into E.coli DH 5. Alpha. By the CaCl2 method
(5) PCR identification of transformed clones
Several colonies were picked and inoculated into 10 mL of LB medium (containing 50. Mu.g/mL of kanamycin) and cultured overnight with shaking at 37 ℃ to select positive clones by the following PCR reaction.
Pre mixtaq 25uL
Upstream primer 1uL
Downstream primer 1uL
Template 1uL
22uL of water
Total volume 50uL
PCR procedure: the procedure of (1) is followed.
Taking 10 mu L of PCR reaction product, carrying out 1% agarose gel electrophoresis, and observing the result under an ultraviolet detector.
The culture of the clone identified as positive by PCR reaction is sent to a sequencing company for sequence determination of the recombinant plasmid, and whether the reading frame of the exogenous gene in the recombinant plasmid is changed or not is analyzed. The sequencing primer is
UBI (forward universal primer): 5'-ACACCCTCTTTCCCCAAC-3' as shown in SEQ _ NO. 5.
E9 TER R (reverse universal primer): 5'-GCAATGAAACTGATGCATTG-3' as shown in SEQ _ NO. 6.
The constructed pCAMBIA1300-UBI over-expression vector is transferred into an agrobacterium EHA105 strain by a liquid nitrogen freeze thawing method, and the gene is transferred into rice by an agrobacterium-mediated genetic transformation method. Transgenic rice plants overexpressing WOX11 gene showed a phenomenon of an increase in tillering angle (FIG. 3). The WOX11 gene is a key regulating factor of the rice tillering angle and has important application value in the research of gramineous plant genetic engineering.
Example 3: cloning of the full-Length ORF of the LAZY1 Gene
Based on the Meng Nong hybrid agropyron cristatum tillering node transcriptome sequencing result, the full-length sequence of the Open Reading Frame (ORF) of the LAZY1 gene is designed and amplified by specific primers.
(1) Amplification of ORF sequence: based on Meng Nong hybrid wheatgrass tillering node transcriptome sequencing result, designing specific primer to amplify the full-length sequence of Open Reading Frame (ORF) of LAZY1 gene, wherein the ORF specific primer comprises: LAZY1 forward primer: 5'-GCCTCTTCAAGATGACGC-3' (SEQ. NO. 7) and LAZY1 reverse primer: 5'-CTTCCAGTTCATCCGTAC-3' (SEQ _ NO. 8). The RNA extraction of the tillering node of the Mongolian hybrid wheatgrass is carried out by adopting a Trizol method, and the First chain of cDNA is synthesized by a RevertAID First Strand cDNA Synthesis Kit of Fermentas company. The ORF sequence of the LAZY1 gene was amplified using KOD FXneo (1U/ul) enzyme, PCR reaction amplification program: 5min at 98 ℃; 10s at 98 ℃; 30s at 55 ℃;72 ℃ for 2min (35 cycles); 10min at 72 ℃. And (3) carrying out electrophoretic separation on the PCR product by using 1% agarose gel, carrying out photographing and gel cutting by using a gel imaging system, carrying out amplification fragment recovery by using a DNA agarose gel recovery kit, carrying out T4 cloning vector connection and transformation of escherichia coli competent cells on a fresh recovery product, and finally sending positive bacteria liquid detected by bacteria liquid PCR to a biological engineering company Limited for sequencing.
(2) And (3) according to a sequencing peak diagram, carrying out sequence splicing, removing a vector sequence, then carrying out alignment on NCBI, and further determining a final result, wherein the final result is shown as SEQ _ NO. 2.
Example 4: construction and genetic transformation of LAZY1 gene plant expression vector
Constructing an overexpression vector of the LAZY1 gene. And (3) carrying out PCR amplification by using a specific PCR primer and cDNA as a template, and constructing the LAZY1 gene open reading frame sequence into a vector pCAMBIA1300-UBI. Selecting positive clones from a screening culture plate to carry out PCR detection and sequencing verification, confirming that the construction of an over-expression vector is successful through the PCR detection and sequencing verification, and naming the gene as pCAMBIA1300-UBI (figure 1), wherein the gene is positioned behind a promoter UBI, and the pCAMBIA1300 can be efficiently expressed in a receptor plant under the drive of the promoter UBI.
The specific method for constructing the vector comprises the following steps:
(1) amplification of a Gene fragment of interest
Designing specific primer according to target gene sequence
Using E.coli DH5 alpha (pGEM-T-easy-target gene) as a template, establishing a PCR reaction system as follows to amplify a pre-expressed target gene fragment;
Pre mixtaq 25uL
upstream primer 1uL
Downstream primer 1uL
pGEM-T-easy-target Gene 1uL
22uL of water
Total volume 50uL
The PCR reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 60 s at 72 ℃ for 35 cycles; extension for 10min at 72 ℃.
The PCR product was electrophoresed through 1% agarose gel, the desired fragment was recovered using E.N.Z.A DNA gel recovery kit, and double digestion was carried out with KpnI/BamHI, as follows:
10×Buffer 5uL
PCR product 35uL
5uL of water
KpnI 2.5uL
BamHI 2.5uL
Total volume 50uL
After 3h is incubated at 37 ℃, 1% agarose gel electrophoresis is carried out, and the target fragment after enzyme digestion is recovered by a DNA gel recovery kit.
(2) Enzyme digestion preparation of overexpression vectors
Selecting a fresh-cultured colony of DH5a (pCAMBIA 1300-UBI), inoculating the colony to 10 mL LB liquid medium (containing 50 ug/mL kalina), and performing shaking culture at 37 ℃ for 12 h; extracting plasmid pCAMBIA1300-UBI by using a small amount of plasmid preparation kit of E.Z.N.A.; double digestion was carried out with KpnI/BamHI, and the reaction system is shown in tables 4-3: after incubation at 37 ℃ for 3h, the samples were subjected to 1% agarose gel electrophoresis, 100V electrophoresis 1 h. And (5) recovering the plasmid after enzyme digestion by using an E.Z.N.A.DNA gel recovery kit.
10×Buffer 5uL
pCAMBIA1300-UBI vector 35uL
5uL of water
KpnI 2.5uL
BamHI 2.5uL
Total volume 50uL
(3) Connection of enzyme-digested vector and enzyme-digested target gene
The following ligation reactions were set up, 16 ℃ overnight.
10 XDNA ligation buffer 1uL
After enzyme digestion, pCAMBIA1300-UBI vector 2uL
After enzyme digestion, the target gene fragment is 6uL
T4 DNA ligase 1uL
Total volume 10uL
(4) Transformation of the ligation products into E.coli DH 5. Alpha. By the CaCl2 method
(5) PCR identification of transformed clones
Several colonies were picked and inoculated into 10 mL of LB medium (containing 50. Mu.g/mL of kanamycin) and cultured overnight with shaking at 37 ℃ to select positive clones by the following PCR reaction.
Pre mixtaq 25uL
Upstream primer 1uL
Downstream primer 1uL
Template 1uL
22uL of water
Total volume 50uL
PCR procedure: the procedure of (1) is followed.
Taking 10 mu L of PCR reaction product, carrying out 1% agarose gel electrophoresis, and observing the result under an ultraviolet detector.
The culture of the clone identified as positive by PCR reaction is sent to a sequencing company for sequence determination of the recombinant plasmid, and whether the reading frame of the exogenous gene in the recombinant plasmid is changed or not is analyzed. The sequencing primer is
UBI (Universal primer in Forward direction) 5'-ACACCCTCTTTCCCCAAC-3'
E9 TER R (reverse Universal primer): 5'-GCAATGAAACTGATGCATTG-3'
The constructed pCAMBIA1300-UBI over-expression vector is transferred into an agrobacterium EHA105 strain by a liquid nitrogen freeze thawing method, and the gene is transferred into rice by an agrobacterium-mediated genetic transformation method. Transgenic rice plants overexpressing the LAZY1 gene showed a phenomenon of an increase in tillering angle (FIG. 2). The LAZY1 gene is a key regulating factor of rice tillering angle and has important application value in gramineous plant genetic engineering research.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. The Meng nong hybrid wheatgrass tillering angle regulating factor is characterized in that the Meng Nong hybrid wheatgrass tillering angle regulating factor is WOX11 gene or LAZY1 gene, and the nucleotide sequences of the regulating factor are shown as SEQ _ NO.1 and SEQ _ NO. 2.
2. A plant over-expression vector containing Meng Nong hybrid wheatgrass tillering angle regulating factor of claim 1.
3. The plant overexpression vector according to claim 2, wherein the plant overexpression vector is pCAMBIA1300-UBI.
4. The plant overexpression vector according to claim 2, wherein the plant overexpression vector uses hygromycin as a selection marker for transgenic plants or screens transgenic plants with kanamycin.
5. The use of Meng Nong hybrid agropyron cristatum tillering angle regulatory factor of claim 1 in plant breeding.
6. The use according to claim 5, characterized in that it comprises the following steps:
1) Constructing a plant over-expression vector of Meng Nong hybrid agropyron cristatum tillering angle regulating and controlling factor;
2) Transforming the constructed plant overexpression vector of the Meng Nong hybrid agropyron cristatum tillering angle regulating factor into plants or plant cells;
3) And culturing and screening to obtain transgenic plants with different tillering angles.
7. Use according to claim 5, wherein the plant is a graminaceous plant.
8. The use of claim 7, wherein the plant is rice.
9. Use according to claim 5, wherein the use comprises modifying the plant type of the plant.
10. The use according to claim 9, characterized in that the use is in particular the regulation of the plant tillering angle.
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