CN115947812A - Chrysanthemum CmULT1 gene and application thereof - Google Patents
Chrysanthemum CmULT1 gene and application thereof Download PDFInfo
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Abstract
The invention discloses a chrysanthemum CmULT1 gene and application thereof, wherein the amino acid sequence of the chrysanthemum CmULT1 is shown as SEQ ID NO. 2. According to the invention, the chrysanthemum CmULT1 gene is expressed in the plant, so that the angles of plant branches and pods are obviously reduced, and the plant type is compact. Therefore, the scheme of the invention provides a new effective method for carrying out genetic improvement on the plants such as rape and wheat, cultivating ideal plant type varieties, improving planting density and promoting mechanical harvesting application.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a chrysanthemum CmULT1 gene and application thereof.
Background
The ideal plant type (ideotype) generally refers to a plant type favorable to plant photosynthesis, growth and development and grain yield traits, and the breeding concept was first proposed by Donald in 1968. In crop production, the ideal plant type is a key factor for improving yield. The method is an urgent research target at present for creating an ideal plant type material with both form and function, and applying the ideal plant type material to breeding to break the situation that the unit yield of crops cannot wander. In rape production, if leaves are erect in the seedling stage, branches and silique included angles in the adult stage are small, so that a single plant occupies less space, the high-density planting is suitable, the light energy utilization rate of a group is improved, the biological yield is increased, the economic coefficient is improved, and the high yield is obtained. Meanwhile, the compact rape has the advantages that the branch parts are properly improved under the condition of close planting, the effective branch number and the branch length are slightly reduced, the maturity period of the rape is relatively concentrated, plants are not crossed, the loss during collection is reduced, and the compact rape is more beneficial to mechanized harvesting. The angle between branches and pedicels of garden and horticultural plants or between carpopodium and inflorescence axim is also of great importance to the morphological characteristics of plant inflorescences, which not only affects the beauty of ornamental plants, but also may affect the yield of plants. Therefore, it is very important to find and use functional genes for controlling the angle of plant branches or pods.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the chrysanthemum CmULT1 protein which can effectively control the plant branching and the angle of the horn fruit.
The invention also provides a nucleic acid molecule for coding the chrysanthemum CmULT1 protein.
The invention also provides a biological material related to the nucleic acid molecule.
The invention also provides a primer for amplifying the nucleic acid molecule.
The invention also provides the application of the chrysanthemum CmULT1 protein, a nucleic acid molecule for coding the chrysanthemum CmULT1 protein, a biological material and a primer.
The invention also provides a method for controlling the branching angle of the plant.
The invention also provides a method for controlling the angle of the silique.
In one aspect of the invention, a chrysanthemum CmULT1 protein is provided, which is a protein of a) or b) or c) or d) as follows:
a) Protein with amino acid sequence shown in SEQ ID NO. 2;
b) Fusion protein obtained by connecting N end and/or C end of protein with amino acid sequence shown as SEQ ID NO.2 with tag;
c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO. 2;
d) Protein with 75% or more than 75% homology with amino acid sequence shown as SEQ ID NO.2 and with the same function.
In a second aspect of the invention, a nucleic acid molecule encoding the above chrysanthemum CmULT1 protein is presented.
In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID No. 1.
In a third aspect of the present invention, there is provided a biomaterial related to the above-mentioned nucleic acid molecule, which is any one of the following 1) to 7):
1) An expression cassette comprising the nucleic acid molecule;
2) A recombinant vector comprising the nucleic acid molecule;
3) A recombinant vector comprising 1) the expression cassette;
4) A recombinant microorganism containing the above-mentioned nucleic acid molecule;
5) A recombinant microorganism comprising 1) said expression cassette;
6) A recombinant microorganism containing 2) the recombinant vector;
7) A recombinant microorganism comprising 3) the recombinant vector.
In a fourth aspect of the invention, a primer for amplifying the above-mentioned nucleic acid molecule is provided.
In some embodiments of the invention, the primer comprises an upstream primer sequence and a downstream primer sequence, wherein the upstream primer nucleotide sequence is shown as SEQ ID NO.3, and the downstream primer sequence is shown as SEQ ID NO. 4.
In the fifth aspect of the invention, the application of the chrysanthemum CmULT1 protein, the nucleic acid molecule for coding the chrysanthemum CmULT1 protein, the biological material and the primer is provided, and the application is the application in plant breeding.
In some embodiments of the invention, the use is in the preparation of a product for plant assisted plant breeding.
In some embodiments of the invention, the use is in modulating the branching angle of a plant.
In some embodiments of the invention, the use is in the modulation of the silique angle of a plant.
In some embodiments of the invention, the use is in the preparation of a product for increasing seed yield in a plant.
In a sixth aspect of the present invention, a method for controlling a plant branching angle is provided, the method comprising the steps of: transferring the nucleic acid molecules and the biological materials into a receptor plant.
In some embodiments of the invention, the plant comprises a crucifer and a gramineae.
In some embodiments of the invention, the cruciferous plant comprises canola, radish, chinese cabbage, violet or arabidopsis thaliana.
In some embodiments of the invention, the gramineae comprises rice, wheat, maize, millet, sugarcane, sorghum, green bristlegrass or rye. In some embodiments of the invention, the transfer is performed by ligating a nucleic acid molecule encoding the ULT1 protein described above to a vector.
In some embodiments of the invention, the vector comprises a pBWA (V) BS-ccdB plasmid.
In a seventh aspect of the present invention, a method of controlling the angle of a plant's silique is presented, the method comprising the steps of: transferring the nucleic acid molecule and the biological material into a receptor plant.
In some embodiments of the invention, the plant comprises a crucifer and a gramineae.
In some embodiments of the invention, the cruciferous plant comprises canola, radish, chinese cabbage, violet or arabidopsis thaliana.
In some embodiments of the invention, the gramineae comprises rice, wheat, maize, millet, sugarcane, sorghum, green bristlegrass or rye.
According to the embodiment of the invention, at least the following beneficial effects are achieved: according to the invention, the CmULT1 gene of chrysanthemum is expressed in plants, so that the angles of plant branches and siliques are obviously reduced, and the plant type is compact. Therefore, the scheme of the invention provides a new effective method for genetically improving the plants such as rape and wheat, cultivating ideal plant types, improving planting density and promoting mechanical harvesting application.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is an electrophoretogram of PCR identification products of transgenic Arabidopsis thaliana in a test example of the present invention; wherein ULT1 is pBWA (V) BS-OECmULT1 control, OE is transgenic Arabidopsis;
FIG. 2 is a graph showing the branching phenotype results of transgenic Arabidopsis plants and wild-type control plants in the test examples of the present invention; wherein Ler is a control group, and OE-CmULT1 is transgenic Arabidopsis;
FIG. 3 is a graph of the silique angle phenotype results of transgenic Arabidopsis and wild-type control plants in the test examples of the present invention, where Ler is the control and OE-CmULT1 is transgenic Arabidopsis;
FIG. 4 is a chart of the statistical results of the silique angles of transgenic Arabidopsis thaliana and wild type control plants in the test example of the present invention, wherein Ler is the control and OE-CmULT1 is transgenic Arabidopsis thaliana.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1
The embodiment provides a chrysanthemum CmULT1 protein, and the amino acid sequence is as follows:
MADGTMMFSEEEVKEMCGFKFCGDGHVEVTCGCTSYCYGDAVGILKVFVNGD
LEITCDCTPGCQEDKLTPAAFEKHSGRETARKWKNNIWVIVDGDKVPLYKTALLKYY
NQALTKTSNKSQSGQLVHRDEFVKCTKCDKLRRFHLHTSEECRLYHDASRDNDWKC
SDMPYEKITCDDEEERASRRVYRGCSRTSTCTGCTSCVCFGCATCRFSDCGCQTCTDF
TSNAKA*(SEQ ID NO.2)。
nucleotide sequence (SEQ ID NO. 1) for coding chrysanthemum CmULT1 protein:
ATGGCGGATGGTACGATGATGTTTTCAGAGGAAGAAGTGAAAGAGATGTGTGGGTTTAAGTTTTGCGGCGATGGTCACGTGGAGGTCACGTGCGGGTGTACTAGCTATTGCTATGGTGATGCTGTTGGTATACTAAAGGTTTTTGTTAATGGTGATCTTGAGATTACCTGTGACTGTACCCCTGGTTGTCAAGAAGACAAATTGACCCCAGCTGCTTTTGAGAAGCATTCGGGCAGAGAAACCGCCAGGAAATGGAAGAACAACATTTGGGTGATTGTTGATGGAGATAAGGTTCCTTTGTACAAAACTGCACTACTCAAGTACTACAACCAAGCTTTAACAAAAACATCCAACAAATCCCAATCTGGACAACTTGTTCATCGAGATGAGTTTGTTAAATGCACAAAATGCGATAAACTTCGCAGGTTTCATCTCCACACAAGCGAGGAATGTCGGCTTTACCATGATGCTTCTCGTGATAATGATTGGAAGTGCTCTGATATGCCTTATGAAAAAATAACCTGTGACGACGAAGAGGAAAGAGCAAGTCGTAGAGTCTACAGAGGCTGTTCGCGCACTTCTACATGCACAGGTTGCACTTCCTGCGTTTGTTTTGGTTGTGCTACCTGTCGTTTTTCTGACTGCGGCTGTCAAACATGCACTGACTTCACAAGTAACGCCAAAGCTTGA。
EXAMPLE 2 preparation of the support
The preparation of the fusion vector comprises the following steps:
(1) Extracting total RNA from a chrysanthemum 'Shenma' inflorescence by using a plant RNA extraction kit of magenta (Meiji) company, and synthesizing a cDNA first chain according to a Novozam reverse transcription kit;
the Chrysanthemum CmULT1 gene sequence (SEQ ID NO. 1) amplification primers are designed by utilizing SnapGene software, and the nucleotide sequences of the primers are shown as follows:
CmULT1F：AACACGGGGGACTTTGCAACATGGCGGATGGTACGATGATGTTTTCAG(SEQ IDNO.3)
CmULT1R：TGAAGACAGAGCTAGTTACATCAAGCTTTGGCGTTACTTGTGAAGTCAG(SEQ IDNO.4)；
(3) Carrying out PCR amplification on the cDNA obtained in the step (1) by using the PCR primer designed in the step (2), carrying out agarose gel electrophoresis on a PCR product, and recovering the PCR product by using a gel recovery kit (purchased from Shanghai Bioengineering Co., ltd.);
(4) The plasmid pBWA (V) BS-ccdB is cut by Bsa I/Eco 31I enzyme (purchased from Saimer Feishale (Shanghai) instruments Co., ltd., the cutting steps are carried out according to the instructions), the cut product is subjected to agarose gel electrophoresis, a gel recovery kit is adopted for recovery, and the recombination cloning method of Boyuan organisms is utilized to connect the chrysanthemum CmULT1 gene segment to pBWA (V) BS-ccdB;
(5) Transferring the ligation product into Escherichia coli DH5 alpha competent cells, coating the cells on an LB culture medium containing 50 mu g/mL kanamycin, selecting positive single colonies from a culture dish after the single colonies grow out to perform colony PCR, selecting the single colonies with the sizes of PCR detection bands conforming to the sizes of the single colonies, performing double enzyme digestion detection reaction after collecting thalli and extracting plasmids, sending the plasmids with the target bands after enzyme digestion detection to a company for sequencing, and naming the plasmids with the target genes connected with the sequencing results as pBWA (V) BS-OECmULT1.
Example 3 acquisition of transgenic plants
1. Transformation of recombinant plasmids
The recombinant vector pBWA (V) BS-OECmULT1 constructed in the example 2 is electrically shocked and transformed into an agrobacterium infection state (purchased from Tiangen biochemistry) to obtain recombinant agrobacterium, the recombinant agrobacterium is inoculated to a solid YEB culture medium containing 50 mu g/mL kanamycin and 50 mu g/mL rifampicin, the recombinant agrobacterium is inversely cultured at a constant temperature of 28 ℃ until a single colony grows out, a positive single colony is randomly selected for PCR detection, the size of a band amplified by the single colony is consistent with that of a positive control, and the condition is met, so that the plasmid containing the CmULT1 gene is successfully transferred into the agrobacterium.
2. Infection by infection
The method comprises the steps of carrying out amplification culture on agrobacterium GV3101 bacterial liquid containing pBWA (V) BS-OECmULT1 recombinant plasmids, completely soaking arabidopsis inflorescences in the bacterial liquid, vacuumizing and impregnating for 5min, horizontally placing the arabidopsis into a tray, keeping wet and dark culture in the tray for 14-16 h, culturing under normal conditions, completely collecting all seeds of transformed plants after the seeds are basically mature, and drying in a drying box at 37 ℃ for later use.
3. Screening to obtain transgenic plant
Spraying 0.001-0.002% Basta herbicide and screening transgenic plants by PCR identification, the PCR identification result is shown in figure 1, which shows that CmULT1 gene is successfully transformed into Arabidopsis thaliana wild-type plants.
Test example plant branching and silique Angle analysis of transgenic plants
The experimental group uses T3 generation transgenic arabidopsis obtained in example 3, the control group uses wild type arabidopsis, and the phenotype of 20 arabidopsis plants in each group, the control group and the experimental group is analyzed. And planting the plants in the soil, and taking a picture after the plants bloom and fruit.
The branching phenotype of the plant is shown in FIG. 2, and the parts of the silique and the rachis are shown in FIG. 3. Meanwhile, the angle between the silique and the rachis is counted, the statistical result is shown in figure 4, and the result shows that the angle between the silique and the rachis of the transgenic plant is obviously smaller than that of the wild type, and the result shows that the CmULT1 gene is expressed in the plant, so that the angle between the plant branches and the silique is obviously reduced, and the plant type is compact.
The ULTRAPETALA1 (ULT 1) protein belongs to one of the trithorax Group (trxG) proteins. In higher eukaryotes, the trxG protein and polycomb group (PcG) act synergistically to regulate gene transcription by regulating the "on" or "off" state of chromatin. The structural domain of plant ULT1 gene is conserved in the evolution process, both of which contain a SAND structural domain and a B-box motif and are commonly expressed in various tissues and organs. In arabidopsis, ULT1 is one of the key factors for inducing accumulation of shoot and flower stem cells and for inducing termination of flower meristems, which regulates the flower development process by activating the floral homeotic regulator gene AG at the right time. The ULT1 gene in chrysanthemum may also be involved in regulating water during flower development. OsULT1 in rice can be combined with cis motif "GAGAG" on OsDREB1b promoter, and is involved in various stress responses of abiotic stress. Phytohormones in saffron can induce the expression of Csult1 gene, and the over-expression of Csult1 gene can lead to the enhancement of PSY, PDS, BCH and CCDs gene expression, which indicates that Csult1 gene is a novel regulator for saffron carotenoid biosynthesis, and also proves that plant SAND domain protein participates in regulating secondary metabolic pathway. In conclusion, in the related art, no research and application report that ULT1 regulates the branch and the horn angle of the plant is found.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A chrysanthemum CmULT1 protein is a protein of the following a) or b) or c) or d):
a) Protein with amino acid sequence shown as SEQ ID NO. 2;
b) Fusion protein obtained by connecting N end and/or C end of protein with amino acid sequence shown as SEQ ID NO.2 with tag;
c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO. 2;
d) Protein with 75% or more than 75% homology with amino acid sequence shown as SEQ ID NO.2 and with the same function.
2. Nucleic acid molecule encoding the chrysanthemum CmULT1 protein of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of said nucleic acid molecule is as set forth in SEQ ID No. 1.
4. The biological material related to the nucleic acid molecule of claim 2, which is any one of the following 1) to 7):
1) An expression cassette comprising the nucleic acid molecule of claim 2;
2) A recombinant vector comprising the nucleic acid molecule of claim 2;
3) A recombinant vector comprising 1) the expression cassette;
4) A recombinant microorganism comprising the nucleic acid molecule of claim 2;
5) A recombinant microorganism comprising 1) said expression cassette;
6) A recombinant microorganism containing 2) the recombinant vector;
7) A recombinant microorganism containing 3) the recombinant vector.
5. A primer for amplifying the nucleic acid molecule of claim 2.
6. The primer according to claim 5, wherein the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.
7. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, the biological material according to claim 4, or the primer according to claim 5 or 6, in any one of the following (1) to (5):
(1) The application in plant breeding;
(2) The application in the preparation of plant-assisted plant breeding products;
(3) The application in regulating and controlling the branching angle of plants;
(4) The application in regulating and controlling the angle of plant siliques;
(5) Use in the preparation of a product for increasing the seed yield of a plant.
8. A method for controlling the branching angle of a plant, comprising the steps of: transferring the nucleic acid molecule of claim 2 or 3, the biological material of claim 4 into a recipient plant.
9. A method of controlling the angle of a plant's silique, the method comprising the steps of: transferring the nucleic acid molecule of claim 2 or 3, the biological material of claim 4 into a recipient plant.
10. The method according to claim 8 or 9, wherein the plant comprises a crucifer and a graminaceous plant; preferably, the cruciferous plants include rape, radish, chinese cabbage, violet or arabidopsis thaliana, and the gramineous plants include rice, wheat, maize, millet, sugarcane, sorghum, green bristlegrass or rye.
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