CN111333708A

CN111333708A - Gene derived from corn and having function of delaying flowering phase and application thereof

Info

Publication number: CN111333708A
Application number: CN202010340742.4A
Authority: CN
Inventors: 李有志; 樊宪伟; 高珺; 雷玲
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2020-06-26
Anticipated expiration: 2040-04-26
Also published as: CN111333708B

Abstract

The invention provides a cryptochrome 2 gene from corn, a protein coded by the cryptochrome 2 gene and application of the cryptochrome 2 gene, wherein the sequence of the cryptochrome 2 gene is shown as SEQ ID NO.1, and the cryptochrome 2 gene is transferred into other plants to obtain transgenic plants, so that the flowering period of the plants can be delayed. The cryptochromene 2 gene provided by the invention is a new gene for regulating plant flowering, provides a new thought for improving premature maize varieties, also provides theoretical support for delaying flowering period of other crops by using heterologous gene technology, can be used for plant molecular breeding, and can solve the problems of low efficiency and long period of traditional breeding.

Description

Gene derived from corn and having function of delaying flowering phase and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a gene with a function of delaying flowering phase from corn and application thereof.

Background

Flowering of plants is an important link in propagation, and therefore flowering control becomes one of the core content research contents in plant production. The structure and function of plant flowering gene are conserved, and the research of molecular genetic mechanism for controlling flowering of model plants such as arabidopsis thaliana and rice is very extensive and intensive, and the research is based and started on photosensitive genes, but the genetic composition of photosensitive systems of monocotyledons and dicotyledons is obviously different.

Corn (Zea mays) is one of the important food crops, having an innate sensitivity to changes in sunlight length due to its origin in tropical regions and its adaptation to flower normally under short-day conditions. After the plant is introduced into a high-latitude long-day area, if the plant is not domesticated, the plant can show vigorous vegetative growth, increased stem node number and leaves, inhibited reproductive growth, delayed androgenesis period and silking period, late maturity, no flowering or delayed flowering. It was investigated that one of the flowering pathways of maize is controlled by photoperiod response genes consisting of photoreceptors, consisting of upstream conz1, gigz1A, gigz1B, and id1 and downstream flowering locus T (FT) -like genes such as ZCN8, wherein the upstream gene components are conserved in both long-day and short-day adaptive maize. Unlike arabidopsis and rice, the maize photosystem consists of 6 genes, but lacks two homologues (pharma et al, 2018) of phyd (e) from arabidopsis, suggesting that maize has unique genes that control flowering. Some progress has been made in the research of genetic characteristics and molecular mechanisms of the photoperiod sensitivity of corn, but a great gap still exists between arabidopsis thaliana and rice, and the research of the molecular mechanisms of the photoperiod sensitivity is particularly poor, and the main reason is that the flowering time gene cloned on the corn is too little.

Disclosure of Invention

The invention aims to provide a cryptochrome 2 gene from corn and application thereof, wherein the gene has the function of delaying the flowering time of plants.

The cryptochromene 2 gene derived from corn has a sequence shown in SEQ ID NO.1 and 1983 basic groups, and an encoded amino acid sequence shown in SEQ ID NO. 2.

The invention also provides an expression vector, which contains the cryptochrome 2 gene from the corn; the expression vector is exemplified by pET-28a, pCAMBIA2301, pSP72, pROKII, pBin438, pCAMBIA1302, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc.

The invention also provides a host cell which contains the prokaryotic cell or the eukaryotic cell transformed by the expression vector.

Another object of the present invention is to provide the use of the cryptochrome 2 gene described above for delaying the flowering phase of plants.

The invention also provides a method for late-flowering plants, which is characterized in that a cryptochrome 2 gene construction recombinant expression vector shown in SEQ ID No.1 is introduced into a receptor plant to obtain a transgenic plant expressing the cryptochrome 2 gene.

Wherein the recombinant expression vector is a vector pCAMBIA1301, and the construction method of the recombinant expression vector pCAMBIA1301 is that the sequence between Nocl I and Pml I recognition sites is replaced by a DNA sequence shown in SEQ ID No. 1.

Wherein the recombinant expression vector can be introduced into plant cells or tissues by using conventional biotechnological methods such as Agrobacterium-mediated, Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation, etc.

Wherein the method also comprises the step of screening the plant with the cryptochromene 2 gene expression derived from the corn from a receptor plant introduced with the gene shown in SEQ ID No.1 to obtain a transgenic plant.

Wherein, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the recipient plant with the gene, but also its progeny. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

The invention has the advantages of

The invention provides a cryptochromene 2 gene from corn, a protein coded by the gene and application thereof in delaying the flowering phase of plants. The cryptochromene 2 gene provided by the invention is a new gene for regulating plant flowering, provides a new thought for improving premature maize varieties, also provides theoretical support for delaying flowering period of other crops by using heterologous gene technology, can be used for plant molecular breeding, and can solve the problems of low efficiency and long period of traditional breeding.

Drawings

FIG. 1 is a gel electrophoresis diagram of the product of PCR amplification of cryptochrome 2 gene;

FIG. 2 is a photograph of a resistant transformed Arabidopsis seedling screen;

FIG. 3 is a photograph comparing leaves of transgenic Arabidopsis thaliana and non-transgenic Arabidopsis thaliana after 20 days of transplantation;

FIG. 4 is a photograph showing the comparison of plant height and flowering of transgenic Arabidopsis thaliana and non-transgenic Arabidopsis thaliana after 30 days of transplantation.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 cloning of maize-derived cryptochrome 2 Gene sequences

Extracting total RNA of a maize B73 inbred line, carrying out reverse transcription on the total RNA to synthesize a first strand cDNA, cloning a cDNA fragment of the cryptochrome 2 gene according to a conventional PCR method by using the first strand cDNA as a template and utilizing sequence specific primers 1d045944-F2 and 1d045944-R, wherein the sequence of 1d045944-F2 is shown as SEQ ID NO.3, and the sequence of 1d045944-R is shown as SEQ ID NO. 4; an electrophoresis diagram of a PCR product is shown in figure 1, a cDNA fragment sequence of the obtained cryptochromen 2 gene through amplification is shown in SEQ ID NO.1, has 1983 basic groups, and has 93.89% of consistency with a sorghum cryptochrome 1 (NCBIaccesssion No. XM _002436943.2) gene cDNA sequence on a nucleotide level. (ii) datasets from a gene ontology database [ GeneOntology (GO); http:// geneontology. org /) ], the annotation result for this gene is "dark response".

The protein coded by the cryptochrome 2 gene has 652 amino acid residues, the sequence of the protein is shown as SEQ ID NO.2, and the protein has 91.72% consistency with the amino acid residue sequence of sorghum cryptochrome 1 protein (NCBI accession No. XP _ 002436988.1).

Example 2 Gene function identification

1. Gene of cryptochrome 2 is transferred into arabidopsis thaliana plant

The complete reading frame sequence of the cDNA of the cryptochrome 2 gene is cloned between Nocl I and Pml I enzyme cutting sites of a plant transgenic expression Vector Pcambia-1301Vector to obtain a recombinant expression Vector.

The recombinant expression vector is introduced into an agrobacterium strain to generate a recombinant agrobacterium strain with the recombinant expression vector.

The cryptochrome 2 gene is transferred into arabidopsis thaliana by a dipping transformation method, the method specifically comprises the steps of soaking a flower organ of arabidopsis thaliana in a recombinant agrobacterium strain culture solution, continuously culturing the produced arabidopsis thaliana seeds of T0 generations indoors, carrying out resistance screening on the arabidopsis thaliana seeds of T0 generations on a kanamycin MS culture medium containing 20mg/mL under the condition of 16h illumination/8 h dark photoperiod to obtain resistance transformation arabidopsis thaliana seedlings (shown in figure 2), identifying the resistance transformation arabidopsis thaliana seedlings as transgenes by PCR amplification on the basis of sequence specific primers 44-2-F (1301) and 44-2-R (1301), and detecting whether a target gene is expressed by a quantitative PCR method. The sequence of the specific primer 44-2-F (1301) is shown as SEQ ID NO.5, and the sequence of the specific primer 44-2-R (1301) is shown as SEQ ID NO. 6. And a parallel control group experiment is non-transgenic arabidopsis, and after the detection and analysis, an arabidopsis transgenic plant of heterologously expressed maize cryptochrome 2 gene is obtained and the function of the transgenic arabidopsis is researched.

2. Functional identification and results

Seeds of an arabidopsis transgenic plant expressing the cryptochrome 2 gene of the corn are planted on two MS culture media without kanamycin and with kanamycin of 20mg/mL respectively, and non-transgenic arabidopsis is used as a control. Transgenic Arabidopsis plants and non-transgenic Arabidopsis plants, which were kanamycin resistant and confirmed by PCR grown on MS medium, were transplanted into potting soil for potting.

Compared with the wild arabidopsis thaliana of the control group, after 20 days of transplantation and cultivation, the arabidopsis thaliana transgenic plant expressing the maize cryptochrome 2 gene is unchanged (figure 3), still cannot flower after 30 days of transplantation and cultivation, but the plant of the control group is bolting and flowering (figure 4), and continuous observation shows that the transgenic arabidopsis thaliana of the maize cryptochrome 2 gene can flower after 40 days of transplantation, and the flowering period is obviously delayed.

Sequence listing

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Claims

1. The cryptochrome 2 gene is derived from corn, and is characterized in that the sequence of the cryptochrome 2 gene is shown as SEQ ID No. 1.

2. The gene of claim 1 encoding a protein characterized by: the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. An expression vector comprising the gene of claim 1.

4. A host cell comprising a prokaryotic or eukaryotic cell transformed with the expression vector of claim 3.

5. Use of the leuco 2 gene of claim 1 in delaying the flowering phase in plants.

6. A method of breeding late-flowering plants using the leuco 2 gene as set forth in claim 1, characterized in that: the cryptochrome 2 gene shown in SEQ ID No.1 is constructed into a recombinant expression vector and is led into a receptor plant to obtain a transgenic plant expressing the cryptochrome 2 gene.

7. The method of claim 6, wherein: the recombinant expression vector is a vector pCAMBIA1301, and the construction method of the recombinant expression vector pCAMBIA1301 is that the sequence between Nocl I and Pml I recognition sites is replaced by a DNA sequence shown in SEQ ID No. 1.

8. The method of claim 6, wherein: the recombinant expression vector is introduced into plant cells or tissues by using conventional biotechnological methods such as Agrobacterium mediation, Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation, and the like.

9. The method of claim 6, wherein: the method also comprises the step of screening the plant expressed by the cryptochrome 2 gene from the receptor plant introduced with the gene shown in SEQ ID No.1 to obtain a transgenic plant.