KR101293756B1 - OsPLIM GENE ISOLATED FROM ORYZA SATIVA, EXPRESSION VECTOR COMPRISING THE GENE, TRANSGENIC PLANTS TRANSFORMED WITH THE EXPRESSION VECTOR AND PRODUCTION METHOD THEROF - Google Patents

Abstract

The present invention relates to an OsPLIM gene isolated from rice, an expression vector containing the gene, a plant transformed with the expression vector, and a method of manufacturing the same.
OsPLIM gene of the present invention is composed of the nucleotide sequence described in SEQ ID NO: 1, as a seed development-related gene derived from rice, after producing an expression vector containing the same, consisting of producing a plant transformed with the expression vector do.
The method for producing a transgenic plant of the present invention comprises the steps of preparing a plant expression vector for transformation comprising the OsPLIM DNA sequence derived from rice consisting of SEQ ID NO: 1, transforming plant cells with the plant expression vector for transformation And regenerating the transgenic plant from the transformed plant cell.
According to the present invention, more efficient crop development using crops having a disadvantage that the number of seeds is more than necessary or tall by using the OsPLIM gene derived from rice, which exhibits the characteristics of increasing the seed size and decreasing the number of grains and decreasing the height. To do it.

Description

OSPLIM gene isolated from rice, expression vector containing the gene, transgenic plant transformed with the expression vector, and method for producing the same AND PRODUCTION METHOD THEROF}

The present invention is a rice ( Oryza OsPLIM gene isolated from sativa ), an expression vector comprising the gene, a transgenic plant transformed with the expression vector, and a method for producing the same, in particular, it can be easily used to develop crops having good seed size or growth state. The present invention relates to a transgenic plant and a method for producing the same, wherein the rice-derived OsPLIM gene is analyzed and overexpressed.

Rice is a very economical crop that is used as a staple food by people in Asia, including Korea, and various breeding methods, including conventional breeding, molecular breeding, and transformation methods, are mobilized to improve yield and quality.

Among them, the transformation method has the advantage of developing new genetically improved varieties by directly introducing agriculturally useful genes to crops, compared to conventional breeding methods.

In other words, the transformation technology in the agricultural field is a breeding method that can be used more directly and actively to improve the yield-related traits of crops, such as resistance to various biological and non-biological stresses such as insect drought, wet and ultraviolet rays.

In particular, rice has been in the spotlight as a crop having high developmental value and practical value due to its economical efficiency in agriculture and its utility as a monocotyledonous plant for genome research.

In order to improve the seed of rice, it is important to identify the seed production mechanism.

However, the function of PLIM genes involved in seed size and growth in monocotyledonous plants (including rice) has not been found. As a technique for improving the size and growth of conventional seeds, cell cycle control proteins in plant cells ( a method of controlling plant cell growth by denaturing the concentration or catalytic activity of a cell cycle control protein (US Pat. No. 6,087,175) or a target gene to enhance nucleotides encoding a synthetic zinc finger protein. Only a method of adjusting the expression level of a target gene in plant cells by introducing the combined expression vector into plant cells (US Patent 7,151,201) and the like are disclosed.

US Patent 6,087,175 "Control of plant cell proliferation and growth" US Patent 7,151,201 "Methods and compositions to modulate expression in plants"

In the present invention, to solve the above problems, an object of the present invention is to provide an expression vector containing the OsPLIM gene derived from rice and it is not known.

It is also an object of the present invention to provide a transgenic plant transformed by overexpressing an OsPLIM gene derived from rice, which is most strongly expressed in the medicinal tissues of rice.

OsPLIM gene derived from rice of the present invention consists of a nucleotide sequence described in SEQ ID NO: 1, characterized in that it is specifically expressed in the drug tissue.

In this case, the plant-transformed rice-derived OsPLIM gene primer set is characterized by using a sense primer (sense primer, 5'-ATGTCGTTCACCGGCACGCAGGACA-3 ') and antisense primer (antisense primer, 5'-CTATGTCGCGTCTTGTGGGGGCACT-3'). .

The present invention also provides a plant expression vector for transformation comprising the gene.

In addition, the present invention provides a transgenic plant transformed with an Agrobacterium transformant transformed with a transgenic plant expression vector comprising an OsPLIM gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1, thereby the size of the seed. The aim is to secure useful genetic resources for the development of crops, which are characterized by larger and smaller grains and shorter heights.

The method for producing a transgenic plant of the present invention comprises the steps of preparing a plant expression vector for transformation comprising the OsPLIM DNA sequence derived from rice consisting of SEQ ID NO: 1, transforming plant cells with the plant expression vector for transformation And regenerating the transgenic plant from the transformed plant cell.

According to the present invention, more efficient crop development using crops having a disadvantage that the number of seeds is more than necessary or tall by using the OsPLIM gene derived from rice, which exhibits the characteristics of increasing the seed size and decreasing the number of grains and decreasing the height. To do it.

1 shows OsPLIM gene amplification by RT-PCR.
L: leaf, S: stem, R: root
A: medicines, O: ovary, Ov: ovule
2 is a view showing the manufacturing process of the vector for transforming rice using pCAMBIA vector.
Figure 3 shows the transgenic plants obtained by Agrobacterium infection transformation.
4 shows phenotypic analysis of transgenic plants.
5 is a view showing the analysis of the amount of expression of the transformed plant tissue.
A: ananthers; O: ovary; Ov: ovule
Figure 6 shows the seed size and weight comparison analysis of transgenic plants.

The terms, techniques, and the like described in this specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, there is provided a LIM ( oryza sativa pollen LIM, OsPLIM) gene isolated from rice and expressed in pollen of rice and its nucleotide sequence.

In order to select LIM ( oryza sativa pollen LIM, OsPLIM) gene isolated from rice and expressed in pollen of rice in the present invention, a specific gene is extracted from rice using SUPERSCRIPTII (Takara ver 3.0) reverse transcriptase, and then RT ( Reverse Transcriptase)-by cloning by PCR and sequencing using ABI 3700 sequencer and then by BLAST search of NCBI Respectively.

The gene selected as described above is a gene involved in seed size, number of grains, size, etc., and its expression is amplified specifically in drug tissues. "LIM ( oryza sativa pollen LIM expressed in rice pollen) , OsPLIM) gene. "

The LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in the pollen of rice consists of all or part of the nucleotide sequence described in SEQ ID NO: 1, and is characterized by being composed of an open reading frame (ORF) of 621 bp.

According to another aspect of the present invention, there is provided an expression vector comprising LIM ( oryza sativa pollen LIM, OsPLIM) gene is isolated from rice and expressed in pollen of rice.

By inserting a LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in the pollen of the rice of the present invention into a known appropriate expression vector, an expression vector containing this gene can be obtained.

An expression vector for inserting the LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in the pollen of rice is pCAMBIA Vectors are preferred.

The expression vector containing the gene of the present invention is shown in FIG.

According to another aspect of the present invention, a transgenic plant isolated from rice and transformed with an expression vector comprising an oryza sativa pollen LIM (OsPLIM) gene expressed in a pollen of rice is provided.

At this time, any plant cell that can be used for the transformation may be any one suitable for the expression of the gene of the present invention. In the present invention, rice and rice plants are targeted, and if the plant belongs to rice plants, there is no particular limitation. .

Examples of plants belonging to rice plants include rice, corn, wheat, and the like, and the present invention is particularly applicable to rice, particularly to Dongjin rice.

In addition, the transformation may be mediated by Agrobacterium.

The transgenic plant according to the present invention can be obtained by the following method:

1) preparing a transformed plant expression vector comprising LIM ( oryza sativa pollen LIM, OsPLIM) DNA sequence isolated from the rice consisting of SEQ ID NO: 1 expressed in the pollen of rice

2) transforming a plant cell with the transformed plant expression vector

3) regenerating the transformed plant from the transformed plant cells.

The size of mature seed, especially rice seed, harvested from transgenic rice plants prepared in this way is about 1.3 times larger than that of existing crops. .

Examples of realizing the rice plant and the production method thereof according to the embodiment of the present invention will be described in detail in the following order, taking rice as a representative example.

The procedure described below can be applied to rice and plants other than rice as it is or to change various conditions.

Hereinafter, the present invention will be described in detail with reference to Examples, but these are not intended to limit the scope of the present invention.

<Example 1> Cloning and sequencing of LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in pollen of rice by RT-PCR

1) First-strand cDNA synthesis

Total RNA was extracted from the leaves of rice and first-strand cDNA was synthesized using SUPERSCRIPTII (Takara ver 3.0) reverse transcriptase as follows.

Sense primer (SEQ ID NO: 2): 5'-ATGTCGTTCACCGGCACGCAGGACA-3 'and antisense primer (SEQ ID NO: 3): 5' expressed in rice pollen -CTATGTCGCGTCTTGTGGGGGCACT-3 'was added to the PCR reaction solution, followed by electrophoresis on a 1% agarose gel and amplified genes were extracted (QIAquick gel extraction kit).

2) sequencing

White colony obtained by plating the amplified gene on X-gal-added LB medium was selected and colony PCR was performed on Topo 2.1 vector (Invitrogen, TOPO TA Cloning kit).

As a result, after extracting the colony plasmid of the band identified, the sequence was analyzed using an ABI 3700 sequencer, and then identified as LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in pollen of rice by BLAST search of NCBI.

Example 2 Analysis of Expression of LIM ( oryza sativa pollen LIM, OsPLIM) Gene Expressed in Rice Pollen

Samples are placed in a mortar of liquid nitrogen to separate RNA from leaves (L), stem (S), root (R), medicine (A), ovary (O), and ovule (Ov). After finely added, the sample was placed in a 2 ml tube (tube), an appropriate amount of Trizol (Trizol; Takara, Japan) 800 μl was added and quickly vortex.

400 μl of chloroform was added to the same tube, followed by simple mixing, and centrifuged at 13,000 rpm for 10 minutes at 4 ° C. using a centrifuge.

After repeating the above process once more, 375 μl of isopropanol was added to the finally obtained supernatant and mixed well, followed by centrifugation at 13,000 rpm for 10 minutes at 4 ° C.

The supernatant was discarded and the RNA precipitate was washed with 1 ml of 70% ethanol, centrifuged at 13,000 rpm at 4 ° C., the supernatant was completely removed, and the RNA precipitate was dried at 37 ° C. for 30 minutes.

50 μl of 1 × TE buffer (ddH 2 O may also be used) was mixed and vortexed to completely dissolve RNA, and then incubated in a 60 ° C. incubator for 10 minutes.

Centrifuged at 13,000 rpm at 20 ℃, the supernatant was transferred to a new 1.5ml tube to quantify the amount of RNA by spectrophotometer (Spectrophotometer) and stored at -70 ℃.

These RNA is RT-PCR using the primer used in Example 2, the first-strand cDNA synthesized in Example 1 expression of LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in the pollen of each tissue The amount was analyzed (FIG. 1).

Example 3 Preparation of Expression Vector for Transformation of Rice and Preparation of Agrobacterium Transformant

1) Production of expression vector for rice transformation

Example 1 LIM which is expressed in pollen of rice obtained in the pCAMBIA vectors (oryza sativa pollen LIM, OsPLIM) LIM (oryza which is expressed in pollen of rice plants which are cloned into a Topo 2.1 vector for cloning a gene sativa pollen LIM, OsPLIM ) Isolate LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in pollen of rice from Topo 2.1 vector using EcoRI and SalI enzyme and insert into pCAMBIA vector using ligase kit (TOYOBO LGK-201) Binary vectors were completed.

After confirming the gene by colony PCR and sequencing (sequencing), the transformed by Agrobacterium infection to transform the rice to produce a binary vector (expression vector for rice transformation) (Fig. 2).

2) Agrobacterium transformant preparation

After freeze and thaw 2 to 3 times in order to transform the produced binary vector into Agrobacterium (LBA4404), it was transformed by heat shock at 37 ° C. (heat shock method). Transformant colonies were identified by incubating overnight in YEP medium (Yeast 10g, NaCl 5g, peptone 10g, Agar 15) g / 1L).

The identified colony was transformed into rice in AB medium <AB buffer (K ₂ HPO ₄ 60g, NaH ₂ PO ₄ 20g / 1L), AB Salts (NH ₄ Cl 60g, MgSO ₄ .7H ₂ O 6g, KCl 3g in, CaCl _{2 and 2H 2 O 0.265g, FeSO 4 .7H} 2 O 50mg / 1L), Glucose 5g / 1L> and incubated.

Example 4 Preparation of Rice Transgenic Plant

1) Induction of rice callus

In order to induce rice callus in MS medium (Duchefa's Murashinge and Skoog vitamin medium), the seeds were washed in lax and then dried appropriately and then healed in medium containing 2,4-D hormone in MS medium.

After incubating for 3-4 weeks at 27 ° C., the callus was induced, and then the embryogenesis callus was sub-cultured in a fresh medium to which 2,4-D hormone was added to MS.

2) Agrobacterium Infection

Embryogenesis callus and Agrobacterium transformed genes were incubated in AB liquid medium for 20 min disinfection and then cultured for 3 days.

After adding cefotaxime to sterile water and washing until complete removal of Agrobacterium, cancer culture was carried out for 3 weeks in a medium in which cefotaxime and Hygro antibiotics were added to MS medium.

The callus, which remained unchanged in the medium added cefotaxime and hygro antibiotics to MS medium, was transferred to the medium added cefotaxime and hygro antibiotics to MS medium, and cultured for 2 weeks.

To induce shooting in the callus, it was transferred to a medium containing MSR-cefotaxime and Hygro antibiotics, and cultured for 4 weeks.

3) seed harvesting and posterior fixation

After 5-7 days, the obtained transformed plant (T ₀ ) was grown in a greenhouse to harvest seeds, and then separated at a ratio of 3: 1 in Higro selection medium for subsequent fixation.

The seeds obtained were grown in GMO packaging for phenotypic fixation and phenotypic analysis of the overexpressing transformants, and then the seeds were used for generation generation after harvesting (FIG. 3).

Example 5 Phenotypic Analysis of Transgenic Plants

- LIM which is expressed in pollen of rice (oryza sativa pollen LIM, OsPLIM) a characteristic that appears when a LIM (oryza sativa pollen LIM, OsPLIM) gene which is a gene inserted into the expression in the pollen of rice plants from the identified transgenic plant with is expressed Comparative analysis with Dongjin rice for analysis (FIG. 4).

Example 5 Analysis of LIM ( oryza sativa pollen LIM, OsPLIM) Gene Expression in Rice Pollen of Rice Transgenic Plants

- LIM which is expressed in pollen of rice (oryza sativa pollen LIM, OsPLIM) gene LIM which is expressed in pollen of rice plants transformed rice plants from the conversion plant insert (oryza sativa pollen LIM, OsPLIM) RT-PCR for the expression analysis of the gene As a result, it was confirmed that the expression is strong in all surgery, pistil, baeju (Figure 5).

Example 6 Seed Phenotypic Analysis of Transgenic Plants

-As a result of comparing the seeds of transgenic plants with Dongjin rice for the expression test of LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in the pollen of rice transformed to rice, the size of seeds was 1.3 times larger than that of Dongjin rice. It confirmed that (FIG. 6, Table 1).

Transformant NUMBER
1000 (g) Dongjin Rice PLIM Seed + husk
(Seed + shell) 25.68
/ 1000 27.79
/ 1000

Attach an electronic file to a sequence list

Claims (6)

Consists of the nucleotide sequence set forth in SEQ ID NO: 1,
Related to seed formation and growth from rice plants,
LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in the pollen of rice.
delete delete Plant expression vector for transformation, comprising the gene according to claim 1.
Consists of the nucleotide sequence set forth in SEQ ID NO: 1,
Characterized in that the transformed Agrobacterium transformants transformed by a transgenic plant expression vector comprising a LIM ( oryza sativa pollen LIM, OsPLIM) gene expressed in rice pollen ,
Transgenic Plants.
Consists of SEQ ID NO: 1,
Preparing a plant expression vector for transformation comprising an oryza sativa pollen LIM (OsPLIM) DNA sequence expressed in rice pollen ;
Transforming plant cells with the transformed plant expression vector; and,
And regenerating the transformed plant from the transformed plant cells.
Method for producing a transgenic plant.

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