KR19990030639A - Flowering-time regulating genes, vectors comprising the same and plants transformed thereby - Google Patents

Abstract

The present invention relates to OsMADS5-8, MdMADS3 and 4, which are flowering time control genes isolated from plants such as rice and apple, and expression vectors containing them, and plants such as tobacco, petunia and rice transformed with the expression vectors. By this transformation, it is possible to induce early flowering and dwarfism of plants without being affected by the environment of flowering time, shorten the growth period and increase crop yield.

Description

Flowering-time regulating genes, vectors comprising the same and plants transformed thereby

The present invention relates to a flowering time regulation gene, an expression vector comprising the same, and a plant transformed with the vector, and more specifically, flowering time regulation genes isolated from plants such as rice and apple, expression vectors containing them, and This relates to a transgenic plant produced by transforming plants such as tobacco, petunia, and rice.

Each plant blooms in a particular season, and the timing of flowering is determined by the size, which is an internal condition of the plant, and various environmental factors such as photoperiod and temperature have a decisive influence on the flowering time.

Since the internal and environmental factors of the plant are known to be controlled by controlling the expression of the flowering time regulation gene, if the isolation of the flowering time regulation gene can be artificially regulated its expression, the flowering time of the plant can be controlled.

Thus, Applicant has isolated the flowering time regulation gene called OsMADS1 from Nakdong rice (Chung, Y. Y. et al, Plant Mol. Biol., 26, 657-665 (1994)). OsMADS1 is a gene belonging to the MADS box gene family and is a regulatory gene having a highly homologous MADS box and K box. To study the function of the OsMADS1 gene, the transformed tobacco was expressed not only significantly earlier than the flowering period but also significantly shortened in height. The MADS box gene has been known to be involved in the development of flower organs, but it has been found by the above studies that the MADS box gene is also involved in flowering time regulation. Early flowering and dwarfing of plants transformed to express OsMADS1 genes were proportional to the amount of OsMADS1 mRNA expressed, and other physiological phenomena such as photosynthetic ability did not differ between transformants and untransformed controls. This suggests the possibility of breeding new varieties with shorter growth periods as the production time cannot be accelerated without decreasing the production by genes.

The inventors of the present invention continue to study how to increase the yield by reducing the growth period of the crops by using the flowering period control genes that have the function of accelerating the flowering period and inducing dwarfity. Thus, OsMADS1 from rice and apple In addition to other flowering time regulation genes were isolated and they were expressed in crops such as tobacco to find out that early flowering, dwarfism, etc. regardless of the season was induced to complete the present invention.

It is an object of the present invention to provide a flowering time control gene, an expression vector containing the same, and a plant transformed therein, which can be usefully used to induce early flowering of crops regardless of seasons to shorten the growth period and increase the yield. .

Figure 1 shows the nucleotide sequence and amino acid sequence of OsMADS5 flowering time regulation gene isolated from rice in accordance with the present invention,

2 shows a comparison between the amino acid sequence of OsMADS5 and the amino acid sequence of other MADS box genes.

3 is a photograph (magnification: 1 / 10-fold) of the tobacco transformed with the expression vector pGA1348 containing the OsMADS5 gene and untreated control tobacco,

Figure 4 shows the nucleotide sequence and the amino acid sequence of the flowering time regulation gene OsMADS6 isolated from rice in accordance with the present invention,

Figure 5 is a photograph (magnification: 1/10 times) of the tobacco transformed with the expression vector pGA1349 containing the OsMADS6 gene and untreated control tobacco,

Figure 6 shows the nucleotide sequence and amino acid sequence of the flowering time regulation gene OsMADS7 isolated from rice in accordance with the present invention,

7 is a photograph (magnification: 1 / 10-fold) of the tobacco transformed with the expression vector pGA1350 containing the OsMADS7 gene and untreated control tobacco,

8 shows the nucleotide sequence and the amino acid sequence of the flowering time regulation gene OsMADS8 isolated from rice according to the present invention,

9 is a photograph (magnification: 1 / 10-fold) of the tobacco transformed with the expression vector pGA1351 containing the OsMADS8 gene and the untreated control tobacco,

10 shows the nucleotide sequence and amino acid sequence of the flowering time regulation gene MdMADS3 isolated from apples according to the present invention,

11 is a photograph (magnification: 1 / 10-fold) of tobacco and untreated control tobacco transformed with the expression vector pGA1534 containing the MdMADS3 gene,

12 shows the nucleotide sequence and amino acid sequence of the flowering time regulation gene MdMADS4 isolated from apples according to the present invention,

FIG. 13 is a photograph (magnification: 1 / 10-fold) of tobacco and untreated control tobacco transformed with the expression vector pGA1588 containing the MdMADS4 gene,

14 shows the expression vector pGA1209 containing the OsMADS1 gene,

15 is a photograph (magnification: 1/10) fold of petunias and untreated control petunias transformed with the expression vector pGA1209 containing the OsMADS1 gene,

Figure 16 shows the expression vector pGA1268 containing OsMADS1 gene,

17 is a photograph (magnification: 1 / 5-fold) of untreated control rice of rice transformed with expression vector pGA1268 containing OsMADS1 gene,

Figures 18A and 18B are photographs comparing the degree and size of flowering by growing progeny and untreated controls of Maryland Mammoth transformed with expression vector pGA1209 containing OsMADS1 gene under single dry and long day conditions, respectively (magnification: 1/10 times)

19 is a result of analyzing the accumulation of OsMADS1 mRNA of Maryland mammoth transformed with the expression vector pGA1209 containing the OsMADS1 gene,

20A and 20B are photographs comparing the degree and size of flowering by growing progeny and untreated controls of Nicotiana sylvestris transformed with the expression vector pGA1209 containing the OsMADS1 gene under single and long-term conditions, respectively. (Magnification: 1/10 times),

Figure 21 shows the results of analyzing the amount of OsMADS1 mRNA accumulation of Nicotiana Sylvestris transformed with the expression vector pGA1209 containing the OsMADS1 gene.

In order to achieve the above object, in the present invention, flowering time control genes OsMADS5, OsMADS6, OsMADS7 and OsMADS8 isolated from rice and flowering time control genes MdMADS3 and MdMADS4 isolated from apples, and expression vectors containing each of them, and these vectors To provide a transformed tobacco.

According to the invention there is also provided petunia, rice, Maryland Mammoth and Nicotiana sylvestris transformed with an expression vector containing OsMADS1.

Hereinafter, the present invention will be described in detail.

In the present invention, the λZapII cDNA library of rice flowers was searched using OsMADS1 cDNA of rice reported by the applicant (Chung, YY et al, Plant Mol. Biol., 26, 657-665 (1994) as a probe and similar to OsMADS1. Dog flowering time regulatory genes were isolated.

The rice flower cDNA library is prepared by separating mRNA from early rice flowers, synthesizing a double-stranded cDNA, cloning the phage vector, and packaging.

The cDNA library of the prepared rice flower was searched with OsMADS1 cDNA to select clones that showed positive reaction. Recombinant plasmids pGA1340, pGA1341, pGA1342 and pGA1343 with cDNA fragments were obtained from the selected positive clones, and then Sanger et al. (Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467). (1977) determine the nucleotide sequence of the cDNA fragment.

Thus, the four flowering time control genes whose base sequences were determined from the rice flower cDNA library have nucleotide sequences shown in FIGS. 1, 4, 6, and 8, respectively, which are OsMADS5 (pGA1340), OsMADS6 (pGA1341), and OsMADS7 (pGA1342), respectively. And OsMADS8 (pGA1343).

Genes homologous with at least 80% of 30 consecutive bases of any of these genes also encode proteins having the same flowering time regulatory activity as the proteins encoded by these genes. As a result of estimating amino acid sequences from the found nucleotide sequences, it was found that they transcribed proteins similar to OsMADS1.

A vector pGA748 was constructed by inserting a linker having the following sequence 1 in HindIII, ClaI site of vector pGA643 (An. G. et al., Plant molecular Biology Manual, ppA3 / 1-19 (1988)):

[SEQ ID NO 1]

By inserting the ecoRI fragments of pGA1340, pGA1341, pGA1342 and pGA1343 into the EcoRI site of the constructed vector pGA748, the flowering period control genes were expressed by expression vectors that can be expressed under the CaMV 35S promoter, namely pGA1348, pGA1349, pGA1350 and pGA1351. Produce each. Since the CaMV 35S promoter is expressed in almost all manipulations of the plant, it is always expressed in all tissues of the plant. The produced expression vectors are transferred to tobacco using Agrobacterium sp. Microorganisms and grown in greenhouse conditions.

The vectors containing the flowering time control genes OsMADS5 to OsMADS8 of the present invention, ie, pGA1348, pGA1349, pGA1350, and pGA1351, respectively, were shown to be shorter in flowering and shorter in height than the untreated control.

According to the present invention, in addition, OsMADS1 cDNA was probed in a similar manner to the apple flower cDNA library, and two cDNAs having high homology with OsMADS1 were isolated and named MdMADS3 and MdMADS4, respectively. These genes, as well as the flowering period control genes of rice, produced expression vectors, ie, pGA1534 and pGA1588, which were able to express the genes under the CaMV 35S promoter using the vector pGA748, and then the expression vectors were respectively stored in tobacco. Transplantation and growth in greenhouse conditions resulted in early flowering and dwarfism.

In the present invention, an expression vector pGA1209 capable of expressing the OsMADS1 gene under the CaMV 35S promoter using the vector pGA748 is also prepared by a method similar to the above, and by using the same, a dicotyledonous plant petunia and a monocotyledonous rice, and a single plant Tobacco varieties were transferred to Maryland Mammoth and the long-life plant Nicotiana sylvestris and then grown.

As a result, all of the transformants exhibit early flowering and dwarfism, and the flowering time control gene acts on both dicotyledonous and monocotyledonous plants to control the flowering time, and the flowering period is accelerated regardless of the environment of the flowering time, that is, photoperiod. Appeared to be shortened.

From these results, if these genes are expressed in plants that bloom only in certain seasons using the flowering time regulation genes, it is expected that flowering may occur regardless of the seasons, thereby shortening the growth period and thus increasing production.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1

Isolation of OsMADS5 Gene for Rice Flowering Time and Production of Transformant

(Step 1) Gene Isolation and Sequence Analysis

MRNA (5 [mu] g) was isolated from the earliest rice (Oryza sativa L. cv. M201) flowers with a spike length of 1 cm or less using a poly (A) Quick mRNA Isolation Kit (Stratagene, USA). Using this as a template, a double-stranded cDNA was synthesized to prepare a cDNA library (ZAP-cDNA Gigapack II Gold Cloning Kit, Stratagene, USA).

Subsequently, the OsMADS1 gene was labeled with the isotope 32P, and then, as a probe, a cDNA library of the rice flower was searched to select a clone having a positive reaction.

Hybridization was performed according to the method of Sambrook et al. (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N. Y., 1989). The experimental process will be described in more detail as follows.

250,000 phages of the rice flower cDNA library were transferred to a nylon membrane (Hybond, Amersham), then added to 6 X SSC, 0.2% BLOTTO solution, and the addition of OsMADS1 labeled with radioisotope ³² P. And hybridized by reacting at 55 ° C. for 16 hours. The hybridized nylon membrane was washed twice at 50 ° C. with 2 × SSC, 0.1% SDS solution and then exposed to X-ray film for 24 hours in the dark.

Recombinant plasmid pGA1340 (OsMADS5) into which a cDNA fragment was inserted was obtained from one of the phage clones positively exposed to the film using the f1 helper phage, R408 (Stratagene). E. coli JM83 transformed with the plasmid pGA1340 was deposited on April 22, 1997 as the accession no. KCTC 0329BP to the Genetic Bank of Korea (KCTC).

The base sequence of the cDNA fragment cloned into recombinant plasmid pGA1340 was analyzed according to the method of Singer et al. (Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977). Figure 1 shows the amino acid sequence from the base sequence of the flowering time control gene OsMADS5 and the amino acid sequence of the protein encoded therein Figure 1 shows the position of the base sequence, the number on the right Indicates the position of the amino acid sequence, and the two underlined sites show MADS box and K box sites with high homology with other flowering timing regulator genes.

FIG. 2 shows the amino acid sequences of OsMADS5 and several other MADS box proteins in order to determine the homology between OsMADS5 and other MADS box genes. The same sites as OsMADS5 are shown in black. As shown here, OsMADS5 transcribes a protein similar to OsMADS1.

(Step 2) Preparation of Expression Vector and Transgenic Plant

PGA748 was prepared by inserting a linker containing an EcoRI site into the HindIII, ClaI site of the vector pGA643 (An. G. et al., Plant molecular Biology Manual, ppA3 / 1-19 (1988)), and the CaMV 35S promoter (P35S). The expression vector pGA1348 containing the OsMADS5 gene was constructed by inserting the OsMADS5 cDNA obtained in (Step 1) between the terminator T7) and the terminator T7 using the restriction enzyme EcoRI, which is expressed in various tissues at all times and has a kanamycin resistance gene. It facilitates the selection of transformants.

The expression vector was then Agrobacterium tumefacience LBA4404 (Hoekema, A. et al., Containing binary Ti plasmid pAL4404 (Hoekema et al., Nature, 303, 179-181 (1983))). Nature, 303, 179-181 (1983)) and using this to select the kanamycin resistant callus of tobacco (N. tabacum L. cv. Xanthi) selection medium (MS medium (Life technologies) + 10.8 μM NAA (naphthalene) After inducing in aceticacid) + 2.5μM BAP (benzyl adenine), transformants were differentiated and transferred to soil to obtain seeds.

The experiment below shows that transformed seeds were selected in kanamycin medium and their growth compared to the control. The control was a tobacco obtained by transforming in the same manner as above with the vector pGA748 without the OsMADS5 gene.

The seed of the tobacco transformed with pGA748 and the seed of the control tobacco plant transformed with pGA748 were sown at the same time, and the flowering time and size were observed while growing under the same conditions until flowering. 3 is a photograph of the tobacco plants (two left plants) and the control (two right plants) transformed with the OsMADS5 gene sown and grown at the same time, the control was not flowering, whereas the transformants were flowering and the size compared to the control. You can see that is small.

Example 2

Isolation of Rice Regulatory Gene OsMADS6 and Preparation of Transformant

(Step 1) Gene Isolation and Sequence Analysis

Using the same method as in step 1 of Example 1, OsMADS1 cDNA was used as a probe to search the cDNA library of rice flowers to obtain the plasmid pGA1341 into which the flowering time regulation gene OsMADS6 cDNA was inserted, and analyzed the nucleotide sequence of the gene. Amino acid sequence was estimated. E. coli JM83 transformed with plasmid pGA1341 was deposited on April 22, 1997, with the accession no. KCTC 0329BP to the Genetic Bank (KCTC), an affiliated biotechnology research institute of Korea Institute of Science and Technology.

Figure 4 shows the nucleotide sequence of the flowering period regulatory gene OsMADS6 and the amino acid sequence of the protein encoded therefrom. In Figure 4, the number on the left indicates the position of the nucleotide sequence, the number on the right indicates the position of the amino acid sequence, and the two underlined sites show the MADS box and K box regions having high homology with other flowering time control genes.

(Step 2) Preparation of Expression Vector and Transgenic Plant

An expression vector pGA1349 containing an OsMADS6 gene was prepared by inserting OsMADS6 cDNA obtained in step 1 between the CaMV 35S promoter (P35S) and terminator T7 of vector pGA748 using the restriction enzyme EcoRI in the same manner as in Example 2 protein 2. After that, it was transferred to tobacco.

The seed of the tobacco transformed with pGA1349 and the seed of the control tobacco plant transformed with pGA748 were sown at the same time, and the flowering time and size were observed while growing under the same conditions until flowering. 5 is a photograph of the tobacco plants (two left plants) and the control (two plants on the right) transformed with the OsMADS6 gene sown and grown at the same time. In the control, the flowers were bloomed only on one side, but the transformants were both bloomed. It can be seen that the size is significantly smaller than the control.

Example 3

Isolation of OsMADS7 Gene and Rice Transformation Regulators

(Step 1) Gene Isolation and Sequence Analysis

OsMADS1 cDNA was probed in the same manner as in Step 1 of Example 1 to search for cDNA libraries of rice flowers to obtain the plasmid pGA1342 into which the flowering time regulation gene OsMADS7 cDNA was inserted, and then analyzed the nucleotide sequence of the gene. Amino acid sequence was estimated. E. coli JM83 transformed with plasmid pGA1342 was deposited on April 22, 1997, with the accession no. KCTC 0329BP to the Genetic Bank of Korea (KCTC).

Figure 6 shows the nucleotide sequence of the flowering time regulation gene OsMADS7 and the amino acid sequence of the protein encoded therefrom. In Figure 7, the number on the left indicates the position of the nucleotide sequence, the number on the right indicates the position of the amino acid sequence, and the two underlined sites show the MADS box and K box regions having high homology with other flowering time control genes.

(Step 2) Preparation of Expression Vector and Transgenic Plant

An expression vector pGA1350 containing an OsMADS7 gene was prepared by inserting OsMADS7 cDNA obtained in step 1 between the CaMV 35S promoter (P35S) and terminator T7 of vector pGA748 using the restriction enzyme EcoRI in the same manner as protein 2 of Example 1. After that, it was transferred to tobacco.

The seed of the tobacco transformed with pGA1350 and the seed of the control tobacco plant transformed with pGA750 were sown at the same time, and the flowering time and size were observed while growing under the same conditions until flowering. 7 is a photograph of the tobacco plants (two left plants) and the control (two plants on the right) transformed with the OsMADS7 gene sown and grown at the same time. Noticeably smaller ones.

Example 4

Isolation and Modified Transformation of OsMADS8, a Rice Flowering Regulator

(Step 1) Gene Isolation and Sequence Analysis

Using the same method as in step 1 of Example 1, OsMADS1 cDNA was used as a probe to search the cDNA library of rice flowers to obtain the plasmid pGA1343 into which the flowering time regulation gene OsMADS8 cDNA was inserted, and then analyzed the nucleotide sequence of the gene. Amino acid sequence was estimated. E. coli JM83 transformed with plasmid pGA1343 was deposited on April 22, 1997, with the accession no. KCTC 0329BP to the Genetic Bank (KCTC), an affiliated biotechnology research institute of Korea Institute of Science and Technology.

Figure 8 shows the nucleotide sequence of the flowering time regulation gene OsMADS8 and the amino acid sequence of the protein encoded therefrom. In FIG. 8, the numbers on the left represent the positions of the nucleotide sequences, and the numbers on the right represent the positions of the amino acid sequences. The two underlined regions represent MADS box and K box regions having high homology with other flowering time control genes.

(Step 2) Preparation of Expression Vector and Transgenic Plant

An expression vector pGA1351 containing the OsMADS8 gene was prepared by inserting OsMADS8 cDNA obtained in step 1 between the CaMV 35S promoter (P35S) and terminator T7 of vector pGA748 using the restriction enzyme EcoRI in the same manner as in step 2 of Example 1. After that, it was transferred to tobacco.

The seed of the tobacco transformed with pGA1351 and the seed of the control tobacco plant transformed with pGA748 were sown at the same time, and the flowering time and size were observed while growing under the same conditions until flowering. 9 is a photograph of the tobacco plants (two left plants) and the control (two plants on the right) transformed with the OsMADS8 gene grown and sown at the same time. Can be.

Example 5

Isolation of Apple's Flowering Timing Gene MdMADS3 and Preparation of Transformant

(Step 1) Gene Isolation and Sequence Analysis

Total RNA was isolated from early apple flowers using guanidium solution and ultracentrifuge, and mRNA was isolated using oligo dT column, and double-stranded cDNA was synthesized using the template to prepare cDNA library. Subsequently, the OsMADS1 gene was labeled with the isotope 32P and the probe was used to search for the apple flower cDNA library to select positive clones.

Hybridization was performed for 18 hours in a 55 ° C., 6 × SSC solution according to Sambrook's method (Sambrook et al., Supra). Recombinant plasmid pGA1528 with cDNA fragments was obtained using the f1 helper phage, R408 (Stratagene, USA) from selected positive clones. E. coli JM83 transformed with plasmid pGA1528 was deposited on April 22, 1997, with the accession no. KCTC 0329BP to the Genetic Bank of Korea (KCTC), Institute of Biotechnology.

The nucleotide sequence of the flowering time control gene MdMADS3 cDNA fragment of apple cloned in recombinant plasmid pGA1528 was described by Sanger et al. (Sanger, F. et., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977). The amino acid sequence was estimated from the nucleotide sequences thus analyzed, and Fig. 10 shows the nucleotide sequence of the flowering time control gene MdMADS3 of apples and the amino acid sequence of the protein encoded therefrom. The position of the sequence, the number on the right, indicates the position of the amino acid sequence, and the two underlined regions represent MADS box and K box regions having high homology with other flowering time control genes.

(Step 2) Preparation of Expression Vector and Transgenic Plant

An expression vector pGA1534 containing the MdMADS3 gene was prepared by inserting the MdMADS3 cDNA obtained in step 1 between the CaMV 35S promoter (P35S) and the terminator T7 of the vector pGA748 using restriction enzyme EcoRI in the same manner as in step 2 of Example 1. After that, it was transferred to the bleach.

The seed of the tobacco transformed with pGA1534 and the seed of the control tobacco plant transformed with pGA748 were sown at the same time, and the flowering time and size were observed while growing under the same conditions. 11 is a photograph of the tobacco plants (two right plants) and the control (two left plants) transformed with the MdMADS3 gene grown and sown at the same time, the flowering of the transformant was faster than the control and confirmed that the size is much smaller Can be.

Example 6

Isolation of Apple's Flowering Timing Gene MdMADS4 and Preparation of Transformant

(Step 1) Gene Isolation and Sequence Analysis

Using the same method as in step 1 of Example 5, OsMADS1 cDNA was used as a probe to search the apple blossom cDNA library to obtain the plasmid pGA1587 into which the flowering time regulation gene MdMADS4 cDNA was inserted, and analyzed the nucleotide sequence of the gene. The sequence was estimated. E. coli JM83 transformed with the plasmid pGA1587 was deposited on April 22, 1997, with the accession no. KCTC 0329BP to the Genetic Bank of Korea (KCTC), Biotechnology Research Institute of Korea Institute of Science and Technology.

Figure 12 shows the nucleotide sequence of the flowering time regulatory gene MdMADS4 and the amino acid sequence of the protein encoded therefrom. In Figure 12, the number on the left indicates the position of the nucleotide sequence, the number on the right indicates the position of the amino acid sequence, and the two underlined portions show the MADS box and K box regions with high homology with other flowering time control genes.

(Step 2) Preparation of Expression Vector and Transgenic Plant

The expression vector pGA1588 containing the MdMADS3 gene was constructed by inserting the MdMADS4 cDNA obtained in step 1 between the CaMV 35S promoter (P35S) and the terminator T7 of the vector pGA748 using the restriction enzyme EcoRI in the same manner as in step 2 of Example 5. After that, it was transferred to tobacco.

The seed of the tobacco transformed with pGA1588 and the seed of the control tobacco plant transformed with pGA748 were sown at the same time, and the flowering time and size were observed while growing under the same conditions until flowering. 13 is a photograph of the tobacco plant (left) and the control (right), to which the OsMADS8 gene was transferred and grown at the same time, which shows that the transformants were more flowering and smaller in size than the control.

Example 7

Preparation of an Expression Vector Containing OsMADS1 and the Transgenic Petunia

Restrict OsMADS1 cDNA (Chung, GM et al, Plant Mol. Biol., 26, 657-665 (1994)) between CaMV 35S promoter (P35S) and terminator T7 of vector pGA748 in the same manner as in step 2 of Example 1 An expression vector pGA1209 containing the OsMADS1 gene was constructed by inserting the enzyme EcoRI. Fig. 14 shows the vector pGA1209 produced as described above. Here, npt represents a kanamycin resistance gene commonly used for selecting transformants of plants, and numbers refer to kilo base pairs, and the total length of the vector is 12.9 kbp.

The expression vectors were then subjected to binary Ti plasmid pAL4404 (Hoekema, A. et al., Nature, 303, 179-181 (1983)) and Agrobacterium tumefacience LBA4404 (Hoekema, A. et al., And petunia, a dicotyledonous plant).

The seed of tobacco transformed with pGA1209 and the seed of control petunia transformed with pGA748 were sown at the same time, and the flowering time and size were observed while growing under the same conditions until flowering. Figure 15 is a picture of petunia (left) and control (right) transformed with the OsMADS1 gene sown and grown at the same time, the transformant showed a marked early flowering and dwarfism compared to the control. This proved that the OsMADS1 gene could act on plants other than tobacco to control flowering time.

Example 8

Expression Vectors Containing OsMADS1 and Preparation of Transgenic Rice

Restrict OsMADS1 cDNA (Chung, GM et al, Plant Mol. Biol., 26, 657-665 (1994)) between CaMV 35S promoter (P35S) and terminator T7 of vector pGA748 in the same manner as in step 2 of Example 1 An expression vector pGA1268 containing the OsMADS1 gene was constructed by inserting the enzyme EcoRI and inserting the shl intron of corn, which increases expression in monocotyledonous plants, between the CaMV 35S promoter and the OsMADS1 gene. Fig. 16 shows the vector pGA1268 produced as described above.

Subsequently, the expression vector was fused with herbicide resistance gene (bar) to transform rice (variety IR36) by electroshock method, and 11 transformation lines selected from PPT herbicides were obtained. As a result of DNA blotting, three lines 3, 6, and 11, in which the transformation of the OsMADS1 gene was confirmed in 11 lines, showed early flowering than the control. The plants in lines 3, 6 and 11 blossomed about 10 days earlier and their height decreased by 10 cm compared to the control. FIG. 17 is a photograph of rice transformed with OsMADS1 gene (b: line 3, c: line 6, and d: line 11) and the control (a), and the transformant showed a significant dwarfity than the control. You can check it. This demonstrates that the OsMADS1 gene can act on both dicotyledonous and monocotyledonous plants to control flowering time and induce dwarfism.

Example 9

Preparation of Tobacco Transformed Maryland Mammoth Transformed with OsMADS1 (Nicotiana tabacum cv. Maryland Mammoth)

In order to find out whether early flowering is possible without affecting the environment of flowering time, for example, photoperiod, by the flowering time regulation gene, first of all, a single plant using the expression vector pGA1209 containing the OsMADS1 gene prepared in Example 7 was used. Tobacco variant Merland mammoths were transformed in the same manner as in Example 7. Plants sensitive to photoperiod are divided into two types: monolithic plants and monolithic plants, which are plants that bloom only when the long cycle is long, and monolithic plants promote flowering only in spring or autumn when the cycle is short.

As described above, the seed of the Maryland mammoth transformed with pGA1209 and the control transformed with pGA748 were sown at the same time in single and long days, and then grown until flowering, and the flowering time and size were observed. 18A and 18B are photographs showing the transformants and controls grown to the flowering period under single conditions and long-term conditions, respectively. In each figure, the left plant is the transformant and the right is the control. As shown here, the transformants showed significant early flowering and dwarfism compared to the control regardless of the work. That is, the Maryland mammoth was a single plant, and the control group did not bloom at all in the long-term condition, but when it was transformed with OsMADS1, it bloomed.

FIG. 19 is a result of analyzing RNA accumulation by extracting RNA accumulated in leaf tissues of three independent Maryland mammoth transformants (columns 1, 2, and 3) and control (column C) by RNA blotting. As shown here, OsMADS1 mRNA was expressed in all three transformants, but not in the control.

Transgenic descendants of six individuals from each of the transgenic buckwheat mammoth lines 1, 2, and 3 and the control line were grown under single conditions (day-10 hours) and long-day conditions (day-16 hours), and at the time of flowering and height after flowering. The average value was obtained by measuring and the results are shown in Table 1.

TABLE 1

From the above results, it can be seen that the transformants showed a significant early flowering and dwarfism compared to the control.

Example 10

Preparation of Nicotiana sylvestris Transformed with OsMADS1

To find out whether early flowering is possible without affecting photoperiod by the flowering time regulation gene, this time, the expression vector pGA1209 containing the OsMADS1 gene produced in Example 7 was used to produce tobacco varieties Nicotiana silvestris ( Nicotiana sylvestris) was transformed in the same manner as in Example 7.

As described above, the seed of Nicotiana silvestris transformed with pGA1209 and the seed of the control transformed with pGA748 were sown under single and long-term conditions, respectively, and grown until flowering, and the flowering time and size were observed. 20A and 20B are photographs showing the transformants and controls grown to the flowering period under single conditions and long-term conditions, respectively. In each figure the left plant is the transformant and the right is the control. As shown here, the transformants showed significant early flowering and dwarfism compared to control. In other words, Nicotiana sylvestris was a long-lived plant, but the control group did not bloom at all under a single condition, whereas when transformed with OsMADS1, it bloomed.

Figure 21 shows the analysis of OsMADS1 mRNA accumulation by extracting RNA accumulated in the leaf tissues of five Nicotiana sylvestris transformants (lines 1, 2, 3, 4, 5) and control (line C) by RNA blotting. One result. As shown here, OsMADS1 mRNA was expressed in all five transformants, of which line 2 and line 4 transformed strongly, line 1 and 5 showed moderate expression, and line 3 transformed insignificantly. Expression was shown. It can be seen that it was not expressed at all in the control.

Transgenic descendants of six individuals from the transgenic Nicotiana sylvestris lines 1 to 5 and control lines were grown in single conditions (day-10 hours) and in long-term conditions (day-16 hours) to increase the flowering period and post-flowering height. The average value was measured and the results are shown in Table 1.

TABLE 2

As can be seen from the above results, the transformants of lines 2 and 4, which had the highest expression levels of OsMADS1 mRNA, bloomed first in 85 and 84 days under single conditions, respectively, and had the smallest height of 35 and 36 cm, respectively. On the other hand, the transformants of lines 1 and 5, which have moderate expression levels, took 102 and 97 days to bloom, and their height was much higher than those of lines 2 and 4. Line 3 with the least amount of expression took 146 days to bloom and bloomed the latest. The control did not bloom under single conditions. Under the long day condition, the transformants bloomed 8-11 days earlier than the control group, and showed a significant dwarfity.

From these results, it was demonstrated that early flowering and dwarfism can be induced regardless of seasons by transforming and expressing the flowering season control gene OsMADS1 in plants that bloom only in certain seasons.

According to the present invention, by transforming flowering period control genes OsMADS5-8 and MdMADS3 and 4 isolated from rice and apple into plants such as tobacco, petunia and rice, early flowering and phenomena of plants without affecting the environment of flowering period Induction can shorten growth period and increase yield.

Claims (27)

OsMADS5 having the sequence of FIG. 1 as a flowering time regulation gene isolated from rice and a gene having at least 80% homology among 30 consecutive bases of any region of the gene. An expression vector comprising the gene of claim 1. The method of claim 2, Expression vector pGA1348. Tobacco transformed with the expression vector of claim 1. OsMADS6 having the sequence of FIG. 4 as a flowering time regulation gene isolated from rice and a gene having at least 80% homology among 30 consecutive bases of any region of the gene. An expression vector comprising the gene of claim 5. The method of claim 6, Expression vector pGA1349. Tobacco transformed with the expression vector of claim 7. OsMADS7 having the sequence of FIG. 6 as a flowering time regulation gene isolated from rice and a gene having at least 80% homology among 30 consecutive bases of any region of the gene. An expression vector comprising the gene of claim 9. The method of claim 10, Expression vector pGA1350. Tobacco transformed with the expression vector of claim 11. OsMADS8 having the sequence of FIG. 8 as a flowering time regulation gene isolated from rice and a gene having at least 80% homology among 30 consecutive bases of any region of the gene. An expression vector comprising the gene of claim 13. The method of claim 14, Expression vector pGA1351. Tobacco transformed with the expression vector of claim 15. MdMADS3 having the sequence of FIG. 10 as an flowering time regulation gene isolated from apples and a gene having at least 80% homology among 30 consecutive bases of any region of the gene. An expression vector comprising the gene of claim 17. The method of claim 18, Expression vector pGA1534. A tobacco transformed with the expression vector of claim 19. MdMADS4 having the sequence of FIG. 12 as an flowering time control gene isolated from apples and a gene having at least 80% homology among 30 consecutive bases of any region of the gene. An expression vector comprising the gene of claim 21. The method of claim 22, Expression vector pGA1588. A tobacco transformed with the expression vector of claim 23. Petunia transformed with the vector pGA1209. Expression vector pGA1268 containing flowering time regulator gene OsMADS1. A rice transformed with the expression vector of claim 26.