KR100319395B1 - Arabidopsis GIGANTEA Gene - Google Patents

Abstract

The present invention relates to a gene for gyogenia (GIGANTEA, hereinafter abbreviated as 'GI') of the Arabidopsis thaliana involved in regulating the biological clock and the flowering period, and a method for separating the same. Flowering of other organisms by providing a Zygenthia gene comprising the nucleotide sequence of SEQ ID NO: 1 and a mutant thereof separated by a Map-Based Cloning Method using the Quiering 1 gene (TH1) It can be used as a probe for exploring timing control genes and also useful for improving biological clock-related traits.

Description

Arabidopsis GIGANTEA Gene

The present invention relates to a GI gene of Arabidopsis thaliana and its isolation method, which are involved in biological clock and flowering time regulation, and specifically, a map-based cloning using thiamine liqueuring 1 gene ( TH1 ) of Arabidopsis thaliana. Isolated by the Map-Based Cloning Method, by providing a GI gene comprising the nucleotide sequence of SEQ ID NO: 1 and a mutant thereof, it can be used as a probe to search for flowering time control genes of other organisms. It can also be usefully used for improvement.

The flowering period of a plant depends largely on temperature or length, or both conditions.The length of the plant is usually a long-day plant that blooms under long-term conditions, a single plant that blooms under a single condition, or a mid-day plant that is unaffected by the sun. These flowering characteristics are largely divided and are fundamentally under the control of several genes (Yaron Y. Levy and Caroline Dean (1998) The Plant Cell, Vol. 10, 1973-1989).

One who carried the mutation induced by X- ray irradiation and intended for Arabidopsis in the early 1960s, the growing season is two times more nutrition than the wild-type elongated body is found in late autumn temperate mutation was known the existence of the GI gene. Due to the increasing number of leaves and the size of the leaves due to long nutrient maturation, the gene was named 'GIGANTEA' from 'GIGES' which means 'large' in Latin (Redei, GP (1962). ) Genetics, 47, 443-460. The most characteristic feature of the mutant is that it is full-length in long-day conditions, which show the same flowering period in single and long-term conditions, so that the function of this gene is to recognize the long-term and determine the flowering period. It is believed that mutations in M.A.M. immediately resulted in significant damage to the cognitive mechanisms of the plants, and the damage to plants' sensitivities was destroyed by this injury (Takashi Araki and Yoshibumi Komeda (1993) The Plant Journal, 3 (2), 231-). 239).

In crops, flowering time has an important meaning. For example, in the case of leafy vegetables, the value of commodities decreases rapidly due to rapid aging of leaves after flowering. In cereals, varieties were divided into early, middle and late varieties based on the developmental time from seeding to flowering. For these reasons, flowering time has been an important subject of classical breeding.

In classical breeding, the breeding method uses breeding breeding in most cases, and since it is impossible to introduce only one or two specific genes into a desired crop, a breeding group that does not need to be reused to leave only the desired genetic characteristics after breeding is impossible. The disadvantage is that it usually takes a long time and effort from 5 to 20 years to remove and fix the desired gene. In addition, these fixed varieties often cause problems after dissemination due to disease resistance or other weak features that were not considered in the breeding process.

Breeding at flowering time is also achieved by conventional breeding methods of existing practices, so problems such as long-term effort and instability of varieties remain. However, in recent years, the development of genetic engineering has made it possible to isolate genes related to flowering time and use them for breeding through appropriate genetic manipulation stages, thereby creating new varieties with artificially adjusted or controlled flowering time. Ove Nilsson and detlef Weigel (1997) Current Opinion Biotechnology 8, 195-199).

This requires the first available genes, which can be isolated by applying the latest molecular and biological techniques. Recently, the map-based cloning method has been widely used as a new gene separation method, which can easily find a known gene or a new gene near a molecular label by using a genetic map and a physical map in parallel.

The present inventors have conducted research to isolate a new gene that can control flowering time in plants. As a result, thiamine requiring I (abbreviated as ' TH1 ') in the Arabidopsis is used as a probe. The present invention has been completed by isolating nearby GI genes and revealing that they are involved in flowering time regulation and biological clock.

An object of the present invention to provide a gene for controlling the biological clock and flowering time in Arabidopsis and a method for isolating the same.

Figure 1 shows the results of Southern analysis in the Arabidopsis Ecological Columbia (Columbia).

Figure 2 shows the results of Northern analysis of RNA according to the organ stage, developmental stage in Arabidopsis Ecological Columbia.

Figure 3 shows the linked physical map of three BAC clones obtained by BAC library screening, the location of the GI gene of the present invention and the size and number of exons.

Figure 4 shows the results of performing RFLP analysis of gi -l and gi -2 plant DNA of wild type Arabidopsis and late autumn mutants.

Figure 5 is the analysis of the vertical movement of the leaves, the biological clock phenomenon in the wild type and gi -1 and gi -2 plants.

Figure 6 shows a portion of the mutations in the wild type and the proteins of gi -1 and gi -2.

Figure 7a shows the expression patterns of the GI , CCAI and LHY genes of the Arabidopsis in the bright state continued after the wild-type and gi -1 and gi -2 seedlings, and FIG. It shows the expression pattern of GI , CCAI and LHY gene in.

In order to achieve the above object, the present invention provides a GI gene and its mutant of Arabidopsis including the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a Arabidopsis GI protein or a mutant thereof comprising the amino acid sequence of SEQ ID NO: 2.

In this case, the GI gene and protein are used as a probe when separating the flowering time regulation gene from organisms other than Arabidopsis, and are used to improve physiological clock-related traits.

The present invention also provides a method for separating the GI gene by the map-based cloning method using Arabidopsis TH1 gene.

Hereinafter, the present invention will be described in detail.

The present invention provides a gene for controlling the flowering time in plants.

In the present invention, in order to isolate genes that regulate the flowering time, we studied the Arabidopsis, which is well studied genetically. Arabidopsis has complete genetic and physical maps of five chromosomes.

In order to isolate genes that regulate flowering time from Arabidopsis, the present invention uses a conventional 'map-based cloning' method using a bacterial artificial chromosome (abbreviated as 'BAC') library. Used. The method requires the presence of an existing molecular label or cloned known gene near the gene to be newly searched for.

In the GI case, there is a molecular label called M235 and a gene called TH1, a gene that makes TMP-PPase, a major enzyme in the thiamin biosynthetic pathway (Lijsebettens MV et al. (1994) EMBO J. 13, 3378-3388). However, M235 is located at a relatively long distance, which can be more than 100 Kb away from the physical map, and cannot be directly used for cloning of GI . The TH1 gene is located at the same location on the GI gene and the genetic map, so there are two genes within 200 Kb on the physical map. It was expected. In addition, the TH1 gene was cloned by the inventors for the first time in the world in 1998 (Yang Suk Kim et al. (1998) Plant Mol. Biol. 37, 955-966), and the TH1 gene was used as a probe.

First, Escherichia coli carrying T23L3 (PMB, Patent) plasmid, a BAC clone having the Arabidopsis TH1 gene obtained by a known method, was cultured in a liquid medium under normal conditions, and then plasmids were separated by a conventional plasmid separation method. The isolated plasmid was then used to search for other clones close to the T23L3 clone.

To find BAC clones close to T23L3, a BAC filter was screened by conventional library screening methods to separate two new clones of T14K14 and T22J18. The three clones were linked as shown in FIG. 3, and based on this, each clone was probed for restriction fragment length polymorphism (RFLP) between wild type and mutants.

In the RFLP, the DNA isolated from the Columbia, gi- 1, and gi- 2 plants by conventional methods was cut using various restriction enzymes, and then developed on a gel using a conventional plate agarose electrophoresis method. Conventional Southern assays were performed to confirm the differences in cleaved patterns on wild-type and mutant DNA.

In the present invention, about 20 kinds of 6-base restriction enzymes were used, and the DNA of three previously obtained BAC clones was directly labeled with radioactivity. Using T22J18 as a probe, two bands disappeared and a new band appeared in the lane where the DNA of the gi- 1 plant was digested with Sut I restriction enzyme. Therefore, these two bands were cloned using a conventional molecular biological method and sequence determined according to a conventional method. As a result, it was found that the function of T22J18 is a new gene whose GI gene is unknown.

In order to finally determine whether there are any abnormalities in the actual nucleotide sequence of the gene in gi -1 and gi -2 mutants, PCR is performed according to LA PCR method (Takara Co., Japan), respectively, and the entire base according to a conventional method. As a result of analyzing the sequence, 5 bases were lost in the C-terminal region in gi -1, and 7 bases were lost in amino acid position 142 in gi -2 (see FIG. 6).

In order to determine the nucleotide sequence of the GI gene of the present invention isolated and identified by the above process, the cDNA library is screened according to a conventional method to obtain a full-length cDNA of the gene, and then the sequence is determined by a conventional nucleotide sequence determination method. Decided.

As a result, the gene of the present invention is a GI gene of the Arabidopsis including the nucleotide sequence of SEQ ID NO: 1, the size of the open reading frame is 3522bp, the gene is known in all known gene bank (GeneBank) It did not show homology with any gene. In addition, Southern analysis confirmed that only one gene exists in the Arabidopsis (see FIG. 1).

The amino acid sequence of the protein encoded by the GI gene of the present invention deduced from the base sequence of the open reading frame is shown in SEQ ID NO: 2.

That is, the Arabidopsis GI protein expressed from the gene is composed of 1173 amino acids and has a molecular weight of 127 kDa, and is a computer structure prediction program (TMpred; http://www.isrec.isb-sib.ch/software/TMPRED_form. html) has been identified as a membrane protein with six transmembrane domains.

In addition, the base sequence and the amino acid sequence of the GI mutant showed that 5 bases were lost in the C-terminal region in gi- 1, and 171 amino acids were lost in the C-terminus due to the generation of termination codons. In the case of gi -2, 7 bases were lost at amino acid position 142. As a result, the new frame shifted 16 new amino acids to the gi -2 protein, resulting in the final 158 short proteins.

According to well-known Northern analysis method of total RNA by organ and development stage of the Arabidopsis Eco-type Columbia, GI gene was expressed in root, leaf, cultivar leaf stem and flower. -5, 9-10, flowering (bolting; 5 cm long), full aging (Full bolting), etc., expressed in all genes throughout the Arabidopsis of the Arabidopsis was confirmed in all the organs (Fig. 2) Reference).

In order to examine the function of the GI gene of the present invention in detail, first, a cDNA library was screened according to a conventional method to secure a full-length cDNA of the gene, and a study on various biological clock-related phenomena was performed using a probe. At the end, it was confirmed that this gene is related to the biological clock and is also involved in determining the flowering time of sun-sensitive plants.

In addition, the destruction of the sensitivity to the work, the most characteristic of the GI gene was found to be due to the abnormality of the biological clock known as the work recognition mechanism. In other words, the gene is directly related to the biological clock, and it has been found that other flowering periods are already under the biological clock. This gene is one of the major genes related to the biological clock in the present invention, and it is also found that the gene is involved in the determination of flowering time at the same time.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Arabidopsis BAC Library Screening

Filters made from Arabesque's BAC library were distributed from the Ohiodopsis Biological Resource Center. The screening was performed according to the conventional library screening method of the filter to obtain two consecutive clones of T14K14 and T22J18 (see FIG. 3).

Example 2. RFLP Analysis

Wild-type plants isolated from Columbia plants and mutants, gi- 1 and gi- 2 plants, were isolated by conventional methods (Dellaporta et al. (1985) Maize DNA miniprep.In Molecular Biology of Plants, A Laboratory manual.pp 36). Each 3 ug of DNA restriction enzymes Aat II, Ava I, Bam HI, Cla I, Dra I, Eco RV, Hind III, Hpa I, Kpn I, Nco I, Nhe I, Nsi I, Pst I, Sac I, Sac II, Sal I, Sca I, Spe I, Sph I, Stu I, Xba I, Xho I, cut using a conventional plate agarose electrophoresis method and spread on the gel, conventional nylon filter The developed DNA was transferred by a conventional 'vacume blot' method and then completely fixed to the filter using a UV cross linker.

5 ug of DNA of each BAC clone was digested with Bam HI at a final volume of 100 ul, and 1 ug of DNA was then labeled with radioactive P32-dCTP (Amershan, UK) via conventional 'Nick translation'. Used.

Each labeled BAC clone DNA was used to hybridize with DNA on a pre-prepared nylon filter. At the time of hybridization, the temperature of the hybridization bath was adjusted to 65 ° C. and shaken slowly for about 15 hours.

The actual RFLP pattern analysis was performed by Southern analysis to confirm the difference of the cleaved pattern on wild type and mutant DNA. In the present invention, about 20 kinds of 6 nucleotide restriction enzymes were used, and when T22J18 was used as a probe, the 7.1 kb and 1.1 kb bands disappeared from the lane where the stu I restriction enzyme was cut from the DNA of gi- 1 plant, instead of 8.2 kb. A new band of appeared (see FIG. 4). This means that there is an error in the Stu I cleavage positions of 7.1 kb and 1.1 kb. Two fraud bands were recovered from the gel by a conventional DNA recovery method.

Each of the two fragments was cloned into a pGEM T-easy vector purchased from Promega of the United States, and T7 and Sp6 primers were used to determine the base sequence according to conventional sequencing methods. It has been found that this is an unknown gene. This portion of the T22J18 clone was identified as about 5 Kb in size.

In order to finally determine whether there are actual sequence abnormalities in gi -1 and gi -2 mutants, PCR was performed on the 5Kb genes according to LA PCR method (Takara Co., Japan), respectively. Cloning into the easy vector and determining the nucleotide sequence of the entire DNA showed that gi- 1 lost 5 bases in the C-terminus and 171 amino acids were lost in the C-terminus by the termination codon. It was. In the case of gi -2, seven bases were lost at the amino acid position 142, and as a result of the frame shift, new 16 amino acids were added to the gi -2 protein to express the final 158 short proteins (see FIG. 6). .

Example 3. GI gene and cDNA acquisition

PCR was carried out using a LAC DNA polymerase from Takara, Japan, using T22J18, a BAC clone, as a template to recover a PCR product of about 9 Kb, which was developed on a conventional agarose gel and subjected to conventional electrolysis. Recovered by Electronlulution. In the same manner, only about 5 kb of gene was recovered and cloned into pGEM T-easy vector. CDNA library of Arabidopsis (M Seki et al. (1998) Plant J, 15,), which was used for cloning and the remaining PCR products were labeled with radioactive methods using a conventional method for preparing molecular probes and distributed from Shinozaki's team in Japan. 707) screened 180,000 phages according to a conventional method to secure the final seven candidates. Analysis was performed according to the conventional molecular biology assay of the obtained clones, six of which had the full-length GI cDNA, and the normal sequencing of the last two different clones was performed. As a result of sequencing, the GI gene was found to be a relatively large gene including 13 exons and about 5 Kb from ATG to TAA. Also, the GI gene was about 9 Kb in size including the 3 kb promoter region and the 1 kb region. It consisted of.

The base sequence of the GI gene recovered by the above method was registered in the Gene Bank under the National Institutes of Health (Registration No. AF105064).

Example 4 GI Gene Characteristics

In previous studies on the flowering period and work, the change of work in the plants affected by work is known to be recognized by the intrinsic biological clock. In particular, the mutant is likely to be a gene related to a biological clock that recognizes the length of the work because it has been severely damaged by the decision to open the book by the work. The present inventors have tried to characterize the GI gene in the wild type and mutant.

In order to confirm whether the gene of the present invention has a regulating function of a biological clock, the movement of leaves up and down in Arabidopsis is best known as a representative physiological phenomenon under the control of the biological clock, and a verification method thereof is also known. It is well known (JA Kreps and SA Kay (1997) The Plant Cell, 9,1235), it was used.

First, wild type, gi -1 and gi -2 mutants are fixed at 23 ° C and sterilized by a known sterilization method. After spraying about 30 pieces and growing them for about 5 days, the video was taken while maintaining the light state for 6 days, and the vertical motion of the leaves was expressed as pixels on the screen. As a result, it was found that the vertical movement of the leaves in both types of mutants was faster than the wild type (see FIG. 6).

In addition, some genes related to biological clock are known to increase or decrease in the amount of expression every 24 hours (JC Dunlap (1999) Cel, 96, 271, Z-Y, Wang and EM Tobin (1998) Cell, 93, 1207) In order to determine whether the gene affects the expression cycle or the amount of these genes, and if the mutant is abnormal in biological clock-related mechanisms, the expression patterns of these genes may differ in the 24-hour cycle or the expression amount. The idea is to use a well-known sterilization method to make seeds of 6 cm in diameter and 1.5 cm high in a general plant incubator that is fixed at 23 ° C. and controlled for 12 hours under light conditions and 12 hours under dark conditions according to well-known verification methods. After aseptic disinfection, sprinkled about 50 each for 2 weeks. Two weeks later, the samples were aliquoted and stored at an extremely low temperature of −70 ° C., taking one saar at three hour intervals for four days, while maintaining a bright state to see the expression of various genes related to the biological clock.

After the sampling was completed, the whole RNA was isolated and the RNA expression pattern was observed by 'DOT blot analysis' according to a known method. Overall, if CCAI LHY gene and a gene is slightly shorter in the 24-hour period it had been a little longer gi -1 pattern, gi -2.

In addition, the GI gene was under the control of the biological clock, and the expression level was also regulated at a 24-hour cycle. However, it was confirmed that the expression amount was significantly reduced compared to wild type, which indicates that the abnormality of the GI itself gene in both gi -1 and gi -2 leads to abnormality of other biological clock-related genes. At the same time, it is to confirm that the gene is related to a new mechanism that is not known at all to the present time, which shows an abnormality in its expression as an inverse regulation by abnormalities of the biological clock-related genes (see FIG. 7A).

Based on the above results, the present inventors found that the gene of the present invention is involved in the biological clock, in order to determine whether the gene is a constituent that constitutes the actual clock, the temperature is fixed at 23 ° C. and 12 hours are bright for 12 hours. In a general plant incubator controlled in the dark state, the seeds of 6 cm in diameter and 1.5 cm in height were sterilized according to a known sterilization method, and then sprinkled about 50 each for 2 weeks. Two weeks later, samples were aliquoted and stored at cryogenic temperature at -70 ° C while taking one Charare at three-hour intervals for three days while continuing to look at the expression of various genes related to the biological clock. After sampling, the whole RNA was isolated using the Tri reagent of Sigma, USA, and the expression pattern of the RNA was observed by dot blot analysis according to a known method. It was confirmed that only a slight abnormality appeared in the expression pattern (see FIG. 7B).

If this gene is a component of the clock, the abnormality should appear regardless of the continuous light or dark cycle. In the present invention, the abnormality of the cancer state appears to be much smaller than that of the bright state. Rather, it turned out to be a gene that transmits cognitive signals in the light state to the clock.

When we infer the function of the gene according to the function of the null mutant of the gi -2 mutant, the function of the gene maintains the correct amplitude and the length of the period in the bright state in the expression of the biological clock gene. It seems to be a gene involved in delivering the work signal needed for the clock.

Therefore, this gene was finally identified as a biological clock-related gene known as the mechanism of the work of plants sensitive to the flowering time.

The GI gene of the Arabidopsis of the present invention is a gene for flowering regulation and a biological clock, which is first cloned in plants, and includes living bodies such as hypocotyl length, glucose metabolism control, production of hormones such as gibberellin and ethylene, and determination of flowering period of a long-acting plant. It may be useful in the use of genetic engineering methods for improving clock-related physiological biochemical phenomena. In particular, artificial flowering time control has a great economic ripple effect and can be applied directly to almost all crops, so its range and usefulness will be infinite.

In addition, the GI gene according to the present invention can be used as a direct molecular probe for cloning a homologous gene in various other organisms, or for the preparation of a PCR probe based on the nucleotide sequence and the amino acid sequence of a protein or a conventional PCR cloning method. Can be.

Claims (5)

Zygenthia gene or mutant thereof, comprising the nucleotide sequence of SEQ ID NO: 1. A gyogentia protein of the Arabidopsis comprising the amino acid sequence of SEQ ID NO: 2 or a mutant thereof. Method for isolating Zygenthia gene by Map-Based Cloning Method using Arabidopsis thiamin liqueuring 1 gene. The gygenthia gene or protein according to claim 1 or 2, which is used as a probe for separating the flowering time regulation gene from organisms other than Arabidopsis. The zigenthia gene or protein according to claim 1 or 2, which is used for ameliorating biological clock-related traits.