KR20040110944A - Expansin 5 protein of Arabidopsis thaliana, transgenic plant thereof and its use - Google Patents

Abstract

PURPOSE: An expansin 5 protein(AtEXP5) of Arabidopsis thaliana, and a method for activating and facilitating the length growth and differentiation of plants using the same are provided. The AtEXP5 facilitates the growth and differentiation of plants. CONSTITUTION: The recessive mutant Arabidopsis thaliana to expansin 5 protein(AtEXP5) showing insensitivity to brassinosteroid(BRs) is genetically manipulated to activate the expression of AtEXP5. The AtEXP5 has the amino acid sequence of SEQ ID NO:1, and participates in the rooting of plants and stem growth. A gene encoding the AtEXP5 has the nucleotide sequence of SEQ ID NO:2.

Description

Expansin 5 protein of Arabidopsis thaliana, transgenic plant etc. and its use

The present invention relates to a protein involved in plant growth, and more particularly, to a plant growth protein expand 5 (AtEXP5) of the Arabidopsis plant, genes and plant transformants encoding the same.

Expandin is a protein present in the cell wall that mediates cell wall renal activity in a pH-dependent manner. In 1992, S. McQueen-Mason et al., Plant Cell , 4 , pp1425-1433, 1992, It was first isolated as a protein that promotes elongation of cell walls under acidic conditions. This protein was found to reversibly break non-covalent (hydrogen bonds) between microfibril and substrate polysaccharides in the cell wall, so that the cell wall matrix is not pectin or xyloglucan. (McQueen-Mason and Cosgrove DJ, Plant Physiol., 107 (1) , pp87-100, 1995). Later, it was found that the activity of expand in the cotyledons of oat cotyledons is large, and the gene structure of the expand is elucidated. Currently, these genes are found in most plants such as cucumbers, Arabidopsis, soybeans, rice, and corn. It is reported that it exists and is very similar in structure (Shcherban et al., Proc Natl Acad Sci US A. , 26; 92 (20) , pp9245-9249, 1995).

Expandin is involved in many different processes, including cell division and elongation. Purified protein was added to the apical meristem of tomato leaves, and the extension of the leaf was promoted, and the expansion of the compound was observed in vivo (Fleming et al., Science , 276 , pp1415). -1418, 1997) Cell changes in apical meristem were also observed by electron microscopy (Fleming et al., Planta , 208 , pp166-174, 1999).

The activity of expandin is regulated by plant hormones. It was found that maximal expression of Expand 2 ( LeExp2 ) was achieved and the gene was regulated by auxin (Catala et al., Plant Physiol. , 122 (2) , pp527-34 ) . , 2000). In the pine hypocotyl, the amount of mRNA of alpha-expandin is increased by 50 to 100 times compared to the area where growth is not occurred in the active area of pine hypocotyl, and also by indole-3-butyric acid, a kind of auxin Increased expression revealed the presence of the expanded gene in the seedlings, which was regulated by auxin, the plant growth hormone (Hutchison et al., Plant Physiol. , 120 (3) , pp827-32, 1999). In addition, the expansion is known to be involved in the germination of plants, maturation of fruit, development of water ducts, and erosive reactions.

To date, many proteins with similar amino acid sequences have been known in peas, corn, rice, and Arabidopsis. Expandin is divided into two families, alpha-expand and beta-expand, and is a multigene family with about tens or more kinds of plant species. It shows organ, tissue, and cell-specific expression patterns. To date, little is known about the specific in vivo function of each expanded protein.

Arabidopsis is known to contain 26 alpha-expanding genes and 3 beta-expanding genes. Antisense mutants have been reported to be involved in the growth of leaf cells (Cho HT and Cosgrove,Proc. Nat'l. Acad. Sci. USA,15; 97 (17), pp9783-8, 2000) Recently, Expandin 7 and 18 have been reported to be involved in the generation of Arabidopsis root hairs (Cho et al.,Plant cell,14 (12), pp 3237-53, 2002). However, since the structures of the expanded proteins are very similar and their mutations are caused to have abnormal function of specific expand proteins due to their functional similarity, there is no clear phenotype. The specific function of the expanded protein of dogs is not known at all.

The inventors of the present invention are proteins that are specifically regulated by brassinosteroids through studies through various physiological and molecular biological methods for related mutants and expand 5 mutants to examine the function of Arabidopsis expand protein. In addition, the present invention was completed by confirming that the development of stem elongation and roots can be enhanced by regulating the expression of the expanded 5 protein.

An object of the present invention is to provide a protein of Arabidopsis 5 (AtEXP5) having the above function by revealing a function related to the growth and differentiation of plants of the expanded 5 protein whose function is unknown.

The present invention also provides a pure thermotropic mutant of the expanded 5 protein to confirm the function of the expanded 5 protein.

Another object of the present invention is to provide a method for promoting plant growth by activating the gene.

Figures 1a to 1e shows that a pure recessive mutation for expand 5 (AtEXP5) from the Arabidopsis mutant group prepared by the insertion of T-DNA, Figure 1a is a gene map of the expand 5, Figure 1b Southern blot result confirming the insertion of T-DNA in the mutant groups AtEXP5-1 and -2, Figure 1c is a view showing the position of the T-DNA inserted into the AtEXP5 of the mutant, Figure 1d in the selected AtEXP5 mutant Northern blot result confirming whether AtEXP5 expression, Figure 1e is a result of RT-PCR confirmed whether AtEXP5 expression in the selected AtEXP5 mutants,

Figure 2 is a diagram observing the phenotype according to the growth date of the AtEXP5 mutant (right) compared to the wild type (left),

3A to 3C show the extent of root length of AtEXP5 mutant is suppressed by brassinolide (BL) and auxin (IAA) compared to wild type (Col-0), and FIG. 3A shows 10 ⁻⁹ M After the treatment of auxin and brassinolide of the roots of the seventh day was shown, respectively, Figure 3b shows the length of the root on the seventh day after the treatment of the brassinolide by concentration, Figure 3c is treated with auxin concentration 7 days after the root length was measured,

Figures 4a to 4d shows the length of the root and the degree of inhibition of root growth according to the treatment of brassinolide and auxin in various BRs-related mutants, Figures 4a and 4b shows BRs insensitive mutants grown for 5-9 days The lengths of the roots were compared with those of each wild type, and FIGS. 4C and 4D show the lengths of the roots at 7 days after treatment with bracinolide and auxin concentrations for the mutations related to the receptors of BRs. ,

FIG. 5 shows that the expression change of AtEXP5 gene was observed by RT-PCR after treatment with auxin and brassinolide in Arabidopsis seedlings grown for 5 days, and treated with ^10-6 M auxin and brassinolide. RT-PCR on AtEXP5 was performed on RNA extracted from the roots and confirmed by Southern blot.

6 is a diagram confirming the expression of AtEXP5 in the roots of various BRs related mutants through RT-PCR and Southern blot,

7 is a diagram confirming the expression of BRI1 and BAK1 genes involved in BRs signaling in the cotyledon and root of the AtEXP5 mutant through RT-PCR and Southern blot,

8 is a result of examining the expression of the other expanded genes having high homology with AtEXP5 in the AtEXP5 mutant,

Figure 9 shows the results of observing the formation of aides after 18 days of treatment with concentrations of auxin in the AtEXP5 mutant after concentration.

In order to achieve the above object, the present invention provides a pure thermotropic mutant plant of AtEXP5 exhibiting an insensitive and abnormal phenotype for brassinosteroids (BRs) for the functional analysis of the Expand 5 protein.

The mutants exhibit small rosette leaves, fast bolting, short peduncles, abnormal stem lengths and phenotypes of hypertrophy.

In the present invention, the function of the expanded 5 gene is lost and the pure recessive mutant plant exhibits the above phenotype, thereby revealing that the function of the expanded 5 gene is related to the growth and differentiation of the unknown plant. Came out.

In another aspect, the present invention provides a method for genetically engineering to activate the expansion and differentiation of plants by genetic engineering to activate the expression of the Expand 5 gene in the plant.

Plants that can be activated growth by the present invention, food crops, including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, millet; Vegetable crops including arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers including roses, gladiolus, gerbera, carnations, chrysanthemums, lilies, tulips; Or rygrass, red clover, Orchard grass, Alfalfa, Tall fescue, Perennial ryegrass, and the like.

By revealing the function of the Expand 5 protein and gene in the present invention, it can be usefully used to develop overexpressing plants that promote growth and differentiation, control the timing of planting bolting, stem length and hypertrophy, leaves and peduncles It can be used in the method of stretching plants such as the size of the future can be used for increasing the agricultural production of crop plants and the like and phenotypic manipulation of flower plants.

In another aspect, the present invention provides the Arabidopsis IX paenjin 5 (AtEXP5) a protein comprising the amino acid sequence of SEQ ID NO: 1 which is involved in plant developmental processes and enhance root elongation stem cells of Arabidopsis thaliana (Arabidopsis thaliana).

Preferably, the AtEXP5 protein of the present invention is characterized by encoding the gene of Expand 5 ( AtEXP5 ) of the nucleotide sequence of SEQ ID NO: 2.

The expansion 5 protein is enhanced by the expression and activation of the steroidal plant hormone Brasinosteroids (BRs) can enhance the development of the kidneys and roots of the stem of the plant.

Hereinafter, the present invention will be described in more detail.

In the Arabidopsis, where the nucleotide sequence of the whole genome is known, the sequence of the Expand 5 gene is classified as At3g29030 in the Arabidopsis Information Resource Center (TAIR; http://www.arabidopsis.org, accession number 2087027). The Expanded 5 protein produced by expression has no known function in plants.

Among the Arabidopsis mutants induced by T-DNA insertion, we isolated pure pyrogenic mutants with defective T-DNA insertion into the Expand 5 gene, which was due to a defect in the AtEXP5 gene. It has various phenotypes that appear.

Specifically, the atypical mutant of AtEXP5 has a slightly longer root in the seedling stage compared to the wild type, and when grown in pollen, the rosette leaves are smaller than the wild type, and bolting occurs quickly. In addition, the stem length is 60-70% of the wild type, the peduncle is short, while the thickness of the stem is thin, and the phenotype cannot grow vertically.

Rosette refers to a group of leaves radially lined in contact with the surface of the stem shortened to the end of the stem.

Bolting (bolting) refers to the plant to produce a flower stem, and usually occurs after forming the flower buds, it means that the flower buds are already formed, but sometimes there is no flower buds formed. For example, in the cultivation of vegetables such as cabbage, onion, blush, celery and the like, there is an early extraction phenomenon that greatly reduces the value of commodities before the stem, leaves, or roots are sufficiently grown.

Through the above study of the present inventors, it can be confirmed that the expanded 5 protein is involved in stem growth and differentiation.

In particular, the present invention suggests that expression of expand 5 protein in Arabidopsis can be regulated by brassinosteroids (BRs), a steroidal plant hormone, which can exhibit cellular activity and related actions.

In the present invention, the AtEXP5 mutant did not inhibit the growth of roots by treatment with high concentrations of bracinolide, and the root growth inhibition was also insensitive to auxin (Auxin), which indicates that the AtEXP5 protein is BRs or BRs in the roots. It was confirmed that the signal transduction process involved the growth of root length. In addition, AtEXP5 mutants generally exhibit a phenotype that is not well generated by auxin treatment, as opposed to those known to promote lateral root formation of auxins.

In addition, the present invention confirmed that the transcription of Arabidopsis Expand 5 gene, whose function is not known at all, was increased by BRs (BRs up-regulated). In particular, the transcription of Expand 5 in the treatment of BRs in the roots of Arabidopsis or in various BRs related mutants is specifically regulated through BRs or BRs signaling.

The present invention also provides a mutant of AtEXP5 that exhibits insensitivity to BRs and exhibits an abnormal phenotype compared to the wild type.

The present invention also provides a method of genetically engineering to activate the expression of the Expand 5 gene in a plant to promote the growth and differentiation of the plant.

Based on the above contents, the present invention provides a method for elongating plants, such as timing of bolting, stem length and hypertrophy, size of leaves and peduncles, using plants capable of overexpressing AtEXP5 protein.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are illustrative of the present invention, and the content of the present invention is not limited by the Examples.

Reference example. BRs related mutants

In the present invention, various BRs-related mutants were used as donated by Professor Jianming Li of the University of Michigan, USA.

bri1-201 is a mutant with a relatively weak allele among bri1 (BRs-insensitive 1) mutations that do not recognize BRs due to mutations in BRI1 , known as receptors for BRs .

BRI1-GFP is a mutant having a BRI1 that is twice as large as the wild type by transforming a BRI1 promoter and a conjugate that binds the gene to the GFP (Green fluorescent protein) gene to identify the expression location of BRI1 .

35S :: BAK1 is a mutant that induced overexpression of BAK1 protein.

bak1 is a mutant in which BAK1, a protein that interacts with BRI1 on the cell membrane and has a kinase activity that phosphorylates BRI1, has failed.

det2 is a broken BRs deficient mutant of the early biosynthetic enzymes of BRs.

Example. Cultivation of Samples

All Arabidopsis poles used in the experiments of the present invention were cultivated in a 22 ° C. culture chamber in a MS (Murashige-Skoog) plate or in a greenhouse controlled at a cycle of 70% relative humidity and 16 hours light / 8 hours dark condition. On the other hand, for chemical treatment, young baby poles were cultivated in a square Petri dish containing MS culture at 22 ° C. under a 16 hour light condition / 8 hour dark condition cycle. Various tissue parts of the plant were harvested and frozen in liquid nitrogen.

Experimental Example 1. Isolation of pure recessive mutant of AtEXP5

A group of mutants expected to have T-DNA inserted into the AtEXP5 gene of the Salk Institute was used in the present invention from the Arabidopsis Biological Resource Center (ABRC).

1-1. AtEXP5 Mutant Discovery

In order to obtain a mutant having T-DNA inserted only in the AtEXP5 gene in a mutation group known to have an average of 1.5 T-DNA inserted, Southern blot of genomic DNA was performed.

firstAtEXP5Of the left including As a result of comparing and analyzing the restriction map of the base sequence and the inserted T-DNA sequence, the kanamycin resistance gene in the T-DNA sequenceNPTⅡWhen used as a probeEcoR I andHindThe treatment of III was expected to result in bands of 5.57 kb and 5.7 kb, respectively (FIG. 1A).

Among several AtEXP5 mutant groups, genomic DNA extracted from two individuals, AtEXP5-1 and AtEXP5-2 , was respectively cut into Eco RI and Hind III, and genomic DNA Southern blot was performed.

As a result, as shown in FIG. 1B, only one DNA fragment was identified at 5.57 kb and 5.7 kb in both of the irradiated individuals, and it was confirmed that only one T-DNA was inserted into the examined AtEXP5 mutant AtEXP5 gene ( 1b).

In addition, as a result of checking the segregation ratio for kanamycin resistance to screen for pure heat mutants, AtEXP5-1 showed resistance and AtEXP5-2 had approximately 3: 1 ratio of resistant and sensitive individuals. AtEXP5-1 was found to be a pure recessive mutant with one T-DNA inserted, and AtEXP5-2 was finally confirmed to be a hetero mutant with one T-DNA inserted.

In addition, to confirm the exact position of the T-DNA inserted on the AtEXP5 gene from the AtEXP5 mutant obtained above, the primer and SEQ ID NO: 4 for the left border (LB) of the T-DNA described in SEQ ID NO: 3 The polymerase chain reaction was performed using specific primers of AtEXP5 . DNA fragments of about 1.3 kb obtained through the polymerase chain reaction were cloned and then sequenced.

As a result, as shown in Figure 1c, that from the 1034th bp (base pair) of AtEXP5 gene to the position of the T-DNA of the left boundary region of the nucleotide sequence is shown the second intron of AtEXP5 gene (intron) The T-DNA is inserted into I could confirm it.

In the following the present invention experiments were all used up the AtEXP5-1 recessive mutant with purified water for AtEXP5.

1-2. Observation of Gene Expression in AtEXP5 Mutants

To examine whether the AtEXP5 mutant selected in Example 1-1 is actually a mutant that does not express AtEXP5 mRNA, the whole RNA extracted from the wild type Arabidopsis (Col-0) and AtEXP5 mutants purchased from the Arabidopsis BRC by using AtEXP5 cDNA as a probe, Northern blot analysis was performed (Northern blot).

Northern blots showed that AtEXP5 was expressed only in the wild type (FIG. 1D), and overall AtEXP5 expression was low.

In order to confirm more precisely, the expression of AtEXP5 was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) method.

Reverse transcription reaction was carried out using the same amount of RNA extracted from wild-type and mutant Arabidopsis, respectively, using the reverse transcription reaction product and the primers of SEQ ID NOs: 5 and 6, and annealing temperature was 52 ° C., and the 36 cycles were repeated. A chain reaction was performed. On the other hand, using the primer for confirming the expression of UBQ5 described in SEQ ID NO: 7 and 8 confirmed that the same amount of RNA was used in this RT-PCR, the annealing temperature in the PCR reaction was set to 57 ℃.

As a result, the wild type only showed a strong AtEXP5 expression AtEXP5 mutants in the expression of AtEXP5 mRNA does not appear at all (FIG. 1e), there is a body of AtEXP5 mutants screened through the present invention AtEXP5 expression to determine a complete inhibition of the mutated cheim In the future it was used as a loss-of-function mutant for AtEXP5 .

Experimental Example 2 Investigation of Phenotype of AtEXP5 Mutant

In the seedlings stage in which selected AtEXP5 mutants were grown in MS medium, no distinctive phenotypic differences, such as the length of the hypocotyl or epicotyl, were observed when compared to wild-type Arabidopsis (Col-0). Overall, however, root length was slightly longer than wild type.

Potted AtEXP5 mutants had smaller rosette leaves, bolting started much faster, and stem lengths were only 60-70% of wild type compared to wild type. In addition, the number of inflorescence stems was larger than that of the wild type, while the stem was thinner than the wild type, so it could not stand vertically and grow on the floor with time, and the length of the pedicel was slightly shorter than the wild type. 2).

Experimental Example 3 Investigation of Root Growth Inhibition of Auxin and Brassinolide by AtEXP5 Mutant

The Arabidopsis wild-type (Col-0) and AtEXP5 mutants were treated with 10 ^-9 M auxin (IAA, Indole 3-acetic acid, Sigma ) and brassinolide , and then compared with root length growth. Auxins and brassinolides are generally known to inhibit root growth.

In contrast, the AtEXP5 mutant measured root length on day 7 after treatment with 10 ⁻⁹ M of auxin and brassinolide , indicating that the inhibition of root growth was inferior to that of wild type (FIG. 3A).

In order to investigate this in detail, atEXP5 mutants and wild type were treated with auxin and brassinolide by concentration (10 ^-10 to 10 ^-7 M), respectively, and grown for 7 days (4 days after germination), and then the root length was measured. It was. As a result AtEXP5 mutants whereas the growth of the bra ^10-7 also processes the Sino fluoride length of M that is not inhibited by (Figure 3b), is as much as wild-type by a high concentration of auxin length growth was inhibited (Fig. 3c).

It was confirmed that the root of AtEXP5 mutant showed insensitivity to brassinolide and slightly insensitive to auxin.

Experimental Example 4. Investigation of root growth inhibition of auxin and brassinolide against BRs-related mutants

The above results indicate that the activity of Expand 5 in roots is closely related to the signaling process that inhibits root growth of BRs. In order to confirm this in more detail, the BRs-insensitive mutant of the reference example was identified. Root length was measured as subjects.

To investigate root growth of BRs-insensitive mutants, we compared bri1-201 , BRI-GFP , 35S :: BAK1 and bak1 with wild type Col-0 and Ws (obtained from Arabidopsis Biomass Center), respectively. The root length of the seedlings grown in the vertical direction was measured at intervals of 24 hours from 5 to 9 days.

As a result, bri1-201 was slightly shorter in root length and BRI-GFP was slightly longer than wild type. In contrast, bak1 was slightly longer in root length than Ws, while 35S :: BAK1 was slightly shorter than wild type (FIGS. 4A and 4B).

Next, the susceptibility to root growth inhibition was investigated by treating the BRs-insensitive mutants with auxin and brassinolide by concentration (10 ^-10 to 10 ^-7 M).

As a result of examining the root lengths of bri1-201 and BRI-GFP treated with bracinolide by concentration as shown in FIG. 4C, the root length of bri1-201 was not inhibited even by high concentration of bracinolide and the roots of BRI-GFP . The length was similar to that of the wild type, and the increase of BRI expression did not increase the inhibitory activity of root growth by brassinolide. Also, wild - type and BRI-GFP showed similar sensitivity to auxin, but bri1-201 showed a slightly insensitive response to auxin, similar to the root growth of AtEXP5 mutants was slightly less sensitive to auxin than wild type. (FIG. 4D).

Experimental Example 5 Observation of Transcriptional Patterns of AtEXP5 in Brassinolide Treated Wild-type Arabidopsis

In the present invention, the Arabidopsis AtEXP5 was investigated whether its expression was increased by brassinolide . Incubated for 6 hours in a liquid MS (Murashige-Skoog) medium containing auxin and brassinolide in wild-type Arabidopsis cultivars grown for one week , and divided them into cotyledon areas including roots and hypocotyls . Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using specific probes.

As a result of Southern blot of the reaction product, it was confirmed that the expression of AtEXP5 was different by the treatment of auxin and brassinolide in the root as shown in FIG. . On the other hand, AtEXP5 did not show any difference compared to the control in the cotyledon, except that it was slightly increased by auxin.

As a result, it was confirmed that expand 5 mediates the physiological activity of BRs in the root of Arabidopsis.

Experimental Example 6 At the Roots of BRs Related Mutants AtEXP5 Expression of

Reverse transcription-polymerase chain reaction and Southern blot of AtEXP5 were performed to investigate the expression pattern of AtEXP5 in all RNAs extracted from the four BRs-insensitive mutants and the BRs-deficient mutant det2 . ).

The expression level of AtEXP5 in the roots of the bri1-201 and bak1 mutants was significantly reduced, indicating that the expression of AtEXP5 was significantly reduced compared to the wild type Col-0 and Ws. Whereas overexpression of a chain of BRI-GFP and BAK1 35S :: AtEXP5 expression in BAK1 I've found is higher than the bri1-201 bak1 was slightly lower than the wild type. This is because the expression of AtEXP5 in plants is regulated via the BRs signal transduction pathway through the activity of BRI1 and BAK1 . When expression of BRs decreases, signaling of AtEXP5 is inhibited. On the contrary, even if the signal is too large, it inhibits the expression of AtEXP5 to some extent, which means that BRs may regulate the expression of AtEXP5 , that is, there may be a process of negative regulation. Also BRs- deficient mutant eotneunde AtEXP5 indicate the results of testing the expression of mutation research weight AtEXP5 lowest level of expression of the chain in which det2 BRs if the endogenous amount of an appropriate level of activity is not maintained BRs signaling pathways also low in AtEXP 5 Failure to induce expression also means that the expression of AtEXP5 in plants is specifically regulated by the signaling process of BRs.

Experimental Example 7. AtEXP5 In mutants BRI1 and BAK1 Expression of

AtEXP5 targeting mutants In order to compare the expression levels of BRI1 and BAK1 to the wild type derived respectively from the cotyledon region and roots, including hypocotyls of wild-type and AtEXP5 with the full RNA BRI1 and RT for BAK1 - polymerase chain reaction (RT -PCR) was performed.

BRI1 primers were used as described in SEQ ID NOs: 9 and 10, BAK1 was used as described in SEQ ID NOs: 11 and 12, the annealing temperature was carried out at 60 ℃.

As shown in FIG.AtEXP5In both mutantsBRI1andBAK1of Expression patterns were similar. These results confirm that the AtEXP5 mutant's lack of inhibition of root growth against external treatment of brassinolide was not caused by reduced activity of BRs recognition, at least due to reduced expression of BRs receptors. . On the other hand, in the part of the cotyledonBAK1Expression decreased compared to wild type.AtEXP5It is likely to induce phenotypes for aboveground organs represented by mutants.

Experimental Example 8. AtEXP5 Investigating the possibility of overlapping expand function in mutants

It is known that there are 26 alpha-type expand genes known to be involved in the cell elongation of dicotyledonous plants. There are 26 known genes in the Arabidopsis. Each of these functions is still unclear, but there is a high degree of homology between proteins. Its function is also expected to be similar (Cosgrove et al., Plant Cell Physiol. , 43 (12) , pp1436-44. 2002). The breakdown of this multigene family is often unclear due to the functional redundancy caused by the very similar structures between expanded proteins. In other words, even if one gene breaks out of 26 expand genes, other genes with similar functions exist, suggesting that there may be a kind of compensation that replaces the failed gene.

The AtEXP5 mutants also due to the failure of AtEXP5 other extreme paenjin to investigate whether the expression of the gene can vary 26 Extreme paenjin expression of AtEXP5 of the high homology IX gene paenjin AtEXP2,3,6,8,9 the The change was examined in AtEXP5 mutants.

From the total RNA extracted from the root of the AtEXP5 mutant, RT-PCR was performed using specific primers of AtEXP2,3,6,8,9 described in SEQ ID NOS: 13-22.

Also, as shown in Fig. 8, the expression level was slightly reduced in the case of AtEXP2 AtEXP3 has Despite that the slight increase in aspect to the overall compared to the control showed no significant difference in the expression level.

These results confirmed that the phenotype represented by the AtEXP5 mutant isolated in the present invention was not compensated by the overlap of other expand genes.

Experimental Example 9. AtEXP5 Formation of lateral roots in mutants

The effect on the entourage formation of AtEXP5 mutants by auxin treatment was investigated.

AtEXP5 mutants and wild type auxin was treated at different concentrations (10 ^-10 M to 10 ^-7 M) and observed after 10 days, which is generally the time point at which the Arabidopsis entourage begins to form.

Figure 9 was observed after 18 days of growth , the AtEXP5 mutant was not normally treated with auxin was normally formed as aides like wild type. However, when more auxin treatment, wild-type increases the concentration of the auxin from those that increase the formation of the inner circle contrary AtEXP5 mutant the higher the concentration of the auxin development was inhibited in the inner circle.

aux1, auxin-related mutants such as axr4 are only known to the inner circle that is also a high concentration of auxin treatment is not formed (Bennett et al, Science, 16 ;. 273 (5277), pp948-50 1996;.. Boerjan et al, Plant Cell , 7 (9) , pp1405-19, 1995; Hobbie and Estelle, Plant J. 7 (2) , pp211-20, 1995) They do not form aides even when untreated with auxin, Because of its strong resistance to auxin, this effect of AtEXP5 mutants was clearly distinguished from auxin-related mutants.

The effect of the present invention is that the Arabidopsis AtEXP5 protein mediates cell growth and related activity of BRs that regulate plant growth and differentiation, thereby providing a physiological activity mechanism through the signaling system of BRs. In addition, the expression of AtEXP5 protein can be manipulated through genetic engineering to be used to develop overexpressing plants that promote the growth and differentiation of plants regulated by BRs, which will increase the agricultural production of crop plants and phenotype manipulation of flower plants. Or the like.

<110> KIM, SEONG KI <120> Arabidopsis thaliana Expansin 5 protein, transgenic plant          and its use <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 255 <212> PRT <213> Arabidopsis thaliana <220> <221> PEPTIDE <222> (1) .. (255) Expansin 5 protein amino acid sequence <400> 1 Met Gly Val Leu Val Ile Ser Leu Leu Val Val His Leu Leu Ala Phe   1 5 10 15 Ser Val Cys Val Gln Gly Gly Tyr Arg Arg Gly Gly His His Pro Gly              20 25 30 Gly His Met Gly Pro Trp Ile Asn Ala His Ala Thr Phe Tyr Gly Gly          35 40 45 Gly Asp Ala Ser Gly Thr Met Gly Gly Ala Cys Gly Tyr Gly Asn Leu      50 55 60 Tyr Ser Gln Gly Tyr Gly Leu Glu Thr Ala Ala Leu Ser Thr Ala Leu  65 70 75 80 Phe Asp Gln Gly Leu Ser Cys Gly Ala Cys Phe Glu Leu Met Cys Val                  85 90 95 Asn Asp Pro Gln Trp Cys Ile Lys Gly Arg Ser Ile Val Val Thr Ala             100 105 110 Thr Asn Phe Cys Pro Pro Gly Gly Ala Cys Asp Pro Pro Asn His His         115 120 125 Phe Asp Leu Ser Gln Pro Ile Tyr Glu Lys Ile Ala Leu Tyr Lys Ser     130 135 140 Gly Ile Ile Pro Val Met Tyr Arg Arg Val Arg Cys Lys Arg Ser Gly 145 150 155 160 Gly Ile Arg Phe Thr Ile Asn Gly His Ser Tyr Phe Asn Leu Val Leu                 165 170 175 Val Thr Asn Val Gly Gly Ala Gly Asp Val His Ser Val Ser Met Lys             180 185 190 Gly Ser Arg Thr Lys Trp Gln Leu Met Ser Arg Asn Trp Gly Gln Asn         195 200 205 Trp Gln Ser Asn Ser Tyr Leu Asn Gly Gln Ser Leu Ser Phe Val Val     210 215 220 Thr Thr Ser Asp Arg Arg Ser Val Val Ser Phe Asn Val Ala Pro Pro 225 230 235 240 Thr Trp Ser Phe Gly Gln Thr Tyr Thr Gly Gly Gln Phe Arg Tyr                 245 250 255 <210> 2 <211> 2051 <212> DNA <213> Arabidopsis thaliana <220> <221> 5'UTR (222) (1) .. (291) <220> <221> exon (222) (1) .. (456) <220> <221> intron <222> (457) .. (808) <220> <221> exon (809) .. (1100) <220> <221> intron (1101) .. (1511) <220> <221> exon (222) (1512) .. (2051) <220> <221> 3'UTR (222) (1821) .. (2051) <400> 2 ttaacattca ccttcctttt tgattttcgt cggttctaca tgttaaaccc ttcaaaaatt 60 aaattataat aagtatagtg aagaccaata agcagttgca tggaccctga ttgattcatt 120 accgctaaaa ctttgtctga atccttcttc ctatttatac cactctcttc ttcttctcca 180 gtttcatcac accaaacttc tcacatctcc tcattctgta ttttgacaaa aaaaaaacac 240 cctttgtttt ttctctcctt tgcatgttgt gattgttttt gcttcacaac atgggagttt 300 tagtaatctc gcttctcgtg gttcatctcc tagctttttc cgtctgcgtt cagggcggct 360 accgtcgtgg cggacaccat cccggcggtc acatgggacc ttggatcaac gctcatgcca 420 ctttttacgg cggcggtgat gcttccggca ctatgggtac gcttagcgat aacttcaata 480 atcattttca cttgcatcac cttaatttaa ctaacaaagg gtagtttagt catttcagtt 540 tttcagagct agctttctat taacatgcaa cattgtcaac ggccaatgag aaatatattt 600 attaatgtct caattgttcc gaatgttaat tgttatatat gattaaaaat tacagacttt 660 tgtgatataa acatgcattt tttatttaat ttcatacaaa ttctactgtt taaaacaata 720 aaaataaata caaacgcaaa catataatct aggacctaat tttttgttaa tgattttgaa 780 cattgatctg cctaatgaac taaattaggt ggagcatgcg ggtacggcaa tctgtatagc 840 caaggttatg gactggagac ggcagcgctg agcacagcgt tattcgacca aggacttagt 900 tgtggcgcat gttttgagct gatgtgtgtc aacgatcctc aatggtgcat aaaaggccga 960 tccattgtgg tcactgccac taacttttgt cctcctggtg gtgcatgcga ccctcccaac 1020 caccatttcg atctttctca gccgatctac gagaaaattg ctctctacaa atccggtatc 1080 attcccgtta tgtatagaag gtacaaaatt tactaaccct gtttagaacc ttcacaatgg 1140 tgttatttaa tggtatgcac agaactgaac tatagtgtat aatctatacg aaggcaaagt 1200 aagatttctt cttaccttcc ttagttattg tttctgactt ttccatagaa catatgtgtc 1260 gaaaacacac aaaagccgcg tatagtgaaa catttataat cagcttttgg tcctataaat 1320 acgcatttaa tacttttgga ccagttgata catgtggtag aaataaggta ttaaattatt 1380 taataggccc aaaactgaat aatagtgaca taccaaactt gctagtggtg ctttctcatt 1440 acccctattt cataaatatt gaaaactttc ataacatata gtttttattt aaataatttt 1500 gtgttatata gggttcggtg caagagaagt ggtgggataa ggttcacgat caacggccac 1560 tcatacttta acttggtgtt ggtcacaaac gtgggtggag ccggggacgt acactcggtc 1620 tcaatgaaag ggtcgaggac aaaatggcaa ttaatgtcga gaaattgggg gcaaaattgg 1680 caaagcaact cttatctcaa tggtcaaagt ctgtcgtttg ttgttaccac aagtgatcgt 1740 cgaagtgtcg tctcttttaa tgttgcacca cccacttggt cctttggcca gacctacacc 1800 ggagggcagt ttcggtatta attatatata atcggttcgg tttgttattg tgcttttttt 1860 ttttttttcg gttccaagtt ggggactctt ttttggtaac caattgagat tgggactgat 1920 agtgatatgt agcgaatata tgtgtgggaa cgagaaaaag agaagtttta ttaattctcg 1980 ttttataata taagaaaatg aaagtgatgt tatgtatttt gctttttaat taatttgagt 2040 actaagtggt g 2051 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> T-DNA left border primer <400> 3 ctttgacgtt ggagtccacg ttctttaata 30 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Expansin5 gene primer <400> 4 atgggagttt tagtaatc 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Expansin5 RT-PCR primer1 <400> 5 atgggagttt tagtaatc 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Expansin5 RT-PCR primer2 <400> 6 cccataacat cactttca 18 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> UBQ5 primer1 <400> 7 gaccataacc cttgaggttg aatc 24 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> 223 UBQ5 primer2 <400> 8 agagagaaag agaaggatcg atc 23 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BTI1 primer1 <400> 9 tagagagatg aggaagagac ggagaaa 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BRI1 primer2 <400> 10 gggtcataat tttccttcag gaacttc 27 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> BAK1 primer1 <400> 11 ggaacgaaga ttaatgatcc cttgct 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> BAK1 primer2 <400> 12 atattatctt ggacccgagg ggtatt 26 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Expansin2 primer1 <400> 13 catgaatctt acagaatatt c 21 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Expansin2 primer2 <400> 14 cctgtttcat tgtatttctt tc 22 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Expansin3 primer1 <400> 15 ttatgacggc gactgcgggg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Expansin3 primer2 <400> 16 attacacggt ccattcgact 20 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Expansin6 primer1 <400> 17 ggaaactcat ctatcaat 18 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Expansin6 primer2 <400> 18 caatatggag ttctacaag 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Expansin8 primer 1 <400> 19 atgtacactc catcatactt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Expansin8 primer2 <400> 20 cttgcttttt tgtaggcaca 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Expansin9 primer1 <400> 21 gaatatgaaa aagagtcgtg tg 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Expansin9 primer2 <400> 22 atcataacgt aaaagagagc tg 22

Claims (5)

A pure recessive mutant plant for the Expand 5 gene that is insensitive to brassinosteroids. Genetic engineering to activate the expression of the Expand 5 gene in plants to activate and promote plant growth and differentiation. The plant of claim 2, wherein the plant comprises: food crops, including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, sorghum; Vegetable crops including arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; Or at least one selected from fodder crops, including lygragrass, redclover, orchardgrass, alfalfa, tolsquesco, perennial lygragrass. An expression 5 protein, which is activated by brassinosteroids, is involved in plant root development and stem elongation, and has an amino acid sequence of SEQ ID NO: 1. The protein of claim 4, wherein the Expand 5 protein is encoded by the nucleotide sequence of SEQ ID NO: 2. 6.