KR100722414B1 - Transgenic plants using superoxide dismutase gene of lilium hybrid cultivar marco polo - Google Patents

Abstract

The present invention is Lico Marcopolo Activity in transgenic potatoes prepared using plant transfer vectors recombined so that cDNA is expressed in the forward and reverse directions using Cu / Zn lily-superoxide dismutase (LSOD) genes expressed in scleroderma. The distribution of oxygen species is different, and their effects show opposite growth and tuber formation rates, and have a great effect on the vitrification rate during in-flight culture.

The Cu / Zn LSOD gene of the present invention increases the H ₂ O ₂ content and decreases the superoxide radical content in the plant when overexpressed, which inhibits growth in potatoes, resulting in a very good initial tuberization rate while shortening the length between the shoot and the node. By introducing a gene encoding the same into a plant cell, the development of a crude plant that can shorten the growth period and the reverse expression thereof can provide a potato plant of all species that can increase the growth period. In addition, the vitrification rate, which is a problem during mass cultivation in the cabin, can be drastically reduced, thereby increasing the purifying rate of aseptic seedlings and lowering the mortality rate during sowing of the in-flight plants, and can provide a transformed potato plant that is resistant to excessive moisture stress environment. .

Superoxide Dismutase, LSOD, Potato, Transgenic Potato Plant

Description

Transgenic plants using superoxide dismutase gene of Lilium hybrid cultivar Marco Polo}

Figure 1 shows the amino acid sequence of lily Marco Polo Cu / Zn lily-superoxide dismutase (LSOD) gene, rice, spinach, coastal pine ( Pinus pinaster ) , tomato (A) and schematic (B) compared to Cu / Zn SODs of Kali-Lily,

Figure 2 is a northern analysis result of the lily Marco Polo Cu / Zn lily-superoxide dismutase (LSOD) gene is expressed during the glomeruloid period,

3 is a schematic diagram of a plant transfer vector recombined so that the Cu / Zn lily-superoxide dismutase (LSOD) gene is expressed in the forward and reverse directions.

Figure 4 A was confirmed through Southern analysis that the Cu / Zn lily-superoxide dismutase (LSOD) gene is introduced into the transgenic plants in transgenic plants, B is introduced Cu / Zn lily-superoxide dismutase introduced in B ) Is a photograph of a Northern analysis showing that genes are overexpressed and inhibited in transgenic plants,

5A and 5B are photographs of growth during inflight and pot cultivation of transgenic plants so that Cu / Zn lily-superoxide dismutase (LSOD) genes are expressed in a sense and an antisense.

FIG. 6A is a diagram illustrating the formation of in vitro tuber tubers and B of tuber formation rate so that Cu / Zn lily-superoxide dismutase (LSOD) genes are expressed in the forward and reverse directions.

7 is a diagram showing the vitrification rate of transgenic plants in-flight culture so that the Cu / Zn lily-superoxide dismutase (LSOD) gene is expressed in the forward and reverse directions.

FIG. 8 is a graph showing the results of comparison of survival rates when transgenic plants are transplanted into pot material after in-flight culture so that Cu / Zn lily-superoxide dismutase (LSOD) genes are expressed in a sense and an antisense.

The present invention is a trait prepared using a plant transfer vector recombined so that cDNA is expressed in the forward and reverse directions using Cu / Zn lily-superoxide dismutase (LSOD) genes expressed in the formation of sperm in lily Marcopolo scales. The distribution of reactive oxygen species in transgenic potatoes is different and their effects on the growth and tuber formation rates are opposite to each other, and the effects on vitrification rate during in-flight cultivation.
Under the control of a wide range of influence factors, including growth substances, nutrients, and light, organogenesis or embryonic development, a process of plant tissue development, is complex. Early organogenesis from tissue culture provides a useful system for studying the regulatory mechanisms of plant development (Hicks, GS 1994. In Vitro Cell. Dev. Biol. 30: 10-15.). Much research has been conducted on various tissue culture systems, and information on organ formation has been accumulated. Dedifferentiation and morphogenesis analysis have been performed by physiological experiments and phenotypic examination of mutant deficient mutants. However, the molecular mechanisms responsible for organogenesis are still unknown. Recently, more in-depth research has been conducted on the basics of plant organogenesis at the molecular level, and genes that are thought to play an important role in each cycle of organ formation have been identified through genetic analysis. Already separated (Waites, R., Selvadurai, HR, Oliver, IR, and Hudson, A. 1998. Cell 93: 779-789. Timmermans, MC, Hudson, A., Becraft, PW, and Nelson, T. 1999 Science 284: 151-153. Byrne, ME, Barley, R., Curtis, M., Arroyo, JM, Dunham, M., Hudson, A., and Martienssen, RA 2000. Nature 408: 967-971.Parsons , RL, Behringer, FJ and Medford JI 2000. Planta 211 : 34-42.).
Lilium species belongs to the family Liliaceae , a monocotyledonous plant. 130 native Lilium species and 500 to 600 mutant species are cultivated worldwide. The process of domain formation in lily scales has been the subject of intensive research in large breeding systems. Many researchers have studied the culture conditions for effective domain formation for various lylium scales. The researchers found that the formation and development of the domains were influenced by photoperiod, temperature, physiological stages of explants, nutrients in the medium, and composition of sucrose and hormones (Kim, KW and De Hertogh, AA 1997). Hort. Rev. 18 : 87-165.). Despite much research on the conditions of domain formation, the developers have not yet revealed the molecular mechanism. Cleavage is abiotic stress in living organisms, and lily scale cutting is a common agricultural method for propagation of bulbous plants that favor spontaneous budding. Moreover, the environment in which plant cells grow is a high oxidative stress environment. Thus, plant cell culture or tissue culture is a special system for the production of antioxidants and the study of resistance mechanisms to oxidative stress in plants. Several high activity enzymes, specific proteins and mRNAs have been identified at specific stages before organ development or regeneration of explants. Some researchers are trying to find biochemical markers for glomerular formation (Park, KI, Kim, EY and Kim, KW 1998. J. Kor. Soc. Hort. Sci. 39 : 647-651.) The processes and mechanisms of formation are incompletely understood because there is very little information on the biochemical changes that occur during plant differentiation. Moreover, no molecular genetic studies on the formation of domains have been carried out to date. Therefore, by studying the characteristics of genes expressed during the formation of the locus, it is possible to control the growth and development of the crop if it is confirmed that it affects the development process of the locus formation by human culture.

Accordingly, the present inventors have studied to develop a plant gene that is expected to play an important role in the general regulatory mechanisms for the formation of glomeruli in lily Marcopolo scaly, resulting in the Cu / Zn LSOD from the lily Marcopolo scaly. The coding cDNA was isolated and cloned into the vector. Subsequently, transgenic potato plants expressing forward and reverse Cu / Zn LSODs were prepared using Agrobacterium tumefaciens transformed with this vector, and these potato plants were confirmed to have different distributions of active oxygen species. Potato plants have been confirmed that there is a huge difference in growth and tuber development, and also have a resistance to the vitrification rate and excessive moisture stress environment during in-flight cultivation, to complete the present invention.
Therefore, the object of the present invention is lily Marcopolo Cu / Zn LSOD was expressed in the forward and reverse directions using Agrobacterium tumefaciens transformed with pMBP-S-LSOD and pMBP-AS-LSOD, in which cDNA of Cu / Zn LSOD was cloned in the forward and reverse directions. It is to provide a transgenic plant.
Another object of the present invention is that the transgenic plants expressing the forward Cu / Zn LSOD show a very good initial tuber growth rate, and thus, it is possible to develop early-growth potatoes that can shorten the growth period of potatoes by promoting tuber formation. In the case of flowers, transgenic plants of short-growing and dwarfed varieties are provided.
Another object of the present invention is to transform the potato plants expressing reverse Cu / Zn LSOD to delay tuber formation and to maintain growth for a long time actively to increase the height of the plant or to increase the total starch synthesis yield per area It is to provide a variety of potato plants that can be.
In addition, the transgenic potato plants expressing reverse Cu / Zn LSOD significantly reduced the vitrification rate during tissue culture, which is a problem when in-flight cultivation in the aircraft, thus increasing the purifying rate of aseptic seedlings and lowering the mortality rate when seeding the plants. It is to provide a transgenic plant that is resistant to excessive stress environment.

Hereinafter, the present invention will be described in more detail.
All RNA samples isolated from the scaly shell before and after the formation of the domains of the present invention were subjected to reverse transcription PCR (RT-PCR) using different primer pairs for differential display and separated using polyacrylamide gels. Intensively increasing cDNA bands during glomerular formation were reamplified using the same primer pairs. Rapid amplification of the cDNA ends (RACE) PCR induced complete cDNA sequences. LSOD cDNA fragments were cloned by continuous PCR and 5 μRACE from all RNA isolated from lily scales in the glomerular stage. Complete DNA sequencing of the LSOD showed a protein synthesis command reading frame consisting of a polypeptide encoding a 223 amino acid with a predicted pI of 6.67 and a molecule of 23 kD. NCBI BLAST (Altschul SF, Gish W., Miller W., Myers EW, Lipman DJ 1990. J. Mol. Biol. 215: 403-410.) We compared the sequence homology with SOD of rice, spinach, Pinus pinaster , tomato and Kali-lilies using network service. The Cu / Zn SOD amino acids of lilies have about 70% homology with the genes of other plants (see Figure 1). In order to investigate the expression of genes consistent with LSOD clones, Northern blotting was performed with all RNA isolated at various stages of glomerular formation. Weak expression of LSOD was detected on day 0, increased concentrations were detected on day 3, and strong signals were detected on days 6 and 12. Thereafter it was maintained to a certain degree (see FIG. 2).
The present invention also provides a vector for plant transfer comprising a cDNA of LSOD which can be used to prepare a transgenic potato plant, and a transgenic potato plant transformed with the vector. The binary vector used is a pMBP containing a CaMV and a nos sequence with T-DNA. Cu / Zn SOD sequences of lilies were inserted in the forward and reverse directions with respect to the CaMV promoter. The NPTII gene is also located in T-DNA (see Figure 3). Two reverse-expressing transgenic plants whose expression level was inhibited and four transgenic plants with high overexpression were selected by Northern blot analysis among the lines where the gene introduction was confirmed by PCR. Various copy numbers of T-DNA inserted into potato chromosomes were analyzed by Southern blotting (see Figure 4). Transgenic lines expressing high levels of S-LSOD and AS-LSOD genes were secondary cultured for tuber formation test. During tissue culture of the transgenic potatoes, some differences in tuberculosis or plant morphological characteristics were observed between the untransformed potato control and the transformant.
Selected transgenic lines expressing S-LSOD and AS-LSOD genes show different morphological features and growth patterns during tissue culture. The growth rate during tissue culture under the same environmental conditions showed a significant difference in growth rate in the SOD overexpressing lines compared to the non-transformed potato control, resulting in reduced stature but inhibiting endogenous SOD activity by reverse expression of Cu / Zn SOD. In the transgenic plants, growth was accelerated, and the growth was more intensive than in the non-transformed potato control in the liquid culture in glass bottles. The same result was observed in pot cultivation in glass greenhouse using in-flight tuber. (See FIG. 5) Therefore, the degree of potato growth could be controlled by the regulation of the SOD gene.
To investigate the effect of the introduced SOD gene of lily on the tuber forming ability of the transgenic potato, tuber forming ability experiment was performed with the transgenic potato having forward and reverse expression of the LSOD gene. Tuberification was increased in the SOD overexpression lines but was delayed in the reverse transduction line where SOD expression was inhibited (see FIG. 6). When observed in vitro, the overexpressed forward SOD transduction lines were immediately followed by tuber formation in the flank of the segment segment. In contrast to the results of the forward transduction line, the reverse transduction line preferentially induced the growth of stolon-like slender stems in the flanks, followed by the formation of in vitro tubers after a significant period of time. lost. These results show that overexpressing transformants of lily Cu / Zn SOD genes are more likely to differentiate into tubers than primary growth. It is interesting to note that tubers are delayed in transgenic plants that inhibit endogenous SOD activity by reverse expression of Cu / Zn SOD. Part of the function of the SOD gene is related to the process of tuber hardening in potatoes. Since potatoes and lilies are nutritive plants, they may have a common regulatory mechanism for tuber or locust formation. In the previous results analyzed by Northern blotting at various stages of splenic formation in lilies, it can be inferred that SOD was expressed at the highest level on the 12th day of induction of the spherical spheres, which influenced the spherical sphere formation.
Selected transgenic lines expressing S-LSOD and AS-LSOD genes showed another difference in the degree of vitrification during tissue culture. Vitrification occurs in the in-flight tissue culture of plants, causing the culture plants to lose their vitality and stop growing. This is often due to the increased humidity in the culture vessel and the accumulation of ethylene gas. The degree of vitrification was significantly increased in the SOD overexpressing transgenic lines in the vial containing liquid MS medium 5 days after incubation in the vial containing untransformed potato control, but significantly in the reverse transgenic lines which inhibited SOD expression. (See Figure 7). In the supersaturated state of the water, the reverse gene introduction line is expected to reduce the damage caused by the summer rainy season, which is one of the problems in the cultivation of spring potatoes in Korea. In addition, it was confirmed that the degree of vitrification at the reverse transduction line was increased compared to the non-transformed potato control because of the cultivation of the greenhouse in the tissue house. However, in the SOD overexpression transgenic lines, the vitrification rate was increased and the survival rate after transplantation into the pot was significantly lower than that of the untransformed potato control (see FIG. 8).
Example 1 Preparation of LSOD Forward and Reverse Vectors from Lily Flakes
All RNAs before and after induction of glomerular formation were isolated by CsCl centrifugation and guanidinium thiocyanate (Glisin, V., R. Crkenjko, and C. Byus. 1974. Biochem) 13: 2633.). From this the DD-PCR reaction was carried out. PCR products showing differences from each other were separated, purified, subcloned, and then sequenced. In order to obtain a full length coding sequence, rapid amplification of the cDNA 5 'end was performed using a 5 ′ / 3 ′ RACE kit (Roche Molecular Biochemicals, USA). As in SEQ ID NO: 6, the cDNA sequence of Marco Polo, the finished oriental lily, was registered in NCBI (Genbank Accession Number, AY898945) and the vector was prepared using the same. The 5'-TCTAGATGCAAGCGATTCTCGCCGCC-3 '(SEQ ID NO: 1) and 5'-GGTACCTCAAACAGGAGTCAACCC-3' (SEQ ID NO: 2) primer pairs were used in the forward direction, and the 5'-TCTAGAGCAGTTTTAACAATCCCTC-3 '(SEQ ID NO: 3) and 5 were reversed. A primer pair '-GGTACCGGGTTGATGCAAGCGATTCTCG-3' (SEQ ID NO: 4) was used and cDNA clone was used as a template. At the end of these primers for cloning and put a new restriction enzyme-digit XbaⅠ and KpnⅠ. These PCR fragments were subcloned in a Zeroblunt PCR cloning kit (Invitrogene). Behind the cloning vector with restriction enzymes to cut and place XbaⅠ KpnⅠ, put into the XbaⅠ and LSOD gene fragment in place of KpnⅠ pMBP binary vector. Sequencing confirmed the forward pMBP-S-LSOD vector and the reverse pMBP-AS-LSOD vector.
Example 2 Introduction of Lily SOD Gene into Potato
The binary vector used in this development is pMBP containing CaMV and nos sequence along with T-DNA. Cu / Zn SOD sequences of lilies were inserted in the forward and reverse directions with respect to the CaMV promoter. The NPTII gene is also with T-DNA. Replanted potato shoots are reselected by kanamycin treatment. To demonstrate the transformation lines selected by kanamycin treatment, PCR analysis was performed with chromosomal DNA extracted from transgenic plants. NPT1 and NPT2 primer pairs were used to analyze gene sequences introduced into chromosomal DNA. Two transgenic plants with high inhibition and four transgenic plants with high overexpression were selected by Northern blot analysis on PCR positive lines. Various copy numbers of T-DNA inserted into potato chromosomes were analyzed by Southern blotting. Transgenic lines expressing high levels of S-LSOD and AS-LSOD genes were secondary cultured for tuberification and grown in greenhouses for herbicide methyl biogen (MV) treatment, which is known to cause oxidative stress. During tissue culture of the transgenic potatoes, some differences in tuberculosis or plant morphological characteristics were observed between the untransformed potato control and the transformant.
Example 3 Northern Blotting Analysis of Lily SOD mRNA
The expression of the LSOD gene was examined by Northern blotting analysis using transgenic potato plants and cultivars grown for magnetic domain formation. To examine the expression of the LSOD gene, 25 μg of all RNAs were electrophoretically separated on a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane. The probes used PCR products using 5'-TCTAGATGCAAGCGATTCTCGCCGCC-3 '(SEQ ID NO: 1) and 5'-ATAGTTGCCTCCGCTACTCCTTCAGAG-3' (SEQ ID NO: 5) primer pairs, which were immediately followed by dioxygeninine (DIG) -dUTP (PCR). DIG labeling MIX, BM, Germany). Membranes were hybridized and then detected by the same method as Southern blot analysis.
Example 4 Selection and Analysis of Transgenic Potatoes
The pMBP-S-LSOD vector and pMBP-AS-LSOD vector were transformed into Agrobacterium tumefaciens LBA4404 (Agrobacterium tumefaciens LBA4404) and introduced into potato field varieties by leaf slice transformation. In order to verify the transformation line selected by kanamycin treatment, PCR and Southern blot analysis were performed to determine whether the LSOD gene of chromosomal DNA extracted from the forward and reverse plants was introduced. NPT1 and NPT2 primer pairs were used to detect gene sequences embedded into chromosomal DNA (Choi KH Yang DK Jeon JH Kim, HS Joung YH Joung, H. 1996. Kor. J. Plant Tiss. Cult. 23: 311-315. ). Chromosomal DNA was isolated according to the experimental method for the Southern blot method. Cut with restriction enzyme EcoRI , confirm by electrophoresis and transfer to nylon membrane. Nylon membranes hybridize with probes labeled with DIG. Hybridization was carried out with a hybridization buffer and reacted at 42 ° C. for 16 hours, followed by DIG Easy Hyb. Buffer. The nylon membrane is washed twice with 2 × SSC, 0.1% ((W / V) SDS for 5 minutes at room temperature and then with 0.1 × SSC, 0.1% ((W / V) SDS) for 15 minutes at 68 ° C. DNA was detected using the Roche Moecular Biochemicals DIG Luminescence Detection Kit, the transduction line with high expression of LSOD gene and reverse expression line of endogenous SOD expression were selected by Northern blot analysis and in vitro. Secondary cultures were made for the tuber hardening test.
Example 5 Growth and Tuber Formation of Transgenic Potatoes
In vitro tuberification tests were performed with untransformed potato controls with transgenic lines with high expression levels of the selected LSOD gene and reverse expression lines that inhibited endogenous SOD expression. In the first step, transgenic plants were subcultured in a petri dish with a 16-hour day cycle in a 25 ° C. incubation chamber. In order to examine their growth, in-flight stems of a certain size are collected and incubated in a test tube containing liquid MS medium for about 7 days and then examined for growth. Growth during pot cultivation was planted in a pot using a microtuber produced in-flight from transgenic potatoes and examined in glass greenhouse. Tuber formation rate of each transgenic potato line was collected from MS stems containing 9% of sugar at the end of stem from 4 stems of stems grown in a steady state. Cancer culture at. Ten nodes were healed in one dish and 10 repetitions were performed to examine tuber formation at two-day intervals.
Example 6 Vitrification Rate and Pot Transplantation Rate of Transgenic Potatoes
Calculation of the vitrification rate of transgenic potato was carried out in a single step by subcultured in a petri dish with a 16-hour day cycle in the incubation chamber at 25 ° C. in a step 1, and then collected a constant size in-flight stem containing liquid MS medium. Incubate in vitro. Five days later, the vitrification occurs from the leaf and examines the number of stems that turn into a transparent state. To calculate the survival rate after transplanting to the port, take it out of the tube after 5 days, wash it with water and transplant it into the round port. After that, the leaves were killed to determine the survival rate after port transplantation.

As in the above description,
The present invention shows opposite growth phenomena and tuber formation rates in potato plants recombined to be expressed in the forward and reverse directions using the Cu / Zn lily-superoxide dismutase (LSOD) gene, thereby reducing the growth period or increasing the growth period. Potato plants and endemic potato plants will be developed. In addition, in the seedling industry, the vitrification rate, which is always a problem when mass-producing in-flight seedlings, is drastically reduced, thereby increasing the purifying rate of sterile seedlings and lowering the mortality rate when sowing vegetation, and unlike tubers and leafy vegetables, tubers are formed underground. Even during periods of high rain, it will be possible to develop transgenic potato plants that are resistant to excessive moisture stress environments.

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Claims (4)

Cu / Zn lily-superoxide dismutase (LSOD) genes derived from lily Marcopolo scales were transformed into cloned vectors pMBP-S-LSOD and pMBP-AS-LSOD to express forward and reverse Cu / Zn LSOD. Overgrown Transgenic Potato Plants Grown by Tissue Culture The transgenic potato plant expressing the forward Cu / Zn LSOD is characterized by promoting tuber formation, shortening the growth period, and short stature compared to the untransformed potato. Transgenic Potato Plants Breeding. The method of claim 1, wherein the transgenic potato plants expressing reverse Cu / Zn LSOD is a tissue culture, characterized in that the tuber formation is delayed, the elongation is longer, and the vitrification rate during tissue culture is reduced compared to the untransformed potatoes Transgenic potato plants that are overgrown by. delete

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