KR20210053409A - 140ES91 maize mutant induced by EMS treatment for use in enhancing salt tolerance - Google Patents

Abstract

The present invention relates to a corn 140ES91 mutant for feed with improved salt resistance induced by EMS treatment, and more specifically, to a corn 140ES91 mutant for feed with improved salt resistance and excellent growth when treated with salt as a mutant induced by EMS treatment on a corn variety KS140 line, a food composition comprising the corn 140ES91 mutant, and a feed composition.

Description

{140ES91 maize mutant induced by EMS treatment for use in enhancing salt tolerance}

The present invention relates to a feed corn 140ES91 mutant with improved salt resistance induced by EMS (Ethyl Methane Sulfate) treatment, and specifically, a mutant derived by treatment with EMS in a corn variety KS140 Inbred line, which has excellent salt resistance when salt treatment It relates to the enhanced feed corn 140ES91 mutant, a food composition comprising the corn 140ES91 mutant, and a feed composition.

When irrigation is performed for crop cultivation, the concentration of water-soluble salts such as sodium, calcium, magnesium, potassium, sulfate, and chlorine in the agricultural land increases. When salt accumulates in the soil above a certain level, the ability of the crop to absorb moisture through the roots decreases, and the plant cells cannot perform normal metabolic activities.

The higher the concentration of salt, the less the amount of water absorbed into the plant, the less the production of the crop is, and the crop itself may even die. Such damage caused by salt is one of the factors that seriously limit the productivity of crops, and is one of the difficulties in the agricultural field that is difficult to solve.

In particular, it is necessary to develop crops with improved salt resistance that can be cultivated in areas where salt remains due to not being completely decontaminated in reclaimed lands developed on a large scale.

On the other hand, livestock and the products they produce are major sources of protein necessary for human survival, and the development of breeding and use of livestock is closely related to the improvement of human health and quality of life. Currently, a large part of grain is used as feed for raising livestock, and as the demand for grain increases as the population increases, cultivation of grain for feed is further required.

As feed for livestock, corn, soybean meal, sesame seeds, and pulses are mainly used, and feed crops, including corn, are dependent on imports. Accordingly, in order to increase the self-sufficiency rate of feed crops, it is necessary to develop feed crops with improved salt resistance so that idle lands such as reclaimed lands developed on a large scale in Korea can be utilized.

Therefore, in order to solve the problem of chlorination in agricultural land, many studies are being conducted to develop salt-resistant crops through variety improvement methods such as crossing, but no clear results have been obtained.

Under this background, the present inventors selected the 140ES91 mutant induced by EMS treatment as a result of earnest efforts to provide corn for feed to improve salt resistance, and the 140ES91 mutant was germinated during salt treatment, stem length, and The present invention was completed by confirming that the growth of the root has excellent enhanced salt resistance.

One object of the present invention is to provide a feed corn 140ES91 mutant with improved salt resistance.

Another object of the present invention is to provide a food composition comprising the corn 140ES91 mutant.

Another object of the present invention is to provide a feed composition comprising the corn 140ES91 mutant.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

In addition, those of ordinary skill in the art can recognize or ascertain using only routine experimentation a number of equivalents to the specific aspects of the invention described herein. Also, such equivalents are intended to be included in the present invention.

One aspect of the present invention for achieving the above object provides a feed corn 140ES91 mutant with improved salt resistance.

"Salt resistance" of the present invention is a property that can withstand the concentration in which the salt is accumulated and grows, and refers to the resistance of the plant to a high salt environment. The "salt" refers to a compound which is an ionic substance in which an anion of an acid and a cation of a base are bound by electrostatic attraction, and a salt crystallization salt containing sodium chloride as a main component is also used.

"Feed" of the present invention refers to a source of food and nutrition supplied to livestock in order to raise livestock in the livestock industry, and the individual to which the composition for feed of the present invention can be applied is not particularly limited, and any form can be applied Do. For example, it can be applied without limitation to animals such as chickens, pigs, monkeys, dogs, cats, mice, cows, sheep, goats, etc. Most of the feed is processed into various forms using grains. In a specific embodiment of the present invention, the feed may be corn, more specifically, it may be a feed corn mutant with improved salt resistance, and more specifically, it may be a feed corn 140ES91 mutant, but is not limited thereto.

The "corn 140ES91 mutant" of the present invention was selected as a feed maize mutant induced by EMS treatment. In a specific embodiment of the present invention, the mutant may be a corn mutant induced by treating EMS in the KS140 Inbred line, but is not limited thereto. The corn 140ES91 mutant was deposited with the National Academy of Agricultural Sciences (KACC) and was given an accession number KACC98064P.

The "mutant induced by EMS treatment" of the present invention means that plants are treated with EMS to increase the frequency of mutations, and useful mutants are selected and grown for several generations. It can be used to improve plant diversity by changing one to two main characteristics, and to develop new varieties with improved agricultural characteristics.

"EMS" of the present invention refers to a mutagenic organic compound called ethyl methane sulfate (Ethyl Methane Sulfate). The EMS is a compound represented by the following formula (I).

[Chemical Formula Ⅰ]

The compound EMS can induce random mutations in genetic material by nucleotide substitution. In particular, it may be made by guanine alkylation, but is not limited thereto. The mutation may be a point mutation, but is not limited thereto.

The "mutant" of the present invention means an individual carrying a gene in which a mutation has occurred. The cause of the mutation is that when EMS is treated in a plant, a part of the chromosome or DNA is changed, thereby changing the signaling system of gene information transmission, and when an individual occurs in the cell in which such change occurs, it becomes a mutant.

In a specific embodiment of the present invention, the 140ES91 mutant is characterized by increasing the expression of the genes ABF9-1, ABF9-2, and CIPK21-2 genes related to flame resistance after salt treatment, and decreasing the expression of the genes CIPK21-1 and CIPK31 It was confirmed that there is a difference in the amount of gene expression compared to the non-mutant (Example 4).

In addition, in another specific embodiment of the present invention, it was confirmed that the 140ES91 mutant has superior germination, stem length, and root growth than the non-mutant after salt treatment (Example 5).

Another aspect of the present invention provides a food composition comprising the corn 140ES91 mutant.

The "corn 140ES91 mutant" of the present invention is as described above.

"Food" of the present invention refers to a product in a natural state that can be eaten by humans or in a processed or cooked state, and the kind of the food is not particularly limited and may include all foods in a conventional sense. Non-limiting examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea. , Drinks, alcoholic beverages, and vitamin complexes. When the composition is used as a food additive, the composition may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

The "food composition" of the present invention refers to a composition further comprising a food pharmaceutically acceptable ingredient. The food composition may include additional ingredients that are commonly used to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr); And amino acids such as lysine, tryptophan, cysteine, and valine. The food composition may be prepared by a method commonly used in the art, and during the production, it may be prepared by adding raw materials and ingredients commonly added in the art.

In a specific embodiment of the present invention, the food composition may include a corn mutant, and more specifically, may include a corn 140ES91 mutant, but is not limited thereto.

Another aspect of the present invention provides a feed composition comprising the corn 140ES91 mutant.

The "corn 140ES91 mutant", and "feed" of the present invention are as described above.

The "feed composition" of the present invention may include, but is not limited to, essential nutrients, energy, amino acids, minerals, vitamins, and water that must be supplied to raise livestock. In a specific embodiment of the present invention, corn is used as a carbohydrate source in the feed composition of the present invention. The corn may be a feed composition having a low crude fiber content and high digestibility, and thus palatability.

In a specific embodiment of the present invention, the feed composition may include a corn mutant, and more specifically, may include a corn 140ES91 mutant, but is not limited thereto.

Therefore, the 140ES91 mutant for feed corn with improved salt resistance induced by the EMS treatment of the present invention has excellent growth status after salt treatment compared to the non-mutant, and is cultivated in a large area such as large reclaimed lands where salt remains because it is not decontaminated. It is possible, and it is expected that its utilization will be high by increasing the self-sufficiency rate of feed crops.

The 140ES91 mutant for feed corn with improved salt resistance induced by EMS treatment of the present invention is not decontaminated and can be cultivated in a wide area such as large reclaimed lands where salt remains. It is expected that its utilization will be high.

1 is a diagram showing the growth according to the treatment of EMS in Kang Da-ok (KS140).
Figure 2 is a diagram showing the selection of the line for improving the flame resistance of the EMS mutant line under 0.7% NaCl treatment conditions.
3 is a diagram showing an RT-PCR primer sequence used for analysis of the expression pattern of a gene in which a mutation on the chromosome of a mutant 140ES91 induced by EMS of the KS140 line has occurred.
4 is a diagram illustrating the expression pattern of a gene in which a mutation on the chromosome of the mutant 140ES91 induced by EMS of the KS140 line has occurred.
FIG. 5 is a diagram showing primer sequences used to analyze the expression of genes related to flame resistance of non-mutant and mutant 140ES91.
6 is a diagram illustrating the expression of genes related to flame resistance of non-mutant and mutant 140ES91 by RT-PCR.
7 is a diagram illustrating the expression of genes related to flame resistance of non-mutants and mutants by RT-qPCR.
8 is a diagram illustrating salt resistance analysis of non-mutant KS140 and mutant 140ES91.
9 is a diagram comparing liver and root lengths of non-mutant KS140 and mutant 140ES91.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example 1. Induction of maize mutation for feed by EMS treatment

1-1. Induction of mutation by chemical treatment

After selecting the KS140 (parent) Inbred line as a mutant breed and treating the EMS (Ethyl Methane Sulfate) at concentrations of 0%, 0.3%, 0.5%, and 0.7%, the degree of mutation was the most in 0.5% as a result of germination and growth status investigation. It was confirmed that it was suitable.

1-2. Corn package propagation treated with EMS 0.5%

Seeds treated with 0.5% EMS in the KS140 (parent) Inbred line are sown in the field, cultivated for 2 weeks, and then transplanted to the field to secure fixed lineage seeds for individuals that can grow, and obtain seeds through self-crossing for generational advancement. I did.

Example 2. Selection of strains for enhancing salt resistance of corn for mutant feed

2-1. Development of a selection method in a greenhouse for selection of salt-resistant mutants

A self-crossing M2 line was obtained from the KS140 line by EMS treatment. In the greenhouse, 20 seeds were sown per line in 50 hole pots, and selected by cultivation at a total concentration of 7 L of 0.7% NaCl during cultivation for 3 weeks.

Lines surviving in 0.7% NaCl were selected for mutants obtained by EMS treatment. To secure mutant seeds of the selected Inbred line KS140 677 line, the seeds were harvested after self-crossing.

2-2. Analysis of salt resistance of M2, M3, M4 seeds obtained by EMS treatment

M2, M3, and M4 seeds were obtained by self-crossing for each line obtained by EMS treatment.

Lines with improved salt resistance were selected by treating a 50 hole plate in a greenhouse at a concentration of 0.7% NaCl for 3 weeks.

2-3. Selection of lines with improved flame resistance among mutants obtained by EMS treatment

Among the mutants obtained by EMS treatment, lines with improved salt resistance after 0.7% NaCl treatment were selected (FIG. 2).

Example 3. Selection of mutants for improving flame resistance by in-flight culture

The salt-treated material used for analysis of the expression pattern of genes related to the enhancement of flame resistance of non-mutants and mutants was cultivated in a medium containing 0.5% NaCl at 25° C. for about 10 days, and then sampled and used.

Example 4. Characterization of flame resistance-related genes using corn mutants for improved salt resistance

4-1. Analysis of genetic variation in flame resistance enhancing mutants

In order to analyze the genetic variation of non-mutant and flame-resistant mutant, DNA of each line was extracted and a resequencing analysis was requested. In addition, the mutated regions and related genes in the mutant were searched using the genome re-analysis results of the non-mutant and mutant (Table 1).

For comparison data for genome reinterpretation, Zea mays inbred line B73 ( http://plants.ensembl.org/Zea_mays/Info/Index , AGPv4, RefGen_v4) was used.

As a result of comparing and analyzing the mutant regions of the non-mutant and mutant after the genome re-analysis of the KS140 line of flame resistance enhancing mutants, 39 amino acid mutations and 150 gene-related mutations, as well as 1,016 mutations occurred in the intergenic region. There were 1,166 mutations.

As a result of genomic re-analysis of the mutants, mutations were also found in 71 locations in the intron region, 13 locations in the 5'UTR domain, and 11 locations in the 3'UTR domain in the region where SNP or Indel occurred on the chromosome of feed corn.

4-2. Analysis of the characteristics and functions of genes found in flame resistance enhancing mutants

In order to analyze the expression pattern of the non-mutant and mutant 140ES91 by NaCl treatment, samples grown for 10 days in the untreated group and 0.5% NaCl treatment condition were used.

RNA was extracted from samples treated with 0.5% NaCl grown by in-flight culture for the samples used for RT-PCR to analyze the expression pattern of the gene in which the genetic mutation of the flame resistance enhancing mutant occurred. Total RNA was extracted from the leaves of non-mutant KS140 and mutant 140ES91, and first-strand cDNA was synthesized using Sprint RT Complete-OligodT (Clontech). Using the BioRad C1000 PCR machine, the PCR reaction cycle was dissociated at 98.0°C for 5 minutes, then reacted at 98.0°C for 30 seconds at 58.0°C for 30 seconds again at 72.0°C for 1 minute, repeated 30 times, and finally at 72.0°C for 8 minutes. . After PCR using the primer sequence shown in FIG. 3, the expression level was confirmed by electrophoresis on 1% agarose, and the result analysis was compared and analyzed based on the Actin gene (FIG. 4).

Serine/threonine-protein kinase AGC1-5 gene that caused amino acid mutation in the 140ES91 mutant was confirmed to be mutated from Leucine (L) to Glutamine (Q), and as a result of RT-PCR reaction, it was expressed after salt treatment in the non-mutant. The amount decreased, and it was confirmed that the 140ES91 strain was hardly expressed before salt treatment, but increased after salt treatment.

The ATP synthase subunit gamma mitochondrial gene was mutated from Alanine (A) to Glycine (G), and as a result of RT-PCR reaction, the expression level was slightly higher in the non-mutant before salt treatment in the non-mutant and 140ES91 line. The expression levels between the non-mutant and 140ES91 strains were similar. In addition, as a result of expression analysis by RT-PCR for genes with SNP or InDel mutations in 5'-UTR and Intron, although amino acid mutations were not caused, among the total 150 genes in which genetic mutations occurred, they were related to infectious stress. As a result of analyzing the expression patterns of the genes before and after salt treatment between the non-mutant and mutant 140ES91 strains by RT-PCR, the COP1 gene was expressed more than the mutant in the non-mutant before salt treatment. The expression pattern between lineages did not change.

In the case of the YABBY gene, it was confirmed that the expression level of the non-mutant before salt treatment was higher than that of the mutant, and the expression level of both strains increased after salt treatment. The expression level of the translation elongation factor gene was higher in the mutant than in the non-mutant, and there was no difference in the expression level after salt treatment. Aldo-keto reductase and Myb98 genes were rarely expressed before salt treatment in the non-mutant, but slightly expressed after salt treatment. In the 140ES91 line, there was little difference in expression levels before and after salt treatment. It was confirmed that the expression level of RNA binding protein and Pentatricopeptide protein was slightly lower in the 140ES91 line than in the non-mutant before salt treatment, and the expression level was decreased in the non-mutant after salt treatment, but the expression level of the 140ES91 line was increased after salt treatment. I did.

In addition, Belclin-1-like protein, Xylem bark cystein, and STRUBBELIG-receptor genes were also expressed less in the 140RS91 line before salt treatment than in the non-mutant. After salt treatment, there was no significant change in the expression of the non-mutant, but it was confirmed that the expression level of the mutant 140ES91 line was increased compared to before salt treatment (FIG. 4).

4-3. Analysis of the expression patterns of genes related to inflammation between non-mutants and mutants

In order to analyze the expression patterns of genes related to flame resistance in mutants and non-mutants, each material was cultivated in an in-flight medium containing 0.5% NaCl for about 10 days, followed by RNA extraction, followed by RT-PCR (FIG. 6).

Five genes related to flame resistance (ABF9-1, 2, CIPK21-1, 2, CIPK31) were used, and each of the primer sequences used in PCR is shown in FIG. 5. In order to correct the level of expression of each gene, the expression patterns in the non-mutant and the mutant with improved salt resistance were analyzed based on the RT-qPCR results using the always-expressed Actin gene (FIG. 7).

In the RT-qPCR reaction method, after synthesizing cDNA using a cDNA ^{synthesis kit (cDNA EcoDry TM, Clontech cat# 639543), RNA of each sample is diluted 10-fold.} The gene reaction was performed according to the protocol of the kit, and each was repeated 3 times. The RT-qPCR was used with a BioRad CFX96 instrument, and the reaction was carried out. The PCR reaction cycle was dissociated at 95.0°C for 3 minutes and then reacted at 95.0°C for 10 seconds at 55.0°C for 10 seconds again at 72.0°C for 30 seconds. The results of the analysis repeated 40 times were compared and analyzed based on the Actin gene.

As a result of analyzing the expression pattern, in the case of non-mutant, the expression level of ABP9-1, CIPK21-1, and CIPK21-2 significantly decreased after salt treatment, ABP9-2 slightly increased, and CIPK31 showed little change in expression level. Was confirmed. In the mutant 140ES91 line, the expression levels of ABP9-1, ABP9-2, and CIPK21-2 genes were increased after salt treatment than before salt treatment, and the expression levels of CIPK21-1 and CIPK31 were slightly decreased after salt treatment.

This suggests that there are many differences in expression patterns between non-mutant and mutant 140ES91 lines. In general, it was confirmed that in the mutant with improved flame resistance, the expression level of the flame resistance-related genes was increased after treatment compared to before salt treatment.

Example 5: Analysis of growth characteristics of mutants with improved flame resistance

5-1. Non-mutant KS140 and Mutant 140ES91 Lines Analysis of Flame Resistance by NaCl Treatment

The flame resistance enhancing mutant 140ES91 strain obtained by EMS treatment was treated with 0.5% NaCl and then incubated for 10 days, and then germination rate, liver (stem length) and root length were investigated.

As a result of investigation of germination rate by in-flight culture of non-mutant and mutant 140ES91, half did not germinate in the case of non-mutant, but it was confirmed that an average of 7-8 germinated in the case of mutant 140ES91 (FIG. 8). In addition, it was confirmed that the growth state of the mutant 140ES91 was better than that of the non-mutant even in the development of liver and root (FIG. 9).

Through this, as a result of comparing the germination rate, liver and root growth after NaCl treatment, it was confirmed that the germination rate of mutant 140ES91 is higher than that of the non-mutant, and stems and roots are grown longer. This suggests that the growth of mutant 140ES91 is better when salt treatment.

As described above, the 140ES91 mutant for feed corn with improved salt resistance induced by the EMS treatment of the present invention is not decontaminated and can be cultivated in large areas such as large reclaimed lands where salt remains. As a result, it is expected to increase the self-sufficiency rate of feed crops and increase its utilization.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

National Academy of Agricultural Sciences KACC98064P 20190731

Claims (10)

Corn 140ES91 mutant with improved salt resistance.
The maize 140ES91 mutant according to claim 1, wherein the mutant is induced by Ethyl methane sulfate (EMS) treatment.
The method of claim 2, wherein the EMS is represented by the following formula (I), corn 140ES91 mutant.
[Chemical Formula Ⅰ]

The corn 140ES91 mutant according to claim 1, wherein the corn is corn for feed.
The maize 140ES91 mutant according to claim 1, wherein the maize 140ES91 mutant is mutated by treating the KS140 inbred line with EMS to induce the mutation.
According to claim 1, wherein the corn 140ES91 mutant is that the expression of the flame-resistant genes ABF9-1, ABF9-2 or CIPK21-2 gene is increased after salt treatment, corn 140ES91 mutant.
According to claim 1, wherein the corn 140ES91 mutant is that the expression of the flame-resistant gene CIPK21-1 or CIPK31 gene is reduced after salt treatment, corn 140ES91 mutant.
The maize 140ES91 mutant according to claim 1, wherein the maize 140ES91 mutant has superior germination, stem length, and root growth than the non-mutant after salt treatment.
A food composition with improved salt resistance, comprising the corn 140ES91 mutant of claim 1.
A feed composition with improved salt resistance, comprising the corn 140ES91 mutant of claim 1.

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