JP2012215482A - Method for evaluating ozone influence of rice yield - Google Patents

Abstract

PROBLEM TO BE SOLVED: To more exactly evaluate an influence on rice yields by ozone.SOLUTION: An influence on yields of test rice cultivars by ozone is evaluated based on expression levels of one or more proteins to be selected from a group consisting of CPN-60 (60kDa chaperonin), ATP synthase, and enolase in the test rice cultivars to which ozone exposure processing is performed.

Description

  The present invention relates to a method for evaluating the effect of ozone on rice yield. More specifically, the present invention relates to a method for evaluating the effect of ozone on rice yield, and a method for selecting rice varieties that do not or do not easily decrease the yield by ozone using this method.

  Ozone is a major air pollutant in the troposphere. In recent years, especially in developing countries in Asia, the ozone concentration is increasing rapidly. In addition, the amount of anthropogenic nitrogen oxides that are precursors of ozone in Asia is expected to increase by 350% in 2020 compared to 1990 if no measures are taken in the future (Non-patent Document 1). And Non-Patent Document 2).

  Under such circumstances, in recent years, there is a growing concern about the effects of ozone on the growth and yield of crops. For example, it has been reported that the growth and yield of rice decrease due to an increase in the ozone concentration in the atmosphere (Non-patent Documents 3 and 4). In addition, the inventors of the present application have reported that the decrease in rice yield due to ozone is greater in Indian varieties than Japanese varieties, and that the effect of ozone on rice yield varies greatly between rice varieties ( Non-patent document 5).

  The problem of reduced rice yield that can be caused by an increase in the ozone concentration in the atmosphere is a very serious problem in regions where rice is a staple food, particularly in Asian countries. Therefore, by elucidating the difference in rice yield reduction due to ozone among varieties, and selecting rice varieties that do not or do not easily decrease the yield based on ozone, future foods, especially in Asian countries, will be selected. It is thought that it can greatly contribute to production.

  By the way, conventionally, studies have been made to evaluate the influence of environmental stress on plants at a molecular level, and many molecular markers such as genes and proteins serving as indicators have been found. For example, Patent Document 1 proposes a technique using sakuranetin, which is a kind of antibacterial secondary metabolite collectively called phytoalexin, as a molecular marker for evaluating the influence of high temperature and ozone on rice yield. ing. Specifically, after the test rice is exposed to a complex environment under a high-concentration ozone environment and a high temperature environment that is higher than the ozone concentration in the normal atmosphere, the amount of sakuranetin accumulated in the test rice is measured, Based on the presence / absence of detection and / or the detection amount, the presence / absence and / or degree of the influence on the rice yield due to ozone at a high temperature is determined. As a result, the effect of ozone on rice yield can be evaluated earlier at the laboratory level without growing rice until the stage of actual harvesting. In addition, rice varieties whose yield is not lowered or hardly lowered by ozone can be selected earlier at the laboratory level without growing rice until the stage of actual harvesting.

JP 2011-33533 A

van Aardenne, J., Carmichael, G.R., Levy H., Streets, D., Hordijk, L. 1999. Anthropogenic NOx emissions in Asia in the period 1990-2020. Atmos. Environ. 33: 633-646 Aunan, K., Berntsen, T.K., Seip, H.M., 2000. Surface ozone in China and its possible impact on agricultural crop yields.Ambio 29, 294-301. Kobayashi, K., Okada, M., Nouchi, I., 1995. Effects of ozone on dry matter partitioning and yield of Japanese cultivars of rice (Oryza sativa L.). Agric. Ecosyst. Environ. 53, 109-122. Yonekura, T., Shimada, T., Miwa, M., Arzate, A., Ogawa, K., 2005. Impacts of tropospheric ozone on growth and yield of rice (Oryza sativa L.). J. Agric. Meteor. 60, 1045-1048. Sawada, H., Kohno, Y., 2009. Differential ozone sensitivity of rice cultivars as indicated by visible injury and grain yield.Plant Biol. 11 (Suppl 1), 70-75.

  In the method for evaluating the effect of ozone on rice yield using sakuranetin as a molecular marker proposed in Patent Document 1, if the temperature is lower than 27 ° C, sakuranetin is likely to accumulate even in ozone-sensitive rice varieties, resulting in an erroneous determination. Therefore, it is necessary to go through a process of exposing the rice to be examined to an environment of high temperature (27 ° C. to 50 ° C.) and high concentration ozone. Patent Document 1 describes that the high temperature treatment for exposing the test rice to a high temperature environment is performed for 5 days or more. However, under conditions where the test rice is exposed to a high temperature environment of 27 ° C. to 50 ° C. for 5 days or more, the temperature condition in the environment where rice is actually grown can be greatly different. That is, in an environment where rice is actually cultivated, the temperature is often lower than 27 ° C. at least at night, and exposure of the test rice to a high temperature environment of 27 ° C. or more all day is the high temperature environment itself. It becomes an environmental stress on the test rice, and ozone and high temperature can have multiple effects on the test rice. Therefore, there is a concern that the method described in Patent Document 1 may not be able to accurately evaluate the effect of ozone on rice yield. In addition, there is a concern that rice varieties may not be accurately selected because the yield does not decrease or is unlikely to decrease due to ozone.

  Then, an object of this invention is to provide the method which can evaluate more accurately the influence on the rice yield by ozone.

  Another object of the present invention is to provide a method that can more accurately select rice varieties whose yield is not lowered or hardly lowered by ozone.

  In order to solve such a problem, the inventors of the present application use an open top chamber, temperature conditions and sunshine conditions are almost the same as those in the outdoors, and a variety of rice varieties as conditions where the ozone concentration is higher than the atmospheric ozone concentration. We conducted a long-term ozone exposure test to cultivate the rice until harvest time, and compared the relationship between the amount of protein expression and the rice yield of rice varieties in rice leaves during the long-term ozone exposure test. As a result, the expression of CPN-60 (60 kDa chaperonin), ATP synthase and enolase was suppressed in the varieties whose rice yield was reduced by the long-term ozone exposure test, and the rice yield was not reduced by the long-term ozone exposure test. It was found that its expression did not change or tended to increase.

  Therefore, a short-term ozone exposure treatment for 3 days was conducted for multiple varieties of rice seedlings, and the relationship between the expression levels of CPN-60 (60 kDa chaperonin), ATP synthase and enolase and rice yield was compared. It was found that the same tendency as in the long-term ozone exposure test was observed.

  From these results, by using CPN-60 (60 kDa chaperonin), ATP synthase and enolase as molecular markers, ozone exposure treatment was conducted under temperature conditions similar to or close to the outdoors, and the effect of ozone on rice yield That rice can be more accurately selected in a short period of time, and that rice varieties can be selected more accurately and less easily by ozone, and based on this finding, various studies are repeated to complete the present invention. It came to.

  That is, the method for evaluating the effect of ozone on the yield of rice according to the present invention comprises one or more proteins selected from the group consisting of CPN-60 (60 kDa chaperonin), ATP synthase and enolase in a test rice variety treated with ozone exposure. Based on the expression level, the effect of ozone on the yield of test rice varieties is evaluated.

  In the present invention, “rice yield” means the weight of fermented rice per individual rice. However, the rice yield may be the weight of sperm per part of a rice individual or the weight of sperm of a plurality of rice individuals.

  Here, in the method for evaluating the effect of ozone on the yield of rice of the present invention, it is preferable that the ozone exposure treatment is performed on young seedlings of the rice varieties to be tested.

  Next, the method for selecting rice varieties of the present invention is to select rice varieties that do not or do not easily decrease the rice yield by ozone, using the method for evaluating the effect of ozone on the rice yield of the present invention.

  According to the method for evaluating the effect of rice yield on ozone according to the present invention, it is possible to more accurately evaluate the effect of ozone on rice yield. That is, as in the technique described in Patent Document 1, it is possible to more accurately evaluate the effect of rice alone on rice yield without performing exposure to a high temperature environment that can cause environmental stress due to high temperatures. .

  According to the method for selecting rice varieties of the present invention, it is possible to more accurately select rice varieties in which the rice yield is not reduced or hardly lowered by ozone. That is, as in the technique described in Patent Document 1, rice varieties are selected more accurately without exposure to or difficult to decrease by ozone without exposure to a high temperature environment that can cause environmental stress due to high temperatures. It becomes possible.

It is a figure which shows the influence (rice yield in ozone concentration 37ppb and 75ppb) by the long-term ozone exposure process on the rice yield. It is a figure which shows the difference in the two-dimensional electrophoresis pattern of the rice leaf leaf protein by a long-term ozone exposure process. It is a figure which shows the difference in Ki-13 spot expression level by a long-term ozone exposure process. It is a figure which shows the change of the various protein expression level by a short-term ozone exposure process. It is a figure which shows the relationship between the ozone concentration in several rice varieties, and the relative expression level of protein about short-term ozone exposure processing. It is a figure which shows the relationship between CPN-60 expression level and a rice yield. It is a figure which shows the relationship between an ATP synthetase expression level and a rice yield. It is a figure which shows the relationship between an enolase expression level and a rice yield. It is a figure which shows the relationship between RuBisCo LSU expression level and a rice yield. It is a figure which shows the condition of the external temperature during a long-term ozone exposure process period. It is a figure which shows the ozone exposure density | concentration pattern in a long-term ozone exposure process period. It is a figure which shows the influence (relationship between various ozone concentrations and rice yield) on the rice yield by long-term ozone exposure processing.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings.

  The method for evaluating the effect of ozone on the yield of rice according to the present invention comprises the expression level of one or more proteins selected from the group consisting of CPN-60 (60 kDa chaperonin), ATP synthase and enolase in a test rice variety treated with ozone exposure. Based on this, the effect of ozone on the yield of test rice varieties is evaluated.

  More specifically, the expression level of one or more proteins selected from the group consisting of CPN-60 (60 kDa chaperonin), ATP synthase and enolase in the test rice varieties subjected to ozone exposure, and ozone exposure treatment. The expression level of one or two or more proteins selected from the group consisting of CPN-60 (60 kDa chaperonin), ATP synthase and enolase in a control rice cultivar (the same rice cultivar as the test rice cultivar) is compared. When the protein expression level of the test rice cultivar is smaller than the protein expression level of the control rice cultivar, it is determined that the yield of the test rice cultivar decreases due to ozone. Conversely, if the protein expression level of the control rice cultivar is similar to the protein expression level of the test rice cultivar, or if the protein expression level of the test rice cultivar is higher, the yield of the test rice cultivar decreases due to ozone. It is determined not to. When the protein expression level of the test rice cultivar is greater than that of the control rice cultivar, it is determined that the yield of the test rice cultivar may increase due to ozone.

  The ozone concentration in the ozone exposure treatment is not particularly limited as long as it is a concentration at which a difference from the protein expression level in the control rice varieties not subjected to ozone exposure treatment can be detected in the rice varieties with high ozone sensitivity, but 40 ppb. It is suitable to set it as -120ppb, and it is more preferable to set it as 40ppb-80ppb. If the ozone concentration is too low, the difference from the protein expression level of the control rice varieties not subjected to ozone exposure treatment due to too weak ozone stress is too small or not recognized. If the ozone concentration is too high, the ozone stress is too strong and a significant effect is exerted on the tested rice cultivar, making it difficult to accurately grasp the protein expression level. In the present invention, even if the ozone concentration is 40 ppb, the influence of ozone on the rice yield can be sufficiently evaluated. Therefore, it is possible to evaluate the effect on rice yield at a concentration closer to the ozone concentration in the atmosphere, which will gradually increase in the future.

  Although the ozone exposure treatment varies depending on the ozone concentration, it can be performed for about 1 hour to 30 days and further until the end of growth. However, when the ozone concentration is 40 ppb to 120 ppb, 12 hours to 60 hours, preferably 24 hours to 48 hours, and more preferably about 36 hours are sufficient. If the exposure time is too short, the difference from the protein expression level of the control rice varieties not subjected to ozone exposure treatment due to too weak ozone stress is too small or not recognized. Even if the exposure time is too long, the difference from the protein expression level of the control rice variety does not increase in proportion to the exposure time. In addition, the ozone exposure process does not necessarily need to be performed continuously, and may be an intermittent process. For example, ozone exposure processing may be performed for a certain time (for example, 12 hours) in one day, and ozone exposure processing may not be performed for the remaining time.

  It is preferable that the ozone exposure treatment substantially matches the cultivation environment conditions of the control rice except for the ozone concentration condition. Thereby, the influence on the rice yield by only ozone can be evaluated reliably. In the control rice cultivation environment, the ozone concentration does not necessarily have to be 0 ppb, and the environment containing ozone to the extent that the difference in protein expression level can be recognized between the test rice variety and the control rice variety. I do not care. In addition, in this invention, since the exposure to a high temperature environment is not required like patent document 1, to the rice yield by ozone as a condition which matched or approximated the cultivation environment condition with the actual cultivation environment condition of rice. To understand the impact of For example, similarly to the actual rice cultivation environmental conditions, even if the temperature at night is lower than 27 ° C., it is possible to accurately grasp the effect of ozone on rice yield. Evaluation is possible even in short-term experiments in the air zone where ozone is added to the non-purified air zone or the amount of expression between the air zone purified with activated carbon etc. and the non-purified air zone under temperature conditions that can grow seedlings outdoors It is.

  The growth stage of the test rice varieties that are subjected to the ozone exposure treatment may be at any stage as long as it is in the rice growth stage, but it is particularly preferable to carry out the treatment on young seedlings. In this case, it is possible to reliably and early determine the influence of ozone on the rice yield.

  Expression level of one or more proteins selected from the group consisting of CPN-60 (60 kDa chaperonin), ATP synthase and enolase in test rice varieties treated with ozone exposure, CPN in control rice varieties not treated with ozone exposure The expression level of one or more proteins selected from the group consisting of -60 (60 kDa chaperonin), ATP synthase and enolase is detected by a conventional method. For example, proteins are extracted from tissues such as leaves of test rice varieties and control rice varieties and subjected to electrophoresis by SDS-polyacrylamide gel electrophoresis or the like. The proteins are transferred from the gel to a PVDF membrane or the like, After blocking the transferred PVDF membrane, it is reacted with a specific antibody of each protein, further reacted with a secondary antibody labeled with HRP (Horse Radish Peroxidase), treated with a chemiluminescence reagent, and then with a luminescence image analyzer or the like. An antigen band is detected.

  If it is determined from the detection result that the protein expression level of the test rice cultivar is less than the protein expression level of the control rice cultivar, it is determined that the rice yield of the test rice varieties is reduced by ozone. Conversely, if it is determined that the protein expression level of the control rice cultivar is similar to the protein expression level of the test rice cultivar, or the protein expression level of the test rice cultivar is greater, the test rice cultivar is determined by ozone. It is determined that the rice yield does not decrease. When the protein expression level of the test rice cultivar is greater than that of the control rice cultivar, it is determined that the yield of the test rice cultivar may increase due to ozone.

  And by using this determination result, rice varieties can be selected in which the rice yield does not decrease or is unlikely to decrease due to ozone. That is, as a result of the determination, rice varieties determined that the yield of rice does not decrease by ozone can be determined to be rice varieties that have low ozone sensitivity and that do not decrease or are unlikely to decrease the yield of rice. Conversely, rice varieties that have been determined to have reduced rice yield due to ozone can be determined to be rice varieties that are highly ozone sensitive and that have a reduced protein expression level due to ozone. Therefore, for rice varieties, only rice varieties that are determined to have low ozone sensitivity, that is, rice yields that are determined not to decrease by ozone are selected by selecting only those varieties that are determined not to decrease rice yield by ozone. can do.

  The above-described embodiment is an example of a preferred embodiment of the present invention, but is not limited thereto, and various modifications can be made without departing from the gist of the present invention. For example, 1 or 2 selected from the group consisting of CPN-60 (60 kDa chaperonin), ATP synthetase and enolase after ozone exposure treatment at a plurality of ozone concentrations for rice varieties determined to reduce rice yield due to ozone A correlation between the expression level of the protein and the rice yield at a plurality of ozone concentrations may be obtained in advance, and the amount of rice yield change may be predicted quantitatively based on this correlation. In this case, even if it is not possible to accurately grasp the ozone exposure status of rice during cultivation, it is possible to predict the amount of rice yield change due to ozone exposure by measuring the expression level of the protein in this rice There is. And about the case where this quantitative prediction is performed, it is preferable to analyze especially using CPN-60 (60 kDa chaperonin) as a parameter | index.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

<Experiment method>
(1) Test rice As the test rice, Japanese type varieties Kirara 397, Koshihikari and Nipponbare were selected, and Indian type varieties Takanari, Kasalath and Suanburi 90 were selected.

  Kirara 397, Koshihikari and Nipponbare rice seeds were obtained from the National Institute for Agro-Environmental Sciences. Takanari rice seeds were obtained from Agricultural Research Organization and Crop Research Institute. Casalas rice seeds were obtained from the National Institute for Environmental Studies. Swanburi 90 rice seeds were obtained from Naresuan University (Thailand).

  For Kirara 397, Koshihikari and Takanari, a proteome analysis of the flag leaf was performed.

(2) Long-term ozone exposure treatment Open top chamber (frontage 3.6m, depth 3.6m, height 2.4m, below) installed at Akagi Test Center, Power Research Institute, Maebashi City, Gunma Prefecture A 1 / 2000a Wagner pot was placed in a block in advance in OTC), and rice seedlings grown in a glass room for about 3 weeks were planted in this Wagner pot, and exposure to high-concentration ozone was started.

  Ozone exposure treatment was carried out from May 31 to October 16, 2007, June 2 to October 31, 2008, May 11 to October 22, 2009, May 26 to 10, 2010. It took place on the 28th of a month. The average temperature from June to October from 2008 to 2010 was 20.7 to 22.5 ° C. FIG. 10 shows the change in the outside air temperature during the ozone exposure treatment.

  In the experiment, I used black soil extracted from the premises of Akagi Test Center as the soil, and planted 4 individuals per pot and placed 5 pots in each chamber.

  After planting, Kumai LP coated composite 444-D80 (14-14-14; manufactured by Chisso Asahi Fertilizer) was fertilized with 5.36 g (15 kg N / 10a equivalent) per pot. In addition, topdressing was not performed.

  The ozone exposure treatment was performed by introducing air purified by an activated carbon filter into the OTC, then introducing ozone to control the ozone concentration, and the ozone exposure treatment was carried out until harvest. Hereinafter, the treatment area where the ozone exposure treatment is performed is referred to as an “ozone treatment area”. The standard ozone concentration was obtained by calculating the time average value from the May-September ozone concentration observed in 2000-2007 at Saitama Environmental Science International Center. The value is set to 2.5 times. FIG. 11 shows an ozone exposure concentration pattern in the ozone exposure treatment.

  Moreover, the control process which introduce | transduces only the air purified with the activated carbon filter into OTC, without introduce | transducing ozone as a control process of the exposure process of high concentration ozone. Hereinafter, the treatment section in which this control process is performed is referred to as a “purified air section”. The average ozone concentration during 12 hours in the purified air zone was 4 ppb.

  In each experiment, 5 pots were used for each cultivar for each treatment section, and the chambers were duplicated.

(3) Proteome analysis Rice leaflets were collected one week after heading for each variety, frozen in liquid nitrogen, and stored at -80 ° C until analysis. Soluble protein was extracted from the flag leaves and subjected to two-dimensional electrophoresis. Using image analysis software, two-dimensional electrophoresis patterns were compared, and the difference in protein expression level between the ozone treatment zone and the purified air zone was analyzed for each variety. A protein spot having a significant difference in expression level was cut out from the gel, converted into a peptide fragment by in-gel digestion, and analyzed by a mass spectrometer to identify the protein.

(4) Yield measurement Harvesting was performed at a stage where the rice straw became 80% or more yellow. The whole pods removed from the ears were sorted into cocoon seeds and sterilized seeds using an automatic seed sorter (FV-459A manufactured by Fujiwara Seisakusho), and the weight of the cocoon seeds was used as the yield.

(5) Short-term ozone exposure treatment The above six varieties of rice were used for raising seedlings (Kureha Horticulture, Kureha Co., Ltd.), artificial climate chamber (12 hours light period, light period 28 ° C / dark period 23 ° C, relative (Humidity 60-70%, light intensity 400 μmol / m ² / s). Rice seedlings 14 days after sowing were exposed to ozone at an ozone concentration of 0 ppb, 40 ppb, and 80 ppb for 3 days. However, exposure to ozone was limited to the light 12 hours of the day. For Nipponbare, Kasalath, and Suanburi 90, ozone exposure treatment was performed only at ozone concentrations of 0 ppb and 40 ppb.

  After the 3-day ozone exposure treatment was completed, the third leaf from the bottom was cut out, immediately frozen in liquid nitrogen, and stored at −80 ° C. until analysis.

(6) Immunoblotting Proteins were extracted from 0.1 g of rice leaves treated with short-term ozone exposure, and after SDS-polyacrylamide gel electrophoresis, they were transferred from the gel to a PVDF membrane using a transfer device. After blocking the transferred PVDF membrane, it was reacted with the following specific antibody against each candidate protein.
Anti-Heat Shock Protein 60 (Acris Antibodies GmbH, Herford, Germany)
・ Anti-ATP Synthase, CF1β Subunit (AntiProt, Pullach i. Isartal, Germany)
・ Anti-Enolase (Aviva Systems Biology, San Diego, USA)

  After reacting with the specific antibody, washing was performed, and the resultant was reacted with a secondary antibody labeled with HRP (horse radish peroxidase). As a control, Anti-RuBisCO large subunit (Zhang, Z. and Komatsu, S. 2000. Molecular Cloning and Characterization of cDNAs Encoding Two Isoforms of Ribulose-1,5-Biosphosphate Carboxylase / Oxygenase Activase in Rice (Oryza sativa L. ) The response to J. Biochem. 128: 383-389) was also analyzed.

  The PVDF membrane reacted with the antibody was washed, treated with a chemiluminescence detection reagent, and an antigen band was detected with a luminescence image analyzer. The density of this band was quantified by image analysis software and quantified.

<Experimental result>
(1) Changes in rice yield due to ozone
Fig. 1 shows the results of studies on the effect of long-term ozone exposure treatment conducted between 2007 and 2010 on rice yield. In addition, the value of the vertical axis | shaft of FIG. 1 is an ozone treatment area (average ozone concentration 37ppb, 75ppb for 12 hours in the day) with respect to the rice yield (refined rice yield) in the air purification area (average ozone concentration 4ppb for 12 hours in the day). It is the relative value of rice yield (refined rice yield). FIG. 12 shows the results of studies on various rice varieties for rice yield (serum yield) at various ozone concentrations (average ozone concentration for 12 hours during the day). The value on the vertical axis is the same as in FIG. The average yield in the ozone treatment zone decreased to 80% for Kirara 397, 86% for Takanari and 88% for Kasaras, compared with the yield in the purified air zone. On the other hand, in Koshihikari, Nipponbare, and Suphanburi 90, no change was observed in the yield.

  From these results, it was considered that Kirara 397, Takanari and Kasalath are cultivars with high ozone sensitivity, and Koshihikari, Nipponbare and Suanburi 90 are cultivars with low ozone sensitivity.

(2) Image analysis result of two-dimensional electrophoresis pattern FIG. 2 shows the result of examining the difference in two-dimensional electrophoresis pattern of rice leaf-leaved protein with and without long-term ozone exposure treatment. As a result of image analysis, a significant difference was found in the expression level of the protein. Furthermore, 31 protein spots with a change amount of 1.5 times or more were detected in Kirara 397, 18 in Koshihikari, and 11 in Takanari. Of these altered protein spots, Ki-13 was the only spot commonly detected in the three varieties.

  Next, FIG. 3 shows the difference in Ki-13 spot expression level depending on whether or not long-term ozone exposure treatment is performed. In varieties (Kirara 397, Takanari) whose rice yield was reduced by long-term ozone exposure treatment, the expression level of Ki-13 was lower in the ozone treatment zone than in the purified air zone. Conversely, in the cultivar (Koshihikari) in which the rice yield was not reduced by the long-term ozone exposure treatment, the expression level of Ki-13 was higher in the ozone treatment zone than in the purified air zone.

(3) Identification of proteins in Ki-13 spots Table 1 shows the results of analysis of peptide fragments digested and extracted from Ki-13 spots with a mass spectrometer. In Table 1, “score” is Mascot score, “C” is the degree of amino acid sequence match, “MP” is the number of matched peptide fragments, “MW” is molecular weight, “pI” is equal Electric point.

  As a result of analysis, it was revealed that the Ki-13 spot contains a 60 kDa chaperonin (CPN-60), a chloroplast-type ATP synthase CF1β subunit, enolase 1, and a protein with unknown function.

  From the above results, it was suggested that 60 kDa chaperonin, ATP synthase and enolase may be used as novel molecular markers for the chronic effect of rice yield due to ozone.

(4) Examination of the expression level of various proteins by short-term ozone exposure treatment In order to confirm whether 60 kDa chaperonin, ATP synthase and enolase are markers that can be used at the laboratory level at an early stage, the test was performed in an artificial weather chamber. Rice seedlings were subjected to a short-term ozone exposure treatment to examine changes in the expression levels of 60 kDa chaperonin, ATP synthase and enolase.

  The experimental results are shown in FIG. Three-leaf stage Kirara 397, Koshihikari, Takanari, Kasaras, Nihonbare, Suphanburi 90 were exposed to ozone for 3 days. The expression levels of all proteins of chaperonin, ATP synthase, and enolase were decreased. On the other hand, in Koshihikari, Nipponbare and Suphanburi 90, which are varieties that do not decrease due to ozone, the expression level of all proteins of 60 kDa chaperonin, ATP synthase, and enolase increases when exposed to 40 ppb ozone, or the same level as the purified air zone of 0 ppb It was kept in.

  Moreover, about the change of the protein expression level by a short-term ozone exposure process, each band shown by FIG. 4 was measured with the densitometer, and what showed by the relative value is shown in FIG. In Kirara 397, Takanari, and Kasalath, which are varieties that lose sales due to ozone, the expression levels of all proteins of 60 kDa chaperonin, ATP synthase, and enolase were already reduced by exposure to 40 ppb of ozone. On the other hand, in Koshihikari, Nipponbare and Suphanburi 90 which are varieties that do not decrease by ozone, 40 ppb ozone exposure increases the expression level of all proteins of 60 kDa chaperonin, ATP synthase, and enolase when exposed to 40 ppb ozone. Or it was kept at the same level as the purified air zone of 0 ppb. In Koshihikari, the amount of ATP synthase expressed in ozone exposure at 80 ppb was less than that in the purified air zone of 0 ppb. However, the expression level of 60 kDa chaperonin and enolase was maintained at the same level as that in the purified air zone of 0 ppb. It was.

  As a control, the expression level of RuBisCO large subunit (LSU), a major protein in plant leaves, was also analyzed. The expression level of RuBisCO LSU was 40 ppb in all varieties except Nipponbare. It was suggested that it was not related to varietal differences in sensitivity to yield.

  From the above results, it was confirmed that 60 kDa chaperonin, ATP synthase and enolase are early markers available at the laboratory level. In addition, for example, in determining the protein expression level by increasing the ozone concentration in the ozone exposure treatment to more than 80 ppb, it has become clear that it is particularly preferable to use 60 kDa chaperonin and enolase as indices.

(5) Examination of the correlation between the relative expression level of protein and the relative yield of rice Correlation with the relative yield of long-term ozone exposure until harvest at the same concentration as the relative expression level of protein after short-term ozone exposure treatment in all varieties Was calculated. As a result, in the 60 kDa chaperonin, an extremely high positive correlation with r = 0.8597 was recognized (FIG. 6). ATP synthase and enolase were also highly positively correlated with r = 0.7413 and r = 0.7502, respectively (FIGS. 7 and 8). On the other hand, no significant correlation was observed with RuBisCO LSU (FIG. 9). Among these proteins, it was revealed that 60 kDa chaperonin is particularly important as a yield prediction marker.

  Moreover, from these results, by analyzing the expression levels of 60 kDa chaperonin, ATP synthase, and enolase in rice seedlings by short-term ozone exposure treatment, it is possible to evaluate the effect of ozone on rice yield at an early laboratory level. It became clear that these proteins could be very effective molecular markers for diagnosing ozone sensitivity in yield.

  According to the method for evaluating the effect of ozone on rice yield of the present invention, it is possible to predict the effect of stress on the yield of rice seedlings. Therefore, it is very useful for crop breeding.

Claims (3)

Based on the expression level of one or more proteins selected from the group consisting of CPN-60 (60 kDa chaperonin), ATP synthase and enolase in the test rice varieties treated with ozone exposure, the yield of the test rice cultivars by ozone A method for assessing the effect of ozone on rice yield, characterized by assessing the impact on rice. The method for evaluating the effect of ozone on rice yield according to claim 1, wherein the ozone exposure treatment is performed on seedlings of the test rice varieties. A method for selecting rice varieties, comprising selecting rice varieties whose yield is not reduced or hardly lowered by ozone using the method for evaluating the effect of ozone on rice yield according to claim 1 or 2.