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Proteins imparting boron-tolerance and genes thereof

外国特許コード F110005473
整理番号 K05327WO
掲載日 2011年9月6日
出願国 アメリカ合衆国
出願番号 52216806
公報番号 20070079394
公報番号 7666678
出願日 平成17年3月15日(2005.3.15)
公報発行日 平成19年4月5日(2007.4.5)
公報発行日 平成22年2月23日(2010.2.23)
国際出願番号 JP2005004553
国際公開番号 WO2005087928
国際出願日 平成17年3月15日(2005.3.15)
国際公開日 平成17年9月22日(2005.9.22)
  • 特願2004-073324 (2004.3.15) JP
  • 2005JP004553 (2005.3.15) WO
発明の名称 (英語) Proteins imparting boron-tolerance and genes thereof
発明の概要(英語) The present invention provides genes and proteins having possibilities to generate plants having tolerance against excessive boron, which can confer a boric acid tolerance to organisms. 5 types of genes that can confer a boric acid tolerance to yeast, such as AtPAB2, AtRBP47c′, AtRPS20B, AtMYB13 and AtMYB68, AtRBP45a, AtRBP45b, AtRBP45c, AtRBP45d, AtRBP47a, AtRBP47b, AtRBP47c, AtUBP1a, AtUBP1b and AtUBP1c which were found by expressing several genes of higher plant Arabidopsis thaliana in yeast that is a organism model of eukaryote.
Further, a key to the toxicity mechanism of boric acid exists in the specific inhibition of splicing, and a gene related to enhancement of splicing efficiency also confers a boric acid tolerance.
Boron is one of the essential trace elements for higher plants (e.g., see nonpatent document 1).
As boron also has toxicity, by over ingesting it, plant growth is inhibited and animal dies of acute intoxication.
Boron exists in uncharged molecule state in soil solution.
Therefore, boron eluviates with relative ease and boron deficiency is easily developed in agricultural crops.
Lowering of yield point and quality in agriculture caused by boron deficiency is reported in 130 varieties in 80 or more countries worldwide including Japan (e.g., see nonpatent document 2).
Boron is also known to have a restricted range of optimal concentration compared with other elements, and has little difference between the concentrations at which deficiency symptoms develop and excess symptoms develop.
Therefore, the quantity adjustment of boron fertilizer application in agriculture is considered to be difficult.
Especially, when boron is fertilized excessively, removal of the boron is difficult and crop production in the agricultural land would be affected.
Further, as boron is contained in tap water, damages caused-by excessive boron often become a problem in drylands when irrigated agriculture is performed.
In addition to agricultural lands over-fertilized with boron in this way, land areas with high concentration of boron are found worldwide.
Countries having such areas have an important agenda for taking measures against damages caused by excessive boron in agricultural policy.
Further, as boron is also present in agents for treating metal surface and bleaches, wastewater from factories using these agents and bleaches contains boron in appreciable quantities.
Although lethal dose of boron for human is 15-20 mg, it is known that various disorders involving digestive organs and nervous systems are developed with less than the lethal dose of boron.
At present, the amount of boron contained in wastewater from factories is becoming an issue.
Recently, a role of boron in plants has been elucidated.
It was elucidated that boron bridges pectic polysaccharides in cell walls (e.g., see nonpatent document 3), and showed that the crossbridges are essential for plant growth (e.g., see nonpatent document 4).
This is the first knowledge regarding the physiological function of boron at a molecular level in plants.
On the other hand, many unclear points remains to be elucidated in the boron transportation mechanism in plants.
It was thought for a long time that boron enters into cells by passive diffusion of lipid bilayer, and is transported in plant body by transpiration stream (e.g., see nonpatent document 5).
In the meantime, it was known that nutrient conditions of boron, which are suited for growth, differ significantly among species and cultivars.
Although absorption, translocation and difference of use efficiency were exemplified as possible causes, molecules of the contributing factors were unknown.
In recent years, transportation via channels has been proposed (e.g., see nonpatent document 6), but the evidence was only in vitro experiments using an expression system or a membrane vesicle in Xenopus laevis oocytes, and it was not shown whether these channel molecules were involved in the boron transportation in actual individual plants.
Further, the presence of active transport by a transporter was suggested from absorption experiments in roots of sunflower roots (e.g., see nonpatent document 7), however, the responsible transporter was not identified.
The present inventors isolated an efflux boron tolerance protein BOR1 from a plant model, Arabidopsis thaliana for the first time in animate nature (e.g., see patent document 1).
It is thought that BOR1 is responsible for an active boron transportation to vessels under nutrient conditions of lower boron (e.g., see nonpatent document 8).
Further, YNL275w of yeast, aside from BOR1 is known as tolerance being responsible for boron transportation (e.g., see nonpatent document 9).
Further, as described above, Boron (B) is an essential trace nutrient for plants (e.g., see nonpatent document 10) and animals (e.g., see nonpatent document 11), but toxic at high concentrations (e.g., see nonpatent documents 12 and 13) .
Naturally occurring soils containing high concentration of B are distributed across the world and human activities such as fertilization with B, fossil combustion, and irrigation using B-containing water created an environment of high boron concentration (e.g., see nonpatent documents 12 and 13).
Symptoms of B toxicity in plants include chlorosis in leaf margin (e.g., see nonpatent document 13) and fruit disorder and/or bark necrosis (e.g., see nonpatent document 14).
Excess B reduces the yield and quality of crops.
B toxicity is a major obstruction of agricultural production worldwide.
B is also toxic to animals and microorganisms at high concentration.
The lethal dose of B is estimated to be about 140 mg/kg for adults and about 270 mg/kg for infants (.e.g., see nonpatent documents 15 and 16) .
Long term-high B intake leads to poor appetite, nausea, weight loss, and decreased sexual activity for humans (e.g., see nonpatent document 17).
At present, the acceptable safe intake of B for adults is suggested to be 13 mg per day (e.g., see nonpatent document 18).
B has been contained in food preservatives for its sterilization effect on microorganisms (e.g., see nonpatent document 19) .
In addition, B has been used as insecticides for many years, especially against cockroaches (e.g., see nonpatent document 20).
In the last several decades since B toxicity has been recognized, a number of studies were conducted to investigate toxic effects of B. Those were mostly physiological studies.
For example, in soybean leaves, the activity of allantoate amidohydrolase is decreased by boric acid (e.g., see nonpatent document 21).
The inhibitions of malate dehydrogenase and isocitrate dehydrogenase activities by B were observed in Chara corallina (e.g., see nonpatent document 22).
A negative correlation between placental B levels and delta-aminolevulinic acid dehydratase activities involved in synthesis of porphobilinogen (an intermediate of porphyrin synthesis) in newborns has been also reported (e.g., see nonpatent document 23).
Solubilized borates are thought to play a major role in B toxicity.
Boric acids in cells are partially converted into borates due to the higher internal pH.
When boric acids with high concentration are supplied to cells, intracellular borate concentration rises to form borate complexes with a variety of cis-diol containing intracellular molecules.
These cis-diols containing molecules include NAD+, ATP, S-Ado Met, RNA and several sugars (e.g., see nonpatent documents 24 and 25).
Since these molecules are used as coenzymes and/or substrates for a number of enzymes, binding of borates is likely to induce loss of function or alteration of enzyme activities, inhibition of biochemical reactions, and finally metabolic disorders.
Despite of the accumulation of biochemical and physiological analysis and speculation related to the toxic effect of B, molecular mechanism of B toxicity that leads to cell death has not been elucidated.
Patent document 1: Japanese Laid-Open Patent Application NO.2002-262872
Nonpatent document 1: Loomis, W. D.; Durst, R. W. (1992) Chemistry and biology of boron.
Biofactors 3: 229-239
Nonpatent document 2: Shorrocks, V. M. (1997) The occurrence and correction of boron deficiency.
Plant and Soil 193: 121-148
Nonpatent document 3: Matoh, T.; Ishigaki, K. I.; Ohno, K; Azuma, J. I. (1993) Isolation and characterization of a boron-polysaccharide complex from radish roots.
Plant Cell Physiol. 34: 639-642
Nonpatent document 4: O'Neill, M. A.; Eberhard, S.; Albersheim, P.; Darvill, A. G. (2001) Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth.
Science 294: 846-849
Nonpatent document 5: Marschner, H. (1995) Mineral Nutritin of Higher Plants, 2nd ed.
Academic Press, San Diego, Calif.
Nonpatent document 6: Dordas, C.; Chrispeels, M. J.; Brown, P. H. (2000) Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from Squash roots.
Plant Physiol. 124: 1349-1362
Nonpatent document 7: Dannel, F.; Heidrun, P; Romheld, V. (2000) Characterization of root boron pools, boron uptake and boron translocation in sunflower using the stable isotope 10B and 11B.
Aust. J. Plant Physiol. 156: 756-761
Nonpatent document 8: Takano, J.; Noguchi, K.; Yasumori, M.; Kobayashi, M.; Gajdos, Z.; Miwa, K.; Hayashi, H.; Yoneyama, T.; Fujiwara, T. (2002) Arabidopsis boron transporter for xylem loading.
Nature 420 (6913): 337-340
Nonpatent document 9: Zhao, R. M.; Reithmeier, R. A. F. (2001) Expression and characterization of the anion transporter homologue YNL275w in Saccharomyces cerevisiae.
American Journal of Physiology-Cell Physiology 281 (1): C33-C45
Nonpatent document 10: Warington, K. (1923) Ann. Bot. 37, 629-672
Nonpatent document 11: Park, M., Li, Q., Shcheynikov, N., Zeng, W., & Muallern, S. (2004) Mol. Cell 16, 331-341
Nonpatent document 12: Gupta, U. C., Jame, Y. W., Campbell, C. A., Leyshon, A. J., & Nicholaichuk, W. (1985) Can. J. Soil Sci.65, 381-409
Nonpatent document 13: Nable, R. O., Banuelos, G. S., & Paull, J. G. (1997) Plant Soil 193, 181-198
Nonpatent document 14: Brown, P. H., & Hu, H. (1996).
Ann. Bot. 77, 497-505
Nonpatent document 15: Young, E. G., Smith, R. P., & MacIntosh, O. C. (1949) Can. Med. Assoc. J. 61, 447-450
Nonpatent document 16: Arena, J. M., & Drew, R. H. (1986) in Poisoning, (C. C. Thomas, Splingfield). pp. 131
Nonpatent document 17: Hunt, C. D. (1993) in Encyclopedia of Food Science, Food Technology and Nutrition, vol. 1, eds. Macrae, R., Robinson, R. K. & Sadler, M J. (Academic Press, London), pp 440-447
Nonpatent document 18: WHO/FAO/IAEA (1996) in Trace Elements in Human Nutrition and Health, (World Health Organization, Geneva), pp. 175-179
Nonpatent document 19: Nielsen, F. H. (1997) Plant Soil 193, 199-208
Nonpatent document 20: Cochran, D. G. (1995) Experientia 51, 561-563
Nonpatent document 21: Lukaszewski, K. M., Blevins, D. G., & Randall, D. D. (1992) Plant Physiol. 99, 1670-1676
Nonpatent document 22: Reid R. J., Hayes J. E., Post A., Stangoulis J. C. R., & Graham R. D. (2004) Plant Cell Environ. 27, 1405-1414
Nonpatent document 23: Huel, G., Yazbeck, C., Burnel, D., Missy, P., & Kloppmann. W. (2004) Toxicol.
Sci. 80,304-309
Nonpatent document 24: Ralston, N. V. C., & Hunt, C. D. (2000) FASEB J. 14, A538
Nonpatent document 25: Ricardo, A., Carrigan, M. A., Olcott, A. N., & Benner, S. A. (2004) Science 303, 196
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

特許請求の範囲(英語) [claim1]
1. A method for conferring boric acid tolerance to a yeast comprising: i) introducing the DNA of SEQ ID NO: 3 operably linked to a promoter into the yeast;
ii) culturing the yeast in the presence of boric acid and expressing the DNA of SEQ ID NO: 3 to confer boric acid tolerance to the yeast; and
iii) selecting the yeast in which tolerance to 80 mM boric acid is conferred.
  • 発明者/出願人(英語)
  • 435/483
参考情報 (研究プロジェクト等) PRESTO Conversion and Control by Advanced Chemistry AREA