產生RNase是植物對抗缺磷逆境的一種方式。已知TN5-9水稻懸浮培養細胞，在缺磷培養下，可誘導產生一17kDa之RNase。為了解是否在缺磷培養下，僅產生一種RNase？而被誘導產生之RNase其基因表現特性為何？本研究中採用分生方法，以subtractive cDNA PCR、RT-PCR方式選殖出缺磷處理下，水稻被誘導表現之RNase基因。在16個轉型株中，並沒有發現T2/S family RNase基因，但找到有一可能具有RNase活性之pathogenesis-related protein 10a (PR-10a)基因。之後以北方轉漬法及RT-PCR方式，可以驗證水稻在缺磷狀況下，會誘導PR-10a基因大量表現。為確認PR-10a是否具有RNase活性，將此一基因重組後，轉型至大腸桿菌E. coli BL21 (DE3)，進行重組蛋白的表現。純化所得之重組蛋白，在Nus tag片段切除後，可分離得到17kDa之PR-10a蛋白質。進行RNase活性檢測，發現唯有重組蛋白之native form不具有RNase活性。將重組蛋白以β-ME、DTT、EDTA、CIAP處理後，發現CIAP處理之重組蛋白可表現出RNase活性，可分解rRNA及tRNA。

In the previous study, we have known that rice TN5-9 suspension cultured cells will secrete a 17kDa RNase to the medium upon phosphate-starvation. It indicates that RNase production can be induced to reuse the organic phosphate during phosphate-starvation. However, it is unclear, how many RNase genes in the rice genome and what is the gene expression pattern of RNases upon phosphate-starvation.
In this study, we try to clone RNase gene from rice by subtractive cDNA PCR and RT-PCR. Sixteen clones induced by phosphate-starcation were isolated. After sequences alignment, none of 16 clones belonged to RNase of T2/S family, but one of them was a putative RNase gene shich was very similar to pathogenesis-related protein 10a gene (PR-10a). Northern and RT-PCR results indicate that rice PR-10a gene is highly expressed during phosphate-starvation, especially from 6 hour to 24 hour after treatment of phosphate-starvation. In order to check whether PR-10a protein has RNase activity. We use E. coli BL21 (DE3) to expressed recombinant PR-10a protein. After protein purification and enterokinase digestion for removing of Nus tag fragnment, a 17kDa recombinant rice PR-10a protein was obtained. However, this mature recombinant PR-10a protein was inactive for RNA digestion. After β-ME, DTT, EDTA, or CIAP treatment, only recombinant PR-10a protein that treated with CIAP exhibited RNase activity in digestion of rRNA and tRNA.

Abel S, Glund K (1986) Localization of RNA-degrading enzyme activity within vacuoles of cultured tomato cells. Physiol. Plant. 66: 79-86
Abel S, Glund K (1987) Ribonuclease in plant vacuoles: purification and molecular properties of the enzyme from cultured tomato cells. Planta 172: 71-78
Bantignies B, Seguin J, Muzac I, Dedaldechamp F, Gulick P, Ibrahim R (2000) Direct evidence for ribonucleolytic activity of a PR-10-like protein from white lupin roots. Plant Mol. Biol. 42: 871-881
Bariola PA, Howard CJ, Taylor CB, Verburg MT, Jaglan VD, Green PJ (1994) The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J. 6: 673-685
Biesiadka J, Bujacz G, Sikorski MM, Jaskolski M (2002) Crystal structures of two homologous pathogenesis-related proteins from yellow lupine. J. Mol. Biol. 319: 1223-1234
Blank A, McKeon TA (1989) Single-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated. Proc. Nat. Acad. Sci. USA 86: 3169-3173
Borsics T, Lados M (2002) Dodder infection induces the expression of a pathogenesis-related gene of the family PR-10 in alfalfa. J. Exp. Bot. 53: 1831-1832
Brown PH, Ho T-HD (1986) Barley aleurone layers secrete a nuclease in response to gibberellic acid. Plant Physiol. 82: 801-806
Bufe A, Spangfort MD, Kahlert H, Schlaak M, Becker W-M (1996) The major birch pollen allergen, Bet v 1, shows ribonuclease activity. Planta 199: 413-415
Dodds PN, Bonig I, Du H, Rodin J, Anderson MA, Newbigin E, Clarke AE (1993) S-RNase gene of Nicotiana alata is expressed in developing pollen. Plant Cell 5: 1771-1782
Dodds PN, Clarke AE, Newbigin E (1996) Molecular characterisation of an S-like RNase of Nicotiana alata that is induced by phosphate starvation. Plant Mol. Biol. 31: 227-238
Green PJ (1994) The ribonucleases of higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 421-445
Hashimoto M, Kisseleva L, Sawa S, Furukawa T, Komatsu S, Koshiba T (2004) A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant Cell Physiol. 45: 550-559
Hugot K, Ponchet M, Marais A, Ricci P, Galiana E (2002) A tobacco s-like RNase inhibits hyphal elongation of plant pathogens. Mol. Plant Microbe Interact. 15: 243-250
Jwa N, Agrawal GK, Rakwal R, Park C, Agrawal VP (2001) Molecular cloning and characterization of a novel jasmonate inducible pathogenesis-related class 10 protein gene, JIOsPR10, from rice (Oryza sativa L.) seedling leaves. Biochem. Biophys. Res. Commun. 286: 973-983
Khush GS (1997) Origin, dispersal, cultivation and variation of rice. Plant Mol. Biol. 35: 25-34
Koeck M, Loeffler A, Abel S, Glund K (1995) cDNA structure and regulatory properties of a family of starvation-induced ribonucleases from tomato. Plant Mol. Biol. 27: 477-482
Koistinen KM, Kokko HI, Hassinen VH, Tervahauta AI, Auriola S, Karenlampi SO (2002) Stress-related RNase PR-10c is post-translationally modified by glutathione in birch. Plant, Cell and Environ. 25: 707-715
Kondo K, Yamamoto M, Matton DP, Sato T, Hirai M, Norioka S, Hattori T, Kowyama Y (2002) Cultivated tomato has defects in both S-RNase and HT genes required for stylar function of self-incompatibility. Plant J. 29: 627-636
Liu C, Muchhal US, Uthappa M, Kononowicz AK, Raghothama KG (1998) Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus. Plant Physiol. 116: 91-99
McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama F, Clarke AE (1989) Style self-incompatibility gene products of Nicotiana alata are ribonucleases. Nature 342: 955-958
McGee JD, Hamer JE, Hodges TK (2001) Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea. Mol. Plant Microbe Interact. 14: 877-886
Mittler R, Lam E (1995) Identification, characterization, and purification of a tobacco endonuclease activity induced upon hypersensitive response cell death. Plant Cell 7: 1951-1962
Moiseyev GP, Beintema JJ, Fedoreyeva LI, Yakovlev GI (1994) High sequence similarity between a ribonuclease from ginseng calluses and fungus-elicited proteins from parsley indicates that intracellular pathogenesis-related proteins are ribonucleases. Planta 193: 470-472
Moiseyev GP, Fedoreyeva LI, Zhuravlev YN, Yasnetskaya E, Jekel PA, Beintema JJ (1997) Primary structures of two ribonucleases from ginseng calluses: New members of the PR-10 family of intracellular pathogenesis-related plant proteins. FEBS Letters 407: 207-210
Park CJ, Kim KJ, Shin R, Park JM, Shin YC, Paek KH (2004) Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J. 37: 186-198
Perez-Amador MA, Abler ML, De Rocher EJ, Thompson DM, Van Hoof A, LeBrasseur ND, Lers A, Green PJ (2000) Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis. Plant Physiol. 122: 169-180
Puhringer H, Moll D, Hoffmann-Sommergruber K, Watillon B, Katinger H, Da Camara Machado ML (2000) The promoter of an apple Ypr10 gene, encoding the major allergen Mal d 1, is stress- and pathogen-inducible. Plant Sci. 152: 35-50
Rogers SW, Rogers JC (1999) Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells. Plant Physiol. 119: 1457-1464
Sacher JA, Tseng J, Williams R, Cabello A (1982) Wound-induced RNase activity in sweet potato. Evidence for regulation at transcription. Plant Physiol. 69: 1060-1065
Sims TL, Ordanic M (2001) Identification of a S-ribonuclease-binding protein in Petunia hybrida. Plant Mol. Biol. 47: 771-783
Stepinski D (2002) Ribonuclease activity in roots of soybean seedlings subjected to chilling stress and recovery. Acta Physiologiae Plantarum 24: 297-301
Taylor CB, Bariola PA, del Cardayre SB, Raines RT, Green PJ (1993) RNS2: A senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc. Nat. Acad. Sci. USA 90: 5118-5122
Taylor CB, Green PJ (1991) Genes with homology to fungal and S-gene RNases are expressed in Arabidopsis thaliana. Plant Physiol. 96: 980-984
Walles B, Han SP (1998) Ribosomes in incompatible pollen tubes in the solanaceae. Physiol. Plant. 103: 461-465
Wu F, Yan M, Li Y, Chang S, Song X, Zhou Z, Gong W (2003) cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus. Biochem. Biophys. Res. Commun. 312: 761-766
Ye XY, Ng TB (2002) A novel and potent ribonuclease from fruiting bodies of the mushroom Pleurotus pulmonarius. Biochem. Biophys. Res. Commun. 293: 857-861
Ye Z-H, Droste DL (1996) Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans. Plant Mol. Biol. 30: 697-709
Yen Y, Green PJ (1991) Identification and properties of the Major Ribonucleases of Arabidopsis thaliana. Plant Physiol. 97: 1487-1493
Zhou XJ, Lu S, Xu YH, Wang JW, Chen XY (2002) A cotton cDNA (GaPR-10) encoding a pathogenesis-related 10 protein with in vitro ribonuclease activity. Plant Sci. 162: 629-636
陳志郎 (1997) Study on the phosphate-starvation induction of excreted ribonucleases in suspension-cultured cells of rice. Master thesis. Dep. Biology, NTNU