摘要
随着植物抗病基因工程的发展,越来越多的抗病基因被人们所发现。2004年,Taler等从印度野生瓜的霜霉病抗性品种中克隆得到两个具有丝氨酸乙醛酸转氨酶(serineglyoxylate aminotransferase,SGT)活性的甜瓜霜霉病抗性基因At1和At2。Taler等发现At1和At2具有丝氨酸乙醛酸转氨酶活性与植物光呼吸途径相关,不属于任何一类已知R基因,对病原菌的抵抗没有种属专化性,并且它们的抗病作用与H_2O_2相关,基于此种现象,他们提出一种新的抗病机制“酶抗病性”,这是首次报道的通过改变酶的表达量而赋予植物抗病性。据推测At1和At2还可能对霜霉病以外的其它多种植物叶部病害具有抗性作用,是很有研究价值的抗病基因,并且,“酶抗病性”作用机制的提出还需要更多的基因证据和试验支持。据此本论文从光呼吸作用非常强的大豆中克隆At1和At2的同源基因GmSGT,对其进行序列分析、酶活性中心预测、原核表达分析,并将其转入受体植物中进行抗病性鉴定。现将本论文的主要研究结果及创新点总结如下:
1.利用大豆EST序列和5'-Race技术,首次从大豆霜霉病抗性品种中克隆了编码丝氨酸乙醛酸转氨酶的GmGGT1基因序列(已获得国家发明专利1项,专利号:ZL2005100887 83.4),同时从受SA诱导的大豆霜霉病感病品种黑农10号中克隆得到了两条GmSGT1的同源基因GmSGT2,GmSGT3。序列分析表明,GmSGT1与Taler等报道的甜瓜霜霉病抗病基因At1、At2氨基酸序列同源性达88.03%和87.78%;同时,GmSGT1与拟南芥(Arabidopsis thaliana L.)、水稻(Oryzae sativa L.)、贝母(Fritillariaagrestis)、紫萍(Spirodela polyrrhiza)的丝氨酸乙醛酸转氨酶同源性达83.33%-85.79%。GmSGT1推导蛋白序列分析显示它具有一个5磷酸吡哆醛结合位点GSQKAL和一个强的过氧化物体定位信号SRI,表明该基因可能在植物的过氧化物体中通过光呼吸途径起作用。GmSGT2和GmSGT3与GmSGT1核苷酸序列同源性高达96.38%和99.17%,推导的氨基酸序列同源性为97.03%和99.25%。利用生物信息学方法对GmSGT1,GmSGT2,GmSGT3蛋白的酶学活性中心进行了分析,结果显示三个蛋白序列均具有丝氨酸乙醛酸转氨酶活性。
2.首次通过存大肠杆菌中模拟植物光呼吸途径的技术,证实了GmSGT1具有丝氨酸乙醛酸转氨酶活性。光呼吸途径中发生的反应:乙醇酸(?)H_2O_2+乙醛酸;乙醛酸(?)甘氦酸。因大肠杆菌中含有乙醇酸氧化酶(GOX),所以向原核表达菌株中加入乙醇酸,可完成上述反应,通过检测H_2O_2量的变化,可确定GmSGT1基因的丝氨酸乙醛酸转氨酶功能。本研究结果显示,只有表达GmSGT1蛋白的组分能诱导大量H_2O_2产生,对照组分无,因此,可以确定GmSGT1具有丝氨酸乙醛酸转氨酶活性。
3.分析了GmSGT蛋白与大豆不同品种的抗性相关性。Western杂交结果显示,抗霜霉病品种早丰5号和九农9号中可检测到有目的蛋白的表达,而感病品种黑农10号中无表达。SA诱导前、后半定量RT-PCR结果表明,感病品种黑农10号在SA诱导前未检测到表达,而SA诱导后有微量表达,大豆对霜酶病的抗病性也有大幅度提高。因此,我们推断,GmSGH的表达确实和SA诱导途径相关,并且随着GmSGT1表达水平的提高,大豆对霜霉病的抗病性也明显提高。
4.构建了GmSGT1植物表达载体,进行烟草转化,获得了转基因植株,并对其进行烟草赤星病,黑胫病,青枯病的抗性鉴定,结果表明转基因烟草显著提高了烟草对赤星病,黑胫病,青枯病的抗性。本研究为Taler等提出的植物“酶抗性基因”提供了新的实验证据。
5.利用抗病品种早丰5号探索了GmSGT1基因在大豆中的时空表达特性,该基因在大豆叶片中有表达,而根,茎中无表达,并且随着生育期的增强而表达增强,生殖生长期最强,然后随着细胞的老化而表达减弱,直至消失。这表明GmSGT1的变化趋势与植物光呼吸的变化趋势相符。同时检测了光呼吸途径中在丝氨酸乙醛酸转氨酶上游起作用的乙醇酸氧化酶(GOX)的表达特性,结果表明GOX的表达水平与GmSGT1表达水平的变化具有一致性,说明GmSGT1表达水平提高的同时,它的上游反应也增强,H_2O_2表达也提高。这与我们推测的GmSGT蛋白在植物光呼吸途径中起作用的结论相符。
6.研究确定了大豆遗传转化体系。建立了大豆早丰5号,黑农10号的胚芽尖组培体系,并利用农杆菌LBA4404介导法将构件好的植物表达载体pIM1.1-GmSGT-plus转化早丰5号,RNAi植物表达载体pIM1.1-GmSGT-plusF转化黑农10号,获得了再生植株。同时,对早丰5号的胚芽尖,子叶节,胚轴三种外植体在再生频率,再生时间,K筛选浓度,农杆菌不同侵染时间对重生芽再生频率的影响进行了研究,为早丰5号组培,转化体系的进一步优化提供了实验依据。
With the development of genetic engenier of plant resistance, more and more resistant genes were descovered. Taler et al. (2004) have cloned two genes At1 and At2 encoding peroxisomal serine glyoxylate aminotransferase (SGT) from the PI line of wild melon (Cucumis melo) resistant to downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis. The study has provided solid evidence that the expression of SGT gene confers protection against disease, by which they proposed a new type of defense mechanism called "enzymatic disease resistance (eR)", since the amino acid identity analysis revealed that these gene products differ from all the previously known R-proteins. eR is valuable and it is worth to be coloned from other plants to gain more evidence. So we coloned the eR gene from soybean, sequences analysis, expressed in E. coli, and the resistance identify of transgenec plant. The result and originality are shown as below:
1. A GmSGT gene encoding serine glyoxylate aminotransferase has been cloned from a soybean variety resistant to downy mildew disease by using a short salicylic acid (SA)-inducted soybean EST and the 5'-RACE method. The full-length of GmSGT gene is 1206 bp encoding a serine glyoxylate aminotransferase. The ORF and deduced 402 amino acids and the 5'and 3'UTR sequences can be found in the GenBank (accession No. DQ167250). Notably, the ORF possesses two typical motifs for SGT that is a GSQKAL binding site (at position 198-203) for the pyridoxal-5-phosphate cofactor and a SRI peroxisomal targeting site at the 3'end, suggesting that the enzyme occurs in plant peroxisome and participates in the production of glycine during photorespiration. Sequence analysis has shown that the GmSGT does not belong to any known R genes. Comparison of nucleotide sequences between GmSGT and At1, At2 from melon (Taler et al., 2004) showed 80.76%and 78.77% homology, respectively, and the similarity of deduced amino acid sequence was as high as 88.30% and 88.28%. Comparison of GmSGT amino acid sequence with other known SGT or AGT proteins from Cucumis melo, Fritillaria agrestis, Spirodela polyrrhiza, Arabidopsis thaliana, Vitis vinifera and Oryza sativa showed 82.7-88.3% similarity. Interestingly, phylogenic analysis showed that the GmSGT and the melon CmAT1 were clustered in the same group, while the other SGTs or AGTs were formed in different groups.
2. As SGT can catalyze L-serine and glyoxylate into glycine, the GmSGT expressed in E. coli showed enzymatic activity which produced 11.9 to 23 times of more glycine than that of the control, the empty vector without GmSGT gene. Meanwhile, the expression of GmSGT gene in E. coli dramatically increased H_2O_2 content and inhibited bacterial growth.
3. Western blot analysis showed that the GmSGT protein was only produced in the downy mildew-resistant variety Zaofeng5 and Jiunong9, while not in the susceptible variety Heinong10. Transcriptional RT-PCR analysis showed that the GmSGT could be detected in the resistant variety (Zaofeng5) prior to SA treatment and it was increased six times more after SA induction. However, for the susceptible variety, the GmSGT could not be detected prior to or was only slightly increased after SA induction. Results indicated that the function of GmSGT gene was closely correlated with the soybean downy mildew resistance, which provided new evidence in supporting the enzymatic disease resistance (eR) proposed by Taler et al. in 2004.
4. Set up the expression carrier of GmSGT and transfored it into tobacco. The resistance of the transgenic plants to Alternaria alternata, Phytophthora parsitica, Pseudomonas solanacearum Smithis notability increased.
5. RT-PCR analysis of differrent parts during differrent periord showed that the GmSGT could be detected in the leaves but not in the root and stem. It also show that the expression is increased with the growth of soybean and the tendency is identical with photorespiration.
6. Established the embryonic tip regeneration system of Zaofeng5 and Heinong10 and compaired the regeneration frequency of the embryonic tip, cotyledonary node and hypocotyl segment.Transformed the expression carrier of GmSGT and the RNAi carrier of GmSGT into Heinong10 and Zaofeng5 seperetly using Agrobacterium tumefaciens-medisted transformation and got the regeneration plant.
引文
1 Aaron H. Liepman and Laura J. Olsen Alanine Aminotransferase Homologs Catalyze the Glutamate:Glyoxylate Aminotransferase Reaction in Peroxisomes of Arabidopsis. Plant Physiol, January 2003, Vol. 131, pp. 215-227
2 Alfano J.R., Charkowski A.O., Deng W.L., Badel J.L., Tanja P.O., and Alan C. 2000. The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type Ⅲ secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants. Proc. Ntal. Acad. Sci. USA 97:4856-4861.
3 Alexander D , Goodman R M, Gut-Rella M et al. 1993. Proc Natl Acad Sci USA. 90: 7327-7334. 123 Alexandre, H., L. Plourde, C. Charpentier, and J. Fran(?)ois. 1998. Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae. Microbiology 144:1103-1111
4 Arnold, D.L., Gibbon, M.J., Jackson, R.W., Wood, J.R., Brown, J., Mansfield, J.W., Taylor, J.D. and Vivian, A. 2001. Molecular characterization of avrPphD, a widely-distributed gene from Pseudomonas syringae pv. phaseolicola involved in non-host recognition by pea (Pisum sativum). Physiol. Mol. Plant Pathol. 58,55- 62.
5 Aron H. Liepman and Laura J. Olsen Peroxisomal alanine : glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana The Plant Journal Volume 2001, 25 5-487.
6 Astua-Monge, G. et al. 2000 Xv4-avrXv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and the wild tomato relative Lycopersicon pennellii. Mol. Plant-Microbe Interact. 13,1346-1355
7 Averyanov A A, Lapikova V P, Djawakhia V G. 1993. Active oxygen mediates heat-induced resistance of rice plant to blast disease. Plant Sci, 92:2734-2738
8 Aviv, D. H., Rusterucci, C., Iii, B. F., Dietrich, R. A., Parker, J. E., and Dangl, J. L. 2002. Runaway cell death, but not basal disease resistance, in Isd1 is SA- and NIM1INPR1 -dependent. Plant J. 29:381-391.
9 Betsche T.1983.Aminotransfer from alanine and glutamate to glycine and serine during photorespiration in oat leaves. Plant Physiol 71: 961-965
10 Bfischges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, van Daelen R, van der Lee T, Diergaarde P, Groenendijk J, Tsch S, Vos P, Salamini F, Schulze-Lefert P, 1997.The barley Mlo gene: a novel control element of plant pathogens resistance. Cell, 88 :695-705
11 B.J. Feys and J.E. Parker, 2000.Interplay of signaling pathways in plant disease resistance, Trends Genet. 16, 449-455.
12 Bohman, S., Wang, M., and Dixelius, C. 2002. Arabidopsis thaliana derived resistance against Leptosphaeria maculans in a Brassica napus genomic background. Theory Appl. Genet. 10:498-504.
13 Bonnet, X., Naulleau, G., Shine, R. & Lourdais, O. 2000.What is the appropriate time scale for measuring costs of reproduction in a capital breeder? Evolutionnary Ecology, 13, 485- 497.
14 Bradley DJ, Kjellbom P, Lamb CJ. 1992.Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell. Jul 10;70(1):21-30.
15 Braun C J, Siedow J N, Williams M E, Levings C S, 1989. Mutations in the maize mitochondrial T-urfl3 gene eliminate sensitivity to a fungal pathotoxin. Proc Natl Acad Sci USA, 86:4435-4439.
16 Brisson LF, Tenhaken R, Lamb C. 1994. Function of Oxidative Cross-Linking of Cell Wall Structural Proteins in Plant Disease Resistance. Plant Cell. Dec;6(12):1703-1712.
17 Brickner, D.G., Harada, J.J., Olsen, L.J. (1997) Protein transport into higher plant peroxisomes. In vitro assay provides evidence for receptor involvement. Plant Physiol. 113,1213-1221.
18 Caid G, Klene M, Kifle S, et al. 1997. Positional cloning of a gene for nematode resistance in sugar beet[ J ]. Science, 275:832-834.
19 Cao, H., Li, X., and Dong, X. 1998. Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc. Natl. Acad. Sci. USA 95:, 6531-6536.
20 Cessna et al., Cessna, V.E. Sears, M.B. Dickman and P.S. Low, 2000.Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant, Plant Cell 12 , pp. 2191-2200.
21 Charne, D.G., et al. 1999. Production of pathogen resistant plants. Patent Application WO9904012 (Pioneer Hi-Bred International).
22 Chycoski CI, and ZK Punja. 1996. Characteristics of populations of Phytophthora infestans from potato in British Columbia and other regions of Canada during 1993 to 1995. Plant Dis 80:579-589.
23 Chen, Z., Ricigliano, J. W., and Klessig, D. F. 1993. a. Purification and characterization of a soluble salicylic acid-binding protein from tobacco. Proc. Natl. Acad. Sci. USA 90:9533-9537.
24 Chen, Z., and Klessig, D. F. 1991. Identification of a soluble salicylic acid-binding protein that may function in signal transduction in the plant disease resistance response. Proc. Natl. Acad. Sci. USA 88:8179-8183.
25 Chen Z, Silva H, Klessig DF. 1993. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science. 262(5141):1883-1886.
26 Ciesiolka, L. D., Hwin, T., Gearlds, J. D. & 11 other authors 1999. Regulation of expression of avirulence gene avrRxv and identification of a family of host interaction factors by sequence analysis of avrBsT.Mol Plant-Microbe Interact 12, 35-44.
27 Cohen Y, Gisi U and Niderman T 1993. Local and systemic protection against Phytophthora infestans induced in potato and tomato plants by jasmonic acid and jasmonic-methylester. Phytopathology 83: 1054-1062.
28 Cohen, Y., Eyal, H., Hanania, J., and Malik, Z. (1989). Ultrastructure of Pseudoperonospora cubensis in muskmelon genotype susceptible and resistant to downy mildew. Physiol. Mol. Plant Pathol. 34, 27-40.
29 Corpas, F.J., Barroso, J.B., and del Rio, L.A. (2001). Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci. 6, 145-150.
30 Costet L, Cordelier S, Dorey S, Baillieul F, Fritig B, Kauffmann S 1999. Relationship between localized acquired resistance (LAR) and the hypersensitive response (HR): HR is necessary for LAR to occur, and salicylic acid is not sufficient to trigger LAR. Mol Plant-Microbe Interact 12: 655-662
31 Dangl, J., and Jones, J.D.G. (2001). Plant pathogens and integrated defence responses to infection. Nature 411,826-833.
32 Dangl JL, Jones JDG. 2001. Plant pathogens and integrated defense responses to infection. Nature, 411: 826-833.
33 Danpure C.1997.Variable peroxisomal and mitochondrial targeting of alanine: glyoxylate amino transferase in mammalian evolution and disease. Bioessays 19: 317-326.
34 Delaney, T. P. 1997. Genetic dissection of acquired resistance to disease. Plant Physiol. 106,5-12.
35 DE Wit MJ and Ransome IGD, Editors 1992. Inversion Tectonics of the Cape Fold Belt, Karoo and Cretaceous Basins of Southern Africa. Balkema, Rotterdam, 269 pp.
36 Dorey, S., Baillieul, F., Pierrel, M.-A., Saindrenan, P., Fritig, B., and Kauffmann, S. 1997. Spatial and temporal induction of cell death, defense genes, and accumulation of salicylic acid in tobacco leaves reacting hypersensitively to a fungal glycoprotein elicitor. Mol. Plant-Microbe Interact. 10:646-655.
37 Doke, N. 1987. Release of Ca2+ bound to plasma membrane in potato tuber tissue cells in the initiation of hypersensitive reaction. Ann. Phytopathol. Soc. Jpn. 53:391.
38 Eckardt NA 2004 Aminotransferases confer 'enzymatic resistance' to downy mildew in melon. The Plant Cell 16(1):1-3.
39 Eckardt, N.A. (2003). Viral defense and counter-defense: A role for adenosine kinase in innate defense and RNA silencing. Plant Cell 15, 2758-2761.
40 Feys, B.J., and Parker, J.E. (2000). Interplay of signaling pathways in plant disease resistance. Trends Genet. 16,449-455.
41 Freiberg, C., Fellay, R., Bairoch, A., Broughton, W.J., Rosenthal, A. and Perret, X. 1997. Molecular basis of symbiosis between Rhizobium and legumes. Nature, 387,394- 401.
42 Galperin, M.Y., and A.J.M. Baker 2004. From biofouling to bioremediation: the good, the bad and the vague. Curr. Opin. Biotech. 15:167-169.
43 Gassmann, D. 2000. Revision of the Papuan Idiocnemis bidentata-group (Odonata:Platycnemididae). Zoologische Mededelingen Leiden 74(23): 375-402.
44 Gebhardt, J.S., Wadsworth, G.J., Matthews, B.F. (1998) Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase. Plant Mol. Biol. 37, 99 108.
45 Gietz, R. D., R. H. Schiestl, A. R. Willems, and R. A. Woods. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355-360
46 Gonzales, M. I., R. Stucka, M. A. Blazquez, H. Feldmann, and C. Gancedo. 1992. Molecular cloning of CIF1, a yeast gene necessary for growth on glucose. Yeast 8:183-192
47 Gorlach,A.,G.Camenisch,I.Kvietikova,L.Vogt,R.H.Wenger,andM.Gassmann.2000.Efficienttranslation of mouse hypoxia-inducible factor-la under normoxic and hypoxic conditions. Biochim.Biophys.Acta 1493:125-134.
48 Gorlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H.. and Ryals, J.1996. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8, 629-43.
49 Greenberg, J. T. 1997. Programmed cell death in plant-pathogen interactions. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:525-545.
50 Hagashita, T., Yoshida, T., Izumi, Y., Mitsunaga, T. (1996) Cloning and expression of the gene for serine: glyoxylate aminotransferase from an obligate methylotroph, Hypho microbium methylovorum GM2 . Eur. J. Biochem. 241,15.
51 Hammond-Kosack, K. E., Staskawicz, B. J., Jones, J. D. G., and Baulcombe, D. C. 1995. Functional expression of a fungal avirulence gene from a modified potato virus X genome. Mol. Plant-Microbe Interact. 8:181-185.
52 Hammond-Kosack, K.E. and Jones, J.D.G. 1996 Inducible plant defence mechanisms and resistance gene function. The Plant Cell 8,1773-1791.
53 Hao L, Wang H, Sunter G and Bisaro DM 2003. Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell 15:1034-1048.
54 Herrero, S., A. K. Culbreath, A. S. Csinos, H. R. Pappu, R. C. Rufty, and M. E. Daub. 2000 Nucleocapsid gene-mediated transgenic resistance provides protection against tomato spotted wilt virus epidemics in the field. Phytopathology. 90:139-147.
55 Hogarth, T., C. Hasluck, G. Pierre, M. Winterbotham and D. Vivian. 2000. Work Life Balance 2000: Results from the Baseline Study. Summary Report. London: Department for Education and Employment. 41pp.
56 Hondred, D., Hunter, J.M., Keith, R., Titus, D.E., Becker, W.M. (1985) Isolation of serine: glyoxylate aminotransferase from cucumber cotyledons. Plant Physiol. 79 (95), 102.
57 Johal GS. Briggs SP. 1992. Science, 258:985-987.
58 Jones AM, Dangl JL. 1996. Logjam at the Styx: programmed cell death in plants. Trends in Plant Science 1: 114-119.
59 Joosten M H A J, De Wit P J G M. 1999.The tomato Cladosporium fulvum interaction: a versatile experimental system to study plant-patogen interactions [J]. Annu Rev Phytopathol, 37: 335-379.
60 Kang L, Li J, Zhao T, Xiao F, Tang X, Thilmony R, He SY and Zhou JM 2003. Interplay of the Arabidopsis nonhost resistance gene NH01 with bacterial virulence. Proc. Natl. Acad. Sci. USA 100:3519-3524.
61 Keen, N. T. 1990. Gene-for-gene complementarity in plant-pathogen interactions. Annu. Rev. Genet., 24, 447-463.
62 Keen NT 1999.Plants and microorganisms - listening in on the conversation. Nat Biotechnol 17:958
63 Keller, R, 1999. The origin and morphogenesis of amphibian somites. Current Topics In Developmental Biology 47, 33-96.
64 Kesarwani, M., Azam, M., Natarajan, K., Mehta, A., and Datta, A. 2000. Oxalate decarboxylase from Collybia velutipes: Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato. J. Biol. Chem. 275, 7230-7238.
65 Knudtson, L.M. and P.A. Hartman. 1992. Routine procedures for isolation and identification of enterococci and fecal streptococci. Appl. and Env. Micro. 58:3027-3031.
66 Kogel, Karl H.;Ehrlich-Rogozinski, Sarah.;Reisener, Hans J.; Sharon, Nathan. 1984. Surface Galactolipids of Wheat Protoplasts as Receptors for Soybean Agglutinin and Their Possible Relevance to Host-Parasite Interaction. Plant Physiol. Dec;76(4):924-928.
67 Lauge,R.,and De Wit,P.J.G.M.1998. Fungal avirulence genes:structure and possible functions. Fungal Genet Biol 24:285-297.
68 Lahaye, T., and Bonas, U. 2001. Molecular secrets of bacterial type III effector proteins. Trends Plant Sci. 6:479-485.
69 Leon, J., Shulaev, V., Yalpani, N., Lawton, M. A., and Raskin, I. 1995. Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis. Proc Natl Acad Sci U S A 92, 10413-7.
70 Liepman, A.H., and Olsen, L.J. (2001). Peroxisomal alanine:glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana. Plant J. 25,487-498.
71 Liu HK, Yang C, Wei ZM. Efficient Agrobacterium tumefaciens -mediated transformation of soybeans using an embryonic tip regeneration system[J]. Planta, 2004,219:1042-1049
72 Mahalingam, R., and Fedoroff, N. 2003. Stress response, cell death and signaling: The many faces of reactive oxygen species. Physiol. Plant. 119,56-68.
73 Malamy et al., 1990 J. Malamy, J.P. Carr, D.F. Klessig and I. Raskin, Salicylic acid: a likely endogenous signal in the resistance response of tobacco viral infection, Science 250,pp. 1002-1004.
74 Martin G B, Williams J G K, Tanksly S D. 1991. Rapid identification of markers linked to a pseudomonas resistance gene in tomato by using random primers and near-isogenic lines [J] . Proc. Natl. Acad. Sci. USA.,88:2336-2340.
75 Mas, C. M., Dixon, M. S., Parniske, M., Golstein, C., and Jones, J. D.1998. Genetic and molecular analysis of tomato Cf genes for resistance to Cladosporium fulvum. Philos Trans R Soc Lond B Biol Sci 353,1413-24.
76 M. Dickman, 2000.Impacts of a mosquito selective pesticide, Bti, on the macroinvertebrates of a subtropical stream in Hong Kong, Chemosphere 41, pp. 209-217.
77 Mehta, P.K., Hale, T.I., Christen, P. (1993) Aminotransferases: demonstration of homology and division into evolutionary subgroups. Eur. J. Biochem. 214, 549 561.
78 Mills, S. D., Boland, A., Sory, M., van der Smissen, P., Kerbourch,C., Finlay, B. B. & Cornelis, G. R. 1997. Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein. ProcNatl Acad Sci USA 94,12638-12643.
79 Metraux, J.P., H. Signer, J. Ryals, E. Ward, M. Wyss-Benz, J. Gaudin, K. Raschdore, E. Schmid, W. Blum, and B. Inverardi. 1990. Increase of salicylicacid at the onset of systemic acquired resistance in cucumber. Science 250: 1004-1006.
80 Morel, J.-B., and Dangl, J. L. 1997. The hypersensitive response and the induction of cell death in plants. Cell Death Differ. 4:671-683.
81. Nancy A. Eckardt Aminotransferases Confer "Enzymatic Resistance" to Downy Mildew in Melon The Plant Cell, Vol. 16,1-3, January 2004
82 Napoli, C. and Staskawicz, B.1987. Molecular characterization and nucleic acid sequence of an avirulence gene from race 6 of Pseudomonas syringae pv. Glycinea. J. Bacteriol. 169,572-578.
83 Orbach, M. J., Farrall, L., Sweigard, J.A., Chumley, F.G. and Valent, B. 2000. A telomeric virulence gene determines efficacy for the rice blast resistance gene Pi-ta. Plant Cell 12 : 2019-2032.
84 Palma JM, Sandalio LM, Corpas FJ, et al. 2002. Plant proteases, protein degradation, and oxidative stress: role of peroxisomes. Plant Physiology and Biochemistry, 40:521-530.
85 Paris, H.S. and S. Cohen. 2000. Oligogenic inheritance for resistance to zucchini yellow mosaic virus in Cucurbitapepo.Ann.Appl. Biot 136: 209-214.
86 Paszkowski, A. & Niedzielska, A. (1990) Serine: glyoxylate aminotransferase from the seedlings of rye (Secale cereale L.) .Acta Biochim. Pol. 37, 277 282.
87 Peng, M., and Kuc, J. 1992. Peroxidase-generated hydrogen peroxide as a source of antifungal activity in vitro and on tobacco leaf discs. Phytopathology 82:, 696-699.
88 Pieterse, C. M. J., and L. C. van Loon. 1999. Salicylic acid-independent plant defense pathways. Trends Plant Sci. 4:52-58.
89 Pieterse, C.M.J., van Wees, S.C.M., Hoffland, E., van Pelt, J.A.,and van Loon, L.C. 1996. Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression. Plant Cell 8,1225-1237.
90 Reumann S. 2004.Specification of the Peroxisome Targeting Signals Type 1 and Type 2 of Plant Peroxisomes by Bioinformatics Analyses Plant Physiology, June 1,135(2): 783 - 800.
91 Rohe, M., A. Gierlich, H. Hermann, M. Hahn, B. Schmidt, S. Rosahl & W. Knogge, 1995. The race-specific elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the Rrsl resistance genotype. The EMBO J 14: 4168-4177.
92 Rose, R. G., and Enderson, L. W., 1961."Research and Development Flight Test Program Using the Little Joe Booster, Abort at High Dynamic Pressure (Little Joe No. IB)," March 3.
93 Ryals J A, Neuenschwander U H, Willits M G, Molina A, Steiner H Y, Hunt M D, 1996. Systemic acquired resistance.Plant Cell, 8:1809-1819.
94 Schafer, W. M.. 1993. Mitigation of Acid Mining Waste Using Lime Application in the Upper Clark Fork Basin In Proceedings of the Lime Products Technology and Reclamation Conference, Fairmont Hot Springs, MT.
95 Schlosser, E., Oerter, H., Graf, W. 1999. Snow accumulation on Ekstromisen, Antarctica, 1980-1996. Berichte zur Polarforschung 313, Bremerhaven,137pp.
96 Song W Y, Wang G L, Chen L L, et al. 1995.A receptor kinase-likeprotein encoded by rice disease resistance gene, Xa21. Science, 270: 1804—1806
97 Stenzel K, Steiner U and Schonbeck F 1985. Effect of induced resistance on the efficiency of powdery mildew haustoria in wheat and barley. Physiol Plant Pathol 27: 357-367.
98 Sweigard JA, Carroll AM, Kang S, Farrall L, Chumley FG, Valent B 1995. Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus. Plant Cell 7:1221-1233.
99 Taler D, Galperin M, Benjamin I, Cohen Y and Kenigsbuch D 2004. Plant eR genes that encode photorespiratory enzymes confer resistance against disease. Plant Cell 16:172-184.
100 Taler, D., Galperin, M., Benjamin, I., Cohen, Y., and Kenigsbuch, D. (2004). Plant enzymatic resistance (eR) genes encoding for photorespiratory enzymes confer resistance against disease. Plant Cell 16,172-184.
101 Thomma BPHJ , Tierens FM-J, Penninckx IAMA, Mauch-Mani B, Broekaert WF and Cammue BPA 2001. Different micro-organisms differentially induce Arabidopsis disease response pathways. Plant Physiology and Biochemistry 39: 673-680.
102 Thomas, C.E., Cohen, Y, McCreight, Y.D., Jourdion, E.L., and Cohen, S. (1988). Inheritance of resistance to downy mildew in Cucumis melo. Plant Dis. 72, 33-35.
103 Ucile Plourde-Owobi, Sophie Durner, Jean-Luc Parrou, Roman Wieczorke, Gerard Goma, and Jean Francois AGT1, Encoding an β-Glucoside Transporter Involved in Uptake and Intracellular Accumulation of Trehalose in Saccharomyces cerevisiae Journal of Bacteriology, 1999, p.3830-3832, Vol. 181, No. 12
104 Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E.and Ryals, J. 1992. Acquired resistance in Arabidopsis. Plant Cell 4: 645-656.
105 Van den Ackerveken, G., Marois, E., and Bonas, U. 1996. Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell 87:1307-1316.
106Van Kan JAL, Van den Ackerveken AFJM & DeWit PJGM.1991. Cloning and characterization of cDNAof avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum, causal agent of tomato leaf mold. Mol. Plant-Microbe Interact. 4: 52-59
107 van Peer R, Niemann GJ, Schippers B 1991. Induced resistance and phytoalexin accumulation in biological control of fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology 81: 728-734.
108 Vivian, A., Atherton, G.T., Bevan, J., Crute, I.R., Mur, L.A.J., Taylor, J.D. 1989 Isolation and characterization of cloned DNA conferring specific avirulence in Pseudomonas syringae pv. pisi to pea (Pisum sativum) cultivars, which possess the resistance allele, R2 . Physiol. Mol. Plant Pathol. 34, 335 344.
109 Vogel J, Somerville S, Vogel P, et al. 2000. Isolation and characterization of powdery mildew-resistant Arabidopsis mutants[J]. Proc.Natl. Acad. Sci., 97:1 897-1 902.
110 Wang H, Hao L, Shung CY, Sunter G. and Bisaro DM 2003. Adenosine kinase is inactivated by geminivirus AL2 and L2 proteins. Plant Cell 15:3020-3032.
111 Watts, R.W.E. (1992) Alanine glyoxylate aminotransferase deficiency: biochemical and molecular genetic lessons from the study of a human disease. Adv. Enz. Reg. 32, 309 327
112 White PA, Mclver CJ, Deng Y & Rawlinson WD 2000. Characterization of the two gene cassettes aadA5 and dfrA17.FEMS Microbiol Lett 182:265-269.
113 Wubben JP,Joosten MH,De Wit PJ.1994.Expression and localization of two in planta induced extracellular proteins of the fungal tomato pathogen Cladosporium fulvum.Mol Plant Microbe Interact.7(4):516-524
114 Wu,C.H.;Lin,W.Y.1995."Effects of Metal Ions on the Catalytic and Thermodynamic Properties of the Aminopeptidase Isolated from Pronase",J.Inorg.Biochem.57,79-89.
115 Wu G,Shortt B J,Lawrence EB,Levine EB,Fitzsimmons KC,Shah DM 1995.Disease resistance conferred by expression of a gene encoding H_2O_2-generating glucose oxidase in transgenic potato plants.Plant Cell 7:1357-1368.
116 Xicai Yang,Yin Yie,Feng Zhu,Yule Liu,Liangyi Kang,Xiaofeng Wang and Po Tien.1997.Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes.Proc.Natl.Acad.Sci.USA.94:4861-4865.
117 Yalpani N.,Silverman P.,Wilson M.A.,Kleier D.A.,and Raskin I.1991."Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco",The Plant Ce113:809-0818.
118 Yu,C.,Liang,Z.,Huang,A.H.C.(1984)Glyoxylate transamination in intact leaf peroxisomes.Plant Physiol.75,712.
119 Yu,I.-c.,K.A.Fengler,S.J.Clough and A.F.Bent,2000.Identification of Arabidopsis mutants exhibiting an altered hypersensitive response in gene-for-gene disease resistance.Mol.Plant-Microbe Interact.13:277-286.
120 Zhu Q et al,1996.Currt Opin in Gene and Devel,5:624-630.
121 蔡新忠 徐幼平 郑重 植物病原物无毒基因及其功能 生物工程学报2002,18(1):5-9.
122 胡忠,杨增明,王钧.1988.天麻球茎中一种抗真菌蛋白的分离和部分特性[J].云南植物研究,10:373-380.
123 王志兴;刘昱辉;孙敬三;贾士荣。葡萄糖氧化酶基因的克隆及其在转基因烟草中的表达。J.自然科学进展。2003,3:248-252。