RNA干扰诱导马铃薯多酚氧化酶基因沉默的初步研究
摘要
马铃薯薯块受到损伤使自身所含的多酚氧化酶与酚类物质的空间隔离被打破,多酚氧化酶在空气中氧分子的作用下催化酚类物质为苯醌类物质,苯醌类物质稳定性较差,容易在不依赖酶的作用下,自身聚合后与体内的亲和性氨基酸等发生作用生成褐色、黑色物质,严重影响了马铃薯在加工过程中的品质。
目前,应用于植物中基因沉默的最新策略是体外将目的基因反向重复连接到内含子两端,转入植物基因组中,转录后产生自我互补的双链RNA,诱导同源基因的转录后沉默。这种RNA干扰技术具有沉默效率高、特异性好的优点。在本实验中,我们克隆了马铃薯的多酚氧化酶基因POT33,并将其高度保守的部分序列反向重复连接到载体pKANNABAL上,获得了中间载体pKANNABAL-ihpPOT33;然后将反向重复的序列连同它们之间的内含子序列一同连入植物双元载体pMBW330和pGB121s上,构建了分别由35S启动子启动的组成型沉默载体pMBW330-ihpPOT33和由GBSS启动子启动的薯块特异性沉默载体pGB121s-ihpPOT33,利用农杆菌LBA4404介导,转化加工型马铃薯大西洋。通过胚性愈伤组织诱导再生途径获得75株再生植株。对转化后的植株进行PCR和Western blot检测。结果显示,两株再生植株的叶片中分子量略小于66 kD的多酚氧化酶的表达被抑制。利用软件Bandscan 5.0分析Western检测结果,数据显示两株低表达多酚氧化酶的再生植株的沉默效率分别为86.02 %和66.10 %。关于在薯块中检测多酚氧化酶基因的表达以及表达降低后对马铃薯薯块褐化的影响还需要进一步的研究。
Polyphenol oxidase (PPO) is the major cause of enzymic browning in potato. In vivo, the PPO localized in plastids, while the phenolic substrates of PPO are localized in the vacuole. The browning reaction only occurs as a result of tuber damage leading to a loss of this subcellular compartmentation. PPO catalyzes the conversion of monophenols polyphenols to o-dihydroxyphenols or to quinones. The quinone products can then polymerize and react with amino acid groups of cellular proteins, resulting in brown or black pigment deposits. Such damage causes considerable economic and nutritional losses in the process of potato.
Recent work has focused on designing the constructs which require two copies of the target sequence in an inverted-repeat orientation encoding self-complementary `hairpin' RNA with a intron between the two copies (ihpRNA), in order to produce duplex RNA to efficiently silence the genes in vivo (called RNAi technology). RNAi technology allows silencing all members of a multigene family or homoeologous gene copies in polyploids by targeting sequences that are unique or shared by several genes.
In this study, we cloned one member of PPO genes-POT33 and constructed a constitive RNAi vector promoted by 35S promoter carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pMBW330-ihpPOT33), and a tissue-specifical RNAi vector promated by GBSS carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pGB121s-ihpPOT33). Agrobacterium LBA4404 harboring the binary vector pMBW330-ihpPOT33 or pGB121s-ihpPOT33 was employed to transform Potato 'Atlantic', and 75 potato plantlets were regenerated after transformation. The transformed potato plants were confirmed by PCR and Western hybridization successfully. The results suggested that one member of the target protein was silenced in two transformed potato plants, and the silencing efficiency of them was 86.02 % and 66.01 % analyzed by software Bandscan, respectively.
目前,应用于植物中基因沉默的最新策略是体外将目的基因反向重复连接到内含子两端,转入植物基因组中,转录后产生自我互补的双链RNA,诱导同源基因的转录后沉默。这种RNA干扰技术具有沉默效率高、特异性好的优点。在本实验中,我们克隆了马铃薯的多酚氧化酶基因POT33,并将其高度保守的部分序列反向重复连接到载体pKANNABAL上,获得了中间载体pKANNABAL-ihpPOT33;然后将反向重复的序列连同它们之间的内含子序列一同连入植物双元载体pMBW330和pGB121s上,构建了分别由35S启动子启动的组成型沉默载体pMBW330-ihpPOT33和由GBSS启动子启动的薯块特异性沉默载体pGB121s-ihpPOT33,利用农杆菌LBA4404介导,转化加工型马铃薯大西洋。通过胚性愈伤组织诱导再生途径获得75株再生植株。对转化后的植株进行PCR和Western blot检测。结果显示,两株再生植株的叶片中分子量略小于66 kD的多酚氧化酶的表达被抑制。利用软件Bandscan 5.0分析Western检测结果,数据显示两株低表达多酚氧化酶的再生植株的沉默效率分别为86.02 %和66.10 %。关于在薯块中检测多酚氧化酶基因的表达以及表达降低后对马铃薯薯块褐化的影响还需要进一步的研究。
Polyphenol oxidase (PPO) is the major cause of enzymic browning in potato. In vivo, the PPO localized in plastids, while the phenolic substrates of PPO are localized in the vacuole. The browning reaction only occurs as a result of tuber damage leading to a loss of this subcellular compartmentation. PPO catalyzes the conversion of monophenols polyphenols to o-dihydroxyphenols or to quinones. The quinone products can then polymerize and react with amino acid groups of cellular proteins, resulting in brown or black pigment deposits. Such damage causes considerable economic and nutritional losses in the process of potato.
Recent work has focused on designing the constructs which require two copies of the target sequence in an inverted-repeat orientation encoding self-complementary `hairpin' RNA with a intron between the two copies (ihpRNA), in order to produce duplex RNA to efficiently silence the genes in vivo (called RNAi technology). RNAi technology allows silencing all members of a multigene family or homoeologous gene copies in polyploids by targeting sequences that are unique or shared by several genes.
In this study, we cloned one member of PPO genes-POT33 and constructed a constitive RNAi vector promoted by 35S promoter carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pMBW330-ihpPOT33), and a tissue-specifical RNAi vector promated by GBSS carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pGB121s-ihpPOT33). Agrobacterium LBA4404 harboring the binary vector pMBW330-ihpPOT33 or pGB121s-ihpPOT33 was employed to transform Potato 'Atlantic', and 75 potato plantlets were regenerated after transformation. The transformed potato plants were confirmed by PCR and Western hybridization successfully. The results suggested that one member of the target protein was silenced in two transformed potato plants, and the silencing efficiency of them was 86.02 % and 66.01 % analyzed by software Bandscan, respectively.
引文
[1] 陈平.果蔬制品变色的机理及对策.杭州食品科技[J], 1993,(2):14-15.
[2] 刘开华,邢淑婕.浅析果蔬加工中酶促褐变的控制.信阳农专学报[J] , 1997, 7(3):21-22.
[3] 王曼玲,胡中立,周明全等. 植物多酚氧化酶的研究进展。植物学通报[J], 2005, 22(2): 215-222.
[4] Stewart R J, Sawyer B J B, Bucheli C S, et al. Polyphenol oxidase is induced by chilling and wounding in pineapple[J]. Australian Journal of Plant Physiology, 2001, 28(3): 181-191.
[5] Haruta M, Murata M, Kadokura H, et al. Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees[J]. Phytochemistry, 1999, 50(6): 1021-1025.
[6] Gerdemann C, Eicken C, Galla H J, et al. Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure[J]. Journal of Inorganic Biochemistry, 2002, 89(1-2): 155-158.
[7] Gerdemann C, Eicken C, Magrini A, et al. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity[J]. Biochimica et Biophysica Acta, 2001, 1548(1): 94-105.
[8] Wititsuwannakul D, Chareyhiphakorn N, Pace M. Polyphenol oxidases from latex of Hevea brasiliensis:Purification and characterization[J]. Phytochemistry, 2002, 61(2): 115-121.
[9] Shi C,Dai Y,Xia B,et al. The purification and spectral properties of Polyphenol Oxidase I from Nicotiana tobacum[J]. Plant Molecular Biology Reporter, 2001, 19(4): 381a-381h.
[10] 陈桂信,潘东明,赖钟雄等. 园艺植物多酚氧化酶的分子生物学研究进展[J]. 江西农业大学学报(自然科学版),2003, 25(1):107-110.
[11] 赵东,刘祖生,奚彪. 茶树多酚氧化酶基因的克隆及其序列比较[J]. 茶叶科学,2001,21(2):94-98.
[12] Goodling P S, Bird C, Robinson S P. Molecular cloning and characterization of banana fruit polyphenol oxidase[J]. Planta, 2001, 213(5): 748-757.
[13] Mazzafera P, Robinson S P. characterization of polyphenol oxidase in coffee[J]. Phytochemistry, 2000, 55(4): 285-296.
[14] Newman S M, Eannetta N T, Yu H. Organization of the tomato polyphenol oxidase gene family[M]. Plant Mol Biol,1993.
[15] Cary J W, Lax A R, Flurkey W H. Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase[J]. Plant Mol. Biol, 1992, 20(2): 245-253.
[16] Anderson J V, Morris C F. Purification and analysis of wheat grain polyphenol oxidase(PPO)protein[J]. Cereal Chemistry, 2003, 80(2):135-143.
[17] Amos S, Emma Ne'eman, Steffens J C, et al. Import, targeting, and processing of a plantpolyphenol oxidase[J]. Plant Physiol, 1994, 105(4): 1301-1311.
[18] Thipyapong P, Steffens J C. Tomato polyphenol oxidase. Differential response of the polyphenol oxidase F promoter to injured and wound signals[J]. Plant Physiolog, 1997, 115(2): 409-418.
[19] Shahar T, Hennig N, Gutfinger T, et al. The tomato 66.3-kD polyphenol oxidase gene:Molecular identification and developmental expression[J]. Plant Cell, 1992, 4(2): 135-147.
[20] Kim J Y, Seo Y S, Kim J E, et al. Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple[J]. Plant Science,2001,161(6):1145-1152.
[21] Hak S J, Seo J H, Chun J P. Changes of phenol compounds and occurrence of skin browning and characterization of partially purified polyphenol oxidase in oriental pear fruits[J]. Journal of the Korean Society for Horticultural Science, 2001, 42(2): 184-188.
[22] Kowalski S P, Eannetta N T, Hirzel A T, et al. Purification and Characterization of Polyphenol Oxidase from Glandular Trichomes of Solanum berthaultii [J]. Plant Physiol, 1992, 100(2): 677-684.
[23] Vaidya B K , Suthar H K, Kasture S, et al. Purification of potato polyphenol oxidase (PPO) by partitioning in aqueous two-phase system[J]. Biochemical Engineering Journal, 2006, 28(2): 161-166.
[24] Ono E, Hatayama M, Isono Y, et al. Localization of a flavonoid biosynthetic polyphenol oxidase in in vacuoles[J]. Plant Journal. 2006, 45(2): 133-143.
[25] Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone sythase gene into petunia results in reversible cosuppression of homologous genes in trans[J]. Plant Cell, 1990, 2(4): 279-289.
[26] Cogoni C, Romano N, Macino G. Suppression of gene expression by homologous transgenes [J]. Antonie Van Leeuwenhoes, 1994, 65(3): 205-209.
[27] Cogoni C, Macino G. Post-transcriptional gene silencing across kingdoms[J]. Curr Opin in Genet Dev, 2000, 10(6): 638-643.
[28] Jorgense R A, Cluster D D, Napoli C A. Chalcone synthase co-suppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences[J]. Plant Mol Biol, 1996, 31(5): 957-973.
[29] Guo S, Kemphues K J. par-l, a gene required for establishing polarity in C.elegans embryos, encodes a putative Ser/Thr kinase that is asymmentrically distributed[J]. Cell, 1995, 81(4): 611-620.
[30] Fire A, Xu S, Montgomery M K. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis Elegans[J]. Nature, 1998, 391(6669): 806-811.
[31] Hannon G J. RNA interference[J]. Nature, 2002, 418(6894): 244-251.
[32] Dykxhoorn D M, Novina C D, Sharp P A. Killing the messenger: short RNAs that silencegene expression[J]. Nat Rev Mol Cell Biol, 2003, 4(6): 457-467.
[33] Ingelbrecht I H, Van H M, Van M M, et al. Post-transcriptional silencing of reporter transgenes in tobacco correlates with DNA methylation[J]. Proc. Natl. Acad. Sci, 1994, 91(22): 10502-10506.
[34] Vander Krol A R, Mur L A. Flavonoid genes in petunia: addition of limited number of gene copies may lead to a suppression of gene expression[J]. Plant Cell, 1990, 2(4): 291-299.
[35] Kooter, J M, Matzke M A. Listening to silent gene: transgene silencing, gene regulation and pathogen control[J]. Trends Plant Sci. 1999, 4(9): 340-347.
[36] Francesco D S, Hanspeter S. Sense and antisense mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs[J]. Proc. Natl. Acad. Sci. USA. 2001, 98(11): 6506-6510.
[37] Metzlaff M, O'Dell M, Cluster P D, et al. RNA mediated RNA degradation and chalcone synthase A silencing in Petunia[J]. Cell, 1997, 88(6): 845-854.
[38] Wang M B, Metzlaff M. RNA silencing and antiviral defense in plants[J]. Current Opinion in Plant Biology, 2005, 8(2): 216-222.
[39] Peter M. Construct design for efficient, effective and high throughput gene silencing in plants[J]. The Plant Journal, 2001, 27(6): 581-590.
[40] Yan H, Robert C, Ye J S, et al. New Construct Approaches for Efficient Gene Silencing in Plants[J]. Plant Physiology, 2006, 141(4): 1508-1518.
[41] Coetzer C, Corsini D, Love S, et al. Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase[J]. Agric Food Chem, 2001, 49(2): 652-657.
[42] Rommens C M, Humara J M, Ye J S, et al. Crop Improvement through Modification of the Plants’ Own Genome[J]. Plant Physiology, 2004, 5(135): 421-431.
[43] Marx J. Interfering with gene expression[J]. Science , 2000, 288(5470): 1370-1372.
[44] Carthew R W . Gene silencing by double-stranded RNA[J]. Curr Opin Cell Biol, 2001,13(2): 244-248.
[45] Baulcombe D C. RNA silencing in plants[J]. Nature , 2004, 431(7006): 356-363.
[46] Lipardi C, Wei Q, Paterson B M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs[J]. Cell, 2001, 107(3): 297-307.
[47] Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing[J]. Cell, 2001, 107(4): 465-476.
[48] Travella S, Klimm T E, Keller B. RNA Interference-Based Gene Silencing as an Efficient Tool for Functional Genomics in Hexaploid Bread Wheat[J]. Plant Physiology, 2006, 12(142): 6-20.
[49] Hunt M D, Eannetta N T, Yu H F, et al. cDNA cloning and expression of potato polyphenol oxidase[J]. Plant Mol. Biol, 1993, 21(1): 59-68.
[50] Balasingham K, Ferdinand W .The purification and properties of a ribonucleoenzyme, o-diphenol oxidase from potatoes[J]. Biochem.1970, 118(1): 15-23.
[51] Batistuti J P, Lourenco E J. Isolation and purification of polyphenol oxidase from a new variety of potato[J]. Food Chem. 1985, 18(4): 251-263.
[52] Robinson S P, Dry I B. Broad bean leaf polyphenol oxidase is a 60-kilodalton protein susceptible to proteolytic cleavage[J]. Plant Physiol.1992, 99(1):317-323.
[2] 刘开华,邢淑婕.浅析果蔬加工中酶促褐变的控制.信阳农专学报[J] , 1997, 7(3):21-22.
[3] 王曼玲,胡中立,周明全等. 植物多酚氧化酶的研究进展。植物学通报[J], 2005, 22(2): 215-222.
[4] Stewart R J, Sawyer B J B, Bucheli C S, et al. Polyphenol oxidase is induced by chilling and wounding in pineapple[J]. Australian Journal of Plant Physiology, 2001, 28(3): 181-191.
[5] Haruta M, Murata M, Kadokura H, et al. Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees[J]. Phytochemistry, 1999, 50(6): 1021-1025.
[6] Gerdemann C, Eicken C, Galla H J, et al. Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure[J]. Journal of Inorganic Biochemistry, 2002, 89(1-2): 155-158.
[7] Gerdemann C, Eicken C, Magrini A, et al. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity[J]. Biochimica et Biophysica Acta, 2001, 1548(1): 94-105.
[8] Wititsuwannakul D, Chareyhiphakorn N, Pace M. Polyphenol oxidases from latex of Hevea brasiliensis:Purification and characterization[J]. Phytochemistry, 2002, 61(2): 115-121.
[9] Shi C,Dai Y,Xia B,et al. The purification and spectral properties of Polyphenol Oxidase I from Nicotiana tobacum[J]. Plant Molecular Biology Reporter, 2001, 19(4): 381a-381h.
[10] 陈桂信,潘东明,赖钟雄等. 园艺植物多酚氧化酶的分子生物学研究进展[J]. 江西农业大学学报(自然科学版),2003, 25(1):107-110.
[11] 赵东,刘祖生,奚彪. 茶树多酚氧化酶基因的克隆及其序列比较[J]. 茶叶科学,2001,21(2):94-98.
[12] Goodling P S, Bird C, Robinson S P. Molecular cloning and characterization of banana fruit polyphenol oxidase[J]. Planta, 2001, 213(5): 748-757.
[13] Mazzafera P, Robinson S P. characterization of polyphenol oxidase in coffee[J]. Phytochemistry, 2000, 55(4): 285-296.
[14] Newman S M, Eannetta N T, Yu H. Organization of the tomato polyphenol oxidase gene family[M]. Plant Mol Biol,1993.
[15] Cary J W, Lax A R, Flurkey W H. Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase[J]. Plant Mol. Biol, 1992, 20(2): 245-253.
[16] Anderson J V, Morris C F. Purification and analysis of wheat grain polyphenol oxidase(PPO)protein[J]. Cereal Chemistry, 2003, 80(2):135-143.
[17] Amos S, Emma Ne'eman, Steffens J C, et al. Import, targeting, and processing of a plantpolyphenol oxidase[J]. Plant Physiol, 1994, 105(4): 1301-1311.
[18] Thipyapong P, Steffens J C. Tomato polyphenol oxidase. Differential response of the polyphenol oxidase F promoter to injured and wound signals[J]. Plant Physiolog, 1997, 115(2): 409-418.
[19] Shahar T, Hennig N, Gutfinger T, et al. The tomato 66.3-kD polyphenol oxidase gene:Molecular identification and developmental expression[J]. Plant Cell, 1992, 4(2): 135-147.
[20] Kim J Y, Seo Y S, Kim J E, et al. Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple[J]. Plant Science,2001,161(6):1145-1152.
[21] Hak S J, Seo J H, Chun J P. Changes of phenol compounds and occurrence of skin browning and characterization of partially purified polyphenol oxidase in oriental pear fruits[J]. Journal of the Korean Society for Horticultural Science, 2001, 42(2): 184-188.
[22] Kowalski S P, Eannetta N T, Hirzel A T, et al. Purification and Characterization of Polyphenol Oxidase from Glandular Trichomes of Solanum berthaultii [J]. Plant Physiol, 1992, 100(2): 677-684.
[23] Vaidya B K , Suthar H K, Kasture S, et al. Purification of potato polyphenol oxidase (PPO) by partitioning in aqueous two-phase system[J]. Biochemical Engineering Journal, 2006, 28(2): 161-166.
[24] Ono E, Hatayama M, Isono Y, et al. Localization of a flavonoid biosynthetic polyphenol oxidase in in vacuoles[J]. Plant Journal. 2006, 45(2): 133-143.
[25] Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone sythase gene into petunia results in reversible cosuppression of homologous genes in trans[J]. Plant Cell, 1990, 2(4): 279-289.
[26] Cogoni C, Romano N, Macino G. Suppression of gene expression by homologous transgenes [J]. Antonie Van Leeuwenhoes, 1994, 65(3): 205-209.
[27] Cogoni C, Macino G. Post-transcriptional gene silencing across kingdoms[J]. Curr Opin in Genet Dev, 2000, 10(6): 638-643.
[28] Jorgense R A, Cluster D D, Napoli C A. Chalcone synthase co-suppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences[J]. Plant Mol Biol, 1996, 31(5): 957-973.
[29] Guo S, Kemphues K J. par-l, a gene required for establishing polarity in C.elegans embryos, encodes a putative Ser/Thr kinase that is asymmentrically distributed[J]. Cell, 1995, 81(4): 611-620.
[30] Fire A, Xu S, Montgomery M K. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis Elegans[J]. Nature, 1998, 391(6669): 806-811.
[31] Hannon G J. RNA interference[J]. Nature, 2002, 418(6894): 244-251.
[32] Dykxhoorn D M, Novina C D, Sharp P A. Killing the messenger: short RNAs that silencegene expression[J]. Nat Rev Mol Cell Biol, 2003, 4(6): 457-467.
[33] Ingelbrecht I H, Van H M, Van M M, et al. Post-transcriptional silencing of reporter transgenes in tobacco correlates with DNA methylation[J]. Proc. Natl. Acad. Sci, 1994, 91(22): 10502-10506.
[34] Vander Krol A R, Mur L A. Flavonoid genes in petunia: addition of limited number of gene copies may lead to a suppression of gene expression[J]. Plant Cell, 1990, 2(4): 291-299.
[35] Kooter, J M, Matzke M A. Listening to silent gene: transgene silencing, gene regulation and pathogen control[J]. Trends Plant Sci. 1999, 4(9): 340-347.
[36] Francesco D S, Hanspeter S. Sense and antisense mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs[J]. Proc. Natl. Acad. Sci. USA. 2001, 98(11): 6506-6510.
[37] Metzlaff M, O'Dell M, Cluster P D, et al. RNA mediated RNA degradation and chalcone synthase A silencing in Petunia[J]. Cell, 1997, 88(6): 845-854.
[38] Wang M B, Metzlaff M. RNA silencing and antiviral defense in plants[J]. Current Opinion in Plant Biology, 2005, 8(2): 216-222.
[39] Peter M. Construct design for efficient, effective and high throughput gene silencing in plants[J]. The Plant Journal, 2001, 27(6): 581-590.
[40] Yan H, Robert C, Ye J S, et al. New Construct Approaches for Efficient Gene Silencing in Plants[J]. Plant Physiology, 2006, 141(4): 1508-1518.
[41] Coetzer C, Corsini D, Love S, et al. Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase[J]. Agric Food Chem, 2001, 49(2): 652-657.
[42] Rommens C M, Humara J M, Ye J S, et al. Crop Improvement through Modification of the Plants’ Own Genome[J]. Plant Physiology, 2004, 5(135): 421-431.
[43] Marx J. Interfering with gene expression[J]. Science , 2000, 288(5470): 1370-1372.
[44] Carthew R W . Gene silencing by double-stranded RNA[J]. Curr Opin Cell Biol, 2001,13(2): 244-248.
[45] Baulcombe D C. RNA silencing in plants[J]. Nature , 2004, 431(7006): 356-363.
[46] Lipardi C, Wei Q, Paterson B M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs[J]. Cell, 2001, 107(3): 297-307.
[47] Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing[J]. Cell, 2001, 107(4): 465-476.
[48] Travella S, Klimm T E, Keller B. RNA Interference-Based Gene Silencing as an Efficient Tool for Functional Genomics in Hexaploid Bread Wheat[J]. Plant Physiology, 2006, 12(142): 6-20.
[49] Hunt M D, Eannetta N T, Yu H F, et al. cDNA cloning and expression of potato polyphenol oxidase[J]. Plant Mol. Biol, 1993, 21(1): 59-68.
[50] Balasingham K, Ferdinand W .The purification and properties of a ribonucleoenzyme, o-diphenol oxidase from potatoes[J]. Biochem.1970, 118(1): 15-23.
[51] Batistuti J P, Lourenco E J. Isolation and purification of polyphenol oxidase from a new variety of potato[J]. Food Chem. 1985, 18(4): 251-263.
[52] Robinson S P, Dry I B. Broad bean leaf polyphenol oxidase is a 60-kilodalton protein susceptible to proteolytic cleavage[J]. Plant Physiol.1992, 99(1):317-323.