基于桑树cDNA文库的3个功能基因的诱导表达研究
|
摘要
桑树是一种重要的经济作物,我国拥有世界上最为丰富的桑树种质资源。但是各种逆境条件和病虫害往往会对桑树的产量造成重大影响,研究桑树一些重要的功能基因及其在胁迫条件下的分子适应机制,可以增强桑树对各种胁迫条件的抗逆性,对桑树种质资源的保存和提高桑树产量有重要作用。与其他作物相比,我们对桑树功能基因的研究至今还处于起步阶段,对桑树的一些功能基因及其编码的蛋白质的功能仍然不甚了解。因此,开展桑树功能基因的研究是一项紧迫而有意义的工作。
本文在构建的桑树幼叶cDNA文库的基础上获得了3个ESTs,克隆了这3个基因,并对获得的序列进行了分析和功能推断,用胁迫诱导的方法对这3个基因进行功能验证。本论文的主要研究内容如下:
1、桑树几丁质酶基因M-chitinase的克隆及胁迫诱导表达分析
从已构建的桑树幼叶cDNA文库得到了一个编码桑树几丁质酶(Chitinase)基因的一段序列,通过RACE与RT-PCR结合获得了这个基因的完整序列,命名为M-chitinase(GeneBank登录号:HQ117891)。序列分析表明,M-chitinase全长1392bp,存在60bp的5’端非翻译序列(5’-UTR)和255bp的3’端非翻译序列(3’-UTR),其开放读码框(ORF)长1077bp,编码358个氨基酸,预测蛋白质分子质量为38.52KD,等电点为4.466。同源分析表明,M-chitinase基因在桑树与卡西猪笼草、玉米、二倍体多年生大刍草等各物种间具有很高的保守性。基于桑树和其它19个物种M-chitinase基因的系统进化分析表明,桑树与野生烟草、湖南烤烟、辣椒、南芥的亲缘关系较近。半定量RT-PCR检测表明,幼叶表达量最高。与正常生长环境相比,M-chitinase基因mRNA的转录水平在NaCl、水杨酸、脱落酸条件下均有明显变化。
2、桑树桑树多聚半乳糖醛酸酶抑制蛋白基因M-PGIP的克隆,序列分析及诱导表达研究
从已构建的桑树幼叶cDNA文库得到了一个编码桑树多聚半乳糖醛酸酶抑制蛋白(po1yga1acturonase inhibiting protein,PGIP)基因的一段序列,通过RACE与RT-PCR结合首次获得了这个基因的完整序列,命名为M-PGIP(GeneBank登录号:HM044383)。序列分析表明,M-PGIP全长1274bp,存在93bp的5’端非翻译序列(5’-UTR)和179bp的3’端非翻译序列(3’-UTR),其开放读码框(ORF)长1002bp,编码333个氨基酸,预测蛋白质分子质量为37.29KD,等电点为7.25。同源分析表明,M-PGIP基因在桑树与美洲李、桃花、梅花等各物种间具有很高的保守性。基于桑树和其它19个物种M-PGIP基因的系统进化分析表明,桑树与甜瓜、葡萄、葡萄根、猕猴桃的亲缘关系较近。半定量RT-PCR检测表明,幼叶表达量最高。与正常生长环境相比,M-PGIP基因mRNA的转录水平在水杨酸、脱落酸、盐胁迫条件下均有明显变化。并对所克隆的基因进行了原核表达,为今后的转基因植物抗病工程方面打下了良好的基础。
3.桑树光合系统IIMpsbR基因的克隆,序列分析及诱导表达研究
根据桑树表达序列标签(expressed sequence tags, ESTs),采取RT-PCR方法克隆了一个编码桑树光合系统II PsbR基因的全长cDNA序列,命名为MpsbR(Genbank登录号:GU937873)。序列分析表明,该基因全长623bp,存在56bp的5’-UTR和306bp的3’-UTR,其开放阅读框(ORF)长261bp,编码86个氨基酸,预测蛋白质分子质量为8.95 kD,等电点为11.09。同源分析表明,桑树光合系统II MpsbR基因与花生、大豆编码氨基酸具有较高的同源性;根据MpsbR基因的氨基酸序列,与其它20个物种的氨基酸同源序列构建的系统进化树表明,桑树与花生、海桑、梨、大豆的亲缘关系较近。半定量RT-PCR研究表明,桑树光合系统MpsbR基因在桑树不同部位的表达水平有一定差异,在幼叶(刚展开第一张叶)和顶芽表达量最高,其作用机理有待于进一步研究之中。
Mulberry (Morus alba) is an economically important plant used for sericulture. our country has the most abundant mulberry germplasm resources. The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses. Efforts to investigate the functional genes and their molecular adaptation mechanisms of stresses and to strengthen stress tolerance in this plant are of fundamental importance to the preservation of these genetic resources and to improve the yield of mulberry. However, the research of the functional genes from mulberry is in a backward state in comparison with other plants, and the exact function of some functional genes and their encoded proteins in the stress response in mulberry is still not fully understood yet. Therefore, the research of functional genes in mulberry is one urgent and the meaningful work.
As a first step towards characterization of functional genes in mulberry, in this paper, we constructed a cDNA library from young mulberry leaves. Based on the research above, we cloned three important genes and carried out the analysis of their sequences. Furthermore, we conducted the functional verification of the three genes by the means of stress-induced method based on the prediction of their functions.The main results were summarized briefly as follows:
1、EST encoding Chitinase was isolated from the cDNA library. And it’s full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for Chitinase in mulberry was designated M-chitinase (GenBank accession number: HQ117891). Sequence analysis showed that the M-chitinase is 1392 bp long and contains a 60 bp 5’-UTR (untranslated region) and a 255 bp 3’-UTR. It’s opening frame(ORF) is of 1077 bp,encoding 358 amino acids with apredicted molecular weight of 38.52KD and an isoelectric point of 4.466. Homology analysis revealed that M-chitinase gene are highly conservative in mulberry and other species including N. khasiana, Zea mays and Zea Diploperennis. Phylogenetic analysis based on M-chitinase gene with other 19 species showed that mulberry shows closer relationship with Nicotiana gossei, nicotiana tabacum, Capsicum annuum and rock cress. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-chitinase mRNA in the young leaf. And the transcriptional level of M-chitinase mRNA changed significantly under the conditions of cold, SA and ABA stresses respectively compared to the normal growth environment.
2、EST encoding PGIP was isolated from the cDNA library. And it’s full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for PGIP in mulberry was designated M-PGIP (GenBank accession number: HM044383). Sequence analysis showed that the M-PGIP is 1274bp long and contains a 93bp 5’-UTR (untranslated region) and a 179bp 3’-UTR.It’s opening frame(ORF) is of 1002bp,encoding 333 amino acids with apredicted molecular weight of 37.29KD and an isoelectric point of 7.25. Homology analysis revealed that M-PGIP gene are highly conservative in mulberry and other species including Prunus americana, Prunus persica and Prunus mume. Phylogenetic analysis based on M-PGIP gene with other 19 species showed that mulberry shows closer relationship with Cucumis melo, Vitis labrusca x Vitis riparia, Vitis vinifera and Actinidia deliciosa. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-PGIP mRNA in the young leaf. And the transcriptional level of M-PGIP mRNA changed significantly under the conditions of SA,ABA and salt sresses respectively compared to the normal growth environment.And we had expressed the recombinant protein to lay a good foundation for disease resistance Engineering of transgenic plants.
3、The full length cDNA sequence coding for photosynthesis system II PsbR in mulberry Using the expression sequence label (expressed sequence tags, ESTs)was adopted the RT-PCR method to clone ,and named MpsbR (the Genbank accession number:GU937873 ). The sequential analysis indicated that this gene is 623bp long, and contains a 56bp 5 ' - UTR and a 306bp 3 '– UTR.It’s opening reading frame (ORF) is of 261bp, encoding 86 amino acids with apredicted molecular weight of 8.95KD and an isoelectric point of 11.09. Homology analysis revealed that , MpsbR gene are highly conservative in mulberry and other species including peanut, soybean and Prunus mume.; The phylogenetic analysis based on MpsbR gene with other 20 species showed that mulberry shows closer relationship with peanut, sea mulberry, pear and soybean. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of MpsbR mRNA in the young leaf and the top bud. Its action mechanism is waiting for further studies.
本文在构建的桑树幼叶cDNA文库的基础上获得了3个ESTs,克隆了这3个基因,并对获得的序列进行了分析和功能推断,用胁迫诱导的方法对这3个基因进行功能验证。本论文的主要研究内容如下:
1、桑树几丁质酶基因M-chitinase的克隆及胁迫诱导表达分析
从已构建的桑树幼叶cDNA文库得到了一个编码桑树几丁质酶(Chitinase)基因的一段序列,通过RACE与RT-PCR结合获得了这个基因的完整序列,命名为M-chitinase(GeneBank登录号:HQ117891)。序列分析表明,M-chitinase全长1392bp,存在60bp的5’端非翻译序列(5’-UTR)和255bp的3’端非翻译序列(3’-UTR),其开放读码框(ORF)长1077bp,编码358个氨基酸,预测蛋白质分子质量为38.52KD,等电点为4.466。同源分析表明,M-chitinase基因在桑树与卡西猪笼草、玉米、二倍体多年生大刍草等各物种间具有很高的保守性。基于桑树和其它19个物种M-chitinase基因的系统进化分析表明,桑树与野生烟草、湖南烤烟、辣椒、南芥的亲缘关系较近。半定量RT-PCR检测表明,幼叶表达量最高。与正常生长环境相比,M-chitinase基因mRNA的转录水平在NaCl、水杨酸、脱落酸条件下均有明显变化。
2、桑树桑树多聚半乳糖醛酸酶抑制蛋白基因M-PGIP的克隆,序列分析及诱导表达研究
从已构建的桑树幼叶cDNA文库得到了一个编码桑树多聚半乳糖醛酸酶抑制蛋白(po1yga1acturonase inhibiting protein,PGIP)基因的一段序列,通过RACE与RT-PCR结合首次获得了这个基因的完整序列,命名为M-PGIP(GeneBank登录号:HM044383)。序列分析表明,M-PGIP全长1274bp,存在93bp的5’端非翻译序列(5’-UTR)和179bp的3’端非翻译序列(3’-UTR),其开放读码框(ORF)长1002bp,编码333个氨基酸,预测蛋白质分子质量为37.29KD,等电点为7.25。同源分析表明,M-PGIP基因在桑树与美洲李、桃花、梅花等各物种间具有很高的保守性。基于桑树和其它19个物种M-PGIP基因的系统进化分析表明,桑树与甜瓜、葡萄、葡萄根、猕猴桃的亲缘关系较近。半定量RT-PCR检测表明,幼叶表达量最高。与正常生长环境相比,M-PGIP基因mRNA的转录水平在水杨酸、脱落酸、盐胁迫条件下均有明显变化。并对所克隆的基因进行了原核表达,为今后的转基因植物抗病工程方面打下了良好的基础。
3.桑树光合系统IIMpsbR基因的克隆,序列分析及诱导表达研究
根据桑树表达序列标签(expressed sequence tags, ESTs),采取RT-PCR方法克隆了一个编码桑树光合系统II PsbR基因的全长cDNA序列,命名为MpsbR(Genbank登录号:GU937873)。序列分析表明,该基因全长623bp,存在56bp的5’-UTR和306bp的3’-UTR,其开放阅读框(ORF)长261bp,编码86个氨基酸,预测蛋白质分子质量为8.95 kD,等电点为11.09。同源分析表明,桑树光合系统II MpsbR基因与花生、大豆编码氨基酸具有较高的同源性;根据MpsbR基因的氨基酸序列,与其它20个物种的氨基酸同源序列构建的系统进化树表明,桑树与花生、海桑、梨、大豆的亲缘关系较近。半定量RT-PCR研究表明,桑树光合系统MpsbR基因在桑树不同部位的表达水平有一定差异,在幼叶(刚展开第一张叶)和顶芽表达量最高,其作用机理有待于进一步研究之中。
Mulberry (Morus alba) is an economically important plant used for sericulture. our country has the most abundant mulberry germplasm resources. The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses. Efforts to investigate the functional genes and their molecular adaptation mechanisms of stresses and to strengthen stress tolerance in this plant are of fundamental importance to the preservation of these genetic resources and to improve the yield of mulberry. However, the research of the functional genes from mulberry is in a backward state in comparison with other plants, and the exact function of some functional genes and their encoded proteins in the stress response in mulberry is still not fully understood yet. Therefore, the research of functional genes in mulberry is one urgent and the meaningful work.
As a first step towards characterization of functional genes in mulberry, in this paper, we constructed a cDNA library from young mulberry leaves. Based on the research above, we cloned three important genes and carried out the analysis of their sequences. Furthermore, we conducted the functional verification of the three genes by the means of stress-induced method based on the prediction of their functions.The main results were summarized briefly as follows:
1、EST encoding Chitinase was isolated from the cDNA library. And it’s full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for Chitinase in mulberry was designated M-chitinase (GenBank accession number: HQ117891). Sequence analysis showed that the M-chitinase is 1392 bp long and contains a 60 bp 5’-UTR (untranslated region) and a 255 bp 3’-UTR. It’s opening frame(ORF) is of 1077 bp,encoding 358 amino acids with apredicted molecular weight of 38.52KD and an isoelectric point of 4.466. Homology analysis revealed that M-chitinase gene are highly conservative in mulberry and other species including N. khasiana, Zea mays and Zea Diploperennis. Phylogenetic analysis based on M-chitinase gene with other 19 species showed that mulberry shows closer relationship with Nicotiana gossei, nicotiana tabacum, Capsicum annuum and rock cress. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-chitinase mRNA in the young leaf. And the transcriptional level of M-chitinase mRNA changed significantly under the conditions of cold, SA and ABA stresses respectively compared to the normal growth environment.
2、EST encoding PGIP was isolated from the cDNA library. And it’s full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for PGIP in mulberry was designated M-PGIP (GenBank accession number: HM044383). Sequence analysis showed that the M-PGIP is 1274bp long and contains a 93bp 5’-UTR (untranslated region) and a 179bp 3’-UTR.It’s opening frame(ORF) is of 1002bp,encoding 333 amino acids with apredicted molecular weight of 37.29KD and an isoelectric point of 7.25. Homology analysis revealed that M-PGIP gene are highly conservative in mulberry and other species including Prunus americana, Prunus persica and Prunus mume. Phylogenetic analysis based on M-PGIP gene with other 19 species showed that mulberry shows closer relationship with Cucumis melo, Vitis labrusca x Vitis riparia, Vitis vinifera and Actinidia deliciosa. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-PGIP mRNA in the young leaf. And the transcriptional level of M-PGIP mRNA changed significantly under the conditions of SA,ABA and salt sresses respectively compared to the normal growth environment.And we had expressed the recombinant protein to lay a good foundation for disease resistance Engineering of transgenic plants.
3、The full length cDNA sequence coding for photosynthesis system II PsbR in mulberry Using the expression sequence label (expressed sequence tags, ESTs)was adopted the RT-PCR method to clone ,and named MpsbR (the Genbank accession number:GU937873 ). The sequential analysis indicated that this gene is 623bp long, and contains a 56bp 5 ' - UTR and a 306bp 3 '– UTR.It’s opening reading frame (ORF) is of 261bp, encoding 86 amino acids with apredicted molecular weight of 8.95KD and an isoelectric point of 11.09. Homology analysis revealed that , MpsbR gene are highly conservative in mulberry and other species including peanut, soybean and Prunus mume.; The phylogenetic analysis based on MpsbR gene with other 20 species showed that mulberry shows closer relationship with peanut, sea mulberry, pear and soybean. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of MpsbR mRNA in the young leaf and the top bud. Its action mechanism is waiting for further studies.
引文
[1] Q. JI, L. Zhang, Y. F.,Wang J. Wang. Genome-wide analysis of basic leucine zipper transcription factor families in Arabidopsis thaliana, Oryza sativa and Populus trichocarpa[J].J Shanghai Univ(Engl Ed), 2009,13(2):174-182.
[2] X. L . Fu,Y. G. Lu,X. D. Liu, J. Q. Li. Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization[J]. Rice Science, 2008, 15(2):79-87.
[3] J. W. Richard, K. M. Plummer, M. A. Carpenter, et al. Approaches to functional genomics in filamentous fungi[J]. Cell Research, 2006, 16 (1):31-44.
[4] K. M. Kragh , T. Hendriks, A . Jong.J-Characterization of chitinase able to rescue somatic embryos of the temperature-sensitive carrot variant tsl1[J]. Plant Molecular Biology, 1996, 31(3): 631-645.
[5]高必达.转几丁质酶基因防植物病害研究:进展、问题与展望[J].生物工程进展,1999,19(2):21-28.
[6]郁志芳,陈崇顺,朱雪峰.植物几丁质酶的生物功能[J].生命的化学, 2000, 20(1): 36-37.
[7] K. J. Zhao, M. L. Chye. Methyl jasmonate induce expression of a novel Brassica juncea chitinase with two chitin-binding domains[J]. Plant Mo1 Bio1, 1999, 40: 1009-1018.
[8] Y . Xu, Q. Zhu, W. Panbangred, K. Shirasu, et a1. Regulation,expression and function of a new basic chitinasegene in rice(Oryzasativa L)[J]. Plant Mo1 Bio1, 1996, 30(3): 387-401.
[9]王军,庞广昌.抗菌肽抗菌机理的研究现状及趋势[J].食品科学, 2005, 26(8): 526-529.
[10] W. C. Hon, M. Grifith, A. Mlynarz, et a1. Antifreeze proteins in winter rye/Ire similar to pathogenesis-related proteins[J]. Plant Physiol, 1995, 109: 879-889.
[11] Z. Minic, S. Brown, Y. Kouchkovshyt, Schuhze. Purification and characterization of a novel chjtinase-lysozyme, of another chitinase, both hydrolyzing Rhizobium meliloti nod factors, and of a pathogenesis related protein from Medicago sativa roots[J]. BiochemicalJournal, 1998, 332(2):329-335.
[12] H. Ling, A. D. Recklies. The chitinase 3-like protein human cartilage glycoprotein 39 inhibits cellular responses to the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha[J]. Biochem J. 2004, 380: 651-659.
[13] Z .Zhu, T. Zheng, R. J. Homer, et a1. Acidic mammalian chitinasein asthmatic The inflammation and IL-13 pathway activation[J]. Science, 2004, 304: 1678-1682.
[14] J. R. Stangarlin, S. F. Pascholati.Activities of ribulose-1, 5-bisphosphate carboxylase-oxygenase (rubisco), chiorophyllase,beta-1, 3 glucanase and chitinase and chlorophyll content in bean cultivars(Phaseolus vulgaris)infected with Uromyces appendiculatus[J]. Phytopathologica, 2000,26(1):34-42.
[15] Z. Minic, S. Brown, Y. Kouchkovsky, et a1. Purification and characterization of a novel chitinase 1ysozyme,of another chitinase, both hydrolysing Rhizobium meliloti nod factors, and of a pathogenesis-related protein from Medicago sativa rots[J]. Biochemical Journal, 1998, 32(2):329-335.
[16]单立波,贾旭.几丁质酶及其在抗真菌基因工程中的应用[J].生物工程进展, 1998, 18(3): 333-335.
[17] H. Shinshi, et a1. Regulation of a plant pathogens is related enzyme: inhibitor of chtinase and chtinase mRNA accumulation in cultured tobacoo tissues by auxin and cytokinin[J]. Proc Natl Acad Sci USA, 1987, 84: 89-93.
[18] Q. Zhu. Isolation of a rice gene encoding a basic chitinase[J]. Mol Gen Genet, 1991, 226: 289-296.
[19] H. Shinshi.et al. Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain[J]. Plant Molecular Biology, 1990, 14: 357-368.
[20] M. J. Davis, et al. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels[J]. Plant Molecular Biology, 1991, 17: 631-639.
[21] F. T. Brederode, H. M. Lanthorst, J. F. Bol. Differential induction of acquires resistance and PR gene expression in tobacco by virus infection cthephon treatment, UV light and wonding[J].Plant Molecular Biology, 1991, 17: 1117-1125.
[22] Breiju FJG,Garro R,Conejero U. C7(P32) and C6(P34)PR Proteins induced in tobacco leaves by citrus exocorite virorid infection are chitinases [J]. Physiological and Molecular Plant Pathology, 1990,36:249-260.
[23] Connads-strauch J,Dow M,Milligan DE,et al. Induction of bydrlyuc enzymes in Brassica campe -strica in response to pathowaves of X anthomonas campestris[J]. Plant Physiology,1990,92:238 -243.
[24] L. Berglund, J. Brunsted, K. K. Nielsen, et al. Induction of bydrlyuc enzymes[J]. Plant Mol Blol. 1995,27:211-216.
[25]陈兰凤,刘德虎,李季伦.植物几丁质酶的结构,基因及其表达[J].生物工程进展, 1998, 118(2):33-36.
[26]张志忠,等.植物几丁质酶及其应用研究进展[J].福建农林大学学报, 2005, 12, 34(4): 33-34.
[27]蒋选利,等.几丁质酶与植物的抗病性[J].西北农业学报, 2002, 11(3): 71-75.
[28] A. Diaz-Perales, C. B. Collada,et al. ClaseⅠchitinase with bevein like domain ,but not classⅡenzymes, are relevant chestnul and avocado allergens [J].J Allergy Immunol, 1998, 102(1):127-133.
[29] Harikrishnak.et al. ClaseⅠchitinase [J]. Plant Mol.Biol., 1996, 30: 899-911.
[30] Kragh, K. M. et al. Differential induction of acquires resistance [J]. Plant Moi Biol, 1996, 31(1): 631-645.
[31]马汇泉等.几丁质酶及其在抗植物真茵病害中的作用[J].徽生物学杂志, 2004, 5(24): 3-4.
[32] K .Broglie, J. J. Gaynor, R . Braglie.M-Ethylene-regulated gene expression:Molecular cloning of the genes encoding an e ndo chitinase from phaseolus vulgaris[J]. Proceedings of the National Academy of Science of USA, 1986, 83: 6820-6824.
[33] K. Brogile, I. Chet, M. Holliday, et al. Broglie R.Transgenic Plants with enhanced resistance to the fungal pathogen.Rhizoctonia solani[J]. Science, 1991, 254: 1194-l197.
[34] H. S. Shin. Regulation of a plant pathogenesis related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin [J]. Proc Nail Acad Sci USA, 1987, 84:89-93.
[35]孟亮,李红双,金德敏等.转几丁质酶基因黑杨的获得[J].生物技术通报, 2004(3): 48 -51.
[36]蓝海燕,田颖川,王长海.表达B-1,3 -葡聚糖酶及几丁质酶基因的转基因导入油菜的研究初报[J].中国油料作物学报, 2000, 22(4): 6 -10.
[37]南相日.菜豆几丁质酶基因转化马铃薯及后代表达[J].中国农学通报, 2006, 22(2): 75- 77.
[38]王果萍,王景雪,孙毅等.几丁质酶基因导入西瓜植株及其抗病性鉴定研究[J].植物遗传资源学报, 2003, 4(2): 104-109.
[39] Y. Nishizawa, Z. Nishio, K. Nakazono. Enhancedresistance to blast( Magnaporthe grisea ) in transgenic japonica rice byconstitutive expression of rice chitinase[J]. Te oretical and Applied Genetics, 1999, 99: 383-390.
[40] G. L. De, R. D’Ovidio, F. Cervone. The role of polygalacturonce-inhibiting proteins (PGIPs)in defense against pathogenic fungi. Annual[J]. Review of Phytopathology, 2001, 39: 313-315.
[41] C. X. Di, Zhang M. X , Xu Shijian, et a1. Role of polygalactur-once-inhibiting protein in plant defense[J]. Critical Reviews in Mi-crobiology, 2006, 32: 91-100.
[42] C. Lang, H. D. rnenburg. Perspectives in the biological function and the technological of polygalacturonases[J]. Applied Microbiology and Biotechnology, 2000, 53(4): 366-375.
[43] P. Albersheim, A. J. Anderson. Protein from plant cell wall inhibit polygalacturonases secreted by plant pathogens[J]. Proceedings of the National Academy of Sciences of the United States of America, 1971, 68(8): 1815-1819.
[44] R. D’Ovidio, B. Mattei, S. Roberti, D. Bellincampi. Polygalacturonases, polygalacturonaseinhibitin proteins and pectic oligomers in plant-pathogen interactions[J]. Biochimica et Biop sica Acta, 2004, 1696(2): 237-244.
[45]阮期平,周立,郑远旗. PGIP在植物抗病方面的研究进展[J].植物学通报, 2000, 17(1): 60-63.
[46] Peter Albersheim, J. A. Anne. Proteins from plant cell walls inhibit polygalacturonases secreted byplant pathogens[J]. Proc Nat Acad Sci USA, 1971, 1815-1819.
[47] P. Toubart, A. Desiderio, G. Salvi, et al. Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein(PGIP) of Phaseolus vulgaris L[J]. The Plant Journal, 1992, 2(3): 367-373.
[48] C. G. Simpson, E. MacRae, R.C. Gardner. Cloning of a polygalacturonase inhibiting Protein from Kiwifruit(Actinidia deliciosa) [J]. Plant Physiol, 1995, 108:1748-1748.
[49] B. J. Haas, N. Volfvsky, C. D. Town, et al. Full-length messenger RNA sequences greatly improvegenome annotation[J]. Genome Bhysiol, 2002, 3(6): Research 0029. 01-12.
[50] Y. Gotoh, S. Nalumpang, A. Isshiki, et al. A Cdna encoding polygalacturonase-inhibiting protein induced in citrus leaves by polygalacturonase of Alternaria citri[J]. Journal of General Plant PATHOLOGY, 2002, 68(1): 57-61.
[51] S. Nalumpang, Y. Gotoh, Tsuboi H, et al. Functional characterization of citrus polygalacturonase- inhibiting protein[J]. Journal of General Plant Pathology, 2002, 68(2): 118-127.
[52] S. Nalumpang, Y . Gotoh, et al. Comparison and characterization of zation polygalacturonase inhibiting protein genes from the genus of Cit rus and its close related genera[J]. Thai J Agri Sci, 2002, 35:112-117.
[53] A. L. Powell, K. J. van, H. A. ten, et a1. Tran sgen ic expression of pear PGIP in tomato limits fungal colonization[J]. Molecular Plant Microbe Interact, 2000, (13): 942-950.
[54] A. S. Raghvenda. Photosynthesis: a comprehensive treatise[D]. Cambridge: Cambridge Univ press, 1998, 72-86.
[55]周峰.光合膜蛋白晶体的结构与功能[J].生命的化学, 2007, 27(5): 370-371.
[56]辛越勇,董风琴等.光系统Ⅱ反应中心的晶体结构被确定-膜结构生物学的重大突破[J].植物学通报, 2001, 18(3): 383-384.
[57] Jon Nield, James Barber. Refinement of the structural model for the Photosystem II supercomplex of higher plants[J]. Biochimica et Biophysica Acta, 2006, 1757 (5-6): 353–361.
[58] O. T. no, Y . Inoue. Mn-p reserving extraction of 33-,24-and 16-kDa proteins from O2–evolving PSⅡparticles by divalent salt-washing[J]. FEBS Lett, 1983, 164: 255-259.
[59] M. Miyao, N. Murata. Role of the 33-kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by ehbride ions [J]. FEBS Lett, 1984, 170: 350-354.
[60] B. Hankamer, J. Barber, E. J. Boekema. Structure and membrance organization of photosystemⅡin green plants Annu Rev plant physical [J]. PlantM ol Biol, 1997, 48: 641-671.
[61] L. X. Shi, W. P. Schroder. The low molecular mass subunits of the photosynthetic supracomplex, photosystem II [J]. Biochim Biophys Acta, 2004, 1608: 75-96.
[62] J. Stockhaus, M. Hofer, Renger G, et a1. Anti-sense RNA efficiently inhibits formation of the 10 kdpolypeptide of photosystem II in transgenic potato plants: analysis of the role of the 10 kd protein [J]. EMBO, 1990, 9(30):13-21.
[63] Marjaana Suorsa, Sari Sirpi?, Yagut Allahverdiyeva, et a1. PsbR.-a Missing Link in the Assembly of the Oxygen-evolving Complex of Plant Photosystem II[J].The Journal of Biological Chemistry,2009, 2: 1127-1140.
[64]方荣俊,戚金亮,扈冬青,等.桑树幼叶cDNA文库的构建及部分表达序列标签分析[J] .蚕业科学, 2008, 34(4): 581-586.
[65]赵卫国.桑树cDNA文库构建及种质遗传多样性的分析[D].南京:南京大学博士后流站, 2008.
[66]杨崇林,陈章良.高等植物的LRR蛋白:结构与功能[J].生物工程进展, 1997, 17(6): 43-47.
[67] Di Matteo A, Bonivento D, Tsemoglou D, et a1. Polygalacturonase-inhibiting protein (PGIP)in plant defense: a structural view[J]. Phytochemistry, 2006, 67(6): 528-533.
[68] F. Leckie, B. Mattei, C. Capodicasa, et a1. The specificity of polygalacturonase-inhibiting protein (PGIP):a single amino acid substitution in the solvent-exposed p-strand/13·turn region of the leucine-rich repeats (LRRs) confers a new recognition capability[J]. The EMBO Journal, 1999, 18(9): 2352-2363.
[69] Di Matteo A, Federici L, MaRei B, Salvi G, et a1.The crystal structure of polygalacturonase- inhibiting protein (PGIP), a leucine rich repeat protein involved in plant defense[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(17): 10124-10128.
[70] C. W. Bergmann, Y. Ito, D. Singer, et al. Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding,elicitors and fungal infection[J]. Plant J, 1994, 5(5): 625-634.
[71] Abu-Goukh A A, Strand L L, Labavitch J M, Development related changes in decay susceplibility and PG inhibitor content of‘Bartlett’pear fruit[J], Physial Plant Patal, 1983, 23:101-109.
[72] Johhnston D J, Rananthan V, Willamson B, 1993, J Exp Bot, 44(262): 971-976.
[73] Liang F, Zhang K, Zhou C, et al. Cloning, characterization and expression of the gene encoding polygalacturonase-inhibiting proteins (PGIPs)of peach[prunus persica(L.)Batch]. Plant Science, 2005, 168(2):481-486.
[74] C. W. Bergmann, Y. Ito, D. Singer,et al. Polygalacturonase-inhibiting protein accumlates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection[J]. Plant,1994, 5(5): 625-634.
[75] I. Gazendama, D. Oelofse, D. K. Berger.High-level expression of apple PGIP is not suficient to protect transgenic potato against Verticillium dahliae[J]. Physiological Plant Pathology, 2004, 65(3): 145-155.
[76]安连荣,张洪武,尹家凤,等.桑树光合作用特性的研究[J].蚕业科学, 2000, 26(2): 15-117.
[77]季宏伟等.莲胚芽暗萌发过程中的光合系统发育研究[J].植物学报, 2001, 43(11): 1129-1133.
[2] X. L . Fu,Y. G. Lu,X. D. Liu, J. Q. Li. Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization[J]. Rice Science, 2008, 15(2):79-87.
[3] J. W. Richard, K. M. Plummer, M. A. Carpenter, et al. Approaches to functional genomics in filamentous fungi[J]. Cell Research, 2006, 16 (1):31-44.
[4] K. M. Kragh , T. Hendriks, A . Jong.J-Characterization of chitinase able to rescue somatic embryos of the temperature-sensitive carrot variant tsl1[J]. Plant Molecular Biology, 1996, 31(3): 631-645.
[5]高必达.转几丁质酶基因防植物病害研究:进展、问题与展望[J].生物工程进展,1999,19(2):21-28.
[6]郁志芳,陈崇顺,朱雪峰.植物几丁质酶的生物功能[J].生命的化学, 2000, 20(1): 36-37.
[7] K. J. Zhao, M. L. Chye. Methyl jasmonate induce expression of a novel Brassica juncea chitinase with two chitin-binding domains[J]. Plant Mo1 Bio1, 1999, 40: 1009-1018.
[8] Y . Xu, Q. Zhu, W. Panbangred, K. Shirasu, et a1. Regulation,expression and function of a new basic chitinasegene in rice(Oryzasativa L)[J]. Plant Mo1 Bio1, 1996, 30(3): 387-401.
[9]王军,庞广昌.抗菌肽抗菌机理的研究现状及趋势[J].食品科学, 2005, 26(8): 526-529.
[10] W. C. Hon, M. Grifith, A. Mlynarz, et a1. Antifreeze proteins in winter rye/Ire similar to pathogenesis-related proteins[J]. Plant Physiol, 1995, 109: 879-889.
[11] Z. Minic, S. Brown, Y. Kouchkovshyt, Schuhze. Purification and characterization of a novel chjtinase-lysozyme, of another chitinase, both hydrolyzing Rhizobium meliloti nod factors, and of a pathogenesis related protein from Medicago sativa roots[J]. BiochemicalJournal, 1998, 332(2):329-335.
[12] H. Ling, A. D. Recklies. The chitinase 3-like protein human cartilage glycoprotein 39 inhibits cellular responses to the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha[J]. Biochem J. 2004, 380: 651-659.
[13] Z .Zhu, T. Zheng, R. J. Homer, et a1. Acidic mammalian chitinasein asthmatic The inflammation and IL-13 pathway activation[J]. Science, 2004, 304: 1678-1682.
[14] J. R. Stangarlin, S. F. Pascholati.Activities of ribulose-1, 5-bisphosphate carboxylase-oxygenase (rubisco), chiorophyllase,beta-1, 3 glucanase and chitinase and chlorophyll content in bean cultivars(Phaseolus vulgaris)infected with Uromyces appendiculatus[J]. Phytopathologica, 2000,26(1):34-42.
[15] Z. Minic, S. Brown, Y. Kouchkovsky, et a1. Purification and characterization of a novel chitinase 1ysozyme,of another chitinase, both hydrolysing Rhizobium meliloti nod factors, and of a pathogenesis-related protein from Medicago sativa rots[J]. Biochemical Journal, 1998, 32(2):329-335.
[16]单立波,贾旭.几丁质酶及其在抗真菌基因工程中的应用[J].生物工程进展, 1998, 18(3): 333-335.
[17] H. Shinshi, et a1. Regulation of a plant pathogens is related enzyme: inhibitor of chtinase and chtinase mRNA accumulation in cultured tobacoo tissues by auxin and cytokinin[J]. Proc Natl Acad Sci USA, 1987, 84: 89-93.
[18] Q. Zhu. Isolation of a rice gene encoding a basic chitinase[J]. Mol Gen Genet, 1991, 226: 289-296.
[19] H. Shinshi.et al. Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain[J]. Plant Molecular Biology, 1990, 14: 357-368.
[20] M. J. Davis, et al. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels[J]. Plant Molecular Biology, 1991, 17: 631-639.
[21] F. T. Brederode, H. M. Lanthorst, J. F. Bol. Differential induction of acquires resistance and PR gene expression in tobacco by virus infection cthephon treatment, UV light and wonding[J].Plant Molecular Biology, 1991, 17: 1117-1125.
[22] Breiju FJG,Garro R,Conejero U. C7(P32) and C6(P34)PR Proteins induced in tobacco leaves by citrus exocorite virorid infection are chitinases [J]. Physiological and Molecular Plant Pathology, 1990,36:249-260.
[23] Connads-strauch J,Dow M,Milligan DE,et al. Induction of bydrlyuc enzymes in Brassica campe -strica in response to pathowaves of X anthomonas campestris[J]. Plant Physiology,1990,92:238 -243.
[24] L. Berglund, J. Brunsted, K. K. Nielsen, et al. Induction of bydrlyuc enzymes[J]. Plant Mol Blol. 1995,27:211-216.
[25]陈兰凤,刘德虎,李季伦.植物几丁质酶的结构,基因及其表达[J].生物工程进展, 1998, 118(2):33-36.
[26]张志忠,等.植物几丁质酶及其应用研究进展[J].福建农林大学学报, 2005, 12, 34(4): 33-34.
[27]蒋选利,等.几丁质酶与植物的抗病性[J].西北农业学报, 2002, 11(3): 71-75.
[28] A. Diaz-Perales, C. B. Collada,et al. ClaseⅠchitinase with bevein like domain ,but not classⅡenzymes, are relevant chestnul and avocado allergens [J].J Allergy Immunol, 1998, 102(1):127-133.
[29] Harikrishnak.et al. ClaseⅠchitinase [J]. Plant Mol.Biol., 1996, 30: 899-911.
[30] Kragh, K. M. et al. Differential induction of acquires resistance [J]. Plant Moi Biol, 1996, 31(1): 631-645.
[31]马汇泉等.几丁质酶及其在抗植物真茵病害中的作用[J].徽生物学杂志, 2004, 5(24): 3-4.
[32] K .Broglie, J. J. Gaynor, R . Braglie.M-Ethylene-regulated gene expression:Molecular cloning of the genes encoding an e ndo chitinase from phaseolus vulgaris[J]. Proceedings of the National Academy of Science of USA, 1986, 83: 6820-6824.
[33] K. Brogile, I. Chet, M. Holliday, et al. Broglie R.Transgenic Plants with enhanced resistance to the fungal pathogen.Rhizoctonia solani[J]. Science, 1991, 254: 1194-l197.
[34] H. S. Shin. Regulation of a plant pathogenesis related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin [J]. Proc Nail Acad Sci USA, 1987, 84:89-93.
[35]孟亮,李红双,金德敏等.转几丁质酶基因黑杨的获得[J].生物技术通报, 2004(3): 48 -51.
[36]蓝海燕,田颖川,王长海.表达B-1,3 -葡聚糖酶及几丁质酶基因的转基因导入油菜的研究初报[J].中国油料作物学报, 2000, 22(4): 6 -10.
[37]南相日.菜豆几丁质酶基因转化马铃薯及后代表达[J].中国农学通报, 2006, 22(2): 75- 77.
[38]王果萍,王景雪,孙毅等.几丁质酶基因导入西瓜植株及其抗病性鉴定研究[J].植物遗传资源学报, 2003, 4(2): 104-109.
[39] Y. Nishizawa, Z. Nishio, K. Nakazono. Enhancedresistance to blast( Magnaporthe grisea ) in transgenic japonica rice byconstitutive expression of rice chitinase[J]. Te oretical and Applied Genetics, 1999, 99: 383-390.
[40] G. L. De, R. D’Ovidio, F. Cervone. The role of polygalacturonce-inhibiting proteins (PGIPs)in defense against pathogenic fungi. Annual[J]. Review of Phytopathology, 2001, 39: 313-315.
[41] C. X. Di, Zhang M. X , Xu Shijian, et a1. Role of polygalactur-once-inhibiting protein in plant defense[J]. Critical Reviews in Mi-crobiology, 2006, 32: 91-100.
[42] C. Lang, H. D. rnenburg. Perspectives in the biological function and the technological of polygalacturonases[J]. Applied Microbiology and Biotechnology, 2000, 53(4): 366-375.
[43] P. Albersheim, A. J. Anderson. Protein from plant cell wall inhibit polygalacturonases secreted by plant pathogens[J]. Proceedings of the National Academy of Sciences of the United States of America, 1971, 68(8): 1815-1819.
[44] R. D’Ovidio, B. Mattei, S. Roberti, D. Bellincampi. Polygalacturonases, polygalacturonaseinhibitin proteins and pectic oligomers in plant-pathogen interactions[J]. Biochimica et Biop sica Acta, 2004, 1696(2): 237-244.
[45]阮期平,周立,郑远旗. PGIP在植物抗病方面的研究进展[J].植物学通报, 2000, 17(1): 60-63.
[46] Peter Albersheim, J. A. Anne. Proteins from plant cell walls inhibit polygalacturonases secreted byplant pathogens[J]. Proc Nat Acad Sci USA, 1971, 1815-1819.
[47] P. Toubart, A. Desiderio, G. Salvi, et al. Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein(PGIP) of Phaseolus vulgaris L[J]. The Plant Journal, 1992, 2(3): 367-373.
[48] C. G. Simpson, E. MacRae, R.C. Gardner. Cloning of a polygalacturonase inhibiting Protein from Kiwifruit(Actinidia deliciosa) [J]. Plant Physiol, 1995, 108:1748-1748.
[49] B. J. Haas, N. Volfvsky, C. D. Town, et al. Full-length messenger RNA sequences greatly improvegenome annotation[J]. Genome Bhysiol, 2002, 3(6): Research 0029. 01-12.
[50] Y. Gotoh, S. Nalumpang, A. Isshiki, et al. A Cdna encoding polygalacturonase-inhibiting protein induced in citrus leaves by polygalacturonase of Alternaria citri[J]. Journal of General Plant PATHOLOGY, 2002, 68(1): 57-61.
[51] S. Nalumpang, Y. Gotoh, Tsuboi H, et al. Functional characterization of citrus polygalacturonase- inhibiting protein[J]. Journal of General Plant Pathology, 2002, 68(2): 118-127.
[52] S. Nalumpang, Y . Gotoh, et al. Comparison and characterization of zation polygalacturonase inhibiting protein genes from the genus of Cit rus and its close related genera[J]. Thai J Agri Sci, 2002, 35:112-117.
[53] A. L. Powell, K. J. van, H. A. ten, et a1. Tran sgen ic expression of pear PGIP in tomato limits fungal colonization[J]. Molecular Plant Microbe Interact, 2000, (13): 942-950.
[54] A. S. Raghvenda. Photosynthesis: a comprehensive treatise[D]. Cambridge: Cambridge Univ press, 1998, 72-86.
[55]周峰.光合膜蛋白晶体的结构与功能[J].生命的化学, 2007, 27(5): 370-371.
[56]辛越勇,董风琴等.光系统Ⅱ反应中心的晶体结构被确定-膜结构生物学的重大突破[J].植物学通报, 2001, 18(3): 383-384.
[57] Jon Nield, James Barber. Refinement of the structural model for the Photosystem II supercomplex of higher plants[J]. Biochimica et Biophysica Acta, 2006, 1757 (5-6): 353–361.
[58] O. T. no, Y . Inoue. Mn-p reserving extraction of 33-,24-and 16-kDa proteins from O2–evolving PSⅡparticles by divalent salt-washing[J]. FEBS Lett, 1983, 164: 255-259.
[59] M. Miyao, N. Murata. Role of the 33-kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by ehbride ions [J]. FEBS Lett, 1984, 170: 350-354.
[60] B. Hankamer, J. Barber, E. J. Boekema. Structure and membrance organization of photosystemⅡin green plants Annu Rev plant physical [J]. PlantM ol Biol, 1997, 48: 641-671.
[61] L. X. Shi, W. P. Schroder. The low molecular mass subunits of the photosynthetic supracomplex, photosystem II [J]. Biochim Biophys Acta, 2004, 1608: 75-96.
[62] J. Stockhaus, M. Hofer, Renger G, et a1. Anti-sense RNA efficiently inhibits formation of the 10 kdpolypeptide of photosystem II in transgenic potato plants: analysis of the role of the 10 kd protein [J]. EMBO, 1990, 9(30):13-21.
[63] Marjaana Suorsa, Sari Sirpi?, Yagut Allahverdiyeva, et a1. PsbR.-a Missing Link in the Assembly of the Oxygen-evolving Complex of Plant Photosystem II[J].The Journal of Biological Chemistry,2009, 2: 1127-1140.
[64]方荣俊,戚金亮,扈冬青,等.桑树幼叶cDNA文库的构建及部分表达序列标签分析[J] .蚕业科学, 2008, 34(4): 581-586.
[65]赵卫国.桑树cDNA文库构建及种质遗传多样性的分析[D].南京:南京大学博士后流站, 2008.
[66]杨崇林,陈章良.高等植物的LRR蛋白:结构与功能[J].生物工程进展, 1997, 17(6): 43-47.
[67] Di Matteo A, Bonivento D, Tsemoglou D, et a1. Polygalacturonase-inhibiting protein (PGIP)in plant defense: a structural view[J]. Phytochemistry, 2006, 67(6): 528-533.
[68] F. Leckie, B. Mattei, C. Capodicasa, et a1. The specificity of polygalacturonase-inhibiting protein (PGIP):a single amino acid substitution in the solvent-exposed p-strand/13·turn region of the leucine-rich repeats (LRRs) confers a new recognition capability[J]. The EMBO Journal, 1999, 18(9): 2352-2363.
[69] Di Matteo A, Federici L, MaRei B, Salvi G, et a1.The crystal structure of polygalacturonase- inhibiting protein (PGIP), a leucine rich repeat protein involved in plant defense[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(17): 10124-10128.
[70] C. W. Bergmann, Y. Ito, D. Singer, et al. Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding,elicitors and fungal infection[J]. Plant J, 1994, 5(5): 625-634.
[71] Abu-Goukh A A, Strand L L, Labavitch J M, Development related changes in decay susceplibility and PG inhibitor content of‘Bartlett’pear fruit[J], Physial Plant Patal, 1983, 23:101-109.
[72] Johhnston D J, Rananthan V, Willamson B, 1993, J Exp Bot, 44(262): 971-976.
[73] Liang F, Zhang K, Zhou C, et al. Cloning, characterization and expression of the gene encoding polygalacturonase-inhibiting proteins (PGIPs)of peach[prunus persica(L.)Batch]. Plant Science, 2005, 168(2):481-486.
[74] C. W. Bergmann, Y. Ito, D. Singer,et al. Polygalacturonase-inhibiting protein accumlates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection[J]. Plant,1994, 5(5): 625-634.
[75] I. Gazendama, D. Oelofse, D. K. Berger.High-level expression of apple PGIP is not suficient to protect transgenic potato against Verticillium dahliae[J]. Physiological Plant Pathology, 2004, 65(3): 145-155.
[76]安连荣,张洪武,尹家凤,等.桑树光合作用特性的研究[J].蚕业科学, 2000, 26(2): 15-117.
[77]季宏伟等.莲胚芽暗萌发过程中的光合系统发育研究[J].植物学报, 2001, 43(11): 1129-1133.