摘要
逆境胁迫,包括极端温度(低温、高温)、干旱、盐胁迫、化学毒害和光污染等,严重影响植物生长及产量,并触发植物在生理、形态、生化和分子结构等方面的一系列变化。逆境胁迫对植物生长和发育具有严重影响,传统的育种因为缺乏有效的资源而抵御逆境效果不明显,因此有必要对抗逆植物的基因资源进行深入发掘。作物在长期进化过程中获得一定的适应逆境的能力,当逆境出现时植物会有很多基因被激发表达来提高自身的抗逆能力,这些基因表达量的差异导致植物抗逆能力不同,只有成簇抗性相关基因的转录失活,才能有效提高作物的抗逆能力。沙冬青(Ammopiptanthus mongolicus( Maxim1)Cheng F1)是我国特有的荒漠植物,常年处于极端低温、高温和干旱等逆境环境,具有极强的抗逆性,是开展抗逆研究的理想材料。
本文以沙冬青为研究对象,进行逆境转录表达谱的构建,并对部分差异表达片段进行了初步的验证和生物信息学分析,希望从中发现高效的抗逆调控基因,为转基因育种提供资源。在优化沙冬青种子萌发和幼苗逆境处理条件的基础上,对经逆境处理的沙冬青开展cDNA-AFLP研究。在对PCR反应条件、聚丙烯酰胺凝胶电泳(PAGE)、转录差异表达条带(TDFs)的回收及二次PCR条件等多方面的优化后,对沙冬青在低温、高温和干旱等逆境条件下的转录表达谱进行了分析。共回收了1487条沙冬青逆境胁迫下的TDFs,并对其中536条TDFs进行克隆并获得序列信息。在此536条TDFs中,有287条得到上调表达(up-regulated expression),249条得到下调表达(down-regulated expression)。利用Blastx进行比对后,选择其中201条与NCBI数据库中已报道基因具有明显同源性的片段进行了基因差异表达生物信息学分析。
在这201条TDFs中,在低温逆境中出现85条,在高温逆境中出现175条,在干旱逆境中出现93条。根据同源性最高序列的预测功能,各逆境下的TDFs均可分为细胞代谢类(Metabolism)、信号传导(Signal transduction)、运输(Transporter)、转录(Transcription)、逆境胁迫/防御相关(Cell resecue / Stress-related / Defence)、细胞生长与结构(Cell growth and structure)、光合与能量(Photosyntesis and Energy)和功能未知(Function unknown)等8个部分。这些与抗逆性相关的功能蛋白为从分子水平研究和阐释沙冬青的抗逆机制,进而发掘其中的抗逆基因资源提供了依据。
采取半定量RT-PCR方法对HSPs、CP43、EIN3a和CCR-4相关因子等功能蛋白编码基因同源的TDFs进行了初步验证。结果显示,在半定量RT-PCR实验中,各基因的表达量出现了与cDNA-AFLP实验一致的上调或下调。该结果进一步证实了相关基因在逆境处理下的表达变化。
Abiotic stresses, including extreme temperatures (extremes of cold and heat), drought, salinity, oxidative stress and chemical toxicity, are serious threats to the plant growth and productivity and result in a series of physiological, morphological, biochemical and molecular changes. In view of the serious influences of abiotic stress on plant, it is important to carry out the research in molecular biology of stress-resistant plant and to gain the gene resourses from the plant with resistance. However, traditional breeding methods usually have little effection on the resistance to abiotic stresses because of the lack of effective gene resources. During the evolutionary, crops have gained certain resistance which can only be stimulated when stress occurs. The activation of cluster resistance-related genes transcription can effectively enhance the resistance of crops. Ammopiptanthus mongolicus, mainly distributing in the desert regions of Northwest China, which is capable of surviving unfavorable conditions such as cold/heat weather or lack of moisture, should be paid due attention as a highly adversity-resistant plant in the research of plant stress.
In order to identify stress-regulated genes, A. mongolicus was chosen as the study object to estamblish its transcriptional profile under abiotic stresses, and some transcription derived fragments (TDFs) from the plant were identified and analysed. Based on the optimization of the conditions of seed germination and seeding stresses, a cDNA-based Amplified Fragment Length Polymorphism (cDNA-AFLP) approach was applied in this study. After optimizations of cDNA-AFLP such as the choice of primers combinations, the reaction conditions of polymerase chain reaction (PCR), ways of polyacrylamide gel electrophoresis (PAGE),recovery of the transcript derived fragments (TDFs) and the method of re-amplifications, the transcriptional profile on A.mongolicus with cold, heat and drought treating was then analyzed. A total of 1487 TDFs expressed in various stresses were gained in the study, of which 536 TDFs were cloned and sequenced. Among the sequenced TDFs, 287 had an up-regulated expression while 249 were down-regulated, and 201 which had significant homeology with the sequences in data bank were chosen to establish the transcriptional profile of A. mongolicus under abiotic stresses.
Among the 201 TDFs, 85 could be found in low temperature stress, 175 could be found in high temperature stree, and 93 could be found in drought stress. According the Blast results of the sequenced TDFs, 201 showed significant similarity to the reported genes in the NCBI databases and were functionally annotated to be used in gene difference expression. The TDFs in each stress could be classified as 8 kinds: cellular metabolism, signal transduction, transporter, transcription, cell resecue/ stress-related /defence, cell growth and structure, photosyntesis and energy, and unknown function. The largest category of identified genes was in cellular metabolism.
A semi-quantitative RT-PCR strategy was then carried out to verify the expression of some TDFs under stresses, which have significant homeology with the encoding genes of HSPs, photosystem II CP43 chlorophyll apoprotein, CCR4-associated factor. The results of semi-quantitative RT-PCR showed that the expression patterns of chosen TDFs were same with previous cDNA-AFLP, representing simultaneous up-regulation or down-regulation.
引文
1.丁琼. "沙冬青种子萌发及幼苗生长特性."植物生态学, 2006, 30(4): 633~639.
2.黄骥. " TFⅢA型锌指蛋白及在提高植物耐逆性中的作用."遗传, 2007, 29(8): 915~922.
3.费云标. "沙冬青抗冻蛋白的分离纯化及其理化特性分析."科学通报, 2000,45(20): 2185~2189.
4.费云标. "沙冬青高活性抗冻蛋白的分离与鉴别."植物学报,1994, 36(6): 649~650.
5.郭丽琼. "应用cDNA-AFLP技术分离草菇冷诱导表达基因."园艺学报, 2005, 32(1): 54~59.
6.黄振英. "新疆10种沙生植物旱生结构的解剖学研究."西北植物学报, 1995,15(6): 56~61.
7.江勇. "抗冻蛋白及其在植物抗冻生理中的作用."植物学报, 1999, 41(7): 677~685.
8.蒋志荣. "沙冬青抗旱机理的探讨."中国沙漠, 2000, 20(l): 72~79.
9.李凤琴. "C12对植物叶片结构伤害的解剖学研究."西北农业学报, 2004, l3(3): 35~39.
10.梁宏伟. "聚丙烯酰胺凝胶快速、高效银染方法的建立."遗传,2008, 30(10): 1379~1382.
11.刘耘. "2种方法银染的AFLP片段再扩增效果比较及改进."华中农业大学学报,2008,27(2): 182~185.
12.马淼. "濒危植物新疆沙冬青叶解剖结构及其光合特性."石河子大学学报(自然科学版), 2005, 23(4): 446~448.
13.王雪征. "沙冬青生境调查及人工栽培技术."河北农业科学, 2005,9(4): 51~52.
14.魏令波. "沙冬青叶片热稳定抗冻蛋白特性分析."植物学报, 1999, 41(8): 837~841.
15.吴乃虎.基因工程原理(下册).北京:科学出版社, 2002.
16.夏晗. "低温、干旱和盐胁迫下沙冬青幼苗脯氨酸含量的变化."吉林林业科技,2000, 36(4): 1~2,20.
17.尹林克. "不同环境下沙冬青属植物的蛋白质氨基酸变化."干旱区研究,2004, 21(3): 269~274.
18.云南网. http://www.yunnan.cn/. 2010, 4,16
19. Agarwal, Reddy, et al. (2006). "Role of DREB transcription factors in abiotic and biotic stress tolerance in plants." Plant Cell Rep 25(12): 1263~1274.
20. Allen, Yamasaki, et al. (1998). "A novel mode of DNA recognition by a beta- sheet revealed by the solution structure of the GCC-box binding domain domain in complex with DNA." EMBO J 17: 5485~5496.
21. Avrova, Venter, et al. (2003). "Profiling and quantifying differential gene transcription in Phytophthora infestans prior to and during the early stages of potato infection. ." Fungal Genet Biol 40(1): 4~14.
22. Bachem, V. d. Hoeven, et al. (1996). "Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP : analysis of gene expression during potato tuber development." The Plant Journal 9(5): 745~753.
23. Bachem, Oomen, et al. (1998). "Transcript imaging with cDNA-AFLP: a step-by-step protocol."
Plant Molecular Biology Reporter 16: 157~173.
24. Bachem, Oomen, et al. (1998). "Transcript imaging with cDNA-AFLP: a step-by-step protocol. ."Plant Molecular Biology Reporter 16: 157~173.
25. Barret, Brinkman, et al. (2004). "Identification of candidate genes forin vitroandrogenesis induction in maize." Theor Appl Genet 109(8): 1660~1668.
26. Bassam and Gresshoff (1991). "Fast and sensitive silver staining of DNA in polyacrylamide gels." Anal Biochem, 196: 80~83.
27. Bohert and Sheveleva (1998). "Plant stress adaptations-making metabolism move." Curr Opin Plant Biol 1: 267~274.
28. Boventius and Weller (1994). "Mapping and analysis of dairy cattle quantitative trait loci by maximum likelihood methodology using milk protein gene as genetic markers." Gnrtics 137: 267~280.
29. Boyer, J. S. (1982). "Plant Productivity and Environment." Science 218(4571): 443-448.
30. Burke, Brown, et al. (2006). "Modeling the recent ecolution of global drought and projections for the twenty-first century with the Hadely center climate model. ." J Hydrometeor 7.
31. Catherine, Barreneche, et al. (2001). Molecular differentiation between two European oak species: quercus robur and quercus petraea. Plant &Animal GenomeⅨConference,. San Diego, CA: 17~21.
32. Chen ( 2002). "Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor." Plant Physiol, 129: 706~716.
33. Chen and Murata (2002). "Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes." Curr Opin Plant Biol 5(3): 250~257.
34. Chinnusamy, V., K. Schumaker, et al. (2004). "Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants." J Exp Bot 55(395): 225~236.
35. Choi, Hong, et al. (2000). "ABFs, a family of ABA-responsive element binding factors. ." J Biol Chem 275: 1723~1730.
36. Craciun, A. R., M. Courbot, et al. (2006). "Comparative cDNA-AFLP analysis of Cd-tolerant and -sensitive genotypes derived from crosses between the Cd hyperaccumulator Arabidopsis halleri and Arabidopsis lyrata ssp. Petraea." Journal of Experimental Botany 57(12 ): 2967~2983.
37. Cushman and Bohnert ( 2000). "Genomic approaches to plant stress tolerance." Curr Opin Plant Biol 3: 117~124.
38. Donson, Fang, et al. (2002). "Comprehensive gene expression analysis by transcript profiling." Plant Molecular Biology Reporter 48: 75~97.
39. Dreier, Beerli, et al. (2001). "Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors." J Biol Chem 276: 29466~29478.
40. FAO (2004). FAO production yearbook. A. O. o. t. U. N. Food. Rome.
41. Feng, James, et al. (2004). A cDNA-AFLP profile of cotton genes in response to drought stress. San Diego, CA: Plant &Animal GenomesⅫconferences,: 10~14.
42. Fox, Green, et al. (1998). "Changes in hexokinase activity in echinochloa phyllopogon and echinochloa crus-pavonis in response to abiotic stress." Plant Physiol 118(4): 1403~1409.
43. Gilmour and Sebolt ( 2000). "Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. ." Plant Physiol 124: 1854~1865.
44. Griffith (1992). " Antifreeze Proteins Produced endogenously in winter rye leaves. ." Plant Physiol 100(2): 593~596.
45. Hazen., Wu, et al. (2003). "Gene expression profiling of plant responses to abiotic stress." Funct Integr Genomics 3(3): 105~111.
46. Honde (1992). "Cloning, characterization and expression of a cDNA encoding a 50KD proteins specification induced by cold acclimation in wheat." Plant Physiol 99: 1381~1387.
47. Hong, Jon, et al. (1997). " Identification of a receptor-like protein kinases gene rapidly induced by abscisic acid, dehydration, high salt and cold treatments in Arabidopsis thaliana." Plant Physiol 113: 1203～1212.
48. Hrabak, Dickmann, et al. (1996). "Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana." Plant Mol Biol 31: 405~412.
49. HU, DAI, et al. (2006). "Overexpressing a NAM, ATAF and CUC(NAC) transcription factor enhances drought resistance and salt tolerance in rice." Proc Natl Acad Sci 103(35): 12987~12992.
50. Jagadeeswaran., Saini., et al. (2009). "Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis." Planta 229(4): 1009~1014.
51. Jason, Lilly, et al. (2002). "The Chlamydomonas reinhardtii Organellar Genomes Respond Transcriptionally and Post-Transcriptionally to Abiotic Stimuli. ." The Plant Cell 14: 2681~2706.
52. Jin and Martin (1999). "Multifunctionality and diversity within the plant MYB-gene family. ." Plant Mol Biol 41: 577-585.
53. Johnes and Rower (1998). "A comparison the efficiency of differential display and cDNA-AFLPs as a tools for the isolation of differentialy expressed parasite genes. ." Fundamental and Applied Nemadity 21: 81~88.
54. Katiyar-Agarwal, Agarwal, et al. (1999). "Emerging trends in agricultural biotechnology research: use of abiotic stress induced promoter to drive expression of a stress resistance gene in the transgenic system leads to high level stress tolerance associated with minimal negative effects on growth." Curr Sci 77: 1577~1579.
55. Kazuoka and Oeda (1992). "Heat stable COR(cold-regulated) Proteins associated with freezing tolerance in spinach. ." Plant Cell Physiol 33: 1107~1114.
56. Kim, Y.-J., E.-W. Jung, et al. (2004). "AP2/EREBP Transcription Factors in Rice." Plant Pathol J 20 (1): 41~45.
57. Kimpel, Nagao, et al. (1990). "Regulation of the heat shock response in soybean seedlings." Plant Physiol 94:988~995.
58. Kumar, Chowdhury., et al. (2001). "Interspecific hybridization in Brassica juncea and Brassica tournefortii through embryo rescue and their evaluation for biotic and abiotic stress tolerance." Indian J Exp Biol 39(9): 911~915.
59. Kyong-Hi ( 2001). "PHOTOSYSTEM II: The Solid Structural Era." Annu. Rev. Biophys. Biomol. Struct. 30: 307~328.
60. Lee, Gippert, et al. (1989). "Three-dimensional solution structure of a single zinc finger DNA-binding domain." Science 245: 635~637.
61. Levitt, Ed. (1980). Responses of plants to environmental stresses, Academic Press.
62. Liu, Kasuga, et al. (1998). "Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabidopsis." Plant Cell 10: 1391~1406.
63. Mizoguchi, Ichimura, et al. (1997). " Environmental stress response in plants: the role of mitogen-activated protein kinases." Trends Biotech 15: 15~19.
64. Money, Reader, et al. (1996). "AFLP-based mRNA Finger printing. ." Nucleic Acids Research 24: 2616~2617.
65. Nanjo, Kasuga., et al. (2002). "[Morphological changes responding to abiotic environmental stress]." Tanpakushitsu Kakusan Koso 47(12 Suppl): 1684~1689.
66. Oh, Song, et al. (2005). "Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth." Plant Physiol 138(1): 341~351.
67. Pater, d. (1996). "Characterization of a zinc-dependent transcriptional activator from Arabidopsis." Nucleic Acids Res 24: 4624~4631.
68. Pradeep, Agarwal, et al. (2006). "Role of DREB transcription factors in abiotic and biotic stress tolerance in plants." Plant Cell Rep 25: 1263~1274.
69. Riechmann and Ratcliffe (2000). "A genomic perspective on plant transcription factors. ." Curr Opin Plant Biol 3: 423~434.
70. Rushton and Somssich (1998). "Transcriptional control of plant genes responsive to pathogens." Curr. Opin Plant Biol 1: 311~315.
71. Sabehat and Weiss (1998). "Expression of heat shock protein at low temperature.." Plant Physiology 117: 651~658.
72. Saijo, Hata, et al. (2000). "Overexpression of a single Ca2+-dependent protein kinase cofers both cold and salt/drought tolerance on rice plant. ." Plant J 23: 319~327.
73. Sanguinetti, Dilasneto, et al. (1994). "Rapid silver staining and recovery of PCR products separated on polyacrylamide gels." Biotechniques 17: 915~919.
74. Seki, Satou, et al. (2004). "RIKEN Arabidopsis full-length (RAFL) cDNA and its applications for expression profiling under abiotic stress conditions." J Exp Bot 55(395): 213~223.
75. Serrano, Mulet, et al. (1999). "A glimpse of the mechanisms of ion homeostasis during salt stress." J Exp Bot 50: 1023~1036.
76. Shinozak, K. and K. Yamaguchi-Shinozaki. (2007). "Gene networks involved in drought stress response and tolerance." Journal of Experimental Botany 58(2): 221~227.
77. Shinozaki, K. (2007). "Molecular mechanisms of abiotic stress responses: light and nutrient environment--overview." Tanpakushitsu Kakusan Koso 52(6 Suppl): 585~586.
78. Shirasawa, Takabe, et al. (2006). "Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation of their tolerance to abiotic stress." Ann Bot 98(3): 565~571.
79. Simoes-Araujo, Rodrigues, et al. (2002). "Identification of differentially expressed genes by cDNA-AFLP technique during heat stress in cowpea nodules. ." FEBS Lett 515(1-3): 44~50.
80. Smirnoff (1998). "Plant resistance to environmental stress. ." Curr Opin Biotech, 9: 214~219.
81. Smith, Gaitatzes, et al. (1999). "The WD repeat: a common architecture for diverse functions." Trends Biochem. Sci, 24(5): 181~185.
82. Souer, V. Ingen, et al. (1996). "The No Apical Meristem gene of petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordial boundaries." Cell 85(2): 159~170.
83. Stockinger, Gilmour, et al. (1997). "Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit." Proc Natl Acad Sci 94: 1035~1040.
84. Tran, Nakashima, et al. (2004). "Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress promoter." Plant Cell 16(2481~2498).
85. Urao, Katagiri, et al. (1994). "Two gene that encode Ca2 +-dependent protein kinase are induced by drought and high salt stress in Arabidopsis thaliana." Mol Gen Genet. 244: 331~340.
86. Valliyodan and Nguyen. (2006). "Understanding regulatory networks and engineering for enhanced drought tolerance in plants." Curr Opin Plant Biol 9(2): 189~195.
87. Vijayan., Liu, et al. (2003). "Enhanced activation of mitogen-activated protein kinase and myosin light chain kinase by the Pro33 polymorphism of integrin beta 3." J Biol Chem 278(6): 3860~3067.
88. Vos, Hogers, et al. (1995). "AFLP: a new technique for DNA finger printing." Nucleic Acids Research 23(21): 4407~4414.
89. Waditee, Bhuiyan, et al. (2005). "Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis." Proc Natl Acad Sci U S A 102(5): 1318~1323.
90. Wang, W., B. Vinocur, et al. (2003). "Plant responses to drought, salinity and extreme temperatures:towards genetic engineering for stress tolerance." Planta 218: 1~14.
91. Waters, Lee, et al. (1996). "Evolution,structure and function of the small heat shock proteins in plants.." J Exp Bot 47(3): 325~338.
92. WX, W., B. Vinocur, et al. (2001a). "Biotechnology of plant osmotic stress tolerance: physiological and molecular considerations." Acta Hort 560.
93. Xiong. and Zhu. (2001). "Abiotic stress signal transduction in plants: Molecular and genetic perspectives." Physiol Plant 112(2): 152~166.
94. Yasuda, Ishikawa, et al. (2008). "Antagonistic interaction between systemic acquired resistance andthe abscisic acid-mediated abiotic stress response in Arabidopsis." Plant Cell 20(6): 16781692.
95. Zabeau and Vos (1993). Selective restriction fragment amplification: a general method for DNA fingerprinting. European Patent Application
96. Zhang, Ruan, et al. (2005). "Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana." Bioinformatics 21(14): 3074~3081.
97. Zhu ( 2001a). "Plant salt tolerance. ." Trends Plant Sci 6: 66~71.
98. Zhu, Gong., et al. (2002). "OSM1/SYP61: a syntaxin protein in Arabidopsis controls abscisic acid-mediated and non-abscisic acid-mediated responses to abiotic stress." Plant Cell 14(12): 3009~3028.
99. Zhu, Hasegawa, et al. (1997). "Molecular aspects of osmotic stress in plants." Crit Rev Plant Sci 16(253~277).