王小龙;李节法;张才喜;王世平;许文平;

• 1:上海交通大学农业与生物学院

摘要(Abstract)：

【目的】分离和克隆梨过敏原蛋白基因Ppmal(Gen Bank登录号为KP008110),探讨其在梨果实发育过程中的功能。【方法】以砂梨品种‘翠冠’[Pyrus pyrifolia(Burm.f.)Nakai]为试材,在盛花期后30 d对植株果柄进行涂抹赤霉素处理,利用同源克隆和RACE方法克隆目的基因全长序列,并通过实时荧光定量技术和半定量技术分析该基因在梨不同组织以及梨果实发育过程中的表达变化。【结果】Ppmal的全长是906 bp,含有480 bp的开放阅读框(ORF),编码159个氨基酸,预测的蛋白质相对分子质量为17.56 k D,等电点为5.62。生物信息学分析显示该基因没有信号肽序列,没有跨膜结构域,具亲水性。与其他物种的过敏原蛋白基因核苷酸序列同源性超过75%,与苹果和西洋梨编码的氨基酸序列同源性分别高达97.48%和96.23%,进化树分析表明该基因与苹果的进化关系最近。同时,定量结果显示经过赤霉素处理后的果实中该基因的表达量随着果实的生长发育呈现先降低后升高的趋势,并且具有组织差异表达的特点,其表达量在叶片中最大,其次是嫩叶,再次是花,枝条最低。【结论】获得梨Ppmal基因全长,对该基因在砂梨品种‘翠冠’发育过程中的表达分析显示,Ppmal受到赤霉素的调控,并在砂梨果实膨大期发生上调表达。

关键词(KeyWords)： 砂梨;RACE;荧光定量;赤霉素

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金面上项目(31070245);; 教育部新世纪人才支持项目(NCET-10-0582);; 晨星青年学者奖励计划(AE1500006)

作者(Author): 王小龙;李节法;张才喜;王世平;许文平;

Email:

DOI: 10.13925/j.cnki.gsxb.20140436

参考文献(References)：

• [1]YANG Jianting.Studies on gingko kernel allergic protein and the allergic mechanism[D].Nanjing:Nanjing Forestry University,2010.杨剑婷.白果过敏蛋白及其致敏机理的研究[D].南京:南京林业大学,2010.
• [2]BREITENEDER H,EBNER C.Molecular and biochemical classification of plantderived food allergens[J].Journal of Allergy and Clinical Immunology,2000,106:27-36.
• [3]HOFFMANN S K.Plant allergens and pathogenesis-related proteins:what do they have in common[J].Int Arch Allergy Immunol,2000,122:155-166.
• [4]RIKA N,REIKO T.Proteomics-based allergen analysis in plants[J].Journal of Proteomics,2013,93(20):40-49.
• [5]YANG Chaowei,CHEN Lin,QIAO Liya,GAO Zhongshan.Fruit allergy and allergen genomics research progress[J].Journal of Fruit Science,2010,27(2):281-288.杨朝崴,陈琳,乔丽雅,高中山.水果过敏及其过敏原基因组学研究进展[J].果树学报,2010,27(2):281-288.
• [6]EBNER C,HIRSCHWEHR R,BAUER L,BREITENEDER H,VALENTA R,KRAFT D.Identification of allergens in fruits and vegetables:Ig E crossreactivities with the important birch pollen allergens Bet v 1 and Bet v 2(birch profilin)[J].Journal of Allergy and Clinical Immunology,1995,95:962-969.
• [7]VAN L L,VAN S E.The families of pathogenesis-related proteins,their activities,and comparative analysis of PR-1 type proteins[J].Physiol Mol Plant Pathol,1999,55:85-97.
• [8]BRENNA O V,PASTORELLO E A,FARIOLI L,PRAVETTONI V,POMPEI C.Presence of allergenic proteins in different peach(Prunus persica)cultivars and dependence of their content on fruit ripening[J].Agricultural and Food Chemistry,2004,52:7997-8000.
• [9]BOTTON A,ANDREOTTI C,COSTA G,RAMINA A.Peach(Prunus persica L.Batsch)allergen-encoding genes are developmentally regulated and affected by fruit load and light radiation[J].Agricultural and Food Chemistry,2009,57:724-734.
• [10]KITAGAWA M,MORIYAMA T,ITO H,OZASA S,ADACHI A,YASUDA J,OOKURA T,INAKUMA T,KASUMI T,ISHIGURO Y,ITO Y.Reduction of allergenic proteins by the effect of the ripening inhibitor(rin)mutant gene in an F1hybrid of the rin mutant tomato[J].Bioscience,Biotechnology,and Biochemistry,2006,70:1227-1233.
• [11]ZHANG Caixi,TANABE K J,TAMURA F,MATSUMOTO K,YOSHIDA A.13C-photosynthate accumulation in Japanese pear fruit during the period of rapid fruit growth is limited by sink strength of fruit rather than by transport capacity of the pedicel[J].Journal of Experimental Botany,2005,56:2713-2719.
• [12]KSENIJA G,ALVARO H,SCHUYLER S K.RNA extraction from different apple tissues rich in polyphenols and polysaccarides for c DNA library construction[J].Plant Molecular Biology Reporter,2004,12(22):437a-437g.
• [13]KENNETH J,LIVAKA,THOMAS D,SCHMITTGEN.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
• [14]ZHANG Caixi,TANABE K J,TAMURA F,ITAI A,WANG Shiping.Spur characteristics,fruit growth and carbon partitioning in two late-maturing Japanese pear cultivars with contrasting fruisize.Journal of the American[J].Society for Horticultural Science,2005,130:252-260.
• [15]TROGGIO M,GLEAVE A,SALVI S,CHAGNE D.Apple,from genome to breeding[J].Tree Genetics and Genomes,2012,8:509-529.
• [16]DANIEL J,COSGROVE,PATRICIA B,DANIEL M,DURACHKO.Group I allergens of grass pollen as cell wall-loosening agents[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(12):6559-6564.
• [17]ZHANG Hongyun,SONG Juanjuan,LIU Zhigang,KANG Minxiong.Cloning and sequence analysis of panallergen profilin genes in litchi chinensis sonn[J].Journal of Tropical Medicine,2006,6(6):620-623.张红云,宋娟娟,刘志刚,康敏雄.荔枝果实中泛变应原profilin基因的克隆及序列分析[J].热带医学杂志,2006,6(6):620-623.
• [18]ZOU Ying.Cloning and functional analysis of the pathogenesisrelated protein Vp PR10 genes from chinese wild vitis pesudoreticulata[D].Yangling:Northwest Agriculture and Forestry University,2013.邹莹.华东葡萄病程相关蛋白Vp PR10s基因克隆与功能分析[D].杨凌:西北农林科技大学,2013.
• [19]LUO Chong,PENG Shu’ang,LI Guohuai.Studies on mechanism of calcium uptake by pear fruit and its transferring,affecting factors[J].Journal of Huazhong Agricultural University,2001,20(3):286-288.罗充,彭抒昂,李国怀.梨不同品种果实发育中钙与钙调素的动态研究[J].华中农业大学学报,2001,20(3):286-288.
• [20]CHEN Lin.The location of allergen gene and their expression in different organization in peach[D].Hangzhou:Zhejiang University,2010.陈琳.桃过敏原基因定位及其在桃不同组织的表达[D].杭州:浙江大学,2010.
• [21]FERNANDEZ R M,BOLHAAR S,GONZALEZ M E,ASERO R,VAN L A,BOHLE B,EBNER C,RIGBY N,SANCHO A,MILES S,ZUIDMEER L,KNULST A,BREITENEDER H,MILLS C,HOFFMANN S K,VAN R R.Apple allergy across Europe:How allergen sensitization profiles determine the clinical expression of allergies to plant foods[J].Journal of Allergy and Clinical Immunology,2006,118(2):481-488.
• [22]CHEN Lin,GAO Zhongshan,SONG Lijuan.The analysis of the identity of the characteristics of genome of allergen protein family between prunus and apple[C]//Chinese Society for Horticultural Science.The abstract set of the tenth second council and seminar of Chinese Society for Horticultural Science.Nanjing:CSHS,2007:46.陈琳,高中山,宋丽娟.李属过敏原蛋白家族的基因组特征及其与苹果属的同一性分析[C]//中国园艺学会.中国园艺学会十届二次理事会暨学术研讨会论文摘要集.南京:中国园艺学会,2007:46.
• [23]HOLGER S,CORNELIA D,LORANT K.Functional assessment of the pathogenesis-related protein PR-1b in barley[J].Plant Science,2003,165(6):1275-1280.
• [24]HAMAMOUCH N,LI C,SEO P J.Expression of Arabidopsis pathogenesis-related genes during nematode infection[J].Molecular Plant Pathology,2011,12(4):355-364.
• [25]AGLIKA E.Pathogenesis-related proteins:research progress in the last 15 years[J].Plant Physiology,2005,31(1-2):105-124.
• [26]DATTA S K,MUTHUKRISHNAN S.Pathogenesis-related proteins in plants[M].Boca Raton:CRC Press,1999:21-47.
• [27]FUJIBE T,WATANABE K,NAKAJIMA N.Accumulation of pathogenesis-related proteins in tobacco leaves irradiated with UVB[J].Journal of Plant Research,2000,113(4):387-394.
• [28]SLUSARENKO A J,FRASER R S,VAN L L C.Mechanisms of resistance to plant disease[M].Dordrecht:Kluwer Academic Publisher,2000:371-478.
• [29]GONNEAU M,PAGANT S,BRUN F,LALOUE M.Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 k Da peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens[J].Plant Molecular Biology,2001,46:539-548.
• [30]MOGENSEN J,WIMMER R,LARSEN J,SPANGFORT M,OTZEN D.The major birch allergen,Bet v 1,shows affinity for a broad spectrum of physiological ligands[J].The Journal of Biological Chemistry,2002,277:23684-23692.
• [31]KOISTINEN K,SOININEN P,VENALAINEN T,HAYRINEN J,LAATIKAINEN R,PERAKYLA M,TERVAHAUTA A,KARENLAMPI S.Birch PR-10c interacts with several biologically important ligands[J].Phytochemistry,2005,66:2524-2533.
• [32]MARKOVIC H Z,DEGANO M,LAMBA D,ROEPENACK L E,CLEMENS S,SUSANI M,FERREIRA F,SCHEINER O,BREITENEDER H.Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier[J].Journal of Molecular Biology,2003,325:123-133.
• [33]ARCHAMBAULT A,STROMVIK V.PR-10,defensin and cold dehydrin genes are among those over expressed in Oxytropis(Fabaceae)species adapted to the arctic[J].Functional&Integrative Genomics,2011,11(3):497-505.
• [34]WEI Jifu,HE Shaoheng.The research progress of latex allergens[J].Jiangsu Medical Journal,2008,34(1):81-82.魏继福,何韶衡.乳胶过敏原研究进展[J].江苏医药,2008,34(1):81-82.
• [35]YANG Decui,ZHANG Yuxi,ZHENG Guosheng.Gene cloning and expression analysis of pathogenesis-related protein 1 of Paeonia suffruticosa[J].Horticulturae Sinica,2013,40(8):1583-1590.杨德翠,张玉喜,郑国生.牡丹病程相关蛋白1基因的克隆及表达分析[J].园艺学报,2013,40(8):1583-1590.
• [36]KAZMIERCZAK A.Induction of cell division and cell expansion at the beginning of gibberellin A3-induced precocious antheridia formation in Anemia phyllitidis gametophytes[J].Plant Science,2003,165(5):933-939.