关鹏;曾斌;李疆;罗淑萍;王建友;李伟阳;田嘉;李鹏;

• 1:新疆农业大学农学院
• 2:新疆农业大学林学与园艺学院·新疆农业大学果树学新疆特色果树研究中心
• 3:中国林业科学院新疆分院

摘要(Abstract)：

【目的】克隆新疆野扁桃(Amygdalus ledebouriana Schlecht.)自交不亲和性花柱特异性决定因子编码基因SRNase全长序列,为自交不亲和性的分子调控奠定基础。【方法】以新疆野扁桃花柱为试材,利用RT-PCR和RACE技术克隆S-RNase基因全长,采用BLAST和ORF Finder对核酸序列进行分析,利用CDD、Prot Param、Tmpred、Signal P、Target P、SOPMA、DANMAN、MEGA6和Prot Fun对推导的氨基酸序列进行分析。【结果】克隆到Pt S16-RNase基因和Pt S17-RNase基因,2者均属于RNase T2基因家族,与其他多种植物的S-RNase基因的序列相似度为83%~98%,序列均具有S-RNas蛋白典型结构。Pt S16-RNase基因ORF长690 bp,编码229个氨基酸,Pt S17-RNase基因ORF长678 bp,编码225个氨基酸。预测2个S-RNase蛋白均为亲水性、不稳定的分泌蛋白,二级结构均以α-螺旋、延伸链和无规卷曲为主,在蔷薇科李属植物中具有较高的系统进化一致性,可能的主要功能为水解酶和激素。【结论】获得2个新疆野扁桃自交不亲和性花柱特异性决定因子编码基因S-RNase全长序列。

关键词(KeyWords)： 野扁桃;自交不亲和性;S-RNase基因;生物信息学;RACE

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31260465);; 国家林业公益性行业科研专项(201304701-1,201304701-5);; 新疆维吾尔自治区科技计划项目(201130102-1-5);; 新疆维吾尔自治区果树学重点学科(201007)

作者(Author): 关鹏;曾斌;李疆;罗淑萍;王建友;李伟阳;田嘉;李鹏;

Email:

DOI: 10.13925/j.cnki.gsxb.20150520

参考文献(References)：

• [1]HARING V,GRAY J E,MCCLURE B A,ANDERSON M A,CLARKE A E.Self-incompatibility:a self-recognition system in plants[J].Science,1990,250(4983):937-941.
• [2]KUBO K,ENTANI T,TAKARA A,WANG N,FIELDS A M,HUA Z,TOYODA M,KAWASHIMA S,ANDO T,ISOGAI A,KAO T H,TAKAYAMA S.Collaborative non-self recognition system in S-RNase-based self-incompatibility[J].Science,2010,330(6005):796-799.
• [3]KAO T H,TSUKAMOTO T.The molecular and genetic bases of S-RNase-based self-incompatibility[J].The Plant Cell,2004,16(Suppl.):S72-S83.
• [4]HUA Z H,FIELDS A,KAO T H.Biochemical models for SRNase-based self-incompatibility[J].Molecular Plant,2008,1(4):575-585.
• [5]QIAO H,WANG F,ZHAO L,ZHOU J,LAI Z,ZHANG Y,ROBBINS T P,XUE Y.The F-box protein Ah SLF-S2 controls the pollen function of S-RNase-based self-incompatibility[J].The Plant Cell,2004,16(9):2307-2322.
• [6]SIJACIC P,WANG X,SKIRPAN A L,WANG Y,DOWD P E,MCCUBBIN A G,HUANG S,KAO T H.Identification of the pollen determinant of S-RNase-mediated self-incompatibility[J].Nature,2004,429(6989):302-305.
• [7]USHIJIMA K,YAMANE H,WATARI A,KAKEHI E,IKEDA K,HAUCK N R,IEZZONI A F,TAO R.The S haplotype-specific F-box protein gene,SFB,is defective in self-compatible haplotypes of Prunus avium and P.mume[J].The Plant Journal,2004,39(4):573-586.
• [8]SONNEVELD T,KENNETH R,TOBUTT K R,SIMON P,VAUGHAN S P,ROBBINS T P.Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype specific F-box gene[J].The Plant Cell,2005,17(1):37-51.
• [9]张晓明,朱墨,姜立杰,张开春,闫国华.果树自交不亲和花粉S基因研究进展[J].果树学报,2005,22(3):261-264.ZHANG Xiaoming,ZHU Mo,JIANG Lijie,ZHANG Kaichun,YAN Guohua.A review on the research on the pollen S genes of fruit tree[J].Journal of Fruit Science,2005,22(3):261-264.
• [10]LEE H S,HUANG S,KAO T H.S proteins control rejection of incompatible pollen in Petunia inflata[J].Nature,1994,367(6463):560-563.
• [11]MURFETT J,ATHERTON T L,MOU B,GASSER C S,MCCLURE B A.S-RNase expressed in transgenic Nicotiana causes S-allele-specific pollen rejection[J].Nature,1994,367(6463):563-566.
• [12]ANDERSON M A,CORNISH E C,MAU S L,WILLIAMS E G,HOGGART R,ATKINSON A,BONIG I,GREGO B,SIMPSON R,ROCHE P J,HALEY J D,PENSCHOW J D,NIALL H D,TREGEAR G W,COGHLAN J P,CRAWFORD R J,CLARKE A E.Cloning of c DNA for a stylar glycoprotein associated with expression of self-incompatibility in Nicotiana alata[J].Nature,1986,321(6665):38-44.
• [13]MCCLURE B A,HARING V.Style self-incompatibility gene products of Nicotiana alata are ribonucleases[J].Nature,1989,342(6252):955-957.
• [14]SINGH A,AI Y,KAO T H.Characterization of ribonuclease activity of three S-allele-associated proteins of Petunia inflata[J].Plant Physiology,1991,96(1):61-68.
• [15]HUANG S,LEE H S,KARUNANANDAA B,KAO T H.Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen[J].The Plant Cell,1994,6(7):1021-1029.
• [16]李疆,曾斌,罗淑萍,李海龙,马达尼亚提·吾拉孜汗.我国野扁桃资源的保护及引种繁育[J].新疆农业科学,2006,43(1):61-62.LI Jiang,ZENG Bin,LUO Shuping,LI Hailong,Madaniyati·Wulazihan.Protection and propagation of Amygdalus ledebouriana Schleche in China[J].Xinjiang Agricultural Sciences,2006,43(1):61-62.
• [17]曹青爽,姬玉英.新疆特有植物——野扁桃资源价值及保护利用[J].中国野生植物资源,2007,26(4):41-42.CAO Qingshuang,JI Yuying.Values and protection of wild almonds resources grown in Xinjiang[J].Chinese Wild Plant Resources,2007,26(4):41-42.
• [18]吕志江,李疆,吾买尔夏提·塔汉,曾斌,罗淑萍.新疆野扁桃种质资源遗传多样性的ISSR分析[J].果树学报,2010,27(6):918-923.LüZhijiang,LI Jiang,Omir shat Tahan,ZENG Bin,LUO Shuping.ISSR analysis for genetic diversity of Amygdalus ledebouriana germplasm from Xinjiang,China[J].Journal of Fruit Science,2010,27(6):918-923.
• [19]赵屹,谷瑞升,杜生明.生物信息学研究现状及发展趋势[J].医学信息学杂志,2012,33(5):2-6.ZHAO Yi,GU Ruisheng,DU Shengming.The research status and development tendency of bioinformatics[J].Journal of Medical Informatics,2012,33(5):2-6.
• [20]郭长奎,罗淑萍,何天明,李疆,高启明.扁桃SFB基因的克隆及其生物信息学分析[J].果树学报,2009,26(2):170-175.GUO Changkui,LUO Shuping,HE Tianming,LI Jiang,GAO Qiming.Cloning and bioinformatic analysis of SFB gene in almond[J].Journal of Fruit Science,2009,26(2):170-175.
• [21]李亚兰,刘小芳,刘海楠,李文慧,冯建荣.2个中亚杏S-RNase基因全长的克隆与序列分析[J].果树学报,2015,32(6):1047-1054.LI Yalan,LIU Xiaofang,LIU Hainan,LI Wenhui,FENG Jianrong.Cloning and sequences analysis of 2 S-RNase genes in apricot cultivars belonging to the central Asian ecotype group[J].Journal of Fruit Science,2015,32(6):1047-1054.
• [22]杨芩,付燕,王永清,陶炼,邓群仙,范建新,邓仁菊.4个枇杷品种S基因型鉴定及新基因S31-RNase序列分析[J].果树学报,2015,32(1):37-42.YANG Qin,FU Yan,WANG Yongqing,TAO Lian,DENG Qunxian,FAN Jianxin,DENG Renju.Identification of S-genotypes of four cultivars and sequence analyses of S31-RNase in Eriobotrya Lindl.[J].Journal of Fruit Science,2015,32(1):37-42.
• [23]康乐,王海洋.我国生物技术育种现状与发展趋势[J].中国农业科技导报,2014,16(1):16-23.KANG Le,WANG Haiyang.Status and prospect of agrobiotechnology-based breeding in China[J].Journal of Agricultural Science and Technology,2014,16(1):16-23.
• [24]TAMURA K,STECHER G,PETERSON D,FILIPSKI A,KUMAR S.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729
• [25]张绍铃,吴巨友,吴俊,齐永杰,高永彬.蔷薇科果树自交不亲和性分子机制研究进展[J].南京农业大学学报,2012,35(5):53-63.ZHANG Shaoling,WU Juyou,WU Jun,QI Yongjie,GAO Yongbin.Advance in molecular mechanisms of self-incompatibility in Rosaceae fruit trees[J].Journal of Nanjing Agricultural University,2012,35(5):53-63.
• [26]郭振宇,常凤启,谢华,徐勇,马荣才.扁桃SLF基因和SRNase基因的克隆及表达分析[J].园艺学报,2006,33(6):1185-1192.GUO Zhenyu,CHANG Fengqi,XIE Hua,XU Yong,MA Rongcai.Cloning and expression analysis of the SLF and S-RNase genes in almond[J].Acta Horticulturae Sinica,2006,33(6):1185-1192.
• [27]MENG X,SUN P,KAO T.S-RNase-based self-incompatibility in Petunia inflate[J].Annals of Botany,2011,118(4):637-646.
• [28]FURUYA T,SHITASHIMA Y,KINO K.Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis[J].Journal of Bioscience and Bioengineering,2015,119(1):47-51.
• [29]POLOZNIKOV A A,ZAKHAROVA G S,CHUBAR T A,HUSHPULIAN D M,TISHKOV V I,GAZARYAN I G.Site-directed mutagenesis of tobacco anionic peroxidase:effect of additional aromatic amino acids on stability and activity[J].Biochimie,2015,115:71-77.
• [30]WENG M,DENG X,BAO W,ZHU L,WU J,CAI Y,JIA Y,ZHENG Z,ZOU G.Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation[J].Biochemical and Biophysical Research Communications,2015,465(3):580-586.
• [31]LUHTALA N,PARKER R.T2 Family ribonucleases:ancient enzymes with diverse roles[J].Trends in Biochemical Sciences,2010,35(5):253-259.
• [32]IOERGER T R,GOHLKE J R,XU B,KAO T H.Primary structural features of the self-incompatibility protein in Solanaceae[J].Sexual Plant Reproduction,1991,4(2):81-87.
• [33]MONTGOMERY M K,XU S,FIRE A.RNA as a target of doublestranded RNA-mediated genetic interference in Caenorhabditis elegans[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(26):15502-15507.