梨S12-RNase基因的克隆、表达分析与鉴定方法

张琳; 刘敏; 谭晓风; 宋志波; 龙洪旭; 曹玉芬; 李秀根

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Cloning, expression analysis, and detection method of the S12-RNase gene existing in three pear species (Pyrus bretschneideri, P. pyrifolia, and P. ussuriensis)

首发时间：2012-11-30

Zhang Lin ¹
Zhang Lin, (1978-), male, Ph.D., Associate Professor, Cultivation and breeding of Non-wood Forest Crops.
Liu Min ² Tan Xiaofeng ² Song Zhibo ²
Zhang Lin (1978-), male, Ph.D., Associate Professor, Cultivation and breeding of Non-wood Forest Crops
Long Hongxu ² Cao Yufen ³ Li Xiugen ⁴

1、Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology, ChangSha 410004
2、Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology, Changsha 410004, China
3、Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, China
4、Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, China

Abstract：A specific forward primer was designed based on the principle of highest similarity, and employed in 3' RACE using Chinese white pear (Pyrus bretschneideri) cultivar 'Yingzhiqing' as materials. The full length cDNA of PbS12-RNase was successfully amplified and deposited in GenBank (Accession No. EU081889). At the amino acid level, the PbS12-RNase exhibited the highest similarity (97.3%) with MdSf-RNase of Malus domestica, and only six amino acid differences were present in the two S-RNases. Phylogenetic analysis of rocaceous S-RNases indicated that the PbS12-RNase clustered with maloideous S-RNases, forming a subfamily-specific not species-specific group. Some intraspecific genetic distance of the S-RNases, in addition, was greater than interspecific genetic distance. In 'Yingzhiqing', the PbS12-allele was specifically expressed in style. Moreover, the expression level of this gene was extremely low at the small bud stage, and subsequently increased rapidly at the bid bud stage and bell stage. A method for rapid detection of the PbS12-allele was developed via PbS12-allele-specefic primers design based on multiple sequence comparisons. Application of the PbS12-allele-specefic primers in 59 cultivars from four pear species showed that the PbS12-allele not only existed in P. bretschneideri, but in P. pyrifolia and P. ussuriensis as well. The present study could provide a scientific base for fully clarifying the mechanism of pear GSI at the molecular level.

keywords： gametophytic self-incompatibility reverse transcript-PCR Rapid Amplification of cDNA Ends S-RNase phylogenetic tree spatio-temporal expression patterns

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梨S12-RNase基因的克隆、表达分析与鉴定方法

张琳 ¹
Zhang Lin, (1978-), male, Ph.D., Associate Professor, Cultivation and breeding of Non-wood Forest Crops.
刘敏 ² 谭晓风 ² 宋志波 ²
Zhang Lin (1978-), male, Ph.D., Associate Professor, Cultivation and breeding of Non-wood Forest Crops
龙洪旭 ² 曹玉芬 ³ 李秀根 ⁴

1、中南林业科技大学经济林培育与保护教育部重点实验室，长沙 410004
2、中南林业科技大学经济林培育与保护教育部重点实验室，长沙410004
3、中国农业科学院果树研究所　兴城121600
4、中国农业科学院郑州果树研究所, 郑州450009

摘要：运用最高相似性原理，在5'非转录区设计正向引物，通过3' RACE的方法从白梨'硬枝青'中克隆到了S12-RNase基因的全长cDNA序列（GenBank接受号为EU081889）。在氨基酸序列水平上，梨S12-RNase与苹果MdSf-RNase 的相似性最高(97.3%)，两者仅有6个氨基酸的差异。蔷薇科植物S-RNase基因的系统进化分析表明，梨S12-RNase与苹果亚科的S-RNase基因形成一个亚科特异而非种特异的S-RNase基因类群，且不同S-RNase基因间存在种内遗传距离远于种间遗传距离现象。在'硬枝青'中，S12-RNase基因仅在花柱中表达，且在小蕾期有微量表达，在大蕾期和铃铛期表达量迅速提高。通过特异引物设计，形成了快速检测梨S12等位基因的方法，以59个中国梨品种为材料进行检测，发现S12基因不仅存在于白梨中，同时也存在于砂梨和秋子梨中。本研究为从分子水平上揭示中国梨的自交不亲和性机理机制提供了科学基础。

关键词：配子体自交不亲和性逆转录PCR 快速扩增cDNA末端 S核糖核酸酶系统进化树时空表达模式

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Zhang Lin,Liu Min,Tan Xiaofeng,et al. Cloning, expression analysis, and detection method of the S12-RNase gene existing in three pear species (Pyrus bretschneideri, P. pyrifolia, and P. ussuriensis)[EB/OL]. Beijing:Sciencepaper Online[2012-11-30]. https://www.paper.edu.cn/releasepaper/content/201211-583.

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论文编号	201211-583
论文题目	梨S12-RNase基因的克隆、表达分析与鉴定方法
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