草莓果实不同发育阶段抑制差减文库(SSH)的构建及相关基因的表达分析  

Construction of Suppression Subtractive Hybridization(SSH) cDNA Library from Two Developmental Stages of Fragaria × ananassa Fruit and Expression Analysis of Related Genes

作　　者：周厚成[1,2] 李刚[2] 赵霞[2] 王子成[3] 郭蔼光[1] 

机构地区：[1]西北农林科技大学生命科学学院、陕西省农业分子生物学重点实验室,杨凌712100 [2]中国农业科学院郑州果树研究所,郑州450009 [3]河南大学生命科学学院,开封475004

出　　处：《农业生物技术学报》2013年第6期641-649,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30900973);中国农业科学院基本科研业务费预算增量项目(No.2013ZL021);河南省重点科技攻关项目(No.112102110040)

摘　　要：寻找控制草莓果实硬度、成熟软化和褐化等性状相关基因并进行有效的调控具有现实意义,本研究分别以丰香草莓(Fragaria×ananassa Duch.cv.Toyonoka)果实大绿果期和转色期两个发育阶段的cDNA为实验组(tester)和驱动组(driver),利用抑制差减杂交(suppression subtraction hybridization,SSH)技术构建了正反两个cDNA文库,分别包含516和524个克隆。通过EST测序、序列拼接后,共获得707条uniESTs,其中正反向文库分别为369和338个非冗余序列(uniESTs),包括123条拼接序列(contigs)和584条单一序列(singletons)。在Nt库比对中有537条uinESTs与已知基因匹配,占比75.95%;在Nr库比对中有509条序列有匹配蛋白,占比71.99%。有193个uniESTs能进行Geney ontology(GO)功能注释,其中细胞组分被注释了315次、分子功能被注释了332次、生物过程被注释了409次。uniESTs的功能分析显示,绿果期文库多数与细胞营养生长、分裂、早期胚胎发育和营养运输有关,转色期文库主要与刺激代谢物形成、色素合成、果实软化和种子成熟有关,与草莓果实发育和成熟生理过程基本一致。采用qRT-PCR对果实成熟相关蛋白(putative ripening-related protein)、多聚半乳糖醛酸酶(polygalacturonase,PG)、MYB转录因子(MYB transcription factor,MYB)、类异黄酮还原酶(isoflavone reductase-like gene,IRL)、胚胎发育晚期丰富蛋白(later embryo abundant protein,LEA)、乙烯受体Etr1(ethylene receptor Etr1)等6个基因在草莓果实发育、成熟期在根、叶、花中的表达模式进行了分析,研究结果为进一步研究草莓果实发育和成熟机制提供基础资料。Identification of genes differentially expressed during development, ripening and softening ofstrawberry fruit is needed to improve strawberry fruit quality and to extend shelf life. In this study a forward and a reverse suppression subtraction hybridization（SSH） cDNA libraries with 516 and 524 positive cloneswere constructed respectively using eDNA from strawberry（Fragaria×ananassa Duch. cv.Toyonoka） and large green stage fruit as the tester and eDNA from strawberry turning stage fruit as the driver. After removingrepeat and redundancy sequences, 707 uniESTs including 369 uniESTs in forward library and 338 uniESTs in reverse library were obtained. The assembling provided a total of 123 contigs and 584 singletons by clusteranalyses of the uniESTs. Nucleotide homology searched with Blastn in NCBI non-redundant nucleotide database and 537 uniESTs （75.95% of total uniESTs） were homologous with known genes. Protein homologysearched with Blastx in NCBI non-redundant protein database and 509 uinESTs （71.99% of total uniESTs） were homologous with known proteins. The results of gene ontology（GO） annotation showed that 193uinESTs were involved in biological process with 315 times, molecular function with 332 times and cell component with 409 times respectively. Among these ESTs, putative proteins were related to cell vegetativegrowth, division, early embryonic development and nutrition transport in large green stage library, and related to metabolites biosynthesis and cell wall-related proteins during fruit softening, pigment synthesis, seedmaturation in turning stage library. These results were generally consistent with physiological processes during strawberry fruit development and ripening. In two SSH libraries six genes including ripening-relatedprotein, polygalacturonase（PG）, MYB transcription factor（MYB）, isoflavone reductase-like gene（IRL）, later embryo abundant protein（LEA）and ethylene receptor Etrl（Etrl） genes were analyzed by qRT-PCR in differenttissues and stages of frui

关 键 词：草莓 果实 抑制差减杂交(SSH) 基因表达 QRT-PCR 

分 类 号：S852.7[农业科学—基础兽医学]