苹果mdcbl3基因的克隆与功能鉴定

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1、园艺学报，2017，44(1)：1–10.ActaHorticulturaeSinicadoi：10.16420/j.issn.0513-353x.2016-0489；http：//www.ahs.ac.cn1苹果MdCBL3基因的克隆与功能鉴定*刘亚静，马齐军，李浩浩，路静，郝玉金，由春香（山东农业大学园艺科学与工程学院，作物生物学国家重点实验室，农业部黄淮地区园艺作物生物学与种质创制重点实验室，山东泰安271018）摘要：从‘嘎拉’苹果（Malus×domesticaBorkh.）中克隆MdCBL3（序列号：MDP0000155124），长852bp。系统进化树分析表明，

2、苹果MdCBL3与白梨亲缘关系最近。利用PlantCare数据库进行启动子顺式作用元件预测分析，MdCBL3启动子序列中存在干旱、低温、光、生长素、防御等响应元件。构建MdCBL3表达载体并利用农杆菌介导法侵染苹果愈伤组织和拟南芥。半定量RT-PCR证明MdCBL3在转基因愈伤组织中过量表达，在拟南芥中得到异源表达。表型分析发现，在盐胁迫中，与野生型相比，转基因愈伤组织和拟南芥成龄苗生长更好，盐胁迫的抗性更强。在拟南芥中异源表达MdCBL3，能提高拟南芥对干旱和低温的抗性。关键词：苹果；MdCBL3；基因克隆；功能鉴定；盐胁迫中图分类号：S661.1文献标志码：A文章编号：0

3、513-353X（2017）01-0001-10MolecularCloningandFunctionalCharacterizationofMdCBL3inApple*LIUYajing，MAQijun，LIHaohao，LUJing，HAOYujin，andYOUChunxiang（StateKeyLaboratoryofCropBiology，MOAKeyLaboratoryofHorticulturalCropBiology（HuanghuaiRegion）andGermplasmInnovation，CollegeofHorticultureScienceandEn

4、gineering，ShandongAgriculturalUniversity，Tai’an，Shandong271018，China）Abstract：AgenenamedMdCBL3（MDP0000155124）wasclonedfrom‘Gala’apple（Malus×domesticaBorkh.）.ThelengthofMdCBL3was852bp.AphylogenetictreeanalysisindicatedthatthePyrusbretschneideriexhibitedthehighestsequencesimilaritytoMdCBL3.Th

5、epromoter’scis-actingelementswereanalyzedandpredictedusingPlantCareDatabases，analysisshowedthattheMdCBL3promotersequenceincludeddrought，lowtemperature，light，auxin，defenseresponsivenessetc.ConstructexpressionvectorofMdCBL3anduseAgrobacteriummediatedmethodtoinfectapplecallusandArabidopsisseed

6、.SemiRT-PCRindicatedthatMdCBL3wasgeneticallytransformedintoapplecallusandArabidopsis.PhenotypicanalysisfoundthattransgeniccallusandArabidopsisseedlinggrowbetterandhavestrongerresistanceunderNaClstresscomparedwithcontrast.DemonstratedthatoverexpressionofMdCBL3，heterogenousexpressionofMdCBL3r

7、emarkablyincreasedthetoleranceoftransgenicapplecallusandArabidopsistohighsalinity.ThestudyfoundthatheterogenousexpressionofMdCBL3inArabidopsisenhencedthetoleranceofdroughtandlowtemperature.Keywords：apple；MdCBL3；molecularclone；functionalidentification；sal