植物相关软件与方法数据库搜索gene

1、1.IdentificationofPeachNAPSubfamilyliana, Vitis vinifera, and Solanum lycopersicum NAP gene were used tosearch the peach genome database1with the NCBIBLASTp toolto peachtwerehighlyhomologoustoNAPsubfamily拟南1.IdentificationofPeachNAPSubfamilyliana, Vitis vinifera, and Solanum lycopersicum NAP gene we

2、re used tosearch the peach genome database1with the NCBIBLASTp toolto peachtwerehighlyhomologoustoNAPsubfamily拟南liana）（Vitis vinifera）（Solanum 1NCBIBLASTp工具来识别序列用于搜索NAP高度同源桃组数据库多重序列比对，系统发育分析和外显子/内含子结构The NCBI BLAST tool2 was used to assess imilarities. The open frames of PpNAP genes yzed using the N

3、CBI Open Reading Frame tool3. Multiple sequence yses were conducted using the program, and graphical ions consensus were completed using Weblogo online tool4. A tree was generated using the NJ method 1,000 repeats) of the MEGA 6.06 software. Genetic structure investigations conducted using the Gene

4、Structure Display Server online tool5. Signal peptides yzed with the SignalP program6 3.0; et al., 2004). molecularweightsandpIswerecalculatedusingtheExPASy/MwNCBI BLAST tool2 用于评估序列相似之处。 的开放阅读框架使NCBI 开放阅读框架查找器进行分析工具 3。进行多重序列比对分析使用程序和图形注释的共识序列使用 工具4用生成系统发生完NJ（1,000次重复MEGA进行遗传结构研究程序和图形注释的共识序列使用 工具4用生

5、成系统发生完NJ（1,000次重复MEGA进行遗传结构研究使用Gene Display工具5。信号肽用SignalP 程序（版本3.0; 2004蛋白分子量和使用ExPASy计算pIMw计算pIs工具7234567SignalP：信工SignalP 是一个信号它的功能给定的氨基酸序列中是否存在在的信号肽剪切位点及其所在，原核生物和真核生物都可以。目前服务提供的是SignalP 4.0 服务：3.Molecular Cloning of Peach NAP Subfamily Members 桃子NAP亚科成员的分子隆To clone the PpNAP genes, 6.0 was used

6、to design gene-airsaccordingtothepeachgenomesequenceTable1).为了克隆，使用remier6.0根据特异性引物对（表 1）组序列设Using cDNA was completed with the Phanta Super-Fidelity Polymerase (Vazyme) according to the mended procedure. 使cDNA 模使用remier6.0根据特异性引物对（表 1）组序列设Using cDNA was completed with the Phanta Super-Fidelity Polym

7、erase (Vazyme) according to the mended procedure. 使cDNA 模板，根据制完成PCRThePCRproductswereisolatedandpurifiedwiththeMiniBESTAgaroseGelExtraction Kit Ver. 4.0 (Takara). 使用cDNA模板，根据制造的方法用Super-Fidelity DNA 聚合酶（Vazyme）完成PCRPurified products were o the pMD-19T vector itive were confirmed by blue/white plaque

8、 assays将纯化的产物pMD-载体（Takara）中。通过蓝色/白色噬菌斑测定确认阳性克(qRT)-synthesized by Sangon Biotech , which also completed sequencingreactions.用于克隆和定量逆转录（qRT）-PCR 的引物Sangon （Shanghai）Co。，其也完成了所有 反应iveReverseRAssays定量逆转录PCR测The qRT-PCR was conducted using the iQ5 real-time PCRsystem (Bio-Rad). gene-specificprimersTabl

9、e 1) qRT-PCR使用iQ5实时PCR系统（Bio-Rad）进行特异性引物（表 weredesigned using the Beacon Designer 8.0 software Eachair (Tm 60C) was designed lify an y 200-bp Foreach le,1LcDNA,1Leachprimer,2Ldouble -distilledwater,and5LSYBR Premix ExTaq II (Takara) were used in a volume of 10 L. The two-RT-PCR wasForeach le,1LcDNA,

10、1Leachprimer,2Ldouble -distilledwater,and5LSYBR Premix ExTaq II (Takara) were used in a volume of 10 L. The two-RT-PCR was completed using mended program, but to were heated at 95C for 10 annealingtemperature was Scooled to to （at a rate of 0.1C s1 for for 15 s, and finally 使用BeaconDesignerernationa

11、l）进行计。设计每个引物对（Tm 60）以扩增约 200-bp 片段。对于每个样品，使用 总体积为10L使用制造的程序完成RT-PCR但是退火温度变为60样品在 95加热 10s，冷却至 6515s，最后加热至 95，以 0.1s-1 的速率进熔解曲线分析yzedusingthe22-CT method(Livak Thespecifictranscriptaccumulationittgen, 2001). Peach 18S omal was used to normalize data. lification, melt curve and melt park of18s omal ge

12、ne in all les can be yzed in triplicate.使用2-CT方in Supplementary Figure S1. Each le （Livakittgen，2001）分析特异性转录物积累。使用桃 18S 核糖体 RNA 标准化样品中 18s 核糖的扩增链曲线和融合区可见补充图 S1每个样品一式三份进行分析4.Searchforing hePromoters of Peach NAPGenes 在桃子NAP因的启动子中搜索顺式作用Upstreamregions (2000 bpupstream ofthe transcription start site)of

13、selected NAPgenes were used to search the PlantCARE database for ive ing (Lescotetal2002).选择的桃子的上游区域（转录NAPgenes were used to search the PlantCARE database for ive ing (Lescotetal2002).选择的桃子的上游区域（转录起始位点上游 用于在 PlantCARE 数据库中搜索推定的顺式作用元件（Lescot 等人，2002）yses统计分GenelevelsweresubjectedysisofvarianceusingValues are provided as the mean standard error (n = 3). 使用SAS表达水进行方差分析。提供为平均值标准误差（n3）The overall least significant difference (p 0.05) was calculated and used to means.计算总体最小显着差异（p 0.05），并用于分离