植物相關(guān)軟件與方法數(shù)據(jù)庫搜索gene

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

2、re used tosearch the peach genome database1with the NCBIBLASTp toolto peachtwerehighlyhomologoustoNAPsubfamily擬南liana）（Vitis vinifera）（Solanum 1NCBIBLASTp工具來識別序列用于搜索NAP高度同源桃組數(shù)據(jù)庫多重序列比對，系統(tǒng)發(fā)育分析和外顯子/內(nèi)含子結(jié)構(gòu)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 用于評估序列相似之處。 的開放閱讀框架使NCBI 開放閱讀框架查找器進行分析工具 3。進行多重序列比對分析使用程序和圖形注釋的共識序列使用 工具4用生成系統(tǒng)發(fā)生完NJ（1,000次重復(fù)MEGA進行遺傳結(jié)構(gòu)研究程序和圖形注釋的共識序列使用 工具4用生

5、成系統(tǒng)發(fā)生完NJ（1,000次重復(fù)MEGA進行遺傳結(jié)構(gòu)研究使用Gene Display工具5。信號肽用SignalP 程序（版本3.0; 2004蛋白分子量和使用ExPASy計算pIMw計算pIs工具7234567SignalP：信工SignalP 是一個信號它的功能給定的氨基酸序列中是否存在在的信號肽剪切位點及其所在，原核生物和真核生物都可以。目前服務(wù)提供的是SignalP 4.0 服務(wù)：3.Molecular Cloning of Peach NAP Subfamily Members 桃子NAP亞科成員的分子隆To clone the PpNAP genes, 6.0 was used

6、to design gene-airsaccordingtothepeachgenomesequenceTable1).為了克隆，使用remier6.0根據(jù)特異性引物對（表 1）組序列設(shè)Using cDNA was completed with the Phanta Super-Fidelity Polymerase (Vazyme) according to the mended procedure. 使cDNA 模使用remier6.0根據(jù)特異性引物對（表 1）組序列設(shè)Using cDNA was completed with the Phanta Super-Fidelity Polym

7、erase (Vazyme) according to the mended procedure. 使cDNA 模板，根據(jù)制完成PCRThePCRproductswereisolatedandpurifiedwiththeMiniBESTAgaroseGelExtraction Kit Ver. 4.0 (Takara). 使用cDNA模板，根據(jù)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將純化的產(chǎn)物pMD-載體（Takara）中。通過藍色/白色噬菌斑測定確認陽性克(qRT)-synthesized by Sangon Biotech , which also completed sequencingreactions.用于克隆和定量逆轉(zhuǎn)錄（qRT）-PCR 的引物Sangon （Shanghai）Co。，其也完成了所有 反應(yīng)iveReverseRAssays定量逆轉(zhuǎn)錄PCR測The qRT-PCR was conducted using the iQ5 real-time PCRsystem (Bio-Rad). gene-specificprimersTabl

9、e 1) qRT-PCR使用iQ5實時PCR系統(tǒng)（Bio-Rad）進行特異性引物（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）進行計。設(shè)計每個引物對（Tm 60）以擴增約 200-bp 片段。對于每個樣品，使用 總體積為10L使用制造的程序完成RT-PCR但是退火溫度變?yōu)?0樣品在 95加熱 10s，冷卻至 6515s，最后加熱至 95，以 0.1s-1 的速率進熔解曲線分析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）分析特異性轉(zhuǎn)錄物積累。使用桃 18S 核糖體 RNA 標準化樣品中 18s 核糖的擴增鏈曲線和融合區(qū)可見補充圖 S1每個樣品一式三份進行分析4.Searchforing hePromoters of Peach NAPGenes 在桃子NAP因的啟動子中搜索順式作用Upstreamregions (2000 bpupstream ofthe transcription start site)of

13、selected NAPgenes were used to search the PlantCARE database for ive ing (Lescotetal2002).選擇的桃子的上游區(qū)域（轉(zhuǎn)錄NAPgenes were used to search the PlantCARE database for ive ing (Lescotetal2002).選擇的桃子的上游區(qū)域（轉(zhuǎn)錄起始位點上游 用于在 PlantCARE 數(shù)據(jù)庫中搜索推定的順式作用元件（Lescot 等人，2002）yses統(tǒng)計分GenelevelsweresubjectedysisofvarianceusingValues are provided as the mean standard error (n = 3). 使用SAS表達水進行方差分析。提供為平均值標準誤差（n3）The overall least significant difference (p 0.05) was calculated and used to means.計算總體最小顯著差異（p 0.05），并用于分離