IfyouareusingMCLAB'sDNAsequenceringservices,youdonotneedtopurchaseorsubmitanyprimerslistedhereinyourorders.YouonlyneedtospecifytheprimernamesinyourorderformandMCLABwilltakecareofeverythingrelatedtotheseprimers. MCLABPrimers MCEasyDepository™forDNASequencingGuidelines WeprovidecustomoligonucleotidesynthesisserviceincombinationofourDNAsequencingservicetobetterassistourcustomers.Firsttimeprimersynthesisorderwillbefulfilledwithin24hours.Oncetheprimersaresynthesized,wealiquotthemfortheDNAsequencingandsavetherestintheMCEasyDepository™forpossIBLefutureusagefromourcustomers.Sothatthenexttimewhenyouorderthesequencingusingthesameprimers,youwillsavetimeandmoneybychoosingtheprimerswesavedforyouintheMCEasyDepository ™.Thisisacompletefreeservicefromustosaythanktoourcustomers. WealsoprovideourcustomersfreetemplatesandprimersstorageinMCLAB'sfreezerarchivesforupto12monthsfromthetimeofsubmission.Thiswaywhenyouorderare-sequencingorsubmitothertemplatestobesequencedbythesamegroupofprimers,whatyouneedtodoisjusttelluswhichprimercombinationtouse.It'sjustlikethateasy. CustomerscouldalsogettheirDNAtemplatesand/orprimersbackbysimplyincludesuchinstructionsintheonlineorderformwhensubmittingtheirsequencingrequeststoMCLAB.It'slikethatsimple.   MCFreePrimers™forDNASequencingService # Primer Sequences(5'd~3') Plasmid Manufacturer 1 28glll GTATGGGATTTTGCTAAACAAC Ph.D.glll NewEnglandBiolabs 1a -96glll CCCTCATAGTTAGCGTAACG Ph.D.glll NewEnglandBiolabs 2 Ac5Forward ACACAAAGCCGCTCCATCAG pAc5.1/V5-His Invitrogen 3 ADReverse AGATGGTGCACGATGCACAG AD   4 a-Factor TACTATTGCCAGCATTGCTGC Pichia(pMET) Invitrogen 5 AOX1Forward GACTGGTTCCAATTGACAAGC Pichia Invitrogen 6 AOX1Reverse GCAAATGGCATTCTGACATCC Pichia Invitrogen 7 ASHSVTagPrimer ATCCTCGGGGTCTTCCG pIEx Novagen 8 ASSoTag18merPrimer GTCCATGTGCTGGCGTTC pIEx Novagen 9 AUG1Forward CAATTTACATCTTTATTTATTAACG Pichia Invitrogen 10 AUG1Reverse GAAGAGAAAAACATTAGTTGGC Pichia Invitrogen 11 BacForward TTTTACTGTTTTCGTAACAGTTTT pBlueBac4.5 Invitrogen 12 BacReverse CGGATTTCCTTGAAGAGAGTA pBlueBacHis2 Invitrogen 13 Baculovirus(+15)Reverse ACTTCAAGGAGAATTTCC pMelBac Invitrogen 14 BGHReverse TAGAAGGCACAGTCGAGG Universal   15 BKReverse ACAGGAAACAGCTATGACCTTG BKvirus   16 BKRSV CGCCATTTGACCATTCA pBK-rsv   17 BluescriptKS TCGAGGTCGACGGTATC pBluescript Stratagene 18 BluescriptSK CGCTCTAGAACTAGTGGATC pBluescript Stratagene 19 CBDcenA TCAACGGCACCACCTGCA   Novagen 20 CBDcexLEAD TAGGTGCAACTGTTGTTCTG   Novagen 21 CBDclos CAACACCAGTTGTAAATCCA   Novagen 22 cIForward GGATAGCGGTCAGGTGTT pHybcI/HK Invitrogen 23 Citeprimer GGGGACGTGGTTTTCCTTTG pCITE Novagen 24 CMVForward CGCAAATGGGCGGTAGGCGTG Universal   25 CYC1Reverse GCGTGAATGTAAGCGTGAC Cyc1 Invitrogen 26 DsbA CGAGTATGCTGATACAGTGA DsRed Clontech 27 DsbC GAATTTCTCGACGAACACCA DsRed Clontech 28 DsRed1-C AGCTGGACATCACCTCCCACAACG DsRed Clontech 29 DsRed1-N GTACTGGAACTGGGGGGACAG DsRed Clontech 30 DsRed-C AAGAAGCCTGTGCAGCTACCAGG DsRed Clontech 31 DsRed-N CGCCTTCTATTTCAAACTCGTGCC DsRed Clontech 32 EBVReverse GTGGTTTGTCCAAACTCATC EBV Invitrogen 33 EF-1aForward TCAAGCCTCAGACAGTGGTTC     34 EGFP-C CATGGTCCTGCTGGAGTTCGTG EGFP Clontech 35 EGFP-N CGTCGCCGTCCAGCTCGACCAG EGFP Clontech 36 GAL1Forward AATATACCTCTATACTTTAACGTC Gal1 Invitrogen 37 Glprimer1 TGTATCTTATGGTACTGTAACTG     38 Glprimer2 CTTTATGTTTTTGGCGTCTTCCA     39 gp64promoter CTACTAGTAAATCAGTCACACC     40 gp64Signalprimer GCGCTATTGTTTTATATGTGC     41 IE1promoterprimer TGGATATTGTTTCAGTTGCAAG pIEx,pBIEx Novagen 42 Lamdagt10For CTTTTGAGCAAGTTCAGGCCTGGTTAAG LambdaGT10 Promega 43 Lamdagt10Rev GAGGTGGCTTATGAGTATTTCTTCCAGG LambdaGT10 Promega 44 Lamdagt11For GGTGGCGACGACTCCTGGAGCCCG LambdaGT11 Promega 45 Lamdagt11Rev TTGACACCAGACCAACTGGTAATG LambdaGT11 Promega 46 M13Forward(-20) GTAAAACGACGGCCAG Universal   47 M13Forward(-40) GTTTTCCCAGTCACGAC Universal   47b M13Reverse CAGGAAACAGCTATGAC Universal   48 pUCR/M13Forward CCCAGTCACGACGTTGTAAAACG pUC PGEM-T PGEM- 49 pUCR/M13Reverse AGCGGATAACAATTTCACACAGGAA pUC PGEM-TPGEM- 50 MTForward CATCTCAGTGCAACTAAA pMT/V5-His Invitrogen 51 myc-HisReverse ATGACCGGTATGCATATTCAG     52 OpIE2Forward CGCAACGATCTGGTAAACAC pMIB/V5-His Invitrogen 53 OpIE2Reverse GACAATACAAACTAAGATTTAGTCAG pMIB/V5-His Invitrogen 54 p10Forward GTATATTAATTAAAATACTATACTG pTriEx-2Hygro Novagen 55 pBADForward ATGCCATAGCATTTTTATCC E.coliaraBAD Invitrogen 56 pBADReverse GATTTAATCTGTATCAGG E.coliaraBAD Invitrogen 57 pCDM8Reverse TAAGGTTCCTTCACAAAG pCDM8 Invitrogen 58 pCEPForward AGAGCTCGTTTAGTGAACCG     59 pCMVForward GATCCGGTACTAGAGGAACTGAAAAAC     60 pDABForward ATGCCATAGCATTTTTATCC   Invitrogen 61 pETUpstreamPrimer ATGCGTCCGGCGTAGA(16)     62 pETBlueDOWNPrimer GTTAAATTGCTAACGCAGTCA     63 pETBlueUPPrimer TCACGACGTTGTAAAACGAC     64 pFastBacForward GGATTATTCATACCGTCCCA pFastBac Invitrogen 65 pFastBacReverse CAAATGTGGTATGGCTGATT pFastBac Invitrogen 66 pGAPForward GTCCCTATTTCAATCAATTGAA pGAPz Invitrogen 67 pGENEForward CTGCTATTCTGCTCAACCT     68 pGEX3' GAGCTGCATGTGTCAGAGG Universal   69 pGEX5' GGCAAGCCACGTTTGGTG Universal   70 pHookForward ACGGTGCATTGGAACGGAC pHook-2,-3 Invitrogen 71 pHookReverse GATTGCGTCGCATCGACCC pHook-2,-3 Invitrogen 72 pHybLex/ZeoForward AGGGCTGGCGGTTGGGGTTATTCGC pHybLex/Zeo Invitrogen 73 pHybLex/ZeoReverse GAGTCACTTTAAAATTTGTATACAC pHybLex/Zeo Invitrogen 74 PinPointSequencingprimer CGTGACGCGGTGGAGGGCG PinPointXa-1,-2,-3,PinPointXa-1T-vector Promega 75 pJET1.2Forward CGACTCACTATAGGGAGAGCGGC     76 pJET1.2Reverse AAGAACATCGATTTTCCATGGCAG     77 pJG4-5Forward GATGCCTCCTACCCTTATGATGTGCC pJG4-5   78 pJG4-5Reverse GGAGACTTGACCAAACCTCTGGCG pJG4-5   79 PolyhedrinForward AAATGATAACCATCTCGC pVL1393 Invitrogen 80 PolyhedrinReverse GTCCAAGTTTCCCTG(15) pVL1393 Invitrogen 81 pQE-TriSystemForward GTTATTGTGCTGTCTCATC     82 pQE-TriSystemReverse TCGATCTCAGTGGTATTTGTG     83 pREPForward GCTCGATACAATAAAGCCC pREP4 Invitrogen 84 pRset CTAGTTATTGCTCAGCGGTGG pRset Invitrogen 85 pRHForward CTGTCTCTATACTCCCCTATAG pRH Invitrogen 86 pRHReverse CAAAATTCAATAGTTACTATCGC pRH Invitrogen 87 pRSETReverse TAGTTATTGCTCAGCGGTGG pRSET Invitrogen 88 pTargetSequencingPrimer TTACGCCAAGTTATTTAGGTGACA pTarget Promega 89 pTRCHisForward GAGGTATATATTAATGTATCG pTrcHis Invitrogen 90 pTrcHisReverse GATTTAATCTGTATCAGG pTrcHis Invitrogen 91 pTRE3' CCACACCTCCCCCTGAAC pTRE BDBiosciences 92 pTRE5' CGCCTGGAGACGCCATCC pTRE   93 pTriplEx3' ACTCACTATAGGGCGAATTG pTriplEx Clontech 94 pTriplEx5' CTCGGGAAGCGCGCCATTGTGTTGGT pTriplEx Clontech 95 pUniForward CTATCAACAGGTTGAACTG pUni Invitrogen 96 pUniReverse CAGTCGAGGCTGATAGCGAGCT pUni Invitrogen 97 pYESTrpForward GATGTTAACGATACCAGCC pYESTrp Invitrogen 98 pYESTrpReverse GCGTGAATGTAAGCGTGAC pYESTrp Invitrogen 99 QEPromoter CCGAAAAGTGCCACCTG pQE Qiagen 100 QEReverse GTTCTGAGGTCATTACTGG pQE Qiagen 101 R-20merPrimer CAGCTATGACCATGATTACG pSTBlue-1 Novagen 102 RsaAReverse GCCGCGCCAGCGACGCGGAGGG pCX Invitrogen 103 RVprimer3 CTAGCAAAATAGGCTGTCCC pGL/pCAT3 Promega 104 Rvprimer4 GACGATAGTCATGCCCCGCG pGL/pCAT3 Promega 105 SeqL-A(proximaltoattL1) TCGCGTTAACGCTAGCATGGATCTC pDONR201/pDONR207 Invitrogen 106 SeqL-B(proximaltoattL2) GTAACATCAGAGATTTTGAGACAC pDONR201/pDONR207 Invitrogen 107 STag18merPrimer GAACGCCAGCACATGGAC pIEx-1 Novagen 108 STagPrimer CGAACGCCAGCACATGGACA pIEx-1 Novagen 109 Sp6Promoter GATTTAGGTGACACTATAG Universal   110 SV40-pArev CCTCTACAAATGTGGTATGG pRL-SV40   111 SV40-Promoter GCCCCTAACTCCGCCCATCC pRL-SV40   112 T3Promoter ATTAACCCTCACTAAAGGGA Universal   113 T7EEV ATGTCGTAATAACCCCGCCCCG pAlterMAX,pSI,pCI,pCI-Neo,pCMVTnT,pTnT,phMGFPVector,HaloTagpHT2,psiCHECK-1,-2 Promega 114 T7gene10Primer TGAGGTTGTAGAAGTTCCG     115 T7Promoter TAATACGACTCACTATAGGG Universal   116 T7Reverse TAGTTATTGCTCAGCGGTGG Universal   117 T7Terminator GCTAGTTATTGCTCAGCGG Universal   118 U6Primer GGGCAGGAAGAGGGCCTAT     119 U-19merPrimer GTTTTCCCAGTCACGACGT M13mp18,pCITE Novagen 121 V5ReversePrimer ACCGAGGAGAGGGTTAGGGAT V5Epitope Invitrogen 122 VP22Forward GGCCACGGCGACTCGA     123 XpressForward TATGGCTAGCATGACTGGT XpressEpitope Invitrogen 124 VLH TTGTGTGGAATTGTGAGCGG     125 VF2 TGCCACCTGACGTCTAAGAA     126 VR ATTACCGCCTTTGAGTGAGC     127 TKpARev CTTCCGTGTTTCAGTTAGC     128 pQE60-FW CCCGAAAAGTGCCACCTG     129 pQE60-Rv GTTCTGAGGTCATTACTGG     130 SeqL-A(ATTL1) GCGAGAGTAGGGAACTGC pENTR Invitrogen 131 SeqL-B(ATTL2) AACATCAGAGATTTTGAGACAC pENTR Invitrogen 132 pmirGloF(7205bp) GAGGTGCCTAAAGGACTGAC pmirGlo promega

Molecular cloning and function analysis of PsMYB18 in 'Akihime' plum (Prunus salicina Lindl.) FANG Zhizhen;JIANG Cuicui;ZHOU Danrong;PAN Shaolin;LIN Yanjuan;YE Xinfu;Fruit Research Institute,Fujian Academy of Agricultural Sciences/Fujian Engineering and Technology Research Center for Deciduous Fruit Trees,Fujian Academy of Agricultural Sciences;   【Objective】Temperature and light are important environmental factors that affect the biosynthesis and accumulation of anthocyanin in the fruit peel. Our previous study indicated that 20 ℃ temperature and light treatment(150 mol·m~(-2)·s~(-1)) could induce anthocyanin accumulation in the peel of postharvest'Akihime'plums. Comparative transcriptomic analysis results demonstrated that the expression of a R2 R3-MYB gene(designated as PsMYB18) was significantly upregulated in the fruit peel treated at20 ℃+ light. The objective of the study was to isolate and elucidate the function of PsMYB18 gene during anthocyanin accumulation in the peel of'Akihime'plum.【Methods】The total RNA of the plum fruit peels was extracted using EZNA Plant RNA Kit(Omega Bio-tek, USA) according to manufacturer's instructions. Real-time quantitative PCR(qRT-PCR) was employed to determine the expression of PsMYB18 in the peel of'Akihime'plum fruits treated at 20 ℃ + light, 20 ℃+ dark, 30 ℃+ light and30 ℃+ dark. First-strand cDNA was synthesized from 500 ng of total RNA using the PrimeScript RT reagent kit with gDNA Eraser(Takara, China). The qRT-PCR was performed using the Eppendorf Realplex4 real-time PCR system(Hamburg, Germany) in a total volume of 20 μL in each well containing 10μL of 2×SYBR~? Premix Ex Taq? II(Tli RNaseH Plus, TaKaRa), 1 μL of cDNA(in 1:10 dilution), and0.4 μL 10 μmol primers. The qPCR conditions were 30 s at 95 ℃, followed by 40 cycles of 5 s at 95 ℃,15 s at 60 ℃, and 30 s at 72 ℃, followed by 60 ℃ to 95 ℃ melting curve detection. Actin gene was used as the reference. For gene cloning, total RNA was extracted from the fruit peel treated with 20 ℃and light. The first-strand cDNA was synthesized using First Strand cDNA Synthesis Kit(Thermo Scientific, USA). PsMYB18 gene was isolated from the skin of'Akihime'plums by reverse transcription polymerase chain reaction(RT-PCR). Phylogenetic tree was constructed using the MEGA 5.02 software to investigate the evolution relationship between PsMYB18 and MYB proteins from other plant species.Multiple sequence alignments of PsMYB18 protein with MYB repressors from peach, apple, grape, poplar, Medicago truncatula, Fragaria × ananassa and Antirrhinum majus were performed using DNAMAN7.02. p CAMBIA1302 vector, which was linearized by restriction enzyme NcoI and BstEII was used to construct overexpression vectors. The coding regions of PsMYB18, PsMYB10.1 and PsbHLH3 were isolated using I-5? 2×High-Fidelity Master Mix(MCLAB, San Francisco, CA) and then cloned into the pCAMBIA1302 vector using One Step Seamless Cloning kit(Aidlab, China). All constructs were introduced into Agrobacterium tumefaciens GV3101. Tobacco(Nicotiana tabacum L.) leaf transient expression assay was performed to verify the function of PsMYB18 in anthocyanin biosynthesis. The Agrobacterium strain GV3101 containing overexpression vectors were grown at 28 ℃ in LB medium containing with antibiotics. Then Agrobacterium cells were harvested and resuspended in the infiltration buffer(10 mmol·L~(-1) MgCl2, 10 mmol·L~(-1) MES, pH 5.6, 100 μmol·L~(-1) acetosyringone) to a final OD600 of 0.8. The bacteria were incubated at room temperature for 3 h before infiltration. Digital photographs of anthocyanin development in tobacco leaves were taken at 7 days after infiltration.【Results】The qRTPCR analysis revealed that the expression of PsMYB18 in the fruit peel treated at 20 ℃ and light was significantly enhanced after 3 d of treatment and remained at a high level thereafter. However, no obvious expression upregulation of PsMYB18 was detected in the fruit peel treated at 20 ℃ + dark, 30 ℃ +light and 30 ℃ + dark. These results were consistent with RNA-seq data. The cDNA sequence of PsMYB18 was isolated from the peel of'Akihime'fruits treated at 20 ℃ and light. PsMYB18 contained open reading frame(ORF) of 702 bp in length, which was predicted to encode a protein of 539 amino acid residues. The results of phylogenetic analysis revealed that PsMYB18 and repressors of anthocyanin and proanthocyanidin biosynthesis, including FaMYB1, MtMYB2, PtMYB182, VvMYBC2-L3, VvMYBC2-L1 and PpMYB18, from other plants belonging to the same class. It had the closest relationship with PpMYB18 from peach. Multiple alignment of the deduced amino acid sequences of PsMYB18 and MYB repressors showed that PsMYB18 shared similarity of 96.57% with the PpMYB18 protein and contained the conserved R2 and R3 domains. It also had C1 and C2 repression motifs in the C-terminus. These results implicated that PsMYB18 could be a repressor of anthocyanin biosynthesis.To investigate the role of PsMYB18 in anthocyanin biosynthesis, the ORF of PsMYB18 was amplified and ligated into the pCAMBIA1302 vector. Transient color assays in tobacco leaves showed that coinfiltration of PsMYB18 with PsbHLH3 could not induce anthocyanin accumulation. Furthermore, coinfiltration of PsMYB10.1 with PsbHLH3 resulted in red pigmentation after 7 days, while no pigmentation was observed when PsMYB10.1 and PsbHLH3 were co-infiltrated with PsMYB18.【Conclusion】In this study, PsMYB18 was isolated and its expression was enhanced by the treatment at 20 ℃ and light. Phylogenetic and sequence analysis suggested that Pp MYB18 was likely a repressor of flavonoid biosynthesis. Transient color assay results showed that PsMYB18 was a negative regulator of anthocyanin biosynthesis.