Characterization of LpGPAT Gene in Lilium pensylvanicum and Response to Cold Stress

LpGPAT was obtained fromL. pensylvanicumusing RT-PCR and rapid amplification of cDNA ends. The cloned full-length cDNA was 1544 bp; it encoded 410 amino acids and had a molecular size of 46 KDa. The nucleic acid sequence analysis showed that it
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  Research Article Characterization of LpGPAT Gene in  Lilium pensylvanicum  andResponse to Cold Stress Shao-kun Sun, 1,2 Ni-na Yang, 1 Li-jing Chen, 1,2 Muhammad Irfan, 1 Xing-hua Zhao, 3 and Tian-lai Li 2 󰀱 Key Laboratory of Agricultural Biotechnology of Liaoning Province, College of Biological Science and Technology,Shenyang Agriculture University, 󰀱󰀲󰀰 Dongling Road, Shenyang, Liaoning 󰀱󰀱󰀰󰀸󰀶󰀶, China 󰀲 Key Laboratory of Protected Horticulture, College of Horticulture, Ministry of Education, Shenyang Agriculture University,󰀱󰀲󰀰 Dongling Road, Shenyang, Liaoning 󰀱󰀱󰀰󰀸󰀶󰀶, China 󰀳 Liaoning Academy of Agricultural Sciences, Shenyang 󰀱󰀱󰀰󰀱󰀶󰀱, China Correspondence should be addressed to Li-jing Chen; chenlijing󰀱󰀹󰀹󰀷@󰀱󰀲󰀶.com and ian-lai Li; litianlai@󰀱󰀲󰀶.comReceived 󰀱󰀵 June 󰀲󰀰󰀱󰀴; Revised 󰀲󰀴 October 󰀲󰀰󰀱󰀴; Accepted 󰀸 November 󰀲󰀰󰀱󰀴Academic Editor: Yong-Pyo LimCopyright © 󰀲󰀰󰀱󰀵 Shao-kun Sun et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.LpGPA was obtained from  L. pensylvanicum  using R-PCR and rapid ampli󿬁cation of cDNA ends. Te cloned full-length cDNAwas󰀱󰀵󰀴󰀴bp;itencoded󰀴󰀱󰀰aminoacidsandhadamolecularsizeof󰀴󰀶KDa.Tenucleicacidsequenceanalysisshowedthatitsharedhigh homology with other known GPAs. SMA result suggests that there is a PlsC that exists in 󰀱󰀷󰀶-󰀳󰀲󰀲 amino acid sequence of LpGAP; it means LpGPA protein is a member of the family of acyltransferase and has acyltransferase enzymatic activity. Resultof real-time quantitative PCR and semiquantitative PCR support LpGPA gene is de󿬁nitely induced by low temperature stress. 1. Introduction Lilium pensylvanicum  belongs to Liliaceae,  Lilium , whichsrcinates in North China including Hebei, Heilongjiang,Jilin and Liaoning provinces, North Korea, Japan, Mongolia,and Russia [󰀱]. It is one of the best parents to cultivate novelanti-cold  Lilium  varieties for strong cold-resistance. Besides,itpossessessigni󿬁cantresistanceagainstheat,drought,alkali,salt, sand burial, and aeolian erosion. It even can surviveunder contions with temperature ranging from  − 󰀷󰀰 ∘ C to − 󰀴󰀰 ∘ C and annual rainfall less than 󰀲󰀰󰀰mm [󰀲]. Hence,it is precious and valuable rescorce for drought-resistanceand cold-resistance, as well as ideal material for resistancestudying.Hypothermia is the main factor limiting plant growth,development, and geographical distribution. One adaptationofplantstotemperaturestressisbybiomembranes,especially the effect of plasma and thylakoid membranes. Plants canregulate the level of unsaturated fatty acids to cold throughthe acyl lipid desaturase and glycerol-󰀳-phosphate acyltrans-ferase (GPA) activity [󰀳]. A large number of studies indicatethat the saturation degree of phosphatidyl glycerol (PG) inmembraneiscloselyrelatedtocoldtoleranceinplants.WhileGPA is the 󿬁rst acyltransferase during the PG synthesis andit initiates the reaction by the acylation of the glycerol-󰀳-phosphate.TesubstrateselectionofGPAplaysakeyroletodetermine the unsaturation degree of the PG molecules [󰀴].Different GPAs in plants with low-temperature tolerancehave different substrate selectivity of acyl, that is, GPAsfrom plants with strong cold-resistance prefer oleic acid,while GPAs from cold-sensitive plants utilize the palmiticacid and oleic acid equally [󰀵]. Such selective difference of GPA to substrate affects the saturation level of the PG inplant membrane, which consequently determines the plantresistance to low temperature [󰀶]. Later, the study in therelationship between plant cold-tolerance and the fatty acidsaturation in membrane achieves a breakthrough [󰀴, 󰀷]. Te fatty acids (saturated and unsaturated) of PG in thechloroplast membrane have in󿬂uence on the synthesis of cold-sensitive plants [󰀸].Hypothermia is inevitably a limitation to achieve annualproduction for the climate feature of China and behind handhorticulture facilities with contrast to Europe and America.Terefore, it is necessary to study the cold tolerance of  Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 792819, 8 pages  󰀲 BioMed Research International 󰁡󰁢󰁬󰁥 󰀱: Te sequences and use o the primers.Primer name Sequences ( 5  → 3  ) Primer useGPAF󰀱 AAGC(/A)G(A/C)AGGAAGGA(G/A)GA For middle ragmentsGPAF󰀲 AAGGA(G/A)GA(A/G)(C/A)GA(C/)AGAA(/C)A For middle ragmentsGPAR󰀱 GGAGG(G/A)GGCA(A/G)A(A/G)CA For middle ragmentsGPAR󰀲 G(A/C)CCAGG(G/A)ACACCAG(C/A)AG For middle ragmentsLpGPA F󰀱 AAGCCGCGGAGACA For 󰀳  RACE o LpGPALpGPA F󰀲 CCAAACCACAGACGGAAGC For 󰀳  RACE o LpGPALpGPA R󰀱 AAGGGAGCCCAAGAGA For 󰀵  RACE o LpGPALpiGPA R󰀲 GGGCCCGCGAGC For 󰀵  RACE o LpGPALpGPA R󰀳 CGCCCGCGAGGGA For 󰀵  RACE o LpiGPALpGPA R󰀴 CAAAGAAGACCCCCAACG For 󰀵  RACE o LpGPAEGPAF GCAGGGGAAGGGAACAGA For expression o LpGPAEGPAF AGGACCAAGGCAGCAGA For expression o LpGPAactinF CCCCCAGCCC For control geneactinR CCGCCAACGGCA For control geneActinF GCCACCACGCCACAG For real-time quantitative PCR ActinR CCCAACAAGCCACCACC For real-time quantitative PCR GPN󰀱 CCCACCCCCAAAGGAGAAA For real-time quantitative PCR GPN󰀲 GGCAGCAGAACCAGCGA For real-time quantitative PCR  L. pensylvanicum  in order to save energy, reduce the costso winter cultivation and storage, and improve the stress-resistance to achieve the large-scale cultivation o   L. pen-sylvanicum . Additionally, the bad natural environment andweather o wild  L. pensylvanicum  coners its very strong coldtolerance.Hence,thereareabundantexcellentcold-resistant-related genes. Exploration and utilization o these genes haveimportant signi󿬁cance or the research on cold toleranceo plants and improvement. In this study, the conservativeragment o   L. pensylvanicum  GPA gene was obtained by R-PCR. Te whole cDNA o the LpGPA gene was clonedbyRACE,whichwasthenanalyzedorexpression.Tisstudy will supply some basis or the deep research on the cold-resistance o   L. pensylvanicum . 2. Materials and Method 󰀲.󰀱. Plant Materials and Pretreatment. Lilium pensylvanicum bulbswerekindlyprovidedbyDaxinganlingForestryBureau,Inner Mongolia province, China, which had been domesti-cated rom wild species over three years.  L. pensylvanicum possessed strong cold tolerance that was stored with 󰀱󰀰cmcovered soil in winter and could survive with 󰀵󰀰 ∘ C to below 󰀰 ∘ C.Te bulbs rom  L. pensylvanicum  were separated andwashed by water, then soaked in 󰀷󰀵% ethanol or 󰀳󰀰s,and then put in 󰀰.󰀱% HgCl 2  or 󰀱󰀰min. Subsequently, they were rinsed with sterilized water 󰀳 times. At last, they wereplaced on dry sterile 󿬁lter-paper or use. Te sterile bulbswere cultured on MS medium adding agar powder (󰀷gL −1 )and sucrose (󰀳󰀰gL −1 ), pH 󰀵.󰀸. Te 󿬂uorescent light wason 󰀱󰀴h everyday with light intensity o 󰀱󰀰󰀰󰀰 ∼ 󰀱󰀲󰀰󰀰lx. Tetemperature was rom 󰀲󰀳 to 󰀲󰀶 ∘ C. wo months later, theadventitiousbudswereremovedromthebulbsandplacedin󰀴 ∘ C or 󰀲h, 󰀴h, 󰀶h, 󰀸h, 󰀱󰀲h, 󰀲󰀴h, 󰀴󰀸h, and 󰀷󰀲h, which werelater used or RNA extraction or were immediately rozenin liquid N 2  and stored in ultra-low temperature reezer at − 󰀸󰀰 ∘ C until needed.otal RNA samples used or cDNA 󿬁rst strand synthesiswere extracted rom the above treated resh young leaves.Afer that semiquantitative PCR and real-time quantitativePCR reactions were perormed using these samples to detectrelativedifferentialexpressionsoLpGPAgeneunderdiffer-ent low temperatures stress.Te RNA extraction kit, vector PGM-, miniplasmid-DNA extraction kit, DNA polymerase, dNPs, DNA MarkerD󰀲󰀰󰀰󰀰, and  E. coli Top 󰀱󰀰  used in the experiment werepurchased rom iangen Biotech Co., Ltd. (Beijing, China).TeagarosegelandDNAGelextractionkitwereboughtromAxyGen(Beijing,China).M-MLVReverseranscriptasewasrom Promega (Beijing, China). All the other chemicals wereo analytical grade. All the primers were synthesized by SBSGenetech Co., Ltd. (Beijing, China). 󰀲.󰀲.RNAExtractionandFirst-StrandcDNASynthesis.  Leaveswere ground in liquid N 2  with a mortar and pestle. otalRNA was extracted rom samples with the plant tissue RNAextraction kit ollowing the manuacturer’s instructions. TeRNA quality was assessed by using 󰀲  g o the total RNA on a󰀱.󰀲% agarose gel. Te total RNA was stored at  − 󰀸󰀰 ∘ C. cDNAsynthesis reactions were perormed with M-MLV reversetranscriptase according to the manuacturer’s instructions. 󰀲.󰀳. Isolation of the Middle Fragments of the LpGPAT Genes. Tehomologousprimersormiddlesegmentsweredesignedbased on conserved sequences o GPA genes rom someplants in NCBI GenBank (GPAF󰀱, F󰀲, R󰀱, and R󰀲, able 󰀱).PCR reactions were perormed in a total reaction volume o   BioMed Research International 󰀳󰀲󰀵  L containing 󰀵󰀰ng o genomic DNA, 󰀱 ×  aq DNA poly-merase buffer, 󰀱.󰀵mM MgCl 2 , 󰀰.󰀵  M each primer, 󰀲󰀰󰀰  Meach dNP, and 󰀱U aq DNA polymerase. Te program orPCRampli󿬁cationwasasollows:initialdenaturationat󰀹󰀴 ∘ Cor 󰀵min, 󰀳󰀵 cycles o 󰀹󰀴 ∘ C or 󰀳󰀰s, 󰀵󰀳 ∘ C or 󰀴󰀵s, 󰀷󰀲 ∘ Cor 󰀶󰀰s, and a 󿬁nal extension at 󰀷󰀲 ∘ C or 󰀱󰀰min. Te PCR products were separated on 󰀱.󰀰% agarose gels, and then thetargeted DNA ragments were recovered and cloned into thepGEM-Easy vector. Te ligated products were transormedinto  Escherichia coli  (DH󰀵 󽠵 ) cells and the resulting plasmidswere used as a sequencing template. 󰀲.󰀴. Ampli󿬁cation of the Complete Coding Sequences of theLpGPAT Genes.  Primers or 󰀵  - and 󰀳  -end cDNA ampli󿬁ca-tion were designed based on the middle ragment sequences(able 󰀱). Te 󰀳  and 󰀵  sequences o cDNA were obtainedby RACE with the 󰀳  - and 󰀵  -RACE System or RapidAmpli󿬁cation o cDNA Ends (Invitrogen, USA). Te PCR products were cloned to pMD󰀱󰀸- vector and sequenced.Based on the nucleotide sequences o the 󰀵  - and 󰀳  -RACEproducts, primers LpGPA 󰀱F, LpGPA 󰀱R, LpGPA 󰀲F andLpGPA 󰀲R (able 󰀱) were used or the ampli󿬁cation o thecomplete coding sequences o   GPAT   genes. 󰀲.󰀵.SemiquantitativeRT-PCRAnalysis.  SemiquantitativeR-PCRwasperormedusinggene-speci󿬁cprimersorEGPAFand EGPAR. As an internal control, a ragment rom whiteash actin gene was ampli󿬁ed using the actinF and actinR primers (able 󰀱). Te PCR ampli󿬁cation was programmedas initial denaturation at 󰀹󰀴 ∘ C or 󰀴min, 󰀳󰀵 cycles o 󰀹󰀴 ∘ Cor 󰀳󰀰s, 󰀵󰀶 ∘ C or 󰀱min, 󰀷󰀲 ∘ C or 󰀱min, and a 󿬁nal extensionat 󰀷󰀲 ∘ C or 󰀱󰀰min. Ampli󿬁ed ragments were detected by electrophoresis using 󰀱.󰀵% (w/v) agarose gels. 󰀲.󰀶. Bacterial Expression of LpGPAT.  According to the ull-length cDNA sequence analysis o the cloned LpGPAgene and the prokaryotic expression vector pE󰀳󰀰a (+), wedesigned a pair o primers which were in accordance to thecDNA ORF in LpGPA as ollows:YGpat󰀱 F (󰀵  -GGACCAGCACGCGGGAACCG-󰀳  ),YGpat󰀱 R (󰀵  -GAACCAGGCGAGAGAGAGAAG-󰀳  ). Eco RI and  Kpn I restriction sites were added in the primers(underlinedpart).ArecombinantplasmidpMD󰀱󰀸-LpGPAandtheexpressionvectorweredigestedwith EcoR Iand Kpn I.Ten target gene was ligated to pE󰀳󰀰a (+), which was laterused to transorm  E. coli  BL󰀲󰀱 competent cells. Te correctcolony was identi󿬁ed by screening medium with Kan andPCR. Finally, the recombinant  E. coli  was cultured in liquidmedium and 󰀱mol/L IPG was added to induce the proteinexpression with different time. 󰀲.󰀷. Bioinformatic and Phylogenetic Analysis.  Primer Premier󰀵 sofware ( was usedor all the primer designs. Sequences were aligned usingDNAman 󰀵.󰀲.󰀲 ( and SMAR( and CLUSAL W 󰀱.󰀸󰀱sofware [󰀹]. Te phylogenetic tree was generated based onthe NJ (neighbour-joining) sequences distance method [󰀱󰀰]and depicted and edited by MEGA 󰀳.󰀱 program [󰀱󰀱]. 󰀲.󰀸. Quantitative Real-Time PCR.  otal RNA was extractedrom the rozen leaves and bulbs in liquid nitrogen, removedrom seedlings which treated with 󰀴 ∘ C or 󰀲h, 󰀴h, 󰀶h,󰀸h, 󰀱󰀲h, 󰀲󰀴h, 󰀴󰀸h, and 󰀷󰀲h, using an RNA extraction kit(Bioteke, China) according to the manuacturer’s instruc-tions. Reverse transcription was perormed using MMLVreverse transcriptase (aKaRa, China) with the Oligo-dprimer (aKaRa, China). Quantitative real-time PCR wasperormed or cDNA ampli󿬁cation using SYBR PremixExaq (akara Bio, Inc., Shiga, Japan) and primers listedin able 󰀱, on a Bio-Rad C󰀱󰀰󰀰󰀰 real-time system (Bio-Rad, Hercules, CA, USA), according to the manuacturer’sinstructions and applied international standards. For eachPCR, 󰀲  L cDNA obtained rom 󰀱  g RNA template wasused. Te thermal cycling conditions consisted o an initialdenaturation step at 󰀹󰀵 ∘ C or 󰀳󰀰sec and 󰀴󰀰 cycles o theollowing 󰀳 steps: denaturation at 󰀹󰀵 ∘ C or 󰀵sec, annealing at󰀵󰀷 ∘ C or 󰀳󰀰sec, and elongation at 󰀷󰀲 ∘ C or 󰀳󰀰sec. Actin wasused as the internal control. 3. Results 󰀳.󰀱. Sequence Analysis of LpGPAT Genes.  Initially, a ragmentabout 󰀶󰀸󰀰bp was ampli󿬁ed by R-PCR. By analysis andcomparison o the sequence, the ragment had the similarstructures with those o known GPA genes. Tis wasollowed by 󰀳  - and 󰀵  -RACE analysis, and two ragments,󰀷󰀵󰀰 and 󰀵󰀰󰀰bp, in sizes were obtained. Finally, the ull-length cDNA o a LpGPA gene was obtained by sequenceassemblyandreampli󿬁cation.Sequenceanalysisrevealedthatthe cDNA ragment was 󰀱,󰀵󰀴󰀴bp in length, including anopen reading rame (ORF) o 󰀱,󰀲󰀳󰀳bp along with 󰀳󰀱󰀱bp 󰀵  -and 󰀱󰀰bp 󰀳  -untranslated sequences (Figure 󰀱). Tis cDNAragment that could encode 󰀴󰀱󰀰 amino acids designated asLpGPAhadbeensubmittedinGenBank(accessionnumberJX󰀵󰀲󰀴󰀷󰀴󰀱). 󰀳.󰀲. Comparison of Amino Acid Sequences.  Te compari-son o amino acid sequences showed thatLpGPA had thesimilar typical primary structures to those o known GPAgenes rom other plants such as  Arabidopsis thaliana ,  Oryzasativa ,  Zea mays ,  Elaeis guineensis ,  Solanum lycopersicum, and  Chlamydomonas reinhardtii.  Tere were high levels o homologyinaminoacidsequencesoallGPAproteinsromothers (Figure 󰀲).Te coding regions begin rom the amino acids MALKin all genes rom woody plants. Te conserved regions o allgenes begin rom about 󰀴󰀰th amino acid residues (WIAPSG-GRDRP) (Figure 󰀳). All GPA amino acid sequences havetwo conserved domains belonging to the acyl-CoAs desat-urase amily and the acyl-ACPs amily, respectively. But they were also different rom each other by some substitutions,  󰀴 BioMed Research International 112141 ACGTGTTAGTATGCTACGTCGGGAACCGTGGCATTGCCCATGTGTGGCGTATAGGGCGCG 61  GGCGATGACGGAGCCTACGCGGGCGATGGCGGAGCCGGCGGAGGGGACGGTGGCGAGGGG 121  TTGCAGGGCGGTGCTGGAGTCGGAGGCGAGATCGCGGCCGTTACTGCGTCCGCGGAACGA 181  GCAAGAACTGCTCTCTTATATTCAAAATGAATCTGAAGCTGGAAGGCTTTCTCCAAGTAT 241  TGCTAGTGGCCTTGTGGAACTATATCATAATTATCGACATGCGGTTTTGCAAAGTGGAAA 301  TCCTAATGCGAGTAAAATTATACTCTCCAATATGGCTGTTGTGTTTGATCGCATTTTGTT 361  GGATGTGGAGGATCCTTTTACCTTTTCACCTCATCACAAAGCAATTCGTGAGCCTTTTGA 421  CTACTACATGTTTGGTCAAAATTATATTAAGCCGCTGGTAGATTTCAGGAGATCATACAT 481  TGGGAACATCACACTTTTTGATGACATTGAAGAGAAACTCAAGCAGGGCCACAATATAGT 541  TTTGGTCTCAAACCATCAGACGGAAGCAGATCCTGCAGTCATTGCTTTGTTGCTTGAAAG 601  AACAAACTCATATATTGCTGAGAAGCTGGTATATGTCGCAGGGGATAGGGTTATTACAGA 661  TCCGCTTTGCAAGCCGTTCAGTATGGGAAGGAATCTTCTTTGTGTATACTCGAAAAAGCA 721  TATGAATGATGATCCTGAGCTTGCCGAGATGAAAAGGAGATCAAATACCCGAAGTCTCAA 781  GGAAATGGCTACACTTTTGAGGGCTGGATCACAGATAATATGGATCGCACCAAGTGGTGG 841  GAGAGACCGTCCTGATCCAGTGACAGGGGAATGGTATCCTGCACCTTTTGATGCATCTTC 901  AGTTGACAATATGAGAAGGCTTGTGGAACATTCTAGTTTTCCTGGACATATACATCCCAT 961  GGCATTGCTATGTCATGATATCATGCCTCCACCCCCAAAGGTAGAAAAGCAAATTGGTGA 1021  GCGAAGAAAGATTGCTTTCCATGGAGTTGGCATATCTGTTGCTCCAGAGTTAAATTTCGC 1081  TGAAGTTACTGCTGCCATTGAGAATCCTGAAATGGCAAAGGAAGCTTTCTCACAGGCTGT 1141  GTACAAATCCGTAACTGAGCAATATGCAGTGCTCCAGTCTGCAATACACGGATACCAAGG 61811011211411611812012212412612813013213413613814011201  ATTAAATGCATCAAACTCTGCTATCTCTCTCTCAGAACCATGAAAATTGCCCTCTCATGC 1261  TTGATATATTTTCTTCTCCGATCACATGATTGCAACAACAAAGTATTTGATTAGAAAAGG 1321  GAAAAAAAGGTGGTCAGATTCTGATGGAGGTGCTGCTGGCAGTGTTTCAATTCTTCTCTG 1381  GGTGTTTTCGGGATACAGCAATTGTTATTTTTTTTCCTGGATTATATACATCACTAATTT 1441  AAGTCTACTGTGATTCATCTTGGAAAAAAAAATGTAAAGCGACTCCGAATTAGGGATTAT 1501  TTATGAAATTGAAGAATATGTTGAGAAAAAAAAAAAAAAAAAAAM L R R E P W H C P C V A Y R A RA M T E P T R A M A E P A E G T V A R GC R A V L E S E A R S R P L L R P R N EQ E L L S Y I Q N E S E A G R L S P S IA S G L V E L Y H N Y R H A V L Q S G NP N A S K I I L S N M A V V F D R I L LD V E D P F T F S P H H K A I R E P F DY Y M F G Q N Y I K P L V D F R R S Y IG N I T L F D D I E E K L K Q G H N I VL V S N H Q T E A D P A V I A L L L E RT N S Y I A E K L V Y V A G D R V I T DP L C K P F S M G R N L L C V Y S K K HM N D D P E L A E M K R R S N T R S L KE M A T L L R A G S Q I I W I A P S G GR D R P D P V T G E W Y P A P F D A S SV D N M R R L V E H S S F P G H I H P MA L L C H D I M P P P P K V E K Q I G ER R K I A F H G V G I S V A P E L N F AE V T A A I E N P E M A K E A F S Q A VY K S V T E Q Y A V L Q S A I H G Y Q GL N A S N S A I S L S E P * F󰁩󰁧󰁵󰁲󰁥 󰀱: Nucleotide and deduced amino acid sequences of  LpGPAT. KLVYVAGDRVITDPLOKPFSMGRNLLOVYSKKHMNDDPEL TMVFVAGDRVLTDPLOKPFSMGRNLLOVYSKKHMDDVPEL NLTYIAGDRVITDPLOKPFSMGRNLIOVYSKKHMLDVPEL NLIYVAGDRVVTDTLOKPFSMGRNLIOVYSKKHMADVPEL NTIFVAGDRVLADPLOKPFSIGRNLIOVYSKKHMFDIPEL NLIYIAGGRVITDPLOKPFSMGRNLIOVYSKKHMNDVPEL NMIYVAGDRVIADPLOKPFSIGRNLIOVYSKKHMLDIPEL NTIFVAGDRVLADPLOKPFSIGRNLISVYSKKHMLDIPEL NIVYVAGDRVVTDPLOKPFSMGRNLIOVYSKKHMNDFPEL NIKCVAGDRVITDPLOKPFSMGRNLIOVYSKKHMNDDPEL NLIYVAGDRVITDPLOKPFSIGRNLIOVYSKKHMLDNPEL NLIYVAGDRVITVPLOKPFSIGRNLIOVYSKKHMLDNPEL NIIYVAGDRVITDPLOKPFSMGRNLLOVYSKKHMNDVPEL NIIYVAGDRVITDPLOKPFSMGRNLLOVYSKKHMNDDPEL NLTYVAGDRVITDPLSKPFSIGRNLIOVYSKKHMLDDPAL NIVYVAGDRVVTDPLOKPFSMGRNLIOVYSKKHMNDFPEL NLIYIAGDRVITDPLOKPFSMGRNLLOVYSKKHMYDDPEL NIIYVAGDRVITDPLOKPFSMGRNPLOVYSKKHMNDDPEL ...........SDPLOKPFSMGRNLLOVYSKKHMNDDPEL DVIYVAGDRVVTDPMOKPFSMGRNLFOVHSKKHMDDAPEL kpfs grn v skkhm d p l Lilium pensylvanicumElaeis guineensisCitrus unshiuRicinus communisCucurbita moschata Jatropha curcasCucumis sativusCucurbita 󿬁cifoliaOryza sativa Arabidopsis thalianaVicia fabaPisum sativumCarthamus tinctoriusCapsicum annuumPhaseolus vulgarisZea maysSpinacia oleraceaSolanum lycopersicumHippophae rhamnoidesChlamydomonas reinhardtiiConsensus 24429230028722929430222925929129128929129629327930330329243 F󰁩󰁧󰁵󰁲󰁥󰀲:Comparisonofthededucedaminoacidsequencesof  LpGPAT   withotherknownGPATgenesfrom󰀲󰀰species.Deep(black)shadingrepresents the identity of amino acid residues that was 󰀱󰀰󰀰%; light (gray) shading represents the identity that was more than 󰀷󰀵%.  BioMed Research International 󰀵 AEMKRRSNTRSLK.EMATLLRAGSQIIWIAPSGGRDRPDP IEMKRRANTRSLK.EMALLLRGGSQIIWIAPSGGRDRPDP IEMKRKSNTRSLK.EMALLLRGGSQIIWIAPSGGRDRPDP TEMKKKANIRSLK.EMVMILRDGSQIVWIAPSGGRDRPDS TETKRKANTRSLK.EMALLLRGGSQLIWIAPSGGRDRPDP TEMKKRANIRSLK.EMAIPLRGGSRIVWIAPSGGRDRPDH AETKRKANTRSLK.EMALLLRGGSQLIWIAPSGGRDRPDP AETKRNANTRTLK.EMALLLRGGSQLIWIAPSGGRDRPDP VDMKRRANTRSLK.EMALLLRGGSQIIWIAPSGGRDRPDP VDMKRKANTRSLK.EMATMLRSGGQLIWIAPSGGRDRPNP IDMKRKANTRSLK.EMATLLRSGSQIIWIAPSGGRDRPVA VDMKRKANTRSRK.EMAMLLRSGSQIIWIAPSGGRDRPVA AEMKKRSNTRSLKGRMALLLRGGSKIIWIAPSGGRDRPDP ADMKKRANTRSLK.EMAMLLRGGSKLIWIAPSGGRDRPDP VEMKRTANIRALK.EMAMLLRNGSQLVWIAPSGGRDRPDA IEMKRRSNTRSLK.EMALLLRGGSQLIWIAPSGGRDRPNP VDVKKRANTRSLK.ELVLLLRGGSKIIWIAPSGGRDRPDA AEMKKRANTRSLK.EMALLLRGGSKIIWIAPSGGRDRPDP AEMKKRANTRSLK.EMALLLRGGSKIIWIAPSGGRDRPDP KAAKMETNRKTLV.AMQRKLNEGGTLMWIAPSGGRDRPNA 28333133932626833334126829833033032833133533231834234268281 k n l g wiapsggrdrp Lilium pensylvanicumElaeis guineensisCitrus unshiuRicinus communisCucurbita moschata Jatropha curcasCucumis sativusCucurbita 󿬁cifoliaOryza sativa Arabidopsis thalianaVicia fabaPisum sativumCarthamus tinctoriusCapsicum annuumPhaseolus vulgarisZea maysSpinacia oleraceaSolanum lycopersicumHippophae rhamnoidesChlamydomonas reinhardtiiConsensus F󰁩󰁧󰁵󰁲󰁥 󰀳: Alignment o deduced amino acid sequences rom 󰀲󰀰 plants GPA cDNA. Te domain o acyl-CoAs desaturase and the domaino acyl-ACPs amily are in black box, respectively. 0 M 72482416842 20001000750500250100 -ActinRNA in L. pensylvanicum 470 bp(bp)Treatment duration (h) F󰁩󰁧󰁵󰁲󰁥 󰀴: Expression o   LpGPAT   in  L. pensylvanicum  under differ-ent cold induced times at 󰀴 ∘ C. insertions, and/or deletions involving single amino acidresidues or motis. Te main regions o variability wereregions about ront 󰀴󰀰 amino acid residues o N-terminaldomains. 󰀳.󰀳.SemiquantitativeRT-PCRAnalysis.  SemiquantitativeR-PCR was employed to con󿬁rm the expression patterns o theLpGPA gene in different induction times. Te result in thisstudy showed that low temperature could induce the expres-sion o LpGPA .  Te expression analysis oLpGPA geneshowed that low temperature could induce the expression o the gene in a short time. With the prolonged cold induction,the expression increased 󿬁rstly and then decreased, a lot o expressions were ound afer 󰀴h, peaked at 󰀱󰀶h, and thendecline gradually and at 󰀷󰀲h basically the same level inkeeping with 󰀰h. (Figure 󰀴). 󰀳.󰀴. Prokaryotic Expression Analysis of LpGPAT Gene.  Terecombinant prokaryotic expression vector pE-LpGPA MA 864210.50 (KDa) 97.266.444.329.046 KDa F󰁩󰁧󰁵󰁲󰁥 󰀵: Te expression o   LpGPAT   protein in  E. coli  BL󰀲󰀱 by SDS-PAGE analysis. was checked by enzyme digestion using  Eco R I and  Kpn  I,which was later transormed into  E. coli  BL󰀲󰀱. Te correcttransormants were cultured and expressed the LpGPAproteins by adding IPG. A predicted extra protein band o 󰀴󰀶kDa was observed in the transormants, while no suchbandwasseeninthenegativecontrols(Figure 󰀵).Besides,theexpression level increased with the treatment time rom 󰀰 to󰀸 hours. 󰀳.󰀵. Evolutionary Relationship Analysis.  Evolutionary rela-tionship was determined by sequences o GPAs obtainedrom BLAS and alignment using Clustalx 󰀱.󰀸󰀳 sofware. Atree was constructed by Mega 󰀴.󰀰 sofware using neighbour- joining method (Figure 󰀶). We ound that  L. pensylvanicum was in the cluster with  E. guineensis ,  O. sativa,  and  Z.mays,  and then in the cluster with  Solanum lycopersicum , Capsicum annuum ,  C. tinctorius ,  C. moschata ,  Cucumissativus ,  Arabidopsis thaliana,  and  C. reticulata , and 󿬁nally with  Spinacia oleracea  and  Chlamydomonas reinhardtii . Inconclusion, the result o the cluster analysis showed that
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