苦蕎中查爾酮合成酶全長基因的克隆及序列分析_英文_

1、Cloning and Analysis of a Chalone Synthase Gene from Fagopyrum tataricumKai LIU 1,2,Yaohui HU 1,Guan WANG 3,Hansong YU 1*1.College of Food Science and Engineering,Jilin Agricultural University,Changchun 130118,China;2.Jilin Agricultural Comprehensive Development Office,Changchun 130118,China;3.Acade

2、my of Life Science,Jilin Agricultural University,Changchun 130118,ChinaSupported by 948Program,Ministry of Agriculture of China (2008-z27.AKey words Fagopyrum tataricum ;CHS;RACE;Intron;CladogramAgricultural Science &Technology,2012,13(4:708-710,726Copyright 訫2012,Information Institute of HAAS.A

3、ll rights reservedAgricultural BiotechnologyTatar buckwheat (Fagopyrum tat -aricum ,commonly known as buckwheat,belongs to family ofPolygonaceae,genus of Fagopyrum esculentum 1-2.Previous studies show that buckwheat has a variety of physio -logical functions including prevention of diabetes and coro

4、nary heart disease,anti -cancer,prevention of internal blee -ding,blood lipids -lowering,blood -glu -cose -lowering and anti -aging 3-7,these physiological functions and medicinal value were found closely related to the abundant flavonoid in buckwheat 8.CHS (chalcone synthaseis the first enzyme in t

5、he flavonoid biosyn -thetic pathway and also a key enzyme in the secondary metabolic pathway of plants,which catalyzes condensation between three acetoxy of malonyl -coenzyme A and a acetoxy of A4-cou -maroyl -CoA 9and synthesizes ch -al -cone with 4-coumaric acid -CoA as the best substrate 10.Chalc

6、one is the sy -nthetic precursor for plant flavonoid 11.Improving the expression level of this key enzyme can increase the chalcone content in plants or plant cells,thus in -creasing the content of flavonoid.Therefore,cloning CHS gene of buck -wheat has important significance for regulating the synt

7、hesis of flavonoid at the molecular level.So far,many researches have been conducted on the determination,synthesis pathway and regulation of flavonoid in buckwheat.However,no research has been reported on regu -lating the synthesis of flavonoid at the molecular level and cloning of full -length CHS

8、 gene sequence from buckwheat.Therefore,in this study,buckwheat was used as experimental material for cloning the full -length cDNA sequence of CHS gene by using RACE (rapid -amplification of cDNA endstechnology and CODEHOP (consensus -degenerate hybrid oligon -ucleotide primersprimer design method

9、and bioinformatics analysis,toprovide an initial basis for regulation of the flavonoid content in buckwheat at the molecular level.Materials and MethodsMaterialsExperimental material Buckwheat from Ukraine Irina was used as the experiment material in this study.Strains and reagents Escherichia coli

10、DH5,which was stored in our laboratory,was used as the recipient strain for transformation.M -MLV re -verse transcriptase was the product of Promega Corporation;Ex Taq DNA polymerase,dNTPs,DNA Marker and pMD18-T vector kit were purchased from TaKaRa Biotechnology Co.,Ltd.MethodsExtraction of total R

11、NA from buck -wheat leaves The total RNA from buckwheat leaves were extracted by using modified CTAB -LiCl method and the specific steps were in accordance with the previous literature 12.Primer design Design of degenerate primers for 5-RACE Design of upstream primer:bioinformatics analysis showed t

12、hat 5sequence of buckwheat CHS was rel -atively conservative,so the primer was designed according to the conserva -tive sequence and named J5CHSup,with sequence of 5-ATGGCACCGA -CGGTCCAGGA -3.Design of down -stream primer:downstream J5CHS -down was designed according to the cloned 3cDNA,with sequenc

13、e of 5-CAGACGACGAGGACACGAGC -3.Design of specific primers for am -plification of full -length RaCHS gene According to the open reading frame (ORFof the cDNA sequence,specific primers for amplification of full -length gene were designed.Upstream primer CHSup:5-ATGGCACCGAC -GGTCCAGGA -3;downstream pri

14、mer CHSdown:5-AGTTGGCTAGGGTG -GTGG -3.PCR amplification conditions First strand cDNA synthesis by reverse transcription was used as the template for 3-RACE experiment.The 3-RACE PCR amplification was started with predenaturation at 94for 3min,fol -lowed by 30cycles of denaturation at 94for 30s,annea

15、ling at 56for 45s,and extension at 72for 1min;the amplification was completed by holding the reaction mixture at 72for 10min to allow complete extension of PCR products.The 5-RACE PCR amplification was started with prede -naturation at 94for 3min,followed by 30cycles of denaturation at 94for 30s,ann

16、ealing at 60for 45s,and extension at 72for 1min;the amplification was completed by hold -ing the reaction mixture at 72for 10min to allow complete extension ofPCR products.The PCR amplification for full -length RaCHS gene was started with predenaturation at 94for 3min,followed by 35cycles of de -nat

17、uration at 94for 30s,annealing at 58for 45s,and extension at 72for 1min;the amplification was com -pleted by holding the reaction mixture at 72for 10min to allow complete extension of PCR products.Recovery,ligation,transformation and identifica -tion of the target gene were conducted in accordance w

18、ith routine method 13,the sequencing was completed by Shanghai Sangon Biological Engi -neering Technology Services Co.,Ltd.Bioinformatics analysis of RaCHS Clustalxl.81and MEGA4software were used for sequence analysis and construction of phylogenetic tree;NCBI Blastn and Blastp programs were applied

19、 for homology analysis of nucleic acid and protein.Results and AnalysisRNA extractionWith the extracted total RNA of buckwheat leaves as the template for reverse transcription by using primers P1and PCR amplification by using primers P2and J3CHS.Subsequently,the products were detected by using agaro

20、se gel electrophoresis,the re -sults(Fig.2showed that an 800bp band was obtained by the 3-RACE experi -ment.The obtained fragment was in -serted into pMD18-T simple vector and transformed into E.coli ,and the posi -tive clones were selected and sent for sequencing.The sequencing results showed that

21、the length of this se -quence was 891bp,which shared 88%homology with Rheum palmatum (accession number:DQ205352.1ac -cording to the sequence alignment by Blastn program,indicating a new gene,which was named RaCHS .5-RACE resultsWith the extracted total RNA of buckwheat leaves as the template for rev

22、erse transcription by using primers P1and PCR amplification by using primers J5CHSup and J5CHSdown,the products were detected by using agarose gel electrophoresis (Fig.3,which showed that a 550bp band was obtained by the 5-RACE experiment.The obtained fragment was inserted into pMD18-T simple vector

23、 and trans -formed into E.coli ,and the positive clones were selected and sent for se -quencing.The sequencing resultsFig.1Electrophoresis of the total RNA ofbuckwheat leavesM,DL 2000marker;1,PCR amplification product.Fig.23-RACE results of RaCHSM,DL 2000marker;1,PCR amplification product.Fig.35-RAC

24、E results of RaCHS showed that the length of this se -quence was 572bp,which shared 87%homology with Rheum palmatum (accession number:DQ205352.1ac -cording to the sequence alignment by Blastn.RaCHS gene merger and full -length cDNA cloningBioinformatic analysis of RaCHS geneDomain analysis of RaCHS

25、gene By using Bankit tool on the NCBI web -site,the obtained DNA sequence of RaCHS gene was submitted to Gen -Bank (accession number:HQ434624.The cDNA sequence was translated into amino acid sequence for analysis by using the NCBI Blastp program,and the results (Fig.5indicated this gene belonging to

26、 the CHS superfam -ily.Translated RaCHS protein had BcsA -type domain,indicating that RaCHS might belong to chalcone syn -thase -like protein.Evolutionary analysis of RaCHS gene Clustal X software was used for multiple sequence alignment be -tween RaCHS gene -deduced amino acid sequence and CHS amin

27、o acid sequences from other plant species with identified function 14-16.Neighbor-Joining algorithm in Bootstrap test of MEGA4software was used for con -struction of phylogenetic tree (Fig.6,number of branches indicated the Bootstrap supporting rate of the branch.As can be seen from Fig.6,RaCHS gene

28、 showed the highest ho -mology to Rheum palmatum and rela -tively high homology to Gypsophila paniculata ,while it was distantly ho -mologous with other plant species.Conclusion and DiscussionChalcone synthase (CHSis a key enzyme in flavonoid biosynthetic pathway,which catalyzes the synthe -sis of c

29、halcone with 4-Coumaroyl -CoA as the best substrate,thereby synthe -sizing flavonoid through a series of pathways.Improving the expression level of CHS can increase the chal -cone content in plants or plant cells,thus increasing the content offlavonoid.Therefore,cloning CHS gene of buckwheat has imp

30、ortant sig -nificance for regulating the synthesis of flavonoid at the molecular level.References1GU RC(顧堯臣.Processing technologyfor non -staple cereals buck wheatprocessing(小宗糧食加工(四蕎麥加工J.Cereal &Feed Industry(糧食與飼料工業(yè),1999(7:19-23.2LIN RF(林汝法.Chinese buck wheat(中國蕎麥M.Beijing:China Agriculture Pr

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34、g.6Cladogram analysis of RaCHS gene and some identified CHS genesFig.5Structure and function domain analysis of RaCHS proteinM,DL 2000marker;1,PCR amplification of full -length cDNAFig.4PCR amplification of full -length cDNAof RaCHS(Continued on page 726苦蕎中查爾酮合成酶全長基因的克隆及序列分析劉凱1,2,胡耀輝1,王冠3,于寒松1*(1.吉林

35、農(nóng)業(yè)大學(xué)食品科學(xué)與工程學(xué)院,吉林長春130118;2.吉林省農(nóng)業(yè)綜合開發(fā)辦公室,吉林長春130118;3.吉林農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院,吉林長春130118摘要目的該研究旨在克隆苦蕎中查爾酮合成酶全長基因。方法選用烏克蘭伊琳娜苦蕎為試驗(yàn)材料,以從葉片中提取的RNA 為模板,應(yīng)用RACE 技術(shù)結(jié)合CODEHOP 引物設(shè)計(jì)方法克隆苦蕎中查爾酮合成酶cDNA 序列,通過電子合并獲得其全長。設(shè)計(jì)基因全長特異性引物,以DNA 為模板進(jìn)行PCR 擴(kuò)增出基因序列。應(yīng)用Clustalxl.81和MEGA4軟件進(jìn)行序列分析和進(jìn)化樹的建立;核酸和蛋白質(zhì)序列同源性分析應(yīng)用NCBI 的Blastn 和Blastp 完成。

36、結(jié)果生物信息學(xué)分析表明,該基因全長1906bp ,具有一個(gè)463bp 的內(nèi)含子序列,編碼區(qū)長度為1188bp ,編碼395個(gè)氨基酸。Blastn 序列比對發(fā)現(xiàn)該試驗(yàn)所獲得的CHS 基因序列與相近物種Rheum palmatum(登錄號:DQ205352.1的CHS 基因同源性達(dá)86%。結(jié)論該研究為闡明苦蕎生物類黃酮合成的分子基礎(chǔ),探索提高苦蕎生物類黃酮含量的有效途徑奠定基礎(chǔ)。關(guān)鍵詞苦蕎;查爾酮合成酶;RACE ;內(nèi)含子;進(jìn)化樹基金項(xiàng)目國家農(nóng)業(yè)部“948”項(xiàng)目(2008-z27。作者簡介劉凱(1970-,男,河北滄縣人,在讀博士,研究方向:植物生物反應(yīng)器與功能性食品。*通訊作者,博士,副教授,研

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