Study On Sucrose Synthase Gene Family And Transcription Regulators Of MeSus1 In Cassava(Manihot Esculenta)

Cassava,Manihot esculenta Crantz,which was derived and domesticated from the tropical lowlands along the southern rim of Amazon basin,is an important starchy root crop.It was widely distributed in the tropical and subtropical of Africa,South America,Southerneast Asia,and characterized as high efficiency photosynthesis,high carbohydrate accumulation potential,and extremely adaptability to diverse environments.Cassava is the staple food in some countries and the storage root is rich in starch.Apart from food usage,it could be used as industrial raw materials,e.g.starch extraction,bioethanol,green chemical materials.Under daylight,assimilate that is mainly sucrose has been transported to the storage root underground by vascular system,and then hydrolyzed by sucrose synthase that is the biochemical marker of sink strength,the hydrolyzate,UDPG or ADPG,would be the substrate for the starch biosynthesis.The sucrose synthase is the first key enzyme of starch biosynthesis pathway,however,details about sucrose synthase is lacking in cassava.Understanding the molecular mechanism of transcriptional regulation will be very important steps to clarify environmental adaptability and efficiency starch accumulation benefit to genetic improvement in cassava.This study was mainly focused on the sucrose synthase family and transcriptional regulation of MeSusl,the results are as followings:1 There are seven members in the sucrose synthase family of cassavaThe gene family could be divided into three groups.Noticeable,about 1 kb leader intron separated the 5'UTR of MeSusl and MeSus4.Internal group,the sequence of exons was.conserved,meanwhile,the introns displayed diverse.All MeSus members were classified into dicot subgroup,and were closely related to Sus proteins from Hevea brasiliensis and Ricinus communis in the Euphorbiaceae family.2 Cloning and characterized the full-lerngth of MeSusl-7The UTR sequences of MeSus 1-7 were cloned by RACE-PCR,and then the full-length transcripts from transcription start site to polyA tail were assembled respectively.Meanwhile,the leader intron exist in the 5'UTR had been confirmed.All cassava MeSus isoforms had at least one Sucrose_synth domain and one Glycos_fransf₁ domain,MeSus6 had a transmembrane structure at carboxyl terminal.3 MeSusl and MeSus4 mainly expressed in cassavaMeSusl and MeSus4 exhibited relatively higher transcript levels in cassava plant and the other five members had remarkably lower transcript levels.The redundancy and differentiation between MeSusl and MeSus4 were noticeable on the transcription level.The transcript of MeSusl could be upregulated by ethylene,indoleacetic acid andcold,heat stress,and downregulated by abscisic acid,gibberellin,salicylic acid,drought,salt stress,respectively.The expression of MeSus4 was increased under treatments of abscisic acid,ethylene,gibberellin,cold stress,and decreased by salicylic acid,indoleacetic acid,heat,drought,salt stress.4 Successfully constructed a full-length cDNA library that could be amplified limitless used for Y1H or Y2H library screeningIn the study,high quality total RNA from storage root sample in three develoment stages was isolated,and then,the mRNA was purificated.Followed the SMART protocol,the double strand cDNA was amplified by LD-PCR.Combined improved prey vector,pGADT7-Rec3,full-length cDNA library was constructed in yeast,the capacity was 4± 1.7×106 cfu.Yeast plasmid mix was isolated by alkaline lysis after digested by Westase,then was transformed into the E.coli competent cell which had quite high transform efficiency.The capacity of cDNA library in E.coli was 1.4±0.3×107 cfu.Before prepared the storage tubes,hundreds colones were detected by PCR,and about 70%colones habored 750 bp inserted.5 Cloning and expression profile of six candidate transcription factors regulate MeSuslThe 5' flank sequence of MeSusl named MeSusl pro was cloned using genomic DNA as template.The bait vector,pMeSuslpro-AbA,was constructed and linearized by Bsp119 I.The backgroud of bait strain was tested after the linearized pMeSus1pro was integrated into genome of YlHGold.Yeast one-hybrid library screening was carried out and details about 24 transcription factors were obtained.Six condidates were selected by the results of expression profile,i.e.bHLHl,ERF72,GRF2,GT1,TALE1,WRKY2.The transcript level of 6 transcription factors in tip leaf and mature leaf were remarkably lower than other tissues and the expression of candidates were affected by phytohormone signal and abiotic stress.6 It is verified that five of these candidate transcription factors can bind to MeSuslproThere were eight DNA binding domain,DBD,in the candidate transcription factors.Six transcription factors were cloned by PCR using first strand cDNA as template and the new prey vectors,AD-TF and AD-TFDBD,were constructed and transformed into bait strain.The results of Y1H assay showed that all the candidates can interact with MeSuslpro in vivo.As the full length coding sequence of 6 transcription factors could not expressed or exist in inclusion body in E.coli,however,6 DBDs of 5 transcription factors were expressed in the supernat.Follow the DPI-ELISA work:flow,the interactions between TFs and MeSuslpro were confirmed in vitro.7 All the candidates were located in the nucleusVectors,CaMV35S pro::TF-GFP,were constructed using new designed vector named pSL1 plus for subcellular location assay,and transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation.The fused protein,TF-GFP,of each candidate was observed in the nucleus of onion epidermal cells under laser scanning confocal microscope.8 Four transcription factors can active the downstream genesVectors,BD-TF,were prepared and transformed into yeast strain Y2HGold.Four reporters were test one by one on the selected medium.The results showed that four TFs including bHLH1,ERF72,GRF2 and GT1 can active at least one reporter,and TALE1 and WRKY2 can not.The transcriptional activity rank from strong to weak is as:ERF72>bHLH1>GT1>GRF2.9 Four transcription factors act as homodimerY2H assays were processed after AD-TF and BD-TF were cotransformed into Y2HGold.The results on the select medium showed that bHLH,1 ERF72,GT1,TALE1 could form homodimer,i.e.these TFs interact with itself,and GRF2,WRKY2 acted as monomer.It could be observed that the protein interactions of bHLH1 and TALE 1 were very strong,the others were weaker.10 Relationships of transcriptional regulation between 6 TFs and MeSusl were confirmedIn the study,Plant one-hybrid assay was designed to confirm relationship between TF and promoter based on dual luciferase assay.Hence,new vector was designed for P1H,named pLuc2.A series vectors which both reporter and effector were assemblied were constructed and transformed.The enzyme activity of luciferase and REN were both tested in one tube after strains were infiltrated into tobacco leaves.The ratio values of luciferase and REN showed that bHLH1 positive regulate the transcription of MeSusl and the others were negative regulators.So far,the following work were going on,including coregulators interact with candidate transcription factors were screening by Y2H,relationships between coregulators,TFs and MeSuslpro were confirmed by P3H,gennetic modified lines of the 6 TFs and coregulators will be obtained,and so on.