Isolation And Functional Characterization Of MeCWINV3 Promoter From Manihot Esculenta Crantz

Cassava(Manihot esculenta Crantz)is a very important energy source,which is a starch storage root crop.Cassava is a kind of C3 plants,but it has the characteristics of C4 plants and their photosynthetic efficiency is much higher than C3 plants.The amount of the storaged energy source by root in the cassava is less than one fifth of the amount of photosynthate synthesis by leaves,so it is meanful to improve energy transferring rate from leaves(source)to roots(library)for cassava starch accumulation.Sucrose cell wall invertase is the key enzyme of the sucrose-metabolizing enzymes,which transfers photosynthate synthezied energy from leaves to roots.The promoter,plays a vital role in regulating gene transcription,is also an important element of genetic engineering,so it is very significant to research the function of promoter for gene regulation mechanism.The MeCWINV3 gene potential promoter region was amplified by PCR using cassava genomic DNA as template and specific primers designed according to the CDS sequence of cassava cell wall invertase gene MeCWINV3 and the predicted gene sequence in cassava genome database.A 1351 bp sequence was isolated,which contains 191 bp CDS sequence and 1160 bp potential promoter sequence.PlantCARE and PLACE software analysis indicated that the promoter contains both typical TATA box and CAAT box and other cis-acting elements that control plant responses to hormone,high and low temperature stress and light.Online bio-informatic analyses of the promoter sequence find some cis-acting elements that relate to stress responses in the promoter of MeCWINV3.The expression mode to different stresses of MeCWINV3 including low temperature stresses of 4?,16 ?and four hormone treatments:100 ?MSA,30 ?M GA3,1%ethephon and 30 ?M ABA were studied in tissue culture seedling of cassava plants by Real-time PCR.The results showed that MeCWINV3 gene expressions were regulated by stresses,which infered that the cell wall invertase promoter of MeCWINV3 may have cis-element ralated to adversity response.A set of 5'deletion mutant promoters were obtained by PCR methods,and six pVKH-CW3-GUS expression vectors were constructed by replacing CaMV35S promoter with MeCWINV3 deleted promoters to fuse with the GUS and then were stably expressed in transgenic Arabidopsis by Agrobacterium-med iated vacuum infiltration.The histochemical GUS staining analysis revealed that MeCWINV3 promoter has the promoter function,which can drive the expression of target gene in transgenic Arabidopsis.The upstream transcription regulatory proteins were screened by yeast one-hybrid method,which is combined with MeCWINV3 promoter.The core sequence of MeCWINV3 promoter was selected and cloned according to the analysis of GUS activity of mutational promoters.The cloned CW3 promoter is connected with the report carrier pAbAi to construct the bait vector CW3-pAbAi.We got the bait strains YIHGold[CW3-pAbAi]by transforming bait plasmids CW3-pAbAi into yeast cells.The ds cDNA library was constructed by extracting total RNA from cassava.The ds cDNA library and pGADT7-Rec vector were transformed into yeast cells.After double screening in SD/-Leu/AbA medium,the positive clones were obtained and sequenced.BLAST homology analysis in NCBI database showed that these proteins are involved in hydrogen peroxide enzyme CAT1,abscisic acid protein5,abscisic acid receptor protein PYL8,Metal sulfur protein,proline PRCC,drought stress factor,vacuole protein,etc.