| Soluble starch synthases(SSSs)is an important component of starch synthases(SS),and also the core enzyme of synthetic starch of plant organism.Soluble starch synthases and branching enzyme(BE)are considered to be the major enzymes taking part in the synthesis of starch cellulose.What's more,soluble starch synthases is also deemed as a participant in the production of starch granule structures of various starch crops.Cassava starch is widely used as the raw material of many kinds of food,fodder as well as industrial starch products.However,at present,there is no promoter research report of cassava soluble starch synthases gene in China.In this research,by inquiring the first exon of MeSSS ?b as well as the upstream sequence in cassava genome database,which are taken as reference sequence,through the expansion of general PCR in cassava cultispecies KU50,268bp sequence of the upstream is achieved.Carrying out promoter structure and element analysis on the achieved upstream sequence,and comparing it with the promoter of homologous gene of other species,the upstream 1566bp promoter region of translation initiation site is determined.Then expression vectors of protein plants combining 5' end gradient loss promoter,including the entire promoter,and GUS is designed as well as constructed.After function loss analysis of promoter by transferring tobacco in the way of leaf disc,it is discovered that 138bp of upstream translation initiation site is the core promoter region.Loss from-1566bp to-1356bp in the upstream of translation initiation site will lead to the lost activity of promoter.The region from-1356bp to-531 bp is rich in AT basic group,especially from-531 bp to-453bp,where there may be unknown element or secondary structure that can strongly inhibit the activity of promoter.The region from-387bp to-138bp causes the expression activity of the missing promoter to decrease;while missing in the region from-453bp to-387bp leads to an increase of the expression activity of promoter,which implies that silent hormone regulating element may impose certain influence on the activity of promoter.In order to further explore the response activity of sequence region with different elements to environmental factors,a treatment experiment directing at tissue culture seeding of cassava group KU50 as well as promoter transgenosis tobacco.After implementing an expression pattern analysis of MeSSS ?b in tissue culture seeding of cassava group KU50 with fluorescent quantitation PCR,it is confirmed that MeSSS II b has response activity towards ethylene treatment as well as drought stress.In addition,in 3 hours after ethylene treatment,the expression value in the maximum in transcriptional level;and in 0.5 hours after drought treatment,the expression value gets the peak in transcriptional level.After determining the response activity of MeSSS ?b towards ethylene treatment and drought treatment as well as the time point of expression value peak,the same treatments are carried out on transgenosis tobacco.In 4 hours after ethylene treatment and 1 hour after drought treatment,the activity of promoter is analyzed through GUS protease activity.It is discovered that both the response activity of MeSSS ?b towards ethylene and drought and its basic expression activity sequence are in the conservative core promoter region.The response regulation of MeSSS ?b towards ethylene is not limited within the regulating elements in 1566bp sequence region of the upstream of the translation initiation site.Corresponding signal cascade regulating also exists in the more upstream sequence region.In MeSSS ?b promoter and in the sequence region from-387bp to-138bp,there may be unknown response regulating elements towards drought stress. |
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