Molecular Cloning And Functional Analysis Of Sucrose Synthase Gene CpSUS In Chimonanthus Praecox

Wintersweet[Chinonanthus Praecox（L.）Link],one of the few winter-flowering plants,is popular in landscape.A certain amount of researches on molecular biology have been carried out,but few studies on sugar metabolism-related genes have been reported.Sucrose is a vitally important photosynthate of plants,and the sucrose synthase,being one of the critical enzymes that promote sucrose to participate in various metabolisms in plants,affects the formation of various tissues and organs by regulating the synthesis and utilization of carbohydrates.So it is particularly important to study the function of sucrose synthetase gene for cultivating improved varieties of wintersweet.In this experiment,the CpSUS gene was successfully cloned from wintersweet,and the anlysis of bioinformatics and transcription level were carried out;the 35S::CpSUS tansgenetic lines were obtained by floral dip transformation in A.thaliana Columbia-0,the phenotypic observations and stress treatments were performed subsequently.Based on the above research results,the possible biological function of CpSUS gene during the growth and development was predicted in wintersweet.The main results are as follows:1.Cloning of CpSUS cDNABased on the partial gene sequence of sucrose synthase obtained from the transcriptome database of wintersweet,the full-length cDNA sequence,namely CpSUS,was successfully obtained by RACE.CpSUS is 2920 bp in length and has an ORF of2418 bp which encodes a predicted polypeptide of 805 aa with 5’/3’-UTR of 170 and322 bp.The 3’-end has a typical tailing signal and an obvious ploy A structure.2.Bioinformatics analysis of CpSUSStructure of CpSUS was predicted by bioinformatics softwares,which contained a sucrose synthase domain and a glycosyltransferase GT-B domain,indicating that it belongs to the sucrose synthase gene family.Its molecular formula is C₄₁₅₁H₆₄₆₄N₁₁₁₆O₁₁₉₁S₂₇ and molecular weight is 91.93 k Da,with no transmembrane domain and signal peptide sequence.So it is speculated that CpSUS protein belongs to the protein located in the cytoplasmic matrix or organelle matrix,not to membrane proteins or secreted proteins.The secondary structure of the CpSUS protein is mainly composed ofα-helix（Alpha helix,53.54%）,extended strand（Extended strand,12.80%）,β-turn（Beta turn,6.46%）and random coil（Random coil,27.20%）.By multiple sequence alignment,the putative CpSUS protein shares high similarity with other SUS-related proteins,e.g.,it shows 91 and 83%identity with the products of the SUS in Cinnamomum micranthum and Nelumbo nucifera,respectively.Phylogenetic tree revealed that CpSUS clustered closely to SUS from to C.micranthum.3.Expression analysis of CpSUSWhen analyzing the expression characteristics of CpSUS gene in different tissues of wintersweet,it was found that the gene was highly expressed in the stems,and the expression level in the leaves reached the lowest,and in the middle petals,outer petals,inner petals,pistils,stamens and young fruits all have different expressions.The results of q RT-PCR experiment indicated that CpSUS transcripts were principally found in flower buds at different developmental stages.And the expression level was relatively low before December,which increased significantly after 9-Dec,peaking on 3-Jan in the fully open period.Because of dramatically increasing expression during flowering,it was inferred that CpSUS gene might play a certain role in the flowering process in wintersweet.In addition,the results of qPCR also showed that CpSUS was induced to express at low temperature（4℃）and salt stress（150mM）,while up-regulation and down-regulation both appeared at different time points under waterlogging condition.CpSUS may be involved in plants stress regulation under low temperature,waterlogging and salt stress.4.Phenotypic observation and resistance study of 35S::CpSUS/A.thaliana Col-0The constructed plant over-expression vector pCAMBIA1300-CpSUS was transferred into wild-type A.thaliana Col-0 using floral dip transformation mediated by Agrobacterium.After a series of screening and identification,homozygous T₂generation transgenic A.thaliana seeds were obtained.Based on the results of qPCR,three transgenic lines with high,medium,and low expression levels of CpSUS and Col-0 were selected for the later phenotypic observations and resistance study.The results showed that there was a significant increase in rosette leaves,plant height and fresh weight in the transgenic lines,and an obvious reduction in total sucrose content,the fruit pods also appeared earlier,It is speculated that CpSUS mainly catalyzes the decomposition of sucrose,so that more sucroses are converted into UDPG and fructose,thus leading to a reduction in the sucrose level in the plant.It provided more precursor materials for the plant to synthesize more cellulose and starch,and finally increasing the number of rosette leaves,plant height and fresh weight of the transgenic plants significantly,and also making fruit pods appear earlier.Comparing with wild-type A.thaliana,less tolerant to wateriogging condition was detected in 35S::CpSUS/Col-0 submerged in water for simulating hypoxic stress.There is no significant increase of salt tolerance in 35S::CpSUS/Col-0 treated with 300 mM Na Cl.It is speculated that the CpSUS gene play an important role in hypoxic stress caused by waterlogging condition.Further studies should be carried out to verify whether it responds to salt stress or not.