| Cestrum nocturanum is a species of plant belonging to cestrum genus, solanaceaefamily, and could be used for development of bio-pesticide, landscape design,pot-cultivation, flower arrangement, yard-laying-out, decroration of window, pavilionand pondside. Terefore, it was a high-valued garden plant with a broad applicationaland developmental prospect.The main characteristics distinguished from conventionalplants was that petals opened and sended out strong smell at night, and that the twobehavours happened in the meantime and controlled by circuradian clock Cestrumnocturanum was a model plant for research on flowering and smell release at nightand control of circuradian clock.A Normalized full-length cDNA library was constructed by combination ofSMART technology and DSN digestion with petals of flowers opening and releasingstrong smell at night as materials, and some single clones were sequenced for ESTanylases and function annotation, three genes related to formation of flower fragnacesnamed CnNAC,CnMYB and CnGCS,were selected for full-length sequencing andsequence analyses according to the results on components of smell, metabolicpathway in fragrance, function forecast of ESTs，and functions of transcription factorsin flowering and formation of fragrance. Plant over-expression vectors wereconstructed by conservative regions of cDNA of the three genes, and transgenictobacco plants were obtained by agrobacterium mediated transformation, thefunctions of the three genes were identified preliminarily, which will provide thetheory basis for research on flowering, formation of smells and control of circuradianclock in night-flowering plants, and has an important significances in theory and agreat value in practice. The main results are as follows:1.The efficient platform of Normalized full-length cDNA library construction wasdeveloped: A Normalized full-length cDNA library of Cestrum nocturanum ataromatic period was constructed by combination of SMART technology and DSNdigestion, the results showed that transformation efficiency of ligation productwithout desalting was7,000colonies per miroliter of ligation product, and theorical recombination rate was96%, and that transformation efficiency of ligation productafter desalting was4.1million colonies per miroliter of ligation product, which ismore585times than that of ligation product without desalting, and theoricalrecombination rate was98%.The results of colony PCR and sequencing proved thatthe insert fragments was contained in blue colonies, and size of insert fragments in allclones averaged was1.6kb. Therefore, theorical recombination rate would bemodified to100%.2.The ESTs in the Normalized full-length cDNA library were sequenced andanalysized: Five hundred of single colonies including50blue colonies were randomlypicked for sequencing, four hundred and sixty four of effective sequences wereobtained by sequence assembly,removal of contaminating and frame-shifting mutativesequences. The results of analysis of contigs showed that the number of unigenes was426containing26contigs accounted for5.6%,and containing400singleton accountedfor94.4%, the redundancy rate was8.1%, which suggested that the results ofnormalization in constructing cDNA library was better than that of ordinary methods.ESTs were annotated and classified by online Blast2go software, the results showedthat genes related to formation of fragrance and circadian clock were27, includinggenes encoding transcription factors such as NAC, MYB, etc.3.The sequences of genes related to formation of fragrance were analysized: Threegenes (two transcription factor genes:NAC named CnNAC and MYB namedCnMYB, gene named CnGCS encoding glutamylcysteine synthase) that were relatedto formation of fragnance and circadian clock were selected,according to the results ofhomological analyses of amino acids and previous research on fragnance. The resultsof bioinformatical analysis showed that full-length of CnNAC was1,624bp, whichcontained179bp of5’-UTR,200bp of3’-UTR,1,245bp of ORF encoding414aminoacids whose homology with NAM transcription factor family of Petunia and that ofNAC2transcription factor of Arabidopsis, was82%and66%, respectively. Theresults of analyses of protein domains indicated that a conserved sequence containingDNA-binding domain existed in N terminus of CnNAC protein, which could concludethat CnNAC gene was a member of the NAC gene family. The full-length of gene encoding CnMYB was1,439bp, which contained178bp of5’UTR,286bp of3’ UTR,975bp of ORF encoding324amino acids whose homology with MYB133transcription factor of Soybean and that of MYB1transcription factor ofChimonanthus praecox was57%, and that of MYB40transcription factor of Alfalfawas53%. The results of analyses of protein domain indicated that a conservedsequence containing DNA-binding domain(R2and R3) of CnMYB protein,whichcould conclude that CnMYB gene was a member of the MYB gene family.Thefull-length of gene encoding CnGCS protein was2,177bp, which contained374bpof5’UTR,246bp of3’UTR,1,557bp of ORF encoding518amino acids whosehomology with the GSH1gene of Tomato and the AtGCS of Arabidopsis and theLjGCS of Lotus, was89%,78%and86%respectively. The results of analyses ofprotein domain indicated that a conserved sequence containing DNA-binding domainof CnGCS protein, which could conclude that CnGCS gene was a member of the GCSgene family. It concluded that the CnNAC, CnMYB and CnGCS were closely relatedto formation of fragrance in Cestrum nocturanum.4. The genes related to fragnance formation genes were transformed into tobacco:The conservative regions of the above three genes were inserted into the plantoverexpressive vectors, transgenic tobacco plantlets were obtained by agrobacteriummediated transformation, the results of genomic PCR showed that the above threegenes didn’t exist in genome of control K326, that efficiency of transformation ofCnNAC, CnMYB and CnGCS was77%,100%and71%, respectively. Which wouldprovide referrence to further research on mechanism of formation of fragnance inplants flowering at night. |
