Cloning Of BnCER7 From Brassica Napus And Its Over-expression Analysis In Arabidopsis Thaliana

ECERIFERUM7(CER7)/AtRRP45B is a core subunit of the exosome.It is a positive regulator of cuticular wax biosynthesis in inflorescence and stems in Arabidopsis thaliana,and encodes the main cellular 3' to 5' exoribonuclease.CER7-dependent exosome activity determines stem wax load by controlling transcript levels of the wax-related gene CER3.In this study,the function and expression of the gene BnCER7 were analysed.Firstly,the whole length cDNA of BnCER7(homologous to Arabidopsis CER7)was cloned from Brassica napus cultivars "ZhongShuang 11";its encoding protein function was predicted by analysis and prediction of 1-,2-and-3D structure of BnCER7 protein;on the other hand,the function was analysed by cloning it into the expression vector pCXSN,and transformed it into Arabidopsis;at the same time,the overexpression construct is also used for transformation of B.napus "ZhongShuang 11".In this study,we also analyzed the expression patterns of BnCER7-3(from A genome)and BnCER7-1(from C genome)in different tissues or organs of B.napus,and their expression changes under high and low temperature stresses and SA treatment.The main results are summarized as followings:1.Bioinformatics analysis of B.napus BnCER7 genesFirst,we obtained the CER7 gene sequence from the NCBI website,and compared it to the homologous sequences in B.napus.Five homologous B.napus genes were found in the rapeseed database,named BnCER7-1,BnCER7-2,BnCER7-3,BnCER7-4 and BnCER7-5,respectively.The location of BnCER7-1 and BnCER7-3 in B.napus genome is known,corresponding to BnaC08g30710D and BnaA09g3 8540D,while the location of other copies on B.napus chromosomes is not yet determined.The genes BnCER7-1,-3 and-4 consist of 7 exons and 6 introns,while genes BnCER7-2 and-5 consist of 9 exons and 8 introns.As the GRAVY values of the BnCER7 proteins are negative,the members of the BnCER7 family proteins are all hydrophilic proteins.From the analyses of 3D protein structure instability coefficient(II)and isoelectric point,BnCER7-3 is acid stable protein;BnCER7-4 protein is not stable alkaline protein,but an unstable acidic protein.Protein structure prediction analysis showed that the 2-and 3-dimensional structures of the 5 copies of B.napus BnCER7 as well as that of the Arabidopsis CER7 were similar,indicating that their molecular function may also conserved during evolution.The phylogenetic tree analysis of BnCER7 homologous protein sequences showed that the BnCER7 genes were highly conserved in plant kindow.Analysis of promoter sequence showed that two regulation elements,HSE(high temperature stress induced cis regulatory element)and WUN-motif(wound-responsive element),are present in the promoter region of BnCER7 genes.2.Construction of overexpression vector of BnCER7-1 and transformation of A.thalianaThe full-length CDS sequence of BnCER7-1 was amplified by PCR from from B.napus cDNA,and successfully cloned into the overexpression vector pCXSN.A.thaliana col-0 line was transformed by floral-dip method,and the TO seed seedlings of the A.thaliana were analyzed by PCR detection.The seeds of the T1 generation of successfully transmormed A.thaliana plants were collected,and the plants were analyzed to examine whether the transformed plants were homozygote or not.The T2 plants from a homozygous T1 plant(T1-4)were observed for stem and pod phenotypic changes,such as organization of epicuticular wax.The results showed that under the same growth conditions,there was a certain difference between the BnCER7-1 transformed plants and wild type of A.thaliana T3 generation of homozygous line was produced for further study on the wax phenotypic analysis.3.Construction of overexpression vector of BnCER7-1 and transformation of B.napusThe genetic transformation of B.napus hypocotyl by pCXSN::BnCER7-1 overexpression vector was carried out.One plant of TO generation was successfully obtained.4.Expression pattern analysis of B.napus BnCER7 genesThe expression patterns of B.napus BnCER7-3 and-1 genes in different tissue or organs were analysed.Using real-time fluorescence quantitative PCR(qRT-PCR)analysis method,It was found that both the BnCER7-3 and-1 genes were expressed in root,stem and leaf,mature leaf,bud,flower and silique,but significant difference in expression level was observed.The expression level of BnCER7-3 was highest in pod,followed by bud,the lowest expression level was observed in mature leaves,while the expression level of BnCER7-1 was relatively high in stems,pods and flowers,and relatively lower inbud,the expression level is lowest in the mature leaves.The expressions of BnCER7-3 and BnCER7-1 genes were detected by cDNA in T2 generation seedlings of transgenic A.thaliana,while BnCER7-3 expression was detected in cDNA seedlings of wild type A.thaliana.Analysis of the expression patterns under high temperature stress showed that BnCER7-3 and BnCER7-1 had different responses.Under the 40 ? high temperature treatment,BnCER7-3 was up-regulated and its expression reached the highest level at 2 h of treatment and showed an significant difference between treated plants and control group.The expression level was significantly decreased after 2 h treatment,but the difference was not significant between treated and control plants;the expression of BnCER7-1 was immediately inhibited under high temperature stress.After 2 h of treatment,the expression level of both treated and control plants showed a significant decrease.Under the treatment of low temperature at 4 ?,the expression of BnCER7-3 and BnCER7-1 showed a different tendency.The expression of BnCER7-3 showed no significant difference between the treated and control plants during 0-24 h,but was up-regulated after 48h of treatment at 4?;the expression of BnCER7-1 was down regulated during 0.5-12 h of treatment at 4 ?,but was up-regulated during 24-48 h of low temperature at 4 ?,but no significant difference was observed when compared with the control.Under the treatment of 200 p.M SA,BnCER7-3 and BnCER7-1 showed different expression patterns,the expression of BnCER7-3 reached the highest level at 3h of SA treatment,and was significantly different from the control plants,and then down-regulated at 12h of treatment followed by another round of up-regulation;the expression level of BnCER7-1 gene was slightly increased at 3h of treatment,but not significantly different from that of the control plants.The expression level decreased significantly after 6h of SA treatment,showing inhibition.These results indicate that two genes of BnCER7-3 and BnCER7-1 are responsive to high and low temperature stresses as well as to SA hormone treatment.