Cloning And Characterization Of Three Developmental Genes In Brassica Napus

Developmental genes play a significant role in plant architecture formation,and recently there are few research reports in Brassica napus.In this study,to regulate the morphological development,three developmental genes were cloned from B.napus,which were fasciclin-like arabinogalactan protein gene(FLA),sterol carrier protein gene(SCP-2)and carboxylesterase gene(CXE),respectively.The sequence characteristics and expression patterns of these genes were analysed,and their overexpression vectors were constructed and transformed into B.napus or Arabidopsis thaliana,and transformants were obtained.The main results are as follows:1)Fasciclin-like arabinogalactan protein genes(FLAs):These genes have been implicated in cell division and elongation,cell adhesion,plant morphogenesis and other biology processes,which impact on plant morphological development significantly.Study on FLAs will contribute to the regulation of plant architecture.? In this paper,a FLA gene was cloned and designated as BnFLA.The open reading frame(ORF)of BnFLA is 1200 bp in length,and encodes 399 amino acids with a predicted molecular mass of 42.89 kD and a pI of 6.37.BnFLA protein,which belongs to FLAs superfamily,harbours an N-terminal signal sequence,two AGP-like domains,two fasciclin-like domains and a GPI-anchor sequence in the C-terminus.The length of two fasciclin-like domains is between 110-150 bp,which both contain three conserved regions H1,H2 and[YF]H.BnFLA is closer to BrFLA17,AtFLA2 and AtFLA1 than other FLAs,sharing 98%,87%and 57%identity,respectively.? BnFLA is expressed in all B.napus tissues analysed,with a highest expression level in 7-day-old hypocotyls,moderately expressing in 7-day-old cotyledons,and lowest expression in stems at flowering stage.? BnFLA expression is up-regulated by GA3,BR,IAA,ABA and NaCl,but down-regulated by 6-BA,sucrose,cold and PEG.This study suggested that BnFLA might be involved in regulation of growth and development of B.napus through participating in the hormone transduction pathways and response to abiotic stresses.? Overexpression of BnFLA plant expression vector was constructed and introduced into Columbia-0 Arabidopsis via an Agrobacterium-mediated floral-dip method.We obtained 19 T1 transgenic plants of A.thaliana overexpressing BnFLA gene.2)Sterol carrier protein gene(SCP-2):SCP-2 is a small,basic protein involved in intracellular lipid circulation,fatty acid metabolism and peroxisomal ?-oxidation,and relevant to metabolism of plentiful matters;also,it influences plant morphogenesis.? We cloned a SCP-2 gene,and named it as BnSCP-2.The ORF of BnSCP-2 is 372 bp in length,and encodes 123 amino acids with a predicted molecular mass of 13.52 kD and a pI of 9.35.BnSCP-2 has 4 exons(126 bp,61 bp,103 bp,82 bp)and 3 introns(80 bp,102 bp,144 bp).The amino acids of SCP-2 protein are highly conserved,which also contain a peroxisomal targeting signal(PTS1)in the C-terminus.BnSCP-2 is closest to AtSCP-2,with an identity of 95.12%.? BnSCP-2 transcript is expressed in all B.napus tissues analysed,with highly expressing in floral organs,moderately expressing in stems and young siliques,and lowest expression in 7-day-old hypocotyls.? BnSCP-2 expression level is up-regulated by treatments of GA3,BR,IAA,6-BA,ABA and sucrose in hypocotyls.In cotyledons,it increases after 6-BA,ABA and sucrose stresses.These results suggested BnSCP-2 was response to various hormones.? We constructed overexpression of BnSCP-2 plant expression vector,and transformed it into B.napus by means of Agrobacterium-mediated floral-dip infection,finally obtained 10 positive transformants of TO generation overexpressing BnSCP-2 gene.3)Carboxylesterase genes(CXEs):CXEs belong to the ?/? hydrolase fold superfamily,and can hydrolyze esters of short-chain fatty acids.Many signal molecules and ester form inactive conjugates stored in plants,and CXEs can regulate their biological activity through selectively hydrolysis.? We cloned a CXE gene,designated it as BnCXE,The ORF of BnCXE is 969 bp in length,which encodes 322 amino acids with a predicted molecular mass of 35.18 kD and a pI of 5.65.BnCXE contains common conserved motifs in CXEs family,including three glycine residues creating the oxyanion hole whose function is stabilizing the substrate-enzyme intermediate during hydrolysis and catalytic triad made up of a serine,an aspartate and a histidine;besides,BnCXE has conserved hormone-sensitive domains of HGG and GXSXG.Phylogenetic analysis showed BnCXE was closest to AtCXE20.? BnCXE transcript is expressed in all B.napus tissues analysed,with highest expression in leaves at flowering stage,moderately expressing in roots,stems and young siliques.? Overexpression of BnCXE plant expression vector was constructed,and introduced into B.napus through an Agrobacterium-mediated floral-dip method.10 plants of T0 generation B.napus overexpressing BnCXE gene were obtained.