Isolation And Characterization Of Nonspecific Lipid Transfer Protein Genes From Chimonanthus Praecox (L.) Link And Their Involvement In Stress Tolerance

Nonspecific lipid transfer protein(nsLTP) is one kind of the low molecular weight basic proteins, which is widespread in higher plants.The genes encoding the nsLTP are usually in the form of gene family in many plants,such as arabidopsis,barley,et al.The nsLTP memebers have the different biological functions mainly involving synthesis of cutin,regulation of theβ-oxidation in the lipid storage catabolism,formation of somatic embryo,plant signal conduction,plant sexual reproduction,pollen development,defense against the pathogen and the response to the biotic and abiotic stresses,et al.In this paper,four genes encoding the nonspecific lipid transfer protein and two promoters were cloned from Chimonanthus praecox(L.) Link,and the bioinformatics,real time quantitative PCR, prokaryotic expression were used to analyze the difference of the stress resistance among the four Chimonanthus praecox nonspecific lipid transfer protein gene family members.The main results are as follows:(1) Cloning and molecular characteristics of four nsLTP genes from Chimonanthus praecoxWe cloned four nsLTP genes,named CpLTP1,CpLTP2,CpLTP3 and CpLTP4,the Genebank accession numbers of which are FJ889521,FJ904082,FJ904083and FJ904084 respectively,based on the cDNA library construction from Chimonanthus Praecox flower and its EST analysis.The full-length cDNA of the four genes are 611,1016,656 and 997 bp respectively,with similar opening reading frame(ORF) of 360 bp,360 bp,351 and 660 bp without the intron,but have different 5' and 3' untranslated region length and different regulation motifs.Analysis of the structure characteristics and properties of the four nsLTP genes by bioinformatics: With similar molecular weight of 9KD,the CpLTP1,CpLTP2 and CpLTP3 have the significant features of the plant nonspecific lipid transfer protein I(nsLTPI),containing four conversedα-Helix,eight cysteine residues and lipid-binding motifs,obvious signal peptide sequence.All the three proteins are most probably localized in extracellular,and can form the tubular hydrophobic cavity by further tertiary structure prediction.On the contrary,CpLTP4 is very different from the other three encoding proteins.It has the high molecular weight(19.7KD).Although CpLTP4 also has the eight conversed cysteine residues,the position of the eighth cysteine residues exhibits some differences from the nsLTPI type. The CpLTP4 is most likely to be located in the plasma membrane,which is different from the other three members.The tertiary structure prediction indicates that it can form the wide top and narrow bottom triangle-shaped hydrophobic cavity.Cluster Analysis shows that it can get together with the tamarix hispida and ricinus communis,maybe they can form a new type of the lipid transfer protein.(2) Prokaryotic expression and analysis of antibacterial activity against product of four nsLTP genes from Chimonanthus praecox in Escherichia ColiThe Prokaryotic expression vector LTP1-pET,LTP2-pET,LTP3-pET and LTP4-pET were constructed and transformed into the expression strain Origami(DE3).A fairly good soluble expression can be gained under the conditions of 28℃,0.5m MIPTG and 6h induced by optimizing the induced conditions.Four purified recombinant protein were obtained by the His-Bind protein Purification Kit.The results of the bacteriostatic test in vitro sbow that both of the four recombinant protein have the different antibacterial activity.The LTP4-pET recombinant protein has the most strong ability to inhibit the wheat phytoalexin where as the LTP1-pET recombinant protein has the weakest ability on it.The antibacterial activity of the four recombinant proteins against the bacteria should be verified by further research.(3) Abiotic stresses respond analysis of four nsLTP genes from Chimonanthus praecox by real time quantitative PCR.The Chimonanthus praecox plants were treated by cold temperature,drought,high salt and ABA. Then real-time quantitative RT-PCR was used to analyse the expression of four nsLTP genes in Chimonanthus praecox leaves.The results revealed that the four genes were differently regulated by drought,salinity,cold and ABA(abscisic acid).The expression of CpLTP1,CpLTP2 and CpLTP3 genes were general down-regulated by the stress treatment.But the expression of CpLTP4 was general up-regulated and had the highest expression under the cold treatment.The results suggest that although CpLTP1,CpL TP2 and CpLTP3 genes are from the same gene family,they have the distinct differences in functions and probably play different roles in water balance,chilling tolerance, metabolism and other physiological processes of Chimonanthus Praecox.CpLTP4 may play a more important role in resisting the abiotic stress of Chimonanthus Praecox.(4) Isolation and transient expression assays analysis of Chimonanthus praecox CpLTP3 and CpLTP4 promoterThe promoter fragment of 1298bp and 838bp upstream from the 5' upper of the CpLTP3 and CpLTP4 genes was isolated from the genomic DNA of Chimonanthus praecox respectively by hiTAIL-PCR.There is great difference between the two sequences with the identity value of 27.75%. The sequencing and soft analysis suggest that the two sequences have the typical promoter structure. Besides the basic promoter elements like TATA-box,CAAT-box and so on,both of the CpLTP3 and CpLTP4 genes contain many light responsive elements,cis-acting element involved in the plant abiotic stress such as ABRE,G-box and HSE.Some elements such as GARE-motif,TATC-b0x,MBS and TC-rich repeats were only found in the CpLTP4pro sequence,while the cis-acting element MSA-like involved in cell cycle regulation,and cis-acting regulatory element Skn-1_motif required for endosperm expression were found in CpLTP3pro promoter sequence.In order to research the transient expression assays,plant expression vector pBI121- CpLTP3pro and pBI121- CpLTP4pro were constructed by replacing the CaMV35S promoter in pBI121 vector by the Chimonanthus praecox CpLTP3 and CpLTP4 promoterUsing leaf disc transformation method,pBI121-CpLTP3pro and pBI121-CpLTP4pro were transfered into tobacco(W38).The results show that under the induction of GA3,CpLTP4pro promoter can drive the expression of GUS gene,but the CpLTP3pro promoter doesn't have the same ability.In Agrobacterium-mediated transient expression assay,activation of the CpLTP3 and CpLTP4 promoter region(1298bp and 838bp long) occurs in tobacco leaves after treatments with ABA,Nacl,PEG,cold temperature(4℃) and high temperature(37℃) which indicates that the Chimonanthus praecox CpLTP3 and CpLTP4 promoter have the potential of driving the expression of the target genes under the induced condition.To sum up the above results,four nsLTP Genes CpLTP1,CpLTP2,CpLTP3 and CpLTP4 from Chimonanthus praecox(L.) Link have the different stress resistance,CpLTP4 gene probably has the closest relationship with the stress resistance of Chimonanthus praecox(L.) Link.In order to identify the difference among the Chimonanthus praecox(L.) Link nsLTP gene family members,the other gene members and their promoters should be cloned.It is necessary to do some deep research on exploring the differences of the encoding proteins' stress resistance and promoter inducing activity by transgenic and other related technologies.