Cloning And Expression Analysis Of Non-specific Transfer Protein Gene From Polygonum Sibiricum Laxm

Non-specific lipid transfer proteins(nsLTPs) is a kind of Pathogenesis-related(PR) protein. They have been suggested to be involved in different aspects of plant physiology and cell biology.Also nsLTPs have been suggested to be important in Several types of Plant stress response.So it is very significant.PsnsLTPs genes have been isolated from SSH library and has been carried on the sequence analysis.By real-time PCR analysis,the differential expression of PsnsLTPs in various tiussue and stress stage were investigated under salt stress and without stress.In order to confirm the function PsnsLTPs expression vectors have been constructed.The main research as fellow:1.Cloning of Coding Sequence of Non-specific Transfer Protein and sequence analysis The full length nsLTPs,a non-specific lipid transfer protein gene,was cloned using RACE (rapid amplification of cDNA end) technology based on a nsLTPs partial sequence obtained from a random clone in SSH library of Polygonum sibiricum Laxm..Bioinformatics analysis indicated that The acquired sequence includes a 5'untranslated region of 65bp,a3' untranslated region of 227 bp with poly(A),and an open reading frame(ORF)302bp,encoding 103 amino acids.31-100 is nsLTP-like function region.The sequencing result revealed that the gene, contained a N-terminalsignal peptide that was a typical conservative region of the nsLTP family.This Protein theory molecular weight is respectively 10.658kD,the isoelectric point 5.01.2.Expression analysis under 3%NaCO₃ Salinity Stress and 1%.0.1%.H₂O without stessThe expression analyses by RT-PCR showed that PsnsLTPs was expressed in leaves,stems and rhizomes of Polygonum sibiricum Laxm.Under the induction of salt stess and without stress.The results showed that the expression of PsnsLTPs has a significantly higher transcript amount in 1 rhizomes,demonstrating a tissue-specific expression property.3.Construct the expression vectorsThe recombinant plasmid pROKâ…¡/PsnsLTPs was constructed by inserting PsnsLTPs gene (cDNA ) into plant expression vector pROKâ…¡.Acquired PsnsLTPs gene by PCR was cuted with Xbaâ… and Kpnâ… enzyme.Finally be translated into pROKâ…¡vector.Construct successfully express vector verified by PCR and enzyme digest detection.