Study On The Function Of ClKptA/Tpt1 Gene In Cunninghamia Lanceolate(Lamb.) Hook

KptA/Tptl is a kind of phosphotransferase,which is ubiquitous in organisms.Its function is to transfer the phosphate group to NAD+during the splicing process of tRNA,and then form ADP-ribosylation.However,the regulatory network involving KptA/Tptl gene and whether it is regulated by phosphorus in plants is still unknown.Phosphorus play an essential role in plant growth and development,not only for nucleic acid formaton and signal transduction pathway.In addition,phosphorus is also an important factor affecting lignin synthesis,which can increase lignin content and improve wood properties.Cunninghamia lanceolate(Lamb.)Hook is the main timber tree species in south China.However,the ClKptA/Tpt1 gene of C.lanceolata has not been cloned or studied yet.Therefore,the objective of this study is cloning ClKptA/Tpt1 gene based on the transcriptome data of phosphorus treated C.lanceolata,and studied the following contents by using biotechnology such as genetic engineering:(1)We Cloned ClKptA/Tpt1 gene based on transcriptome database from phosphorus treated C.lanceolate.The results showed that the total length of CDS(code sequence)of ClKptA/Tpt1 gene was 1143 bp,encoding 380 aa.By comparison amino acid sequence had found that the protein was highly conserved in different species and had characteristic HGT motif.Functional domain analysis revealed that the functional domain of PTS-2-RNA superfamily was ubiquitous in KptA/Tpt1 gene from prokaryotes to eukaryotes.Evolutionary tree analysis showed that ClKptA/Tpt1 genes had the closest relationship with the KptA/Tpt1 of Hevea brasiliensis and the furthest relationship Abrus precatorius.The results showed that KptA/Tpt1 genes in woody and herbaceous plants were distantly related.ClKptA/Tpt1 protein tertiary structure prediction found that mainly in the form of α-helical and β-folded.(2)We extracted RNA of C.lanceolata different organs and transcribed into cDNA.The expression pattern of ClKptA/Tpt1 has been investigated in root,stem,leaf,flower,and fruit of C.lanceolata.The result indicated that the expression level of ClKptA/Tpt1 highest in C.lanceolata leaves and the lowest in stems.After 60 d of C.lanceolata was treated with 0.0 mmol/L,0.067 mmol/L,0.133 mmol/L and 0.20 mmol/L of different concentrations of phosphorus,the study showed that different concentrations of phosphorus affected the expression level of ClKptA/Tpt1,and the expression level of ClKptA/Tpt1 also increased during soil phosphorus concentration increasing.The lignin content also increased with the expression level of ClKptA/Tpt1.The frozen sections showed that with the increase of phosphorus concentration,the xylem became darker and denser.The results showed that phosphorus regulated the expression of the ClKptA/Tpt1,which in turn affected the biosynthesis of lignin.In addition,we cloned 500 bp promoter sequence of ClKptA/Tpt1 by using genome walking method.Analysis of PlantCare showed that it contained 8 TATA-box and 12 CAAT-box,two important cis-acting components.Therefore,the promoter sequence satisfies the basic core promoter element.In addition to the important cis-acting elements mentioned above,there ARE the original ARE and the enhancer CAAT-box which respond to oxygen stress.In addition,we has constructed PClKptA/Tpt1::GUS resistant tissue culture seedlings and the subcellular localization vector pE3308-GFP-ClKptA/Tpt1 of ClKptA/Tpt1.(3)We constructed pCold-TF-ClKptA/Tpt1 prokaryotic expression vector,the protein has been expressed in E.coli using the final concentration of 0.0 mmol/L,0.4 mmol/L,0.6 mmol/L,0.8 mmol/L ITPG induced.SDS-PAGE electrophoresis showed that,compared with pCold-TF vector,there was a large amount of protein enrichment at about 100 kD.The activity of the protease was determined by purifying the protein with RNA as the substrate and adding the final concentration of 0.0 μmol/L,0.1μmol/L,0.2 μmol/L,0.4 μmol/L,0.8 μmol/L phosphorus.The results showed that ClKptA/Tptl protein could degrade the substrate RNA.The activity of the enzyme of ClKptA/Tptl protein was the highest at the concentration of 0.1 μmol/L.Subsequently,the concentration of phosphorus decreases with the increase.The results showed that the activity of ClKptA/Tptl protein was inhibited by phosphorus to some extent.We had been constructed pBI121-ClKptA/Tpt1 expression vector,by using the method of agrobacterium mediated transformation of poplar had expressed the fir ClKptA/Tpt1 gene transgenic poplar somaclone,named ClKptA/Tpt1-1,ClKptA/Tpt1-2,ClKptA/Tpt1-3.And then,the lignin of ClKptA/Tptl transgenic poplar and wild type poplar are analyzed,and the influence of high phosphorus on lignin biosynthesis results show that the lignin content of transgenic poplar is wild type lignin content of 1.52,1.68 times and 1.75 times.In addition,using frozen sections,found that the transgenic poplar xylem dyed dark area is large,the xylem cells closely followed by 24 mg/L of phosphoric treated with wild type poplar transgenic poplar,the determination of lignin content results show that under high phosphorus treatment.The results showed that the lignin content of wild and transgenic plants increased under high phosphorus treatment,and the levels of ClKptA/Tpt1-1,ClKptA/Tpt1-2 and ClKptA/Tpt1-3 of transgenic poplar trees were 1.18 times,1.27 times and 1.34 times of those of the wild type.The lignin content of wild and transgenic poplar increased,but the growth rate of wild poplar was higher than that of transgenic poplar.The results indicated that the overexpression of ClKptA/Tptl in C.lanceolata increased the content of lignin in poplar,but inhibited the expression of ClKptA/Tpt1 gene under high phosphorus content of 24 mg/L,which reduced the growth rate of lignin in transgenic poplar.