Expression And Function Analysis Of BpLTP Gene In Birch

The plant lipid transfer protein(LTP)is a small molecule basic protein.Studies have shown that LTP family genes not only participate in cell wall loosening but also respond to environmental stress.Therefore,this study cloned and screened the salt-tolerance-related Betula platyphylla Suk LTP gene,and initially analyzed its function in resisting salt stress,and obtained the following results:Six LTP gene sequences with complete ORF were obtained by screening and analyzing the whole genome of Betula platyphylla,named BpLTP1-6.The results of bioinformatics analysis showed that the BpLTP1-6 coding region has a sequence length of 350bp-400bp,and all of them have LTP family domains.The BpLTP family proteins are all hydrophobic proteins,and the protein advanced structure prediction analysis shows that the family proteins are composed of α-helix,β-fold,β-turn,and random coils,which are coiled and folded in a three-dimensional conformation.The BpLTP family promoter contains various elements such as ABRE,G-Box,MBS,TAC-element and so on.Quantitative Real-time PCR and subcellular localization analysis results showed that the expression of LTP family genes in leaves was generally higher than that in stems and roots,and only BpLTP3 was expressed in stems higher than other tissues.The six genes responded to salt stress with different degrees and time,but they were all induced by salt stress.BpLTP1,2,5 genes were induced by mannitol stress,BpLTP3,4,6 genes were negatively regulated by mannitol treatment.BpLTP1,2,3 genes are negatively regulated by GA3,BpLTP5 is an early response gene to GA treatment,and BpLTP6 is strongly induced by GA3 treatment.Subcellular localization results showed that BpLTPs proteins were all expressed on the cell membrane.Tobacco transgenic stress test was used to analyze the salt tolerance of BpLTP gene under heterologous transformation.The stress-responsive BpLTP4 gene was transformed into tobacco using Agrobacterium-mediated leaf disc method to obtain overexpressing transgenic lines.After 150 mM NaCl stress treatment,the germination rate,root length,histochemical staining and physiological and biochemical indexes of transgenic lines and wild type controls were measured.The results showed that the germination rate of tobacco seeds of transgenic lines after salt stress treatment was higher than that of WT,and the growth of root length of seedlings of transgenic lines was larger than that of the control.DAB,NTB,and Evans Blue staining results showed that the transgenic lines were lighter colored than the control,and the POD and SOD activities of the overexpressed lines were higher than the control,indicating that the transgenic lines had increased ROS scavenging capacity and decreased ROS accumulation.The cells are less damaged.This shows that BpLTP4 heterologous transformation of tobacco can improve the salt tolerance of transgenic tobacco,and BpLTP4 has a certain salt tolerance function.Agrobacterium-mediated transient transformation was used to study the salt-tolerant function of the homologously transformed BpLTP gene.The pROK Ⅱ-BpLTP3 and pRO K Ⅱ-BpLTP4 vectors were transiently transformed into Betula platyphylla,and then subj ected to NaCl stress treatment.The transiently transformed strains and wild type control s were subjected to histochemical staining and physiological index determination.Histoc hemical staining results showed that DAB and Evans Blue of BpLTP3 were darker than WT after salt stress treatment,and the difference of NBT staining was not obvious.A fter NaCl stress treatment,the DAB and Evans Blue of BpLTP4 were lighter than WT.The results of physiological and biochemical indicators showed that the POD and SO D activities of BpLTP3 overexpressing lines were similar to those of the wild type;the POD and SOD activities of BpLTP4 overexpressing lines were higher than those of th e controls;Salt function.The BpLTP3 gene does not have salt tolerance,and it plays a negative regulatory role in the response of birch to salt stress.This study provides theoretical data for further analysis of the function of LTP fa mily genes of Betula platyphylla,and provides materials for breeding salt-tolerant transg enic Betula platyphylla.