Cloning And Functional Analysis Of LkERF-B2 Transcription Factor Gene From Larix Kaempferi

AP2/ERF transcription factors play important roles in the growth and development of plants.ERF subfamily transcription factors are mainly involved in response to stress.In the present study,Larix kaempferi was selected as material to investigate the function of LkERF-B2.Based on transcriptome sequencing data,an ERF transcription factor gene was amplified by reverse transcription-PCR（RT-PCR）.Phylogenetic and physicochemical characteristics of LkERF-B2 were further analyzed using bioinformatics software.At the same time,plant expression vector was successfully constructed.Functional characteristics of LkERF-B2 were systematically studied via genetic transformation.Main results were as following:（1）Total RNA was extracted from leaves of Larix kaempferi.A full-length coding sequence of ERF transcription factor gene,named LkERF-B2,was obtained by RT-PCR amplification.Sequence analysis showed that the size of LkERF-B2 was 1344 bp,encoding a protein of 447 amino acids.The LkERF-B2 was predicted to contain one AP2/ERF domain.（2）Phylogenetic analysis and homology analysis showed that LkERF-B2 protein had the highest sequence similarity（about 79.0%）and closest genetic relationship to Picea sitchensis.Hydrophilic amino acids and hydrophobic amino acids of LkERF-B2 were 46.4%and 40.5%,respectively.LkERF-B2 was a hydrophilic protein,and had no transmembrane region.Secondary structure of LkERF-B2 contained 5.59%alpha-helix,3.58%beta-sheet and 90.83%loops.（3）Plant expression vector pRI201-AN-GUS-LkERF-B2 was successfully constructed,and transformed to Arabidopsis thaliana via dip flower.Five 35S::LkERF-B2 transgenic Arabidopsis thaliana homozygous lines were obtained.GUS analysis showed that LkERF-B2was mainly expressed in roots and leaf tips of transgenic Arabidopsis thaliana.（4）Under different stress conditions（low temperature,high temperature,salt and drought）,the survival rates of 35S::LkERF-B2 transgenic Arabidopsis thaliana seedlings were14.82%±1.85,44.45%±1.39,52.78%±1.96,23.15%±1.22,which were significantly higher than control Arabidopsis thaliana.The results showed that LkERF-B2 could significantly improve stress resistance in Arabidopsis thaliana.（5）The results of salt stress showed that low concentration of NaCl(100 mmol·L^-1,150mmol·L^-1)could promote Arabidopsis thaliana growth and development,and high concentration of NaCl(200 mmol·L^-1,300 mmol·L^-1)inhibit growth and development.Under low concentration of NaCl treatment,salt injury rate and salt injury index of transgenic plants were significantly lower than control.There was no significant difference between control and transgenic plants under high concentration of NaCl.The activities of SOD and POD in control and transgenic plants were increased with the increase of NaCl concentration.The activities of SOD and POD in transgenic plants were significantly higher than control under 300mmol·L^-1 NaCl treatment.Soluble sugar content of control and transgenic plants increased and then decreased with the increase of NaCl concentration.Under 100 mmol·L^-1 NaCl treatment,soluble sugar content reached to maximum.The content of soluble sugar in control and transgenic plants were 1.50%±0.05 and 1.74%±0.08,respectively.Soluble protein content of wild-type and transgenic plants showed a decreasing trend with the increase of NaCl concentration.When the concentration of NaCl was 0 mmol·L^-1-250 mmol·L^-1,soluble protein content of transgenic plants was significantly higher than control.When NaCl concentration was 300 mmol·L^-1,there was no significant difference between control and transgenic plants.The contents of chlorophyll-a,total chlorophyll and carotenoids in control and transgenic plants showed a decreasing trend with increasing NaCl concentration.When the concentration of NaCl was 250 mmol·L^-1 and 300 mmol·L^-1,the contents of chlorophyll a,total chlorophyll and carotenoids in transgenic plants were significantly higher than control.Chlorophyll b content in control and transgenic plants increased and then decreased with the increase of NaCl concentration,both of which reached to maximum under 150 mmol·L^-1NaCl treatment.