Function Analysis Of BpEIN3 Gene In Betula Platyphylla

EIN3 is an important transcription factor in the ethylene signal transduction pathway.It initiates the expression of a series of genes that regulate ethylene,positively regulating ethylene reaction,which affects the various physiological and biochemical processes of plants through interacting ethylene signal and other signals in the signal transduction pathways.In order to reveal the function of BpEIN3 in Betula platyphylla,this study used birch as a genetic transformation receptor,and analyzed expression characteristics of BpEIN3.Constructing BpEIN3 overexpression,inhibit expression vector,we transferedto birch to obtain BpEIN3 transgenic lines and study the function of this gene,which provided a reference for further revealing the molecular mechanism of BpEIN3 regulation of senescence and programmed cell death of birch leaves,The research results are as follows:We studied on the expression characteristics of BpEIN3 used transgenic PromEIN3::GUS lines as materials identified by PCR.The PromEIN3::GUS lines were treated with ET,IAA,MeJA,SA and GA at different time to analyze GUS expression and activity.The results showed that the expression and activity increased under the treatments with different hormones,indicating that BpEIN3 responded to these hormones in varying degrees.In this study,the plant height,diameter and photosynthetic parameters of BpEIN3 transgenic lines were measured.The number of leaf lesions,histochemical staining,antioxidant enzymes,endogenous hormones and the expression of disease-related genes were also recorded.We identified the lines by PCR and there were obvious amplified bands,which showed they were transgenic lines.We analyzed the expression of BpEIN3 by qRT-PCR,which was down-regulated in BpEIN3inhibited expression lines and up-regulated in overexpression lines.There was no difference in leaf and phenotype of transgenic lines at seedling stage.However,the 2-year-old BpEIN3-inhibited expression lines exhibited brown spots on the leaves,leading to premature senescence and defoliation.The number of leaf spots are significantly different between different transgenic lines based on solid microscope.In particular,EY-1 had the highest number of leaf spots,while EY-3 had the largest area of spots which had the largest plant height compared with other lines.Photosynthetic parameters of BpEIN3 inhibited expression lines such as Pn,Gs and Tr were significantly lower than wild type.The accumulation of H2O2 in the leaves of transgenic lines was higher than wild type based on DAB staining.The content of endogenous SA,ABA,SOD,POD and CAT in BpEIN3 inhibited expression lines were higher than those in WT,except JA content.Meanwhile,BpEIN3 inhibited expression lines showed strong resistance to Alternaria alternate(Fr.)Keissle.We analyzed the expression of genes which were involved in ABA,JA and SA biosynthesis,metabolism and senescence.The results showed that PYR9 and PYL4 related to ABA signal transduction,NPR1 related to SA signal transduction and JAZ10 related to JA negative regulatory were up-regulated,which increased with the senescence of leaves.Meanwhile,SAG12 and NAC2 used to indicate aging and PR1a and PR1b involved in defense were up-regulated in 6th and 7th leaves of transgenic lines.Therefore,we concluded that the suppression of BpEIN3 could lead to lesion and promote premature senescence.