Identification Of Litchi AP2/EREBP Transcription Factors And Its Expression Analysis In The Process Of Somatic Embryogenesis

AP2 / EREBP transcription factor is one of the largest family of transcription factors in plants.AP2 / EREBP transcription factors have known to promote somatic embryogenesis,which can provide a new sight to break through the obstacles of highquality crop breeding.However,there still has not been related report about the applcation of AP2/EREBP transcription factors family in the vitro regeneration of litchi.This study analyzed the AP2 / EREBP gene family derived from the litchi genome and studied the expression patterns of several AP2 / EREBP genes during litchi somatic embryogenesis.The main results are as follows:1.202 litchi AP2 / EREBP transcription factors were identified from the litchi genome.Based on the number of domains and characteristics,they were classified into five subfamilies,of which 105 belonged to ERF,70 to DREB,six to RAV and 19 to AP2 as well as two genes(Litc.04A00760,Litc.14A009930)in solo subfamilies.By bioinformatics analysis,the AP2/EREBPs have a total of 20 conserved amino acid motifs,these AP2/EREBP superfamily genes in different subgroups with different gene structure and were unequally located on the 15 litchi chromosomes.2.To compare expression patterns among 202 genes in ten tissues of L.chinensis Sonn.cv.‘Feizixiao’,we found that gene expression patterns in different tissues with differentially and similarity.And analyzed their expression patterns between different litchi varieties with high-frequency somatic embryogenesis L.chinensis Sonn.cv.‘Xinqiumili’ and low-frequency somatic embryogenesis L.chinensis Sonn.cv.‘Dadingxiang’,we found genes lie in same somatic embryogenesis stage of different cultivar were different genes at the same somatic embryogenesis stage in different cultivar have different expression patterns.Subsequently,verified them by q-pcr.3.The regeneration system in vitro of the L.chinensis Sonn.cv.‘Dadingxiang’ was preliminary established by using orthogonal expermients mothod.The best formula for embryogenic callus proliferation medium were: M3,M4;the best formula for somatic embryogenesis medium was D11.After 30 days of somatic embryo differentiation,the somatic embryo differentiation rate is 998 / g FW,and milky white somatic embryos in normal form were obtained;the best formula for somatic embryos maturation medium was C;the best formula for somatic embryo regeneration medium was DR4,with a somatic embryo germination rate of 7.34%.And the somatic embryogenesis of L.chinensis Sonn.cv.‘Dadingxiang’were divided into five stages by paraffin section method.They were,0 days: embryogenic callus;5 days: the stage of somatic embryos began to germinate;10 days: The vigorous multiplying stage of somatic embryos;15 days: the differentiation maximization stage of somatic embryos;20 days: the end stage of somatic embryogenesis.4.Based on the transcriptome data,five candidate genes related to somatic embryogenesis were selected.The medium cultures of different stages of somatic embryogenesis as materials analyed the five genes expression partterns which revealed that only gene Litc.01B032540 was with higher expression level at the germination stage of the somatic embryogenesis.Furthermore,a candidate gene that play a key role in somatic embryogenesis was seleted and colned.The full-length c DNA was 839 bp.By BLAST sequence alignment analysis,it had high homology with the sweet orange ERF114 gene which was proved to have certain effect on the somatic embryogenesis from the previously studied.Hence,it is speculated that litchi Litc.01B032540 may promote somatic embryogenesis in litchi crops.5.Litc.01B032540 gene was ligased into basic vector pCAMBIA1301 to form the plant overexpression vector.Based on the Agrobacterium-transformation system of the litchi variety L.chinensis Sonn.cv.‘Feizixiao’,the Agrobacterium-mediated genetic transformation system of the L.chinensis Sonn.cv.‘Dadingxiang’ was optimized and established.The conditions are : agrobacterium tumefaciens GV3101 as genetically transformed strain,bacterial concentration OD600 = 0.6,callus infection time is 20 minutes,co-cultivation days are 3 days,and the concentration of acetylsyringone added in the resuspension is 100μmol/L,and hygromycin concentration for 25μmol/L,and Timentin concentration for 100μmol/L was used as the ratio of ’Dadingxiang’ resistant callus screening agent.’Dadingxiang’ callus was transformed by agrobacterium-mediated method,the positive callus was identified by GUS histochemical assays and PCR.