Isolation Of Embryonic Cell Specific Sequences From Pineapple SERK15’ And Screening,Identification Of Their Interaction Transcription Factors

Pineapple is a perennial herb that is one of the four tropical fruit trees in the world and an important tropical ornamental plant.The pineapple SERK1 gene(AcSERK1)is a marker gene for the somatic embryogenesis of pineapple.It plays an important role in the transformation of non-embryogenic cells into embryonic cells and has been proven to effectively enhance pineapple somatic embryogenesis rate.Therefore,in-depth study on the molecular mechanism of AcSERK1 specific expression regulation in the early stage of pineapple somatic embryogenesis is of great significance to reveal the molecular mechanism of embryogenesis and early embryonic degeneration of cell differentiation.Previous studies by the research group showed that the regulatory elements that regulate the specific expression of AcSERK1 in the embryonic cell phase are in the 5’ upstream-983 nt/-881 nt region.Based on this,this study sought more precise embryogenic cell-specific segments by further cleavage and deletion analysis of-983 nt/-881 nt.The bioinformatics predictive analysis and yeast one-hybrid screening library were used to screen the embryogenic specific region interaction proteins,and two important candidate genes were selected to regulate the role and mechanism of somatic embryo development.The main findings are as follows:1.To further reduce the region of the embryonic cell-specific regulatory elements,the AcSERK15’ upstream-983 nt/-881 nt was spliced,and a series of plant deletion expression vectors were constructed to transform the pineapple.The GUS transient expression analysis technique was used to identify the activity of the regulatory sequences.The results showed that the 41 base sequence of-921 nt/-881 nt upstream of AcSERK1 is a necessary segment of the promoter-specific activity of the promoter.Bioinformatics analysis revealed that the major binding sites may interact with the-921 nt/-881 nt region of SEF3 MOTIFGM,CAATBOX1,ROOTMOTIFTAPOX1,and the transcription factors may be associated with the WRKY family members,other types of transcription factors such as HDZip,KANADI;in addition,there is a conserved region in the region that binds to the embryo related factor SEF3 MOTIFGM.2.The bait vector p Ab Ai-SE41 was constructed by using AcSERK1 5′(-921 nt/-881 nt)sequence as a bait.The yeast single-hybrid technique was used to extract from the pineapple somatic embryo.A total of 33 proteins with an interaction relationship with the-921 nt/-881 nt region were screened in the c DNA library.Protein function annotations indicate that among these proteins,transcription factors such as YABBY,LEA,HDZip,and KAN may be involved in somatic embryo development,including some histones and unknown functional proteins.In the further analysis of the expression of somatic embryos,the transcription factors closely related to somatic embryogenesis were mainly Aco002761.1(HDZip)and Aco015964.1(KANADI),named Ac HB13 and Ac KAN2,respectively.3.Based on the information of the pineapple genome sequence,Ac HB13 and Ac KAN2,were cloned.In the induction process of pineapple callus somatic embryos,two genes can be efficiently induced by 2,4-D,and their expression is accumulated in embryogenic callus induced by 2,4-D for 15 days.With compared to non-embryonicity,the expression levels in callus were 6 and 2.8 times.Ac HB13 and Ac KAN2 have different expression levels in different tissues and organs.Ac HB13 and Ac KAN2 were not induced by high temperature/low temperature stress;Ac HB13 could be induced by ABA,MEJA,SA,Na Cl and PEG6000;Ac KAN2 was induced by Na Cl and SA and inhibited by ABA,MEJA and PEG6000.Ac HB13 and Ac KAN2 are localized in the nucleus.Transcriptional activation experiments confirmed that both have transcriptional activation activity,consistent with the characteristics of typical transcription factors.Agrobacterium tumefaciens-mediated transformation of pineapple embryogenic calli obtained 7 and 8 transgenic plants.overexpressing Ac HB13 and Ac KAN2,respectively.The Ac HB13 and Ac KAN2 expression levels of transgenic strains expressing Ac HB13 and Ac KAN2 in excess were significantly increased,and the expression levels of AcSERK1,Ac HB13 and Ac KAN2 in somatic embryo induction were higher than those of the wild type,and the amount of pineapple somatic embryogenesis was also increased.4.Ac HB13 and Ac KAN2 recombinant proteins were obtained after further purification by prokaryotic expression;yeast one-hybrid,double luciferase transient expression analysis and EMSA experiments confirmed that both Ac HB13 and Ac KAN2 could bind to-921 nt/-881 nt upstream of AcSERK1 transcription initiation site.Ac HB13 and Ac KAN2 activate the promoter activity and regulate the expression of AcSERK1 gene by binding to the two cis-elements of CCCATA and AATTAGAC in this segment;Yeast two-hybrid and BIFC experiments demonstrated that there is an interaction between Ac HB13 and Ac KAN2 proteins,forming a binary complex to function.5.The JASPAR online analysis predicted that 38 transcription factors might bind to the-921 nt/-881 nt region,of which there are 20 transcription factors in the WRKY family,but no WRKY transcription factor was screened in subsequent yeast library screening.To verify the reliability of online predictions,we conducted an analysis of WRKY family members in pineapple.Combined with bioinformatics analysis and PCR experiments,54 WRKY family members of pineapple were identified,and their expression patterns were different.Among them,51 WRKY genes were unevenly distributed on 25 chromosomes,and the other 3 were not anchored.Real-time PCR analysis showed that 8 WRKY genes including Ac WRKY4,Ac WRKY9,Ac WRKY18,Ac WRKY30,Ac WRKY32,Ac WRKY34,Ac WRKY35 and Ac WRKY52 may be involved in the occurrence of pineapple somatic embryos.However,further yeast single-hybrid point-to-point verification results show that 8 WRKY protein does not bind to the-921 nt/-881 nt region.In summary,this study further isolated and identified 41 bp of-921 nt/-881 nt upstream of AcSERK15’ as the necessary segment for embryogenic cell-specific regulation in the early stage of the research group.The candidate transcription factors Ac HB13 and Ac KAN2 binding to the 41 bp segment;it was initially confirmed that there is an interaction between Ac HB13 and Ac KAN2,forming a binary protein complex and two key in the-921 nt/-881 nt segment upstream of AcSERK15’ The combination of the elements(CCCATA and AATTAGAC)affects the transcriptional activity of the AcSERK1 promoter and promotes the specific expression of AcSERK1 in embryonic cells,thereby realizing the transcriptional regulation of somatic embryogenesis in pineapple.