Molecular Regulatory Mechanism Of SERK1 During The Early Somatic Embryogenesis In Pineapple

Pineapple(Ananas comosus L.),as perennial herb,is one of the important tropical fruit in the world.Pineapple SERK1 gene(AcSERK1)is a molecular marker gene of pineapple somatic embryogenesis,which plays an important role in aquired embryogenic ability of pineapple somatic cells,which can effectively improve the frequency of somatic embryogenesis of pineapple.The molecular mechanism of AcSERK1 which is specific expression during the early somatic embryogenesis of pineapple is very important to reveal the molecular mechanism of cell differentiation,embryogenesis and embryo early degradation,and to improve the frequency and synchronization level of somatic embryogenesis.Based on the identification of the specific activity of AcSERK1 5' upstream regulatory sequences,our research further identified the embryogenic cell-specific region with biological function in the 5' upstream regulatory sequence.Yeast one-hybrid system was used to screen out 30 candidate proteins interacting with the embryogenic cell-specific region.In addition,the methylation status of CpG island and the effect of methylation on the expression of AcSERK1 were analyzed.Studying the molecular regulation mechanisms of AcSERK1 expression will lay the foundation of pineapple somatic embryogenesis molecular regulation network.The main results are as follows:The 5' upstream sequence of AcSERK1 was cloned from the pineapple by hi-TAIL PCR.NCBI online BLAST results showed that sequence from the location of 2348 nt to 2503 nt relative to the AcSERK1 initiation codon was homologous to the sequence of the pineapple PPO1 gene,indicating that the full length sequence of 5' upstream regulatory seqence of AcSERK1 was obtained(Genbank access number: KM264323).PlantCare analysis showed that the AcSERK1 promoter sequence contained TATA-BOX,CAAT-BOX and other eukaryotic promoter core elements and 9 hormone response elements,19 environmental response elements and so on.EMBOSS Cpg plot predicted AcSERK1 containing 2 CpG islands near TSS.In order to investigate the transcriptional regulation of AcSERK1,the TSS and the promoter activity of 5' upstream regulatory sequences were analyzed and identified.The TSS was identified by 5'-RACE technique,which is located at 258 nt(G)of sequece upstream relative to the start codon(ATG).The plant expression vector pAS2(-2090 ~ +258):: GUS was constructed by replacing the CaMV 35 S promoter in pBI121 with the 5' upstream complete regulatory sequence of AcSERK1.The activity analysis showed that region from-2090 to +258 can initiate the embryogenic cell-specific expression of GUS.The promoter activity of the regulatory sequence is the same as the expression pattern of AcSERK1.The promoter is embryogenic cell-specific promoter.In order to isolate and identify the embryo-specific region of the AcSERK1 promoter,the promoter activity was analyzed by constructing the promoter 5'-terminal subtractive deletion vector and the internal deletion vector.After the transient transformation of embryogenic callus of pineapple,-983 nt ~-880 nt in the AcSERK1 5' upstream regulatory sequence was isolated.It is the embryogenic cell-specific region of the promoter,which is a necessary functional region of the AcSERK1 embryogenic cell-specific promoter.The yeast one-hybrid bait vector was constructed.Embryonic cell-specific region-983 nt ~-880 nt sequence from AcSERK1 5' upstream regulatory sequence was inserted into pAbAi vector.The bait vector was transferred into the competent cells of Y1 H Gold yeast strain.The autophagy activation was detected on SD/-Ura medium supplemented with different concentrations of Aureobasidin A(AbA).The autologous activation was effectively inhibited when the AbA concentration was 600 ng / mL.The proteins that interact with the embryonic cell-specific region were screened from the cDNA library,and the protein coding genes which could interact with the region were selected,including ribosomal protein,DNA binding protein and some unknown genes,which were used as the candidate proteins,in order to further study for the mechanism of expression of AcSERK1.BSP analysis was performed on the methylation status of CpG islands frome the 5' upstream regulation sequence of AcSERK1 in embryogenic callus and non-embryogenic callus.CpG-1 island contains 32 CpG sites.In the embryogenic cells 29 cytosine(C)sites did not occur DNA methylation,only 3 sites occur DNA methylation.In the nonembryogenic,there were 29 sites with DNA methylation,and only 3 sites without DNA methylation.CpG-2 island consisted 16 CpG sites.In the non-embryogenic,there were 11 sites with DNA methylation.In the embryogenic,there were 2 sites with DNA methylation.The number of methylation sites of CpG islands in embryogenic cells was significantly lower than that in non-embryogenic cells.In addition,it was found that suspension of pineapple callus in liquid MS medium with 5 ?M/L methylation inhibitor 5-azaC for 5 days increased the expression of AcSERK1 in nonembryogenic calli.Methylation inhibitor 5-azaC can also accelerate the process of somatic embryogenesis of pineapple.