Screening The Key Gene Of The Albino Of Ananas Comosus Var.bracteatus Leaf And Cloning,Functional Identification Of The AbhemC Gene

Ananas comosus var.bracteatus is an important ornamental plant for its colorful leaves and fruits.Study on the formation mechanism of the chimeric characters are meaningful and important for the regulation of the chimeric character stability.Complete white(CWh)and complete green(CGr)plants derived from in vitro culture were used as typical meterials in this study.The rate-limiting steps and key genes of chlorophyll biosynthesis in albino leaves were screened.The hemC gene(AbhemC)was cloned and its function were verified by transformation.This study provides the theoretical basis for further analysis of the molecular mechanism of the albino phenotype of the leaves.The major study results are as follows:1.Screening of rate-limiting steps and key genes play important role in chlorophyll biosynthesis of albino leavesA total of 1,431 differentially expressed unigenes(DEGs)in between CGr and CWh leaves were identified using RNA-seq.A comparison to the COG,GO and KEGG annotations revealed DEGs involved in chlorophyll biosynthesis,chloroplast development and photosynthesis.The expression patterns of 13 candidate DEGs in chlorophyll biosynthesis pathway between the CGr and CWh leaves were detected by RT-qPCR.The results which showed that only 2 POR genes expression levels in CWh leaves are reduced.verified the reliability of the data of the RNA-seq.Furthermore,the measurement of main precursors of chlorophyll biosynthesis in the CWh and CGr leaves confirmed that the rate-limiting step in chlorophyll biosynthesis.and thus the cause of the albino phenotype of the white cells,was the conversion of pyrrole porphobilinogen(PBG)to uroporphyrinogen ?(Uro ?).The enzyme activity of porphobilinogen deaminase(PBGD)and uroporporphyrinogn ? synthase(UROS).which catalyze the transition of PBG to Uro ?.was significantly decreased in the CWh leaves.Combined with the differences in transcriptome data can be speculated that the hemC gene may be the key gene,and the suppressing function of the protein(PBGD)encoded by hemC gene may be a key factor in the formation of the albino cells.2.Cloning and sequence analysis of the AbhemC geneTwo full length cDNA sequences of AbhemC gene was obtained,defined as AbhemCl and AbhemC2.The AbhemCs was comprised of 1135 bp,containing an 1116bp open reading frame(ORF)which encods 371 amino acids.There are 9 locations with different bases and 2 positions with different amino acids between the two AbhemC genes.They are belonged to the hemC gene family,and shared all the characteristic domains of the hemC family.AbHEMC showed an overall high identity(78%-82%)with other HEMCs at protein level.3.Functional identification of AbhmC gene in tobaccoRNAi recombinanted plasmid of AbhemC were constructed based on the expression vector pFGC5941,the plasmid of pFGC5941-AbhemC and pFGC5941 were transformed to tobacco by Agjrobacterium(EHA105).The positive transgenic plants were identified by PCR.RT-qPCR results showed that the expression level of hemC.in test group(Ri)is reduced significantly compared to negative control group(CK)and positive control group(P).The color of tobacco leaves of Ri group are more yellow than control groups at both seedling and mature stage.The measurement of main precursors of chlorophyll biosynthesis in tobacco of Ri group showed that content of Uro? and Chi reduced significantly compareing to control group(CK)and positive control group(P).The enzyme activity of PBGD was significantly decreased in Ri group.It is suggested that hemC gene and the activity of PBGD protein play a key role in the albinism of the leaf cells.4.Prokaryotic expression of two AbhemC genesProkaryotic expression recombinanted plasmid of two AbhemCs was constructed.and recombinanted protein was expressed in E.coli Rosetta-gami(DE3).The activity of the two recombinanted proteins was measured,but there is no significant differences between the two proteins.