Functional Analysis And Transcriptional Regulation Of AbDVR Gene In Ananas Comosus Var.bracteatus

Ananas comosus var.bracteatus is an important new ornamental plant with colorful green and white chimeric leaves.The chimeric leaves of this plant are composed of normal green tissues and albino white tissues,which is an ideal material for studying the regulation mechanism of chlorophyll synthesis and photosynthesis.Divinyl reductase（DVR）is an indispensable enzyme for biosynthesis of monoethylene chlorophyll（MV-Chl）in plants,and it is of great significance to further study the function and transcriptional regulation of DVR gene for revealing the molecular mechanism of albino leaf.In the study,the key gene of Ab DVR in Chl synthesis was screened by comparative transcriptomic and proteomic analysis,and its function was verified by sequence analysis,expression pattern analysis and genetic transformation.The promoter sequence was cloned and its transcriptional activity was analyzed.The interaction between transcription factor（Ab GNC,Ab GNL）and Ab DVR promoter was verified by yeast one-hybrid.The regulatory effects of Ab GNC and Ab GNL on Ab DVR transcription were analyzed by dual luciferase reporter system.In this paper,the roles of Ab DVR in Chl biosynthesis and the molecular mechanism of albino leaf in A.comosus var.bracteatus were studied.The main results were as follows:1.Comparative transcriptomic and proteomic analysis of white tissues（Whs）at the edge and green tissues（Grs）at the center of chimeric leaves were performed.Using Grs as control,1,683 differentially expressed genes（DEGs）（711 up-regulated and 972down-regulated）were identified by RNA-seq.DEGs were significantly enriched in the cellular energy metabolic pathways such as carbon fixation in photosynthetic and glycolysis pathway.Nine DEGs were annotated in Chl synthesis and all were up-regulated in Whs.The up-regulated DEGs related to Chl synthesis and energy metabolism in albino tissues may be a compensatory mechanism for Chl deficiency.A total of 1,813differentially expressed proteins（DEPs）（1,018 up-regulated and 795 down-regulated）were identified by TMT.Down-regulated DEPs were significantly enriched in photosynthesis and photosynthesis-antenna protein pathways,indicating that photosynthesis of albino tissues was significantly inhibited.The abundance of 9 proteins in Chl anabolism pathway was significantly up-regulated,which also showed compensatory upregulation due to Chl deficiency.The abundance of DVR protein was significantly down-regulated in Whs.Our comparative analysis results showed that protein abundances were positively related with transcriptional levels.DVR was reduced while the expression level of m RNA was up-regulated in Whs.The result suggested that DVR may be a key gene that inhibited Chl synthesis in Whs,and transcriptional and post-transcriptional regulation may play important roles in the albino phenotype of chimeric leaves.2.Bioinformatics analysis showed that Ab DVR was a hydrophilic stable protein with the highest evolutionary homology with DVR of pineapple.The expression of Ab DVR was up-regulated but the enzyme activity was significantly decreased in Whs,which was consistent with the sequencing results.Prokaryotic expression analysis showed that Ab DVR had the ability of encoding complete protein.HPLC results showed that the Whs and Grs of chimeric leaves contained only MV-Chl,but no DV-Chl.And this indicated that Ab DVR had the catalytic function and excluded the possibility of leaf albinism caused by Ab DVR gene mutation.3.The vector of p TCK303-Ab DVR-RNAi was constructed and were transformed to tobacco leaves mediated by agrobacterium.The positive transgenic plants with p TCK303-Ab DVR were identified by PCR.Compared with control plants,transgenic tobacco leaves with p TCK303-Ab DVR-RNAi vector were less green with significantly reduced DVR expression and Chl content,as well as reduced DVR enzyme activity.These results indicated that Ab DVR successfully interfered the expression of tobacco homologous gene,and Ab DVR was a key gene in Chl synthesis.4.We isolated the 5’upstream 2000bp fragment of Ab DVR and named P_{Ab DVR}from A.comosus var.bracteatus.Bioinformatics analysis showed that the promoter fragment contained not only CAAT-box and TATA-box,but also environmental and hormone responsive elements.TFSEARCH result suggested that there were multiple binding sites of GATA family transcription factors in P_{Ab DVR},indicating that the expression of Ab DVR may be regulated by GATA.Transient expression vector P_{Ab DVR}-gus was constructed and infected to the roots,stems and leaves of tobacco by vacuum infiltration method.These results indicated that P_{Ab DVR}had promoter activity but no significant tissue specificity.The activity of P_{Ab DVR}was weaker than that of p BI121,suggesting that the activity of P_{Ab DVR}may be regulated by internal and external environmental factors and transcription factors.5.We cloned the sequences of Ab GNC and Ab GNL genes.Bioinformatics analysis suggested that the amino acid sequences of both genes had typical zinc finger structure(C-X₂-C-X₁₈-C-X₂-C)and LLM domain of B-GATA family.The fusion expression vectors p BI221-Ab GNC-GFP and p BI221-Ab GNL-GFP were constructed,and the results showed that Ab GNC and Ab GNL were located in the nucleus.q RT-PCR analysis showed that Ab GNC was significantly up-regulated in Whs,while Ab GNL was significantly down-regulated in Whs,suggesting that Ab GNC and Ab GNL may have different modes of function.6.Yeast one-hybrid analysis confirmed that both Ab GNCand Ab GNL could bind to P_{Ab DVR}.Transient expression analysis of double luciferase showed that Ab GNC had a significant activation effect on P_{Ab DVR},and regulated the expression of Ab DVR by enhancing the transcriptional activity of P_{Ab DVR}.But Ab GNL inhibited the activity of P_{Ab DVR}significantly.Ab GNC and Ab GNL have different regulatory effects on P_{Ab DVR}.Therefore,the significantly up-regulated expression of Ab GNC and down-regulated expression of Ab GNL in albino tissues of chimeric leaves may be the main reason for the up-regulated expression of Ab DVR gene in albino tissues.