| Ananas comosus var.bracteatus（A.var.bracteatus）is an ornamental herbaceous monocot plant cultivated for its colorful leaves and fruits.The chimeric leaves were consisted of normal green parts and albino white parts.The loss of the green leaf color may be due to the apparent expression of degradation pathway genes,where chlorophyll metabolism process is blocked and white color was expressed.Pheophorbide a oxygenase（Pa O）function as a colorless linear tetrapyrroles in the chlorophyll degradation pathway.The degradation process leading to the chimera leaf character formation mechanism remain un-revealed.The protein analysis revealed that Ab Pa O is Rieske-type coordinated by two cysteines and two histidines.It can be located in about eight plant organelles,including the nucleus,mitochondrion,cytoplasm,chloroplast,Golgi body,plasma membrane,extracellular membrane,and peroxisomes by bioinformatics prediction.Ab Pa O was mainly localized in the chloroplast.The Non-yellow coloring 1（NYC1）,expressed apparently higher in green than in white leaf parts.Chlorophyllase（CLH）up-regulates and 7-Hydroxymethyl Chlorophyll a reductase（HCAR）down-regulate consequently to cause lower chlorophyll content.The down-regulation of CHLM and NOL may cause Slip-regenerated White Leave（SWL）albino.Stay Green（SGR）,Pa O,and PPH apparently up-regulated consequently to cause whitening of Tissue Culture White leaves（TCW）.Under 30 days of dark stress,Slip-regenerated Green Leave（SGL）and Tissue Culture Green（TCG）leaves turned white.Glutamyl-t RNA reductase 1,and Protochlorophyllide oxidoreductase（POR）down-regulation and SGR up-regulated to result in SGL albino color formation.The whitening of TCG may caused by the down-regulation of CHLM,NOL and up-regulation of Pa O.The loss of green leaf color affected down-regulation of PGK and Psa A expression,especially in TCG.The enzyme solution optimal method of A.var.bracteatus revealed a higher yield and efficiency of protoplast isolation,1.6×108and 80%respectively,when the optimal conditions were determined to be the most beneficial at 1.8 g mannitol,0.3 g cellulase in 25ml of enzyme solution,and 12 hours of digestion time.By random amplification of polymorphic DNA analysis,the leaf protoplasts had the same banding patterns as the donor plants of the plant leaves.The protoplast culture and callus induction were best in ammonium liquid MS medium supplemented with 1.0 mg L-1 NAA and 1 mg L-1 of BAP.Using polyethylene PEG-4000 in calcium chloride transfection,the study obtained a higher transfection rate more than 70%than expected.The 35S:GFP transfection efficiency was 5x 105 when the subcellular location of GFP and bright field were combined.The p C2300-35S:Ab Pa O-GFP vector was constructed and transformed into protoplasts of A.var.bracteatus leaves.Subcellular localization revealed that 35S:Ab Pa O-GFP function in the chloroplast and causes leaf color loss.The 35S:Ab Pa O-GFP expression analysis in the transformed protoplasts showed the expression of Ab Pa O was significantly increased by50%of the 35S:Ab Pa O-GFP-transformed protoplasts compared to the 35S:GFP vector protoplasts.The wild-type tobacco leaf tissues were sliced into pieces and digested in 25 ml enzyme solution containing 1.8 g mannitol and 0.3 g cellulase for 6 h,the yield of protoplast was3.5×105.The transformation efficiency increased to 80%apparently when using 30μg/ml p C2300-35S:GFP plasmid.The transfection efficiency of p C2300-35S:GFP plasmid introduced into 2×105 ml-1 wild-type tobacco protoplasts incubated in PEG-4000 mediated medium for 22 h efficiency was at 80%,and showed a clear GFP signal in transfected protoplast.The transfection time efficiency increased significantly at 10 min and climaxed to 80%at 15 min.The 35S:Ab Pa O-GFP protein subcellular localization via transient expression showed the clear location of Ab Pa O in the chloroplast.The expression of Ab Pa O in the 35S:Ab Pa O-GFP transformed protoplast was significantly up-regulated.The transformed protoplast were cultured in the MS medium containing 1 mg/L 6-BA,1 mg/L NAA,0.5 mg/L 2,4-D and 5 mg mannitol for about 12 days in dark.In addition,strong microcalli were obtained in full force MS solid medium supplemented with 0.1 mg/L of GA3（gibberellic acid）and 1 mg/L of 2i P（isopentenyl adenine）.Similarly,the transformed tobacco protoplast cells were cultured MS medium containing 1 mg/L 6-BA,1 mg/L NAA,0.5 mg/L 2,4-D and 5 mg mannitol for about 12 days and in the full force of MS solution to obtain the transgenic shoot.The transformation of 35S:Ab Pa O-GFP caused the green color fading phenotype.This research is significant for the study on the role of Ab Pa O gene in leaf chlorophyll degradation and albino mechanism of Ananas comosus var.bracteatus.And the established protoplast isolation,culture,transformation and regeneration techniques of var.bracteatus and tobacco provided an efficient way to study gene function and effectively regenerate plants of Ananas comosus var.bracteatus. |
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