| The hydrangeas have unique ornamental,which has high value for viewing and research.Under different processing states of hydrangea,the flower organs show different colors.Flower color is an important character in ornamental plants.The formation and the regulation of flower color are affected by many factors.Vacuole p H value is one of the most important factors.This study started from the perspective of vacuolar p H regulated flower color changes.Through the screening of transcriptome data,the gene NHX1,which affects the vacuole p H of hydrangea sepals,was further studied.Additionally,the existing problem in the mechanism of vacuole p H value regulating flower color formation and future prospects were analyzed.This paper would be useful for further research in vacuole p H value related genes function,as well as color regulation and molecular breeding in ornamental plants.1.Technology Improvement of Sepal Colored Protoplast Isolation in Hydrangea macrophyllaIsolation system of sepal colored protoplast in Hydrangea macrophylla is benefit for research on mechanism of color formation.The emzymatic hydrolysis system was established,which included hydrangea sepals treated methods,the component of enzymatic hydrolysate,enzymolysis time,centrifugal rate for protoplast collection.The result showed that the method of toring the epidermis of sepals,4%(w/v)cellulase,0.4%(w/v)macerozyme,0.6 mol.L-1 mannitol,and 0.1%(w/v)BSA,10 mmol.L-1 Ca Cl2,enzymolysis for 2h,and 150*g centrifuge at 4°C are optimal for enzymolysis.This system was used to isolate sepal colored protoplast of four cultivars in Hydrangea macrophylla,and 13-35 protoplasts per microscope vision at 200×were obtained.H+flux measurement showed that the H~+effluxing tendency of blue vacuoles was more obvious than that of pink vacuoles in the same cultivar,which indicated that p H in blue vacuoles was higher than that in pink vacuoles in the same cultivar.2.Reliable reference genes selection for normalization genes expression of controlling hydrangea sepals color in‘Bailmer’and‘Duro’.The accuracy of relative quantitative analysis of RT-q PCR depends on reliable reference genes.However,up to now,a spot of reference genes have been reported in hydrangeas.In different cultivar groups,different development stages groups,an aluminum treatment and a control group,eight candidate genes were selected(Actin7,EF1-α,EF1-β,GADPH,RPL10,RPL34,RPS4,UBI),all of said genes being ranked by ge Norm,Norm Finder,Best Keeper and Ref Finder.EF1-β(elongation factor 1-beta)and RPL34(60S ribosomal protein L34)are the two most stable reference genes in all materials.CCR(cinnamoyl‐Co A Reductase),NHX1(sodium/hydrogen exchanger 1-like)and LODX(leucoanthocyanidin dioxygenase)are used to verify the exactitude of reference genes.EF1-βand RPL34 were preferable for quantifying the target gene expression as reference genes during the experiment,and EF1-β,RPL34 or the double internal reference method could be selected according to the Cq value of target genes during calculation.In the present study,reference genes were screened in Hydrangeas for the first time,thereby providing a technical basis for further experiments.3.Cloning and expression analysis of vacuolar p H regulating gene NHX1NHX1 has an open reading frame of 1626 bp and encodes a total of 541 amino acids.Bioinformatics analysis showed that the gene encodes a Na~+/H~+exchanger located on the vacuolar membrane.Tissue-specific expression analysis showed that the expression level of this gene in the treatment group was higher than that in the control group,and the expression level increase in development stages.The ion currents of the colored vacuoles of hydrangea in the treatment group and the control group were measured respectively,and the results showed that the proton pump encoded by NHX1 was indeed a Na+/H+exchanger. |
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