Establishment Of The Protoplasts Transient Gene Expression System And The Subcellular Localization Of Cryptochromes In Soybean

Cryptochromes are a kind of blue light receptors which mediate the regulation of growth and development in plants by mediating various light responses including de-etiolation of seedlings,flowering time,shade avoidance response,and so on.Many secret of cryptochromes have been revealed in Arabidopsis,however,it is little known about the cryptochromes in soybean.The transient gene expression system using protoplasts has provided a rapid and highly efficient way for the subcellular localization.We want to investigate the subcellular localization of soybean cryptochromes in darkness and under blue light using soybean protoplasts and provide information on the function of cryptochromes in soybean.There is little report about soybean protoplasts transformation,however,we have successfully isolated the protoplasts by enzymatic digestion of mesophyll cells from soybean and transformed the expression vector to the protoplasts for subsequent transient expression,which is mediated by PEG.Moreover,we optimized the main influencing factors including the suitable enzyme digestion system,the molecular weight of PEG,the different growth stages of leaves,the plasmids concentrations and the incubation time,and developed an efficient transient gene expression system using soybean mesophyll protoplasts whose transformation efficiency is over 50 %.We studied the subcellular localization of cryptochromes in soybean in darkness and under blue light using this system.The main results are showed as follows:1)The best growth stages of leaves: the 10-day-old unifoliolates after emergence and the first trifoliolates which is just unfolding.2)The best enzyme digestion system: Celluase 1.0 %,Pectolase Y-230.2~0.4 %.3)The best molecular weight of PEG: 4000.4)The best plasmids concentrations: greater than or equal to 1 μg/μL.5)The best incubation time: 15 min.6)The subcellular localization of cryptochromes in soybean: The GmCRY1a、GmCRY1b、GmCRY1c and GmCRY1 d are located in the nucleus and the cytoplasm of cells in darkness,and formed photobody-like structure in cytoplasm while transferred to blue light.The GmCRY2a、GmCRY2b and GmCRY2c are always present in the nucleus,and it is difficult to observe the photobody formation of the GmCRY2a、GmCRY2b and GmCRY2 c.