Effects Of NaCl Stress On AsA-GSH Cycle And The MicroRNA Targets Degradome Analysis Involved In Sour Jujube Seedlings

A hydroponic experiment was conducted to study the effects of NaCl concentration（0,50,100,150 and 200 mmol·L-1）and CaCl2 on AsA-GSH cycle in different organs of sour jujube seedlings（Ziziphus acidojujuba C.Y.Cheng et M.J.Liu）.Two degradome libraries（CK and NaCl treatment）had also been constructed to identify the targets of known and novel miRNAs involved in sour jujube seedling leaves.The results of degradome sequences provide reference imformations for additional study about miRNA functions.In our study,the expression of eight miRNA and five mRNA candidate reference genes involved in sour jujube seedlings were tested by qPCR under different NaCl treatments（0,6,12,24 and 48 h）.Three softwares geNorm,NormFinder and Bestkeeper were employed to validate the stability of miRNA and mRNA candidate reference genes.Mian research results are as follows:1.The results showed the NaCl treatments significantly increased AsA and DHA concentrations in jujube roots,stems,and leaves.The AsA/DHA ratio and the GSH concentration in leaves increased and then decreased as NaCl stress increased.The 200 mmol?L-1 NaCl treatment significantly increased GSSG concentration in jujube stems.The GSH/GSSG ratio in stems increased and then decreased as NaCl stress increased.The NaCl treatments significantly reduced APX activity in jujube roots,stems,and leaves.In contrast,NaCl had significant effects on the activity of either GR or MDHAR.The addition of CaCl2 to the hydroponic solution increased AsA concentrations in shoots and stems compared with the NaCl treatment.In contrast,NaCl + CaCl2 reduced AsA concentrations and APX activity in leaves.The NaCl + CaCl2 treatment also significantly increased MDHAR activity in roots and DHAR activity in leaves and stem.The GSH concentration in roots,stems and leaves was less in the NaCl + CaCl2 treatment than in the NaCl treatment.In conclusion,high NaCl concentrations increased AsA and DHA concentrations but reduced APX activity in roots,stems and leaves of sour jujube.The NaCl also increased GSSG concentrations in stems and leaves.In the AsA-GSH cycle in sour jujube,MDHAR is the main enzyme that regenerates AsA from DHA.Exogenous CaCl2 improved AsA regeneration in sour jujube roots,leaves and stems by increasing the MDHAR and DHAR activity in roots,stems,and leaves.The CaCl2 also promoted AsA synthesis in sour jujube leaves,stems and roots.The CaCl2 improved the ability of sour jujube seedlings under NaCl stress to eliminate hydrogen peroxide（H2O2）by increasing AsA concentrations in leaves,roots and stems.2.The expressed stability of the 8 miRNA candidate reference genes（5S rRNA、5.8S rRNA、U6 snRNA、miR472a、miR319、miR156q、novel_mir₅0、novel_mir₆2）and 5 mRNA candidate reference genes（GADPH、UBQ、H3、EF1-α、Actin）under different NaCl treatments was detected by qPCR and validated by geNorem,NormFinder,Bestkeeper softwares.Results showed that the best reference genes for mi RNA qPCR detection were 5S rRNA and miR156 q,for mRNA were Actin and H3.3.By using degradome sequencing,634,882,392 and 601,821,464 reads were obtained in CK and NaCl degradome libraries,respectively.10,517,778（77.86%）and 9,947,967（77.69%）were perfectly mapped to jujube genome by using BLAST software.A total of 232 targets,which contained 28 targets for 57 known and 175 targets for 126 novel miRNAs were obtained after pridicting the cleavage sites.Functional analysis of these miRNA targets indicated that their involvement in important activities,such as plant development,transcription regulation,and stress response.A part of these targets may relate to response to salt stress of sour jujube.