Physiological Responses To Cadmium Stress And Regulation Of Related MiRNAs In Roots Of Brassica Juncea

Mustard(Brassica juncea),an accumulator of various heavy metals,belongs to Cruciferae.Cadmium is one of the non-essential heavy metals,which can inhibit plant growth,change the ultrastructure of chloroplasts,inhibit photosynthesis,induce lipid peroxidation,and therefore have toxic effects on plants.miRNAs are a kind of small molecule RNAs that are widely involved in plant stress resistance.At present,the regulatory role of miRNAs in the response of B.juncea roots to cadmium stress is completely unclear.In this study,the physiological response of mustard roots to cadmium stress was analyzed,and found that the cell activity of mustard roots became worse,the production of reactive oxygen species was increased,and the activity of antioxidant enzymes increased to cope with cadmium stress.The miRNA expression levels were significantly changed under cadmium stress.Finally,miR408 overexpression vector was constructed and T1 transgenic plants were obtained by agrobacteriummediated transformation of Mustard.The results are as follows:(1)Physiological response of B.juncea roots to cadmium stress.The effects of cadmium stress on cell activity,active oxygen level,malondialdehyde content and antioxidant enzymes(superoxide dismutase,peroxidase,catalase,ascorbate peroxidase,and glutathione reductase)in B.juncea roots were studied.The results showed that with the increase of cadmium treatment concentration and the prolongation of time,superoxide anion and hydrogen peroxide gradually increased,and the activity of B.juncea roots cells decreased.The activities of different antioxidant enzymes showed different changing rules with the concentration and time of cadmium stress,but on the whole,the activity of each antioxidant enzyme under cadmium treatment were higher than those in the control.The results indicate that cadmium stress leads to the accumulation of reactive oxygen species in B.juncea roots,which in turn leads to a decrease in cell activity.B.juncea roots respond to oxidative stress caused by cadmium stress by increasing the activities of antioxidant enzymes.(2)Responses of 5 miRNAs to cadmium stress in B.juncea roots.The changes of gene expression of 5 miRNA(miR156,miR397,miR398a,miR398b/c,and miR408)in the roots of B.juncea under cadmium treatment were detected by real-time quantitative PCR.The results showed that 5 miRNAs showed significant up-regulation or down-regulation under different Cd stress treatment times and concentrations.Except for miR156,the other genes had the highest relative expression on the 7 th day.The target gene prediction results showed that these five miRNAs may be involved in the response of B.juncea roots under cadmium stress by regulating the expression of target genes such as laccase and SBP-box transcription factors.(3)Construction of miR408 overexpression vector and genetic transformation.The B.juncea miR408 stem-loop sequence was cloned and ligated into pCAMBIA1300 plasmid to construct an overexpression vector.The regenerated seedlings were obtained by Agrobacterium-mediated genetic transformation,and PCR detection of the regenerated seedlings showed that 3 miR408 transgenic plants were positive.The results of realtime fluorescence quantitative detection showed that the relative expression of miR408 in the T1 generation transgenic plants was significantly upregulated,indicating that transgenic B.juncea overexpressing miR408 was successfully obtained.These results lay a foundation for further revealing that miR408 regulates the response of B.juncea to cadmium stress.