Physiological Responses To Cadmium Stress And Related MiRNAs In Brassica Juncea

Heavy metal pollution in soil is a global environmental problem,so it's of great significance to study soil heavy metal pollution remediation technology.Soil phytoremediation techniques for heavy metal pollution include plant extraction,rhizosphere filtration,plant stabilization,and plant volatilization.Among them,the applicability of plant extraction is the strongest,that is using hyperaccumulating plants to absorb and transport heavy metals from soil to the aboveground parts,and remove heavy metals from soil by harvesting plant stems and leaves.Brassica juncea(B.juncea)has great application potential in phytoremediation.In this paper,the physiological changes of B.juncea and the expression levels of related miRNAs under Cadmium(Cd)stress were studied.The main results are as follows:(1)The seedlings of B.juncea were cultivated in 1/4 Hoagland nutrient solution until the plant had 5 euphylla.Then the seedlings were treated with 0?M,10?M,80?M,and 200?M Cd for 0h,6h,48h,and 7d respectively.The contents of Cd,malondialdehyde(MDA),H2O2 and the activity of antioxidant enzymes(SOD,POD,CAT)were determined in different treatments of B.juncea.The results showed that as the concentration and the time of Cd treatment increased,the content of Cd in B.juncea roots and leaves also increased;the content of MDA in leaves increased during 0h-7d of Cd treatment;As the concentration of Cd increased and the treatment time prolonged,DAB staining spots on leaves became more obvious;The activity of SOD,CAT and POD kept rising and reached the highest within 0h-48h,and fell during 48h-7d.This indicated that Cd stress resulted in increased ROS and lipid peroxidation in B.juncea.Moreover,the stress time and concentration of Cd are positively correlated with the degree of oxidative stress,as shown by the higher content of MDA and H2O2 in B.juncea leaves.Antioxidant enzymes were important mechanisms to resist oxidative stress in B.juncea.However,the change of antioxidant enzyme activity was not completely consistent with that of oxidative stress,that is,the antioxidant enzyme activity increased in the short term and decreased with the extension of stress time.(2)The B.juncea seedlings were cultured with 1/2 MS medium containing 100 ?M Cd for 14 days,and the control group didn't contain Cd.RNA was extracted from the roots and leaves separately.After passing the quality inspection,small RNA high-throughput sequencing was performed.The total number of Raw Reads of sRNA in the 4 samples were 21966292,22330210,22022242,and 24168375 respectively.The effective ratios accounted for more than 70%and the proportion of 20?24nt sequences is higher.The differential analysis of miRNA expression showed that under the stress of Cd,the expression levels of miR399,miR172 and miR164 in the leaves were significantly reduced,and the expression levels of miR397 and miR395 in the leaves were significantly increased,while the expression levels of miR172 in the roots were significantly decreased,and the expression levels of miR156,miR169,miR398 and miR408 in the roots were significantly increased.The results of qRT-PCR were consistent with the sRNA sequencing.Target gene GO enrichment analysis shows that the target genes of differentially expressed miRNA are enriched in protein modification process,transport,transcription,phosphorylation and kinase activity under Cd stress.These results suggest that miRNAs(miR164,miR395,miR397,miR399,miR408,miR156,miR398,etc.)may regulate growth,development,phosphorus and copper balance,and oxidation-reduction balance by targerting enzymes and transcription factors in B.juncea under Cd stress.