Identification And Stress-regulating Network Of Cold Response CircRNAs In Soybean

Low temperatures restrict crop production and yield in northeast China.As a thermophilic crop,soybeans are very sensitive to low temperature stress.Circular RNA(Circ RNAs)is a new type of non-coding RNA recently discovered and has been shown to be involved in plant abiotic stress response mechanisms.Mi RNA sponge has the most noticeable function of plant circ RNA.Mi RNAs are endogenous(21-24 nts)single-stranded non-coding RNAs that can cleave target m RNA to cause gene silencing.Mi RNA-sponge refer to the existence of mi RNA complementary sites on circ RNAs.After the combination of circ RNA and mi RNA,its activity to cleave downstream target genes is inhibited.In the present study,soybean variety Kenfeng 16 was used as an experimental material,and the next-generation circ RNA high-throughput sequencing technology was employed to display the related sequence information and differential expression levels of circ RNA in soybean leaves at 4°C.The authenticity of some cold-responding soybean circ RNAs was identified and analyzed for their possible functions.Real-time PCR was used to evaluate the expression of 9 identified soybean circ RNAs as mi RNA sponges.Gma＿circ＿0000313 and gma-mi R1508 a with predicted adsorption were selected for subsequent research.The expression patterns of gma-mi R1508 a and the target gene Gm XTH22 under cold,drought and ABA treatments were evaluated.Using soybean variety Dongnong 50 and wild type Arabidopsis Colombia as experimental materials,the phenotype and stress-related physiological indicators of gma-mi R1508 a overexpressing soybean and Arabidopsis under the above treatments were analyzed.The responses of soybean gma＿circ＿0000313-gmami R1508a-Gm XTH22 regulatory network to abiotic stress were demonstrated.The main results are as follows:1.There were 41 significantly differently expressed circ RNAs in the 4 h cold-treated comparison group,and 35 in the 8 h cold treatment comparison group.Twelve circ RNAs with significant differential expressions were found in both comparison groups.At 4 and 8 h of cold treatment,6 expression levels were significantly up-regulated and 5 were significantly downregulated.Comparing with CK-0 h,at 4 and 8 h of cold treatment,8 expression levels were significantly up-regulated and 11 were significantly down-regulated.The above 4 groups of circ RNA were selected for further research.2.The analysis of the function of the source gene revealed that these source genes responding to cold stress were mainly focused on the catalytic reaction and the binding reaction,the cell component located were mainly at the cytoplasm,and the involved biological process was mainly in the cellular processes.The regulatory pathways responded included nitrogen metabolism,carbon metabolism and photosynthesis.3.Back-splicing sites of 36 soybean circ RNAs were obtained using PCR technology and Sanger sequencing.Through the detection of c DNA and g DNA,the authenticity of 9 back-splicing sites of corresponding circ RNAs was verified.The results of real-time quantitative PCR showed that gma＿circ＿0000313 and gma-mi R1508 a had a complementary relationship under 0°C treatment.The gma＿circ＿0000313 was likely to be an upstream regulator that controls the accumulation of gma-mi R1508 a by adsorption in response to freezing stress in soybean.4.Gma-mi R1508 a had tissue expression specificity,and its expression level was up-regulated by cold treatment,down-regulated by drought treatment,and up-regulated by ABA treatment.Gmami R1508 might be a positive response factor for cold and ABA stress,but a negative response factor for drought stress.Gma-mi R1508 a overexpressed Arabidopsis plants were cold-resistant,ABAsensitive and drought-sensitive.Gma-mi R1508 a overexpressing soybean exhibited a dwarfed and thickened cell wall phenotype,also having a cold-resistant but drought-sensitive phenotype.The overexpression of gma-mi R1508 a in Arabidopsis could resist cold stress and exogenous ABA stress by reducing the damage of active oxygen to plants.5.5’RACE technology was used to explore the precise cleavage sites of gma-mi R1508 a on the target m RNA.The cleavage site of gma-mi R1508 a on Gm XTH22 was located at 6-7 bases downstream of the complementary region.There were two cleavage sites on Glyma.16G162100,both of which were located at the complementary region,respectively at the 2-3 and 9-10 bases.6.Homologous gene of Gm XTH22 in Arabidopsis was At XTH23.The deletion of At XTH23 conferred cold resistance,drought sensitivity and ABA sensitivity to Arabidopsis plants,and the Gm XTH22 restored Atxth23 mutant Arabidopsis plants could alleviate these stress-responsive phenotypes.The lack of At XTH23 conferred the ability to resist active oxygen stress under low temperature conditions in Arabidopsis.Gm XTH22 overexpression in Atxth23 mutant Arabidopsis could attenuate the resistance of Atxth23 mutant to reactive oxygen species under cold treatment.