Identification Of Drought-responsive CircRNAs And Analysis Of Regulating Pathway Under Abiotic Stress In Soybean

Soybean is the important resource for plant protein and edible oil.However,the production of soybean is seriously affected by adverse environmental factors such as drought and salt stresses.CircRNA,a new class of non-coding RNA,has a variety of functions.For example,circRNA with miRNA binding site can bind to miRNA by adsorbing it,thus inhibiting the cleavage of miRNA to its downstream target genes.CircRNA has been proved to be able to encode proteins in animals,and participate in a variety of biotic and abiotic stress response mechanisms in plants.In this study,the soybean variety Kenfeng 16 was treated with simulated drought treatment using 5%PEG 6000.The drought-responsive circRNAs of soybean were excavated by a new generation of high-throughput sequencing technology.The back-splicing sites of circRNAs were verified by PCR technology and Sanger sequencing,and its authenticity was determined.The circRNA-miRNA-m RNA interaction network map was generated by website prediction.The expression pattern of soybean circRNAs as miRNA sponge was analyzed by quantitative real-time PCR.Gma-miR9725 and gma?circ?0000531 which meet the complementary trend were selected for subsequent functional verification.The complementary cleavage site between gma-miR9725 and its target gene was validated by 5'RACE experiment.The expression patterns of gma-miR9725 and its target gene Gm AKT1 were analyzed under drought and ABA treatments.Soybean composite plants with transgenic roots by Agrobacterium tumefaciens-mediated hairy root induction technique were obtained to verify the phenotype and function of gma-miR9725 under stresses quickly.The expression patterns of Gm AKT1 were analyzed under low potassium and high salt treatments.Gm AKT1-overexpressing transgenic plants were obtained using Columbia wild-type Arabidopsis thaliana and Dongnong 50 as experimental materials,and their phenotypes and functions were analyzed under stresses.The gma?circ?0000531-gma-miR9725-Gm AKT1 regulatory pathway of soybean in abiotic stress responses was clarified.The main results were as follows:(1)1,275 drought-related soybean circRNAs,derived from 959 parental genes,were obtained by high-throughput sequencing technology.There were 145 differentially expressed circRNAs in 6h comparison group,and 61 in 12 h comparison group.Twenty differentially expressed circRNAs were both found in the two groups.Compared with the control group at 6 h and 12 h,six circRNAs were significantly up-regulated and eight were significantly down-regulated.(2)The back-splicing sites of differential expressed circRNAs were verified by PCR technology and Sanger sequencing,and it was found that five soybean circRNAs(gma?circ?0000287,gma?circ?0000302,gma?circ?0000095,gma?circ?0000298 and gma?circ?0000531)did exist after excluding gene recombination.Parent genes of the above five circRNAs involved in various abiotic stresses and plant development.(3)Quantitative real-time PCR showed that the expression patterns of gma?circ?0000531 and gma-miR9725 under drought stress were complementary.Therefore,gma?circ?0000531 was the upstream regulator of gma-miR9725 expression level by acting as miRNA sponge in response to drought stress in soybean.(4)There were several stress-responsive elements in the promoter sequence of gma-miR9725.The soybean hairy root mediated by Agrobacterium tumefaciens also showed that gma-miR9725could respond to drought and ABA stresses and affected the scavenging of reactive oxygen species in soybean under stresses.(5)5'RACE analysis showed that gma-miR9725 directed Gm AKT1 m RNA cleavage in vivo.Quantitative real-time PCR also showed that the expression level of Gm AKT1 was down-regulated in the gma-miR9725-overexpressing hairy roots,and the expression patterns of gma-miR9725 and Gm AKT1 showed a complementary trend under drought and ABA treatments.(6)The expression of Gm AKT1 was not only regulated by drought and ABA treatments,but also induced by low potassium and high salt treatments.(7)Gm AKT1 could enhance the tolerance of soybean to drought,low potassium and high salt stresses by regulating the absorption of K~+and maintain Na~+/K~+homeostasis.