Analysis On Molecular Mechanism Of The Endophytic Fungus Metabolite Zearalenone Regulating Root Development Of Tetrastigma Hemslevanum

Tetrastigma hemsleyanum Diels&Gilg ex Diels(T.hemsleyanum)belongs to the genus Tetrastigma of Vitaceae,is a precious medicinal plant in China.The whole plant can be used as medicine,and the underground tuberous root has the best medicinal effect,which has the functions of clearing away heat and detoxifying,anti-virus and anti-tumor.However,under natural conditions,the growth and development of the root system of T.hemsleyanum is slow,and it takes many years to form calabash-shaped roots.In the former study,the research group isolated an endophytic fungus that can promote its growth from T.hemsleyanum.Zearalenone(ZEN)was detected in the metabolites of this endophyte,and experiments have preliminarily verified that a certain concentration of ZEN can promote root development.In order to explore the mechanism of ZEN regulating the root growth and development of T.hemsleyanum,the following study was carried out.Fluorescence quantitative PCR technology was used to detect the changes of expansin gene(Th-exp)expression in roots,stems and leaves of T.hemsleyanum treated with ZEN;microRNA high-throughput sequencing technology was used to detect the distribution of miRNA in roots after ZEN treatment of T.hemsleyanum.Differentially expressed miRNAs were screened out by bioinformatics analysis.Then related miRNA overexpression vectors were constructed,and their potential functions were verified by transgenic hairy roots.The main results are as follows:(1)After seedlings of T.hemsleyanum culturing on medium supplied with ZEN,the expression of Th-exp gene in roots was significantly increased.However,the expression of Th-exp gene in leaves and stems was no significant change.The results preliminarily showed that ZEN affected root growth and development by increasing the expression of Th-exp in roots.(2)Using the control group and ZEN-treated T.hemsleyanum as materials,a total of 61 known miRNAs were identified and 234 new miRNAs were predicted in the roots by small RNA high-throughput sequencing technology.Among them,miRNA differential analysis showed that there were significant differences in the expression abundance of 162 miRNAs between the control and ZEN-treated groups.The target genes of differential miRNAs were predicted by bioinformatics analysis,and 16 differentially expressed miRNAs and their candidate target genes that may be related to root development were screened out.GO enrichment analysis showed that 162 differentially expressed miRNA target genes were significantly enriched in lignin biosynthesis,transcriptional regulation,ADP binding and defense response.KEGG pathway enrichment analysis showed that 162 differentially expressed miRNA target genes were enriched in 20 pathways,including flavonoid and flavonol biosynthesis,secondary metabolite biosynthesis,lipopolysaccharide biosynthesis and MAPK signaling pathway.(3)The differentially expressed precursor gene fragments of miR156b and miR156f were cloned,and the overexpression vectors of miR156b and miR156f were constructed.Transgenic hairy root of overexpressing miR156b and overexpressing miR156f were obtained by Agrobacterium rhizogenes transformation.Wild-type hairy roots and hairy roots overexpressing miR156b and miR156f were taken,and cultured under the same conditions for 30 d.Then,their phenotypic characters were analyzed using a root system analyzer.The results showed that the number of lateral roots and total root length of transgenic hairy roots overexpressing miR156b and overexpressing miR156f were significantly higher than those of wild type,indicating that overexpression of miR156b and miR156f in T.hemsleyanum promoted lateral root development.Fluorescence quantitative PCR was performed on the predicted target genes SPL9 and/or SPL10 of miR156b and miR156f.The results showed that the relative expression of SPL9 gene in the roots of overexpressing miR156b was significantly lower than that of wild-type,while the expression of SPL10 was no significant differences between the wild-type and overexpressing miR156b.The relative expression of SPL10 gene in roots of overexpressed miR156f was significantly lower than that of wild-type,while the expression of SPL9 had no significant difference between the wild-type and overexpressed miR156f.It is suggested that SPL9 may be the target gene of miR156b,while SPL10 may be the target gene of miR156f.miR156b negatively regulates the expression of the target gene SPL9,and miR156f negatively regulates the expression of the target gene SPL10,thereby further regulating the development of the roots in T.hemsleyanum.This study preliminarily revealed the molecular mechanism of the endophytic fungal metabolite ZEN regulating the root growth and development of T.hemsleyanum.