Identification and Expression Analysis of Southern Blight Disease Responding miR171b and miR171b's Target DcGRAS Gene Family in Dendrobium catenatum
LAI Dan-Ni*, CAI Xiang*, CHEN Dong-Hong, HAN Zhi-Gang, WU Ling-Shang, SI Jin-Ping**, LI Cong**
State Key Laboratory of Subtropical Silviculture/National Forestry Dendrobium catenatum Engineering Technology Research Center, Zhejiang A&F University, Lin'an 311300, China
Abstract：Dendrobium catenatum is a perennial herbal medicinal plant, which is beneficial to stomach and lung. Sclerotium delphinii, a necrotrophic pathogen, is responsible for D. catenatum Southern blight disease, which causes widespread loss in the near-wild cultivation of D. catenatum. But use of chemical fungicides is not a good choice as it causes accumulated toxic componds in plant and pollutes environment. A safer management is urgently needed. microRNA171b (miR171b) is an ancient and conserved miRNA gene family which mainly targets GRAS (gibberellic acid insensitive (GAI), repressor of GAI (RGA), and scarecrow (SCR)) family transcription factors (TFs). The miR171-GRAS regulatory model plays an important role in plant response to biological and abiotic stresses. In this study, through Small RNA-seq and RNA-seq analysis of D. catenatum infected by S. delphinii, miR171b was significantly downregulated. Seven of the 10 target genes predicted by miR171b were GRAS TFs. At the same time, the GRAS gene family members of D. catenatum were identified. Phylogenetic analysis of GRAS protein with homologous proteins of Arabidopsis thaliana, Oryza sativa, Phalaenopsis equestris and Apostasia shenzhenica showed that the 51 GRAS genes were divided into 11 subfamilies with protein sequences ranging from 345 to 757 amino acids. The relative molecular weight of the proteins ranged from 39.274 0 to 85.661 6 kD, and the theoretical isoelectric point ranged from 4.59 to 7.52, which all contained GRAS conserved domains. Subcellular localization prediction revealed that all DcGRAS proteins were localized in the nucleus, which was consistent with their function as transcription factors. Motif analysis showed that the each DcGRAS proteins possessed a variable N-termianl and a conserved C-terminal domain. Most GRAS proteins contained Motif5, Motif8 and Motif16 in the C-terminal domain, all members in DELLA subfamily and DoSCL34 contained Motif13 in N-termianl. The exon-intron organization analysis showed that almost all GRAS genes were intronless, and each gene contained 1~3 exons, similar to the lack of introns in Arabidopsis and rice GRAS genes. The cis-acting element analysis showed that the 12 cis-acting elements selected in DcGRAS could be classified into 3 types: hormone response elements, development-related elements and stress response elements. Based on the location and number of cis-acting elements on the genes, it was speculated that DcGRAS genes may has a wide range of effects on plant hormone response, growth and stress response. Transcriptome data showed that DoNSPL2-2 was highly expressed in green root tips (P<0.05), and DoNSPL2-5 was highly expressed in roots of D. catenatum (P<0.05); DoSCL21-2 and DoSCL3-3 expression was significantly upregulated as drought time extended (P<0.05), and DoCIGR1 and DoSCL23-like expression was significantly upregulated under cold stress (P<0.05). DoNSPL2-2 and DoNSPL2-5 expression were significantly upregulated after S. delphinii infection (P<0.05). Therefore, based on the transcriptome data analysis, DoNSPL2-2 and DoNSPL2-5 regulated by miR171b might be mainly respond to S. delphinii in root. This study preliminarily analyzed the miR171b and its target GRAS gene family in D. catenatum, clarified the molecular mechanism of the miR171b-GRAS regulatory model involved in the regulation of D. catenatum defense against S. delphinii, so as to provide unique insight into the molecular mechanism of the immune mechanism of D. catenatum and provide theoretical basis for disease resistance breeding of D. catenatum.
赖丹妮, 蔡翔, 陈东红, 韩之刚, 吴令上, 斯金平, 李聪. 铁皮石斛白绢病响应miR171b及miR171b靶标DcGRAS基因家族的鉴定及表达分析[J]. 农业生物技术学报, 2022, 30(10): 1869-1883.
LAI Dan-Ni, CAI Xiang, CHEN Dong-Hong, HAN Zhi-Gang, WU Ling-Shang, SI Jin-Ping, LI Cong. Identification and Expression Analysis of Southern Blight Disease Responding miR171b and miR171b's Target DcGRAS Gene Family in Dendrobium catenatum. 农业生物技术学报, 2022, 30(10): 1869-1883.
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