| Ramie(Boehmeria nivea(L.)Gaudich.)is a perennial bast fiber crop in Urticaceae.It blooms and bears fruit in the third growth period of each year,which will lead to the decline of fiber yield and quality.Previous studies have shown that the gender expression of ramie is regulated by ethylene.AP2 / ERF gene family members are important transcription factors in ethylene signal transduction pathway.Based on the previous research,this study used sequencing technology to sequence the transcriptome of GBN09 and GBN10 germplasm,so as to screen AP2 / ERF transcription factors closely related to ramie gender expression,and then conduct AP2 / ERF gene mining on the ramie genome data of "Zhongshizhu NO.1".On this basis,the gender related Bn AP2-12 gene was cloned and analyzed.Fried dough twist can be used to lay a foundation for the development and gender differentiation of ramie flowers.It is of great significance to reveal the mechanism of plant flower development and gender expression.The specific results are as follows:(1)The transcriptome data of GBN09(female flower)and GBN10(male flower)were controlled by quality control,expression level,GO/KEGG database annotation of DEGs,transcription factor screening,and AP2/ERF gene family members screening analysis.The results showed that ramie flower transcript group got 11015 DEGs,5499 base genes and5514 down regulated genes.GO classification,KEGG classification and Pathway enrichment of DEGs were mainly concentrated in redox,catalytic activity,carbon metabolism and other pathways.Ethylene related transcription factors are mainly distributed as AP2 / ERF family members.(2)Based on the ramie genome,83 members of AP2 / ERF gene family were identified and divided into four subfamilies: ERF(41),DREB(21),AP2(18)and RAV(3).Evolutionary analysis showed that the structure and conserved motifs of closely related AP2/ ERF family members were more similar,and the existence of unique motifs was helpful to the classification and functional annotation of AP2 / ERF.The number of amino acids of AP2 / ERF family members varies greatly,including 204-773 amino acids.The distribution of each gene on the chromosome is relatively scattered,but there are 8 gene clusters.There are a large number of collinear genes between the genome of ramie and cotton.37 genes may come from genome-wide replication or fragment replication.The expression profile of AP2 /ERF family members at full flowering showed that there were different transcripts of Bn DREB-19 gene and there were differences in germplasm expression,which may be related to flower sex.Bn AP2-12 may play an important role in regulating the gender of ramie flowers.Bn RAV-3 may be related to the characters of unisexual flowers produced by different germplasm.Tissue specific expression analysis of 8 ramie AP2 / ERF family members from different subfamilies.The expression pattern of Bn AP2-12 was male flower >tender leaf > female flower > stem bark > stem tip.It was further verified that the expression of Bn AP2-12 in male flower was significantly higher than that in female flower.(3)A differentially expressed gene Bn AP2-12 in male and female flowers were cloned.The length of open reading frame fragment was 1107 bp.Bioinformatics analysis showed that Bn AP2-12 encoded 368 amino acids.Homologous sequence alignment showed that Bn AP2-12 was evolutionarily conservative.The analysis of its encoded protein domain found that it contained two AP2 conserved domains.The homologous sequence alignment showed that it was closest to Morus notabilis(XP＿024028518.1).Arabidopsis was transformed by flower soaking method to preliminarily verify the function of Bn AP2-12.The preliminary functional verification results of Bn AP2-12 gene showed that the flowering time of Arabidopsis plants overexpressing Bn AP2-12 gene was delayed,the flower organ structure at different maturity stages did not change,and the appearance and morphology of fruit pods increased.Subcellular localization showed that the gene was located in the nucleus and cell membrane. |
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