| Paris polyphylla Sm.var.yunnanensis(Fr.)Hand is a rare and endangered medicinal herb and classified as Paris L.In recent years,with the shortage of wild resources of Paris polyphylla Sm.var.yunnanensis(Fr.)Hand,the artificial planting areas of this plant has been gradually expanding but the cultivation is difficult because of long seed dormancy and very slow growth from seed.At present,The molecular mechanism of seed dormancy is is relatively small,and its molecular mechanism remain unclear.Studies have shown that plant gibberellin plays an important role in germinated seed.GA2-oxidase,GA20-oxidase and GA3-oxidase,belonging to three multi-gene familys,are three key enzylnes involved in GA biosynthetie Pathway,and participants in the regulation of gibberellin level.GA2-oxidase gene,GA20-oxidase gene and GA3-oxidase gene has not been cloned from Paris polyphylla Sm.var.yunnanensis(Fr.)Hand,and there are few reports about the expression of GAox gene family members in plant growth and development.In this study,The germinating seed of Paris polyphylla Sm.var.yunnanensis(Fr.)Hand was used as material first.The GAox gene,which could be involved in seed development and dormancy release,was excavated by high-throughput sequencing.Cloning and identification of members of GAox gene family,and then a series of bioinformatic analysis was carried out including sequence homology search,construction of molecular phylogenetic tree,the Physical characteristics of the protein,domain search,and 3D structure prediction,etc.The expression levels of GAox gene in germinating seed and different organs of Paris polyphylla Sm.var.yunnanensis(Fr.)Hand were analyzed using qRT-PCR.The results are as follows:A total of 23 Unigenes sequences were selected as GAox gene from ranscriptome sequencing data of Polyphylla Sm.var.yunnanensis(Fr.)Hand.There were 6 sequences of complete open reading frame(ORF),basis on the design of specific PCR primers successfully cloned the full-length CDS of six Paris polyphylla Sm.var.yunnanensis(Fr.)Hand GAox genes.Sequencing results showed that the full-length CDS of six Paris polyphylla Sm.var.yunnanensis(Fr.)Hand GAox genes were about 1000bp.which were named PpGA2oxl,PpGA2ox2,PpGA2ox3,PpGA2ox4,PpGA20ox1,PpGA3oxl,And submitted to the GeneBank database.Then the bioinformatic analyses were carried out.The expression patterns of the GAox genes in Seeds at different developmental stages and different organs were detected using quantitative real-time PCR.These genes showed expression patterns that varied based on growth stage.PpGA2ox1 and PpGA2ox3 showed a decreasing trend with the germination of seeds,they may be related to the dormancy of seeds.PpGA2ox2,PpGA2ox4 and PpGA3ox1 increased with the germination of seeds,they may be related to the dormancy release of seeds.Although the expression of PpGA20ox1 fluctuates slightly,the overall expression level remains low,the gene should not play a key role in seed germination and development.The study found that PpGA2ox1,PpGA2ox2,PpGA2ox3,PpGA2ox4,and PpGA3ox1 gene expression changes occurred mainly in 6-9week.6-9week is a critical period for the seeds from the dormant state to break the dormant state.The expression of GAox genes in different organs of Paris polyphylla Sm.var.yunnanensis(Fr.)Hand were obvious different.PpGA2ox1?PpGA2ox2 PpGA2ox3?Pp PpGA2ox4?PpGA20ox1 and PpGA3ox1 genes expression in different organs has significant difference.PpGA2ox1,PpGA2ox2 and PpGA2ox3 were specifically expressed in seeds,and PpGA2ox4 and PpGA3ox1 were specifically expressed in roots,and PpGA20ox1 was specifically expressed in flowers.Theses results suggested that different GAox genes have different expression patterns and may play a different role in the growth process of different organizational development.The above study have great theoretical significance to further explored the role of GAox gene in the germination and development of seeds of Paris polyphylla Sm.var.yunnanensis(Fr.)Hand and clarify the molecular mechanism of seed dormancy. |
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