Cloning And Functional Analysis Of The DiLAC17 Gene From Davidia Involucrata

The dove tree(Davidia involucrata Baill.)is a large deciduous tree in the family Nyssaceae,which is a unique monotypic genus in my country and a national class I key protected plant.Dove tree has high research value in plant phylogeny and evolution,geohistorical changes and so on.The low reproductive capacity of seeds is the main reason for the endangerment of dove tree.The long dormant period and high abortion rate of dove tree seeds make it difficult to regenerate naturally.Revealing the molecular mechanism of dove tree seed development is crucial for the conservation and utilization of this rare species.By analyzing the transcriptome database of dove tree seeds completed earlier,we found that almost all members of the gene family encoding laccase were significantly up-regulated in aborted seeds,suggesting that these genes were involved in the process of dove tree seed abortion.This study focused on the gene with the most significant differential expression,analyzed its expression pattern in different tissues and different developmental stages of seeds,and used genetic transformation to overexpress this gene in Arabidopsis thaliana to observe its effect on plant growth and development.Especially the effect of seed development,to verify the biological function of the dove tree laccase gene.The main results of this study are as follows:(1)A transcript,c39491.graph＿c0,with significant up-regulation in abortive seeds was cloned from the Davidia genome.Sequence analysis showed that the full length of the fragment was 2,926 bp,and the ORF length was 1,755 bp,containing 6 exons and 5 introns.The encoding product was a laccase protein containing 584 amino acids,which had the highest similarity with Ns LAC17 of Laccase protein of Nyssa sinensis,therefore the gene was named DiLAC17.(2)The expression pattern of the DiLAC17 gene in different tissues of Davidia was analyzed by q PCR,and it was found that its expression pattern was tissue-specific.The expression level of the DiLAC17 gene in pre-abortion seeds was the highest,which was significantly higher than that in other tissues,and was 5.3 times that of normal seeds at the same stage.The most significant difference of the expression patterns of DiLAC17 between normal and abortive seeds occurred at the early stage of seed development,suggesting that the gene was highly expressed at the early stage of seed development,thus restricting the continued development of seeds.(3)Phenotypic observation of transgenic Arabidopsis showed that overexpression of the DiLAC17 gene resulted in shortened pods,reduced number of seeds in pods,darkened seed color,and abnormal seed development(showed as shriveled or deformed seeds).q PCR analysis of Arabidopsis seed development-related genes showed that At LEC2,At ANT1,At RGE1,At IKU1,At IKU2,At MINI3,At SHBI and other genes that positively regulate seed development in transgenic Arabidopsis were significantly down-regulated,indicating that the DiLAC17 gene inhibited the expression of these genes in Arabidopsis,thus impacted the development of embryo,endosperm and seed coat,resulting in abnormal seed development..(4)Qualitative analysis of lignin staining of transgenic plants with phloroglucinol showed that transgenic plants had wider color range and deeper staining in stems and pods.Quantitative analysis of lignin content by colorimetric method showed that the lignin content of transgenic lines was higher.This indicates that DiLAC17 gene is involved in regulating the synthesis of lignin and can promote the accumulation of lignin.The expression levels of Arabidopsis thaliana lignin synthesis-related genes in stems,leaves,pods and seeds were analyzed,and it was found that the expression levels of At4CL1,At F5 H,At Co MT1 and At CCo AOMT1 were significantly decreased in transgenic Arabidopsis stems,At F5 H increased in leaves,while At4CL1,At Co MT1 and At CCo AOMT1 decreased in seeds.The above results indicated that DiLAC17 gene could affect the expression levels of genes related to the lignin synthesis pathway in Arabidopsis thaliana,thereby affecting the content and composition of lignin in different tissues.In this study,we cloned a laccase gene that was highly expressed in the early stage of abortive seeds of dove tree.Through genetic transformation of Arabidopsis,it was proved that it has an important regulatory role in the process of seed development,and seed development may be associated with the increase of lignin accumulation.