Cloning Of CesA Gene In Davidia Involucrata And Its Expression Analysis In Seed Development

Davidia involucrata Baill.also known as the dove tree,is a kind of special protection of rare and endangered plants in China.And it is a tertiary ancient relic species of tropical plants that has a high value to ornamental and academic study.Its seed fecundity is week thus difficult to natural regeneration.There was a long dormant period of the seed in the dove tree and it needs a strict survival environment.Seed abortion is one of the important reasons for restricting the serious sexual reproduction of Davidia involucrate,this phenomenon is pervasive in many woody plants.However the explanation at molecular level for it is limited.This experiment had previously constructed the unigene library of fruits and seeds in D.involucrata.Using this library as reference sequences,we screened the transcriptome data and selected 2,361 different expression genes(DEGs)by Digital Gene Expression Profiling(DGE).We observed that the genes encode cellulose synthase are significantly up-regulated in aborted seeds compared with normal seeds of different samples in accordance.In this study,we analyzed the expression pattern of cellulose synthase gene in the process of seed development of Davidia involucrata,the activity of cellulose synthase in Davidia involucrata and its function in the process of seed development were studied preliminarily by the determination of cellulose content.And a gene was selected,through the expression analysis,cloning,protein expression and functional validation to clarify the function of the gene.The purpose of this study is to explore the cellulose synthase gene resources in D.involucrata and to interpret the regulatory mechanism of seed abortion,which might provide theoretical basis for in-depth understanding of the spontaneous seed abortion mechanism.The main research contents are as follows:1.Analysis of transcription data of cellulase synthase gene in Davidia involucrateWe screened 14 cellulose synthase genes from the transcriptome data in Davidia involucrate,a total of 22 transcripts,showing a consistent up-regulation expression in aborted seeds.Among them,7 genes had significant different expressions between normal and abortive seeds.2.Expression analysis of cellulose synthase geneThe results of qPCR showed that CesA gene showed a significant up-regulation expression in aborted seeds,four CesA genes significantly upregulated in abortive seeds were selected for expression analysis.The results showed that the expression patterns of them were similar,and the amount of expression in the abortive seeds was significantly higher than that in the normal seeds,and the expression was up-regulated in the later stage of the development.The expression of c43681.graph_c0 gene,which was the most significant difference,was expressed in different tissues of Davidia involucrate,the results showed that c43681.graph_c0 gene was highly expressed in the reproductive organs such as flowers and fruit,and low expression in vegetative organs such as leaves and shoots,the highest expression was found in aborted seeds.The results show that c43681.graph_c0 gene was preferentially expressed in the reproductive organs.3.Cloning of c43681.graph_c0 gene and analysis of its coding productPrimers were designed to clone the sequence c43681.graph_c0 gene and obtained 882 bp fragments.Through sequences analysis,we know the product contains 292 amino acids,which has 6 transmembrane domains and the isoelectric point is about 9.07,and it is alkaline protein.It has the highest homology with Arabidopsis thaliana cellulose synthase 4 catalytic subunit,which contains the conserved region.4.The target gene expression proteinThe successfully constructed recombinant plasmid pET28/CesA was introduced into E.coli BL21 for prokaryotic expression and analyzed by SDS-PAGE.The expression of cellulose synthase gene was the highest when the induction time was 4h and the IPTG concentration was lmmol/L.5.Gene function verificationThe successfully constructed pBI121/CesA recombinant plasmid was introduced into Agrobacterium,and the target gene was transformed into Arabidopsis thaliana by inflorescence method,the seeds were screened by the culture medium containing kanamycin.The development of Arabidopsis thaliana plants and the second generation of seed abortion were observed.Experiments show that the plants that have not successfully transferred to the target gene can germinate,but they whiten and die before they grow true leaves.