Cloning,Expression And Transformation Of LcCHI And LcANS Genes From Loropetalum Chinense Var.Rubrum

Loropetalum chinense var.Rubruma,a variety of Loropetalum chinense?R.Br.?Oliver,is belonging to the genus Loropetalum in the Hamamelidaceae family.It is characteristic flowering and foliage plant in China due to its red leaves and red flowers.The Loropetalum chinense var.rubrum is a common landscape plants in the south of China since it is easy to reproduce and has strong adaptability and stress resistance,and it is easy to shape and prune,and blooms many times a year.The redness of the leaves of Loropetalum chinense var.rubrum is determined by the content of anthocyanidin.The sustained high temperature in summer can cause the leaves change from red to green,which will seriously affect its ornamental value and economic value.Therefore,it is necessary to investigate the molecular mechanism of the accumulation of anthocyanin in Loropetalum chinense var.rubrum,so as to create conditions for further molecular breeding and variety improvement of the Loropetalum chinense var.rubrum.Based on the results of the transcriptome sequencing of Loropetalum chinense var.rubrum,two important genes of chalcone isomerase?Lc CHI?and anthocyanin synthesis?Lc ANS?in the anthocyanin biosynthesis pathway were screened,and the genes related function studies were carried out in this study.Using transcriptome sequencing data and RT-PCR technology,c DNA sequences of Lc CHI and Lc ANS were cloned from Loropetalum chinense var.rubrum,and open reading frames?ORFs?of those two genes were obtained.The amino acid sequences encoded by Lc CHI and Lc ANS genes were predicted respectively,and analysis of amino acid properties,multiple alignment of amino acid sequences,phylogenetic analysis,and tertiary structure modeling were carried out.The expression pattern of Lc CHI and Lc ANSwas analyzed using real-time fluorescence quantitative PCR technology.Meanwhile,p Lc CHI-SUPER1300 and p Lc ANS-SUPER1300overexpression vectors were constructed and heterologously expressed in Arabidopsis thaliana to obtain transgenic plants.Our research has laid the foundation for the further study of those two genes in the anthocyanin biosynthesis pathway in Loropetalum chinense var.rubrum.The main results are as follows:?1?The gene encoding Lc CHI,a chalcone isomerase gene from Loropetalum chinense var.rubrum,was cloned.Its ORF is 657 bp in length,encoding 218 amino acids.The relative molecular weight is 53.6k Da and the isoelectric point is 5.16.The results of multiple alignments of amino acid sequences show that the amino acid sequences of Lc CHI of Loropetalum chinense var.rubrum are highly identical to CHIs of other plants',such as Rosa rugosa Thunb.,Rosa chinensis Jacq.,Camellia sinensis,etc.,all exceeding 75%.Five amino acid residues?Arg38,Thr46,Tyr108,Asn115,and Ser192?relating to enzyme catalysis in Lc CHI are completely consistent in other species,indicating that Lc CHI is highly conservative in evolution.Phylogenetic study showed that Lc CHI is closely related to Rosa rugosa Thunb.and Rosa chinensis Jacq.The tertiary structure modeling of Lc CHI protein shows that Lc CHI has a high degree of matching with At CHI's crystal structure model derived from Arabidopsis thaliana.It is mainly composed of multiple?-helices and?-sheets to form a globular protein,and its catalytic region has two nitrate ions(NO^3-)Binding site.Real-time quantitative PCR results showed that the Lc CHI gene is expressed in different tissues,which is related to the red color in these tissues.Among them,the expression level in roots is the highest,followed by tender leaves,stems and flowers,and the lowest in mature leaves.?2?The anthocyanin synthase encoding gene Lc ANS of Loropetalum chinense var.rubrum was cloned.Its full-length ORF is 1071 bp and encodes 356 amino acids.The relative molecular weight is 87.5 k Da and the isoelectric point is 5.07.The results of multiple alignments of amino acid sequences showed that the amino acid sequences of Lc ANS from Loropetalum chinense var.rubrum were highly consistent to ANSs from plants,such as Paeonia suffruticosa Andr.,Paeonia lactiflora Pall.,and Acer palmatum,all exceeding 84%.Phylogenetic studies have shown that Lc ANS is more closely related to ANSs of Paeonia suffruticosa Andr.And Paeonia lactiflora Pall.,which are belonging to the Paeonia L.genus of the Ranunculaceae.Tertiary structure modeling analysis shows that the structure model of Lc ANS is highly matched to At ANS derived from Arabidopsis thaliana.The Lc ANS protein forms a globular protein,has many?-helix and?-sheets,and its core region contains 2 ligand(Fe²⁺and 2-ketoglutarate)and a flavonoid substrate binding site.Real-time fluorescence quantitative PCR analysis showed that under high temperature stress,the relative expression of Lc ANS gene in the leaves of Loropetalum chinense var.rubrum was significantly reduced.After 48-96h treatment under high temperature,the relative expression level of Lc ANS gene is only 1/10 to Lc ANS before treatment.?3?The ORFs of Lc CHI and Lc ANS of Loropetalum chinense var.rubrum were amplified.The gene overexpression vectors p Lc CHI-SUPER1300 and p Lc ANS-SUPER1300 were constructed,and heterologous transformated in Arabidopsis thaliana,transgenic plants were obtained.