Cloning And Expression Analysis Of LrANS Gene In Lycium Ruthenicum Murr

Lycium ruthenicum Murr,from the family Solanaceae,is an excellent shrub growing in Northwest China for windproof and sand fixation.It is also an important edible and medicinal plant.The fruit of L.ruthenicum is rich in anthocyanin.Studying the synthesis mechanism of anthocyanin can provide an important theoretical basis for resources development and variety breeding in L.ruthenicum.Therefore,in this study,L.ruthenicum was chosen as the experimental material,and the gene ANS related to anthocyanin synthesis was cloned by RT-PCR combined with RACE technology,and the relative expression level of ANS gene was researched.Main results for this studies were concluded as follows:1.The length of LrANS(Gen Bank accession number MK713977)gene was 1 527 bp,which contained a 1 290 bp ORF encoding 429 amino acids.Comparison of homology amino acid sequence indicated that LrANS had higher similarity with Nicotiana tabacum ANS(77.2%).The prediction of SOPMA demonstated that the LrANS protein inclusive 37.53% alpha helix,5.59%beta turn,40.56% random coil and 16.32% extension strand.The prediction of SWISS-MODEL showed that LrANS protein belongs to the superfamily of PLN03178 protein.q RT-PCR analysis revealed that LrANS gene was expressed in roots,stems,leaves,flowers,green fruits,purple fruits and black fruits,and the expression level in black fruits was significantly higher than other tissues.2.The LrANS gene was linked to pBWA(V)HS-GLosgfp vector using double enzyme digestion gene recombination technique,and the recombinant vector was named as 35S:LrANS-GFP.The recombinant vector was transformed to tobacco mesophyll cells instantaneously by Agrobacterium.The LrANS protein was localized in the cytoplasm and cytomembrane observed by confocal microscopy,which was consistent with the online software prediction results.3.LrANS gene expression was dramatically decreased under UV irradiation.The lowest expression level was at 1 hour which was about one twelveth of control group.After 12 hours stressed by UV irradiation,it return to one fifth of control group.This result suggested that LrANS gene might respond to UV irradiation.4.Using virus-induced gene silencing technology,a VIGS experimental system that infects germinated seeds of L.ruthenicum has been successfully constructed The results of q RT-PCR showed that the anthocyanin-synthesis-related genes LrANS,Lr TTG1,and Lr MYB in L.ruthenicumwere successfully induced silence by VIGS technology.This study provides the basis for the further study on the functions of anthocyanin-synthesis genes in L.ruthenicum.