Effect Of Lycium Ruthenicum LrSUS On The Occurrence Of Branch Thorn And Its Possible Mechanism

Lycium ruthenicum is a shrub belonging to Lycium genus and Solanaceae family.It is an excellent tree species with both ecological and economic value.In this paper,we preliminarily judged that sucrose synthase gene(LrSUS)was related to the occurrence of branch spines by means of bioinformatics analysis and RNA in situ hybridization of LrSUS of L.ruthenicum.Through the comparative analysis of LrSUS overexpression and control L.ruthenicum,we explored the mechanism of LrSUS promoting the growth and occurrence of branch spines of L.ruthenicum,and tried to obtain thorn free or thorn less L.ruthenicum more suitable for production through transgenic RNAi technology.The main research findings are as follows:(1)LrSUS contains 10 introns and 11 exons.LrSUS protein consists of 803 amino acids.It is a weakly acidic protein.It contains sucrose synthase domain and glycosyltransferase domain.It has no transmembrane domain and is mainly located in the cytoplasm.The protein phosphorylation modification showed that it contained 70 phosphorylation sites of serine,tyrosine and threonine.Secondary structure analysis showed that the protein was mainly composed of irregular curl and α helix.The tertiary structure shows that LrSUS protein is composed of two symmetrical tetramers.Phylogenetic analysis showed that LrSUS belonged to SUS class I and was closely related to Solanum tuberosum and Solanum lycopersicum related genes.RNA fluorescence in situ hybridization showed that the expression of LrSUS in the thorn part of thorny branches was significantly higher than that in the thorn primordium of thornless branches.Thus,we speculated that LrSUS promoted the occurrence and growth of visible branches of L.ruthenicum.(2)LrSUS overexpression L.ruthenicum was micro-propagated and transplanted.DNA and RNA identification showed that the foreign gene was not lost after transplantation,and the expression of LrSUS in leaves and stems was higher than that in non-transgenic control.Sucrose synthase activity in stems and leaves of over-expressing LrSUS plants was slightly higher than that of non transgenic control plants.The measurement of growth morphological indexes showed that the thorning rate,leaf length,leaf width,branch diameter and the number of new branches of the overexpressed plants were significantly higher than those of the control plants.This proved that LrSUS not only promoted the growth and stem pumping of L.ruthenicum,but also promoted the occurrence of visible branch thorns.(3)High throughput transcriptome sequencing(RNA-Seq)analysis showed that stem segments and leaves of both LrSUS overexpression and control materials were clustered into two different categories.There were 447 differentially expressed genes(DEGs)between overexpression and control leaves(LSUs vs.LWT)of L.ruthenicum,and 242 DEGs between overexpression and control stem segments(SSUS vs.SWT)of L.ruthenicum.Real-time fluorescence quantitative PCR(q RT-PCR)analysis proved that the results of RNA-seq were reliable.DEGs between overexpression LrSUS and control stem segments were significantly enriched in KEGG pathways such as ABC transporter and Oxidative phosphorylation,and 8DEGs were enriched in KEGG pathway of Starch sucrose metabolism.DEGs between overexpression LrSUS and control leaves were significantly enriched in KEGG pathways such as Plant hormone signal transduction and Plant pathogen interaction,and six DEGs were annotated in KEGG pathway of Starch sucrose metabolism.Thus,it can be seen that LrSUS can promote the growth,stem pumping and visible branch-thorn occurrence of L.ruthenicum by affecting the above KEGG pathways and the expression of its key genes.(4)A pRNAi-SUS vector was designed and constructed to inhibit the expression of LrSUS.Based on the stable genetic transformation system of L.ruthenicum established by our research group,the pre-culture time was extended to 4 days.The leaf tip explants were infected with Agrobacterium EHA105 or LBA4404 engineering bacteria(OD600 = 0.4)for5-15 min,and more L.ruthenicum transgenic plants could be successfully obtained,with an average stable genetic transformation rate of 9.3%.However,the transgenic RNAi(LrSUS)plants of L.ruthenicum suggested short plants and slow root growth in tissue culture bottles,which was contrary to the overexpression LrSUS L.ruthenicum and consistent with the previous research findings.The thorn phenotype cannot be observed in the bottle,so it is impossible to judge whether to obtain thorn free and thorn less plants at present.This study enriched the research on plant branch-thorns,revealed the novel function of LrSUS gene of L.ruthenicum,and explored the mechanism of LrSUS promoting the growth,stem pumping and branch-thorn occurrence of L.ruthenicum,so as to lay a foundation for cultivating L.ruthenicum without thorns.