A Preliminary Study On The Mechanism Of Branch Thorn Occurrence Of Lycium Ruthenicum

Lycium ruthenicum is an important ecological and economic thorny shrub,and its thornless type is more suitable for cultivation as an economic tree.In this paper,plant tissue culture,the pot experiment,scanning electron microscopy(SEM)observation,high-throughput sequencing of transcriptome(RNA-seq),Real-time PCR(q RT-PCR)and high performance liquid chromatography(HPLC)were used to study the thorn occurrence regularity of L.ruthenicum and explore the factors that may affect its occurrence.The main results are as follows:(1)We used the inflorescences of L.ruthenicum to be the explants.The result shows that treatment with 75% alcohol for 45 s and 0.1% Hg Cl2 for 3 min showed the best sterilization effect;the optimum result was obtained on the MS medium supplemented with 6-BA 0.2mg/L and NAA 0.1 mg/L,the explants in the medium showed the best regeneration effect;1/2MS supplemented with 0.2 mg/L IBA was the most suitable for root induction,the rooting ratio can reach 100%;the culture medium with the best regeneration effect was selected for dehyperhydricity,multiple shoots which inoculated into the medium with 40 μM Ag NO3 showed the highest dehyperhydriciton rate.(2)Some plants of L.ruthenicum clone show thornless character when the field water capacity is more than 80%,and when the field water capacity is below 60%,all the plants produce thorns.The result of SEM observation of the thornless and thorny samples of L.ruthenicum showed that the thorns of L.ruthenicum were originated from the axillary meristem in leaf axil,which indicated that the thorns of L.ruthenicum were branch thorns.Meanwhile,three development phases of branch thorns were determined(non-thorn stage,thorn initiation stage and thorn formation stage).This provides a foundation for the collection of the subsequent test samples.The experiment also found columnar protrusions of function unknown on the the surface of leaf primordium,leaves and leafy structure surrounding the thorns of L.ruthenicum.(3)The transcriptome sequencing of L.ruthenicum at non-thorn stage(CK),thorn initiation stage(Init)and thorn formation stage(Form)totally found 62.33 GB clean data,52816 unigenes were spliced,and the total number of unigenes annotated to the seven functional data bases was 32 000.The total annotated rate was 60.59%.The result showed that there were 1 208 differential expression genes(DEGs)between CK and Init,which were significantly enriched in the GO terms of oxidoreductase activity,S-methyltransferase activity,etc.and the KEGG pathway of flavonoid biosynthesis,etc.;there were 2 828 DEGs between Init and Form,which were mainly enriched in the GO terms of oxidoreductase activity,oxidation-reduction process,etc.and KEGG pathway of plant hormone signal transduction,etc..There were 74 common GO terms and five common KEGG pathways annotated in the two comparison groups.The common five KEGG pathways were plant hormone signal transduction,phenylalanine biosynthesis,flavonoid biosynthesis,phenylpropanoid biosynthesis and circadian rhythm – plant,respectively.In addition,we found that the DEGs between Init and Form are significantly enriched in the KEGG pathway of starch and sucrosemetabolism.Also,there were three DEGs between CK and Init annoted in starch and sucrose metabolism KEGG pathway.In the KEGG pathway,a DEG(Sucrose synthetase,SUS)showed a significantly low level of expression in Init,and the content of sucrose in Init is significantly lower than that in both CK and Form.The expression of SUS is found to be very significantly positively correlated with sucrose content and significantly negatively correlated with glucose content.According to the research results of plant lateral branch development,we infer that(a)the KEGG pathway of starch and sucrose metabolism is related to the occurrence of L.ruthenicum branch thorn;(b)the sucrose is the signal to regulate the occurrence of L.ruthenicum branch thorn;(c)the glucose provides energy for the occurrence of the branch thorn and(d)SUS participates in the regulation of the occurrence of L.ruthenicum branch thorn by the sucrose.The research lays a foundation for revealing the occurrence mechanism of L.ruthenicum branch thorn and cultivating varieties of thornless L.ruthenicum.