Screening Of Key Genes Regulating Phellem Formation In Two Quercus Species

Quercus Linn is an important forest resource.The phellem layer of the bark of Quercus suber L.and Quercus variabilis Bl.,also known as cork,is a recyclable green renewable resource with great economic value and application prospects.At present,there are few studies on the formation of Q.suber and Q.variabilis,and the molecular mechanism of regulating phellem formation needs further study.In this study,the seed germination seedlings and two-year-old potted seedlings of Q.suber,Q.variabilis and Q.acutissima Carr were used as experimental materials.Q.acutissima could not continuously accumulate phellem and produce commercial cork,which was used as the control group in this study.By observing the growth state of three Quercus seedlings and the cross section of the basal bark of the stem section at different growth stages,the occurrence and activity of the phellogen of Q.suber and Q.variabilis were analyzed.The relationship between endogenous hormones and phellem formation was explored by measuring the content of endogenous hormones in the stem bark of three Quercus species during the growth period.Through the transcriptome sequencing analysis of Q.suber,Q.variabilis and Q.acutissima barks at different growth stages(dormancy stage,germination stage and growth stage),combined with the difference of endogenous hormone levels,the key genes regulating the formation of Q.suber and Q.variabilis phellem were screened,in order to provide a reference for further study on the molecular mechanism of phellem formation in Quercus plants.The main results are as follows:(1)The results of paraffin section and microscopic observation showed the occurrence of phellogen of Q.suber and Q.variabilis and the accumulation of phellem in two years.When the annual stem segment of Q.suber was about 6.0cm from the bottom of the apical bud,the cells of the lower epidermis began to divide horizontally,and the growth to half a year was a division of the phellogen.When Q.suber grew to 1 year old,there were about 10 layers of phellem cells with a thickness of about 72.04 μm.When it grew to two years,there were about 12 layers of phellem cells in the stem bark,and the thickness of phellem was about 74.87 μm.When Q.variabilis was about 16.5 cm from the bottom of the apical bud,the volume of the subepidermal cells increased significantly.The phellogen of half-year-old Q.variabilis began to differentiate gradually.When it grew to one year,there were about 9-10 layers of phellem cells in the stem base of Q.variabilis,and the thickness of periderm was about 58.80 μm.When it grew to two years,there were about 11 layers of phellem cells,and the thickness of periderm was about 66.40 μm.It shows that the phellogen cells of Q.suber and Q.variabilis occur in the lower epidermis.Q.suber first occurs phellogen,and the accumulation rate of phellem cells of Q.suber is slightly faster than that of Q.variabilis.(2)The endogenous hormones in the bark of two-year-old stems of Q.suber,Q.variabilis and Q.acutissima were determined respectively.A total of 6 plant hormones were detected,among which ethylene synthesis precursor(ACC)and salicylic acid(SA)were highly expressed in the barks of the three oak plants.The content of jasmonic acid(JA)in Q.suber bark was significantly higher than that in Q.variabilis and Q.acutissima.Abscisic acid(ABA)content in bark of Q.acutissima was significantly higher.We speculate that high levels of SA and ACC,as well as significantly different plant hormones JA and ABA play an important role in the regulation of phellem synthesis.(3)A total of 181.30 Gb of filtered data were obtained by transcriptome sequencing.Through Wayne analysis,enrichment analysis,functional annotation and correlation analysis of different growth stages,germination stages and growth stages of the same species,27 genes regulating the synthesis of Q.suber and Q.variabilis phellem were screened out,which were mainly enriched in 11 metabolic pathways.Biosynthesis includes fatty acid synthesis(map00061),biosynthesis of unsaturated fatty acids(map01040),fatty acid elongation(map00062),starch and sucrose metabolism(map00500),cutin,cork and wax biosynthesis(map00073),monoterpene biosynthesis(map00902),diterpene biosynthesis(map00904),sesquiterpene and triterpene biosynthesis(map00909),phenylpropanoid metabolism(map00940),biosynthesis of other secondary metabolites-part 2(map00998)and plant hormone signal transduction(map04075).Among them,LOC111984781,LOC112008862,LOC112027116 and LOC112034079 were up-regulated in plant hormone signal transduction pathway.there were 20 genes related to the regulation of Q.suber phellem synthesis,22 genes related to the regulation of Q.variabilis phellem synthesis There were 15 identical genes,including one AUX1(LOC111984781),one CYP94A5(LOC112004051),four E1.11.1.7(LOC111989638,LOC112023892,LOC112028402,LOC112040194),two FAB2(LOC111989517,LOC111992817),one GH3(LOC112008862),two IAA(LOC112027116,LOC112034079),one PLR(LOC112009314),three TOGT1(LOC112024399,LOC112039851,LOC112039854),may be genes that simultaneously regulate the formation of Q.suber and Q.variabilis phellem layer.(4)Among the 20 genes related to the regulation of Q.suber phellem,one FAB2 gene(LOC111989517),one GH3 gene(LOC112008862),two E1.11.1.7 genes(LOC112023892,LOC112040194)and one IAA gene(LOC112034079)were the key genes regulating the formation of Q.suber Phellem.Among the 22 genes related to the synthesis of Q.variabilis,two FAB2 genes(LOC111992805 and LOC111992817),one GH3 gene(LOC112008862),one PPT gene(LOC112011632),and one LUP4 gene(LOC112029584)play a key role in the formation of phellem.The indole-3-acetic acid-amide synthase gene GH3(LOC112008862)may be a key gene regulating the rapid accumulation of phellem.