Comprehensive Analysis Of Poplar PtrCOBRA And COBRA3/11 Function

COBRA encodes a plant specific GPI anchored protein.The number of COBRA gene family ranges from a few to a dozen.Different COBRA genes play a different role in plant tissues and are characterized by functional diversification.To know more information and function of the COBRA genes in trees,we identified the COBRA gene family in Populus trichocarpaand further made analytic studies.The main research results are as follows:We identified the 14 COBRA genes in the genome of P.trichocarpa.All COBRA members are characterized by a carbohydrate-binding module,a highly conserved central cysteine-rich domain and a hydrophobic C-terminal domain.PtrCOBRAs were divided into two branches of phylogenetic tree.They were grouped with AtCOB and AtCOBL7 of Arabidopsis.The gene structure is the same as that of the phylogenetic tree,the first subbranch of the PtrCOBRA family contains multiple introns,while the second subbranch has only one intron.COBRA3 and COBRA11 were specifically expressed in xylems on the high transcriptional level.COBRA12 and COBRA13 were expressed in root on the high transcriptional level.Five COBRA genes were expressed in all tissues.The rest were not expressed or expressed very low.COBRA3 and COBRA11 were expressed from the third stem section,and gradually increase.COBRA10 was expressed in the first three internodes and was higher.Five COBRA genes were expressed in all internodes.The rest were not detected on the transcriptional level.PtrCOBRA genes were diversely expressed on transcriptional level in different issues,and PtrCOBRA genes expression exist functional redundancy.Cas9/gRNA gene editing technique was used to construct pHSE401-PtrCOBRA3 and pHSE401-PtrCOBRA11 successfully.Agrobacterium-mediated genetic transformation was performed using P.trichocarpa stem as explants for infection,and we produced nine transgenic plants.We carried out genome DNA PCR and detected gene target sequence.The result showed that we produced gene knockout mutants.We obtained four independent ptrcobra3 mutants and five independentptrcobrall mutants.Phenotypes of ptrcobra3 mutants were as follows:dwarf plants and decreased about 12%,narrow and curling leaves,slightly bending stems and brittleness.Phenotypes of ptrcobrall mutants were as follows:dwarf plants decreased about 25%,narrow by about 20%and flat leaves,slightly bending stems and high brittleness.We observed xylem fiber cell phenotype of mutant by means of paraffin section technology and chemical staining method.The result showed that there is no difference to compare with wild type in xylem fiber cell morphology and size,but the cell wall of xylem fiber cell was thin in the higher lignified stem nodes.We used scanning electron microscope for observing the stem nodes cross-sections xylem fiber cell phenotype of ptrcobrall mutants.The result showed that there is no difference to compare with wild type in xylem fiber cell morphology and size,but the cell wall of xylem fiber cell was thin in the higher lignified stem nodes.We calculated fiber cell wall thickness of xylem in the higher lignified stem nodes.Statistics indicated that fiber cell wall thickness of xylem of mutant became visibly thin about 50%.PtrCOBRA3 and PtrCOBRA11 are main members of P.trichocarpa COBRA gene family,which involve in secondary growth of Populus.Genetic data of PtrCOBRA3 and PtrCOBRA11 knockout show they function in Populus cellulose synthesis and stem secondary growth.Additionally,PtrCOBRAll is redundant to the PtrCOBRA3 gene on the function.