Cas9/gRNA-mediated COBRA3 gene mutation in Populus trichocarpa

Objective COBRA plays an important role in the growth and development of plants.Present studies have shown that COBRA genes are associated with plant secondary growth.To know more information and function of the COBRA genes in trees, we identified the COBRA gene family in Populus trichocarpa and further made analytic studies.

MethodWe used phylogenetic tree for analyzing the relationship among the PtrCOBRA members and semi-quantitative RT-PCR for determining transcriptional expression of the PtrCOBRA genes in various tissues of P. trichocarpa.Cas9/gRNA gene editing technique was used to produce PtrCOBRA3 gene mutant. Agrobacterium-mediated genetic transformation was performed using P. trichocarpa stem as explants for infection.

ResultWe identified the 14 COBRA genes in the genome of P. trichocarpa. PtrCOBRAs were divided into two branches of phylogenetic tree. Semi-quantitative RT-PCR analysis showed that PtrCOBRA3 and PtrCOBRA11 were specifically expressed in xylems on the high transcriptional level and their transcription levels were also high in the lignified stems. We knocked out PtrCOBRA3 gene in P. trichocarpa via Cas9/gRNA technique. As a result, we obtained four independent ptrcobra3 mutants. Phenotypes of these mutants were as follows: dwarf plants, narrow and curling leaves and slightly bending stems.

Conclusion PtrCOBRA3 and PtrCOBRA11 are main members of P. trichocarpa COBRA gene family, which involve in secondary growth of Populus trichocarpa. Genetic data of PtrCOBRA3 knockout shows the function of PtrCOBRA3 in Populus trichocarpa stem secondary growth. Additionally, PtrCOBRA11 is redundant to the PtrCOBRA3 gene on the function.