| Aquaporins(AQPs)are proteins that efficiently transport water in plants.It is responsible for the transmembrane transport of water and neutral small molecules.This process is not only closely related to plant physiological activities,but also participates in processes such as plant resistance to adversity stress.At present,the main research is on the role of AQPs in the process of material transport and stress,but there is no report on the effect of AQPs on the secondary growth of wood.In this study,Populus trichocarpa was used as the research material,and PtrPIP2;11,a member of the AQPs subfamily,was used as the research object to study the effect of PtrPIP2;11 on the secondary growth of P.trichocarpa.The main conclusions are as follows:Fourteen PIP2 subgroup members were screened in the P.trichocarpa.database,and it was proved by multiple sequence alignment that they all contained six conserved transmembrane domains and two NPA motifs of AQPs.Semi-quantitative detection of the expression levels of PIP2 subgroup genes in different tissues of P.trichocarpa,the results showed that PtrPIP2;11 was highly expressed in the xylem;Aspwood was used to predict the expression abundance of its stem tissue,and the results showed that PtrPIP2;11 was in the xylem expansion region have the highest abundance expression.By constructing a35S::PtrPIP2;11-YFP expression vector and transforming poplar leaf protoplasts by PEGmediated method,Fluorescence of YFP fusion proteins is mainly concentrated in the endomembrane system by confocal fluorescence microscopy,which proved the localization of PtrPIP2;11 protein on the intracellular membrane system.Based on the CRISPR gene editing strategy,a Cas9/g RNA mutation vector of the PtrPIP2;11 gene was constructed.Five transgenic plants were obtained by Agrobacteriummediated transformation of P.trichocarpa stem segments and DNA molecular identification.Comparing wild type to obtain three target gene biallelic editing plants.After the mutant and wild-type P.trichocarpa were grown in the greenhouse for 90 days,compared with the wildtype,the plant height of the ptrpip2;11 mutant increased by 11%,and the length and diameter of stem nodes and leaf width were significantly increased,indicating that PtrPIP2;11 affected the vegetative growth of P.trichocarpa.In our study,the stem tissues of ptrpip2;11 mutants and wild-type plants were sectioned by shaking,and observed under light microscope after phloroglucinol staining.We found that the secondary phloem region of ptrpip2;11 mutant plants was larger than the wild type and had more layers of xylem cells in the same stem node.The stem segment fibroblasts and ductal cells were isolated by acid and then measured by light microscopy.It was found that the largediameter fibroblasts and ductal cells of the ptrpip2;11 mutant accounted for more,which indicated the changes in the plant height and basal stem of the ptrpip2;11 mutant.It is caused by the enlargement of xylem fibroblasts and vessel cells.The above phenotypes prove that knockout of PtrPIP2;11 can cause xylem cells to expand and the secondary phloem region to become larger,which indeed affects the development of stems to a certain extent.The cell wall thickness of the stem segment was observed by scanning electron microscope after freehand sectioning.Compared with the wild type,the cell wall of the ptrpip2;11 mutant was thinner.Compared with the wild type,the lignin content of the ptrpip2;11 mutant was decreased by 10%,and the cellulose content was increased by 14%,which further proved that PtrPIP2;11 affects the stem Cell wall synthesis and wood component content of cells.This study showed that PtrPIP2;11 knockout affected stem development,indicating that PtrPIP2;11 directly or indirectly affected plant growth. |
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