Study Of Layered Double Hydroxide Nanosheet As Gene Nanocarriers For RNA Molecules Deliver Into Plant Cells

The emerging applications of nanocarriers as gene carriers in plants provide a promising alternative for mild and passive deliver functional genes or nucleic acid of transgene into intact plant cells.Mg-Al layered double hydroxide nanosheets（LDH）are one of the nanocarriers that have attracted the most attention.In the context of interdisciplinary disciplines such as nanomaterials science and plant biology.To develop new methods for LDH based delivery systems that can deliver si RNA,ds RNA or circ RNA into intact plant cells.This methods allows highly efficient and plant intracellular specific delivery that can overcome host-range limitations.It provides advanced research tools for plant biology and enriches the research methods of nanomaterials in the field of biology.The zeta potential,Z-average size,atomic force microscopy（AFM）imaging,mass ratio of ds RAN adsorption,ds RNA adsorption time,ds RNA adsorption efficiency and the durability of ds RNA protection of LDH nanocarriers were measured.It is thought after comprehensive analysis that the LDH nanocarriers prepared with CO₂ free ultrapure water and synthesis temperature of 30℃have the advantages of appropriate Z-average size,high zeta potential,low mass ratio of nucleic acid adsorption,rapid adsorption and long-lasting protection of ds RNA.LDH nanocarriers has excellent protective effect on nucleic acid molecules.Changing p H or adding Na Cl can realize the desorption of nucleic acid molecules.LDH nanocarriers can be stored for 3 months in a sealed CO₂ free environment,but the newly synthesized LDH is better than that stored for a long time.Long term storage of LDH nanocarriers will lead to aging phenomena.For example,the number of monolayer nanocarriers decreases,the stacking of some nanocarriers,zeta potential and Z-average size begins to decreases.The objective of this study is to explore the effect of LDH nanocarriers adsorbing si RNA,ds RNA,and circ RNA molecules to interfere target genes in Nicotiana benthamiana and Arabidopsis.PDS gene of N.benthamiana and Arabidopsis,WUS gene of Arabidopsis and N.benthamiana,RHD6 gene related to rood hair formation of Arabidopsis were studied by leaf injection and co-incubation,and then the results of phenotypic characteristics,target gene transcription level and protein content were analyzed.The results showed that LDH nanocarriers could load a variety of RNA molecules into the leaves,shoot apical meristem and roots of model plants,breaking through the limitations of cell wall and cell membrane obstacles,without affecting the normal function of RNA molecules.It was confirmed that LDH nanocarriers can be used as novel gene nanocarriers to deliver RNA into model plant cells.It provides a new gene nanocarriers and method for gene function detection and functional verification of si RNA,ds RNA,and circ RNA.In order to further broaden the application field of LDH nanocarriers,cassava NBS-LRR proteins were studied.The NBS-LRR is the most widely existing protein in plants.It was found that LDH nanocarriers adsorbing ds RNA had gene silencilg function,similar to virus induced gene silencing.An actual example of cassava gene function research was given to illustrate the concrete application of the new method.The new method provides a methodological reference for the study of gene function in cassava and other plants.In conclusion,Mg-Al layered double hydroxide nanocarriers was optimized and synthesized and proved to be used as gene nanocarriers to diliver a variety of RNA moleculaes to breaking through the limitations of cell wall and cell membrane obstacles and transfer them into plant cells.It is a new,efficient,broad-spectrum and easy to operate method.This approach will have a diverse range of applications in plant gene function,plant biotechnology and plant biology.