Study On Directional Editing Technology Of Rice Sperm Genome Based On Magnetic Nanoparticle Carrier

Rice is one of the most important food crops in China and occupies a very important position in agricultural production.CRISPR/Cas9 gene editing technology can realize precise editing of the target gene,and has advantages of simple operation,short cycle and high efficiency.In rice research,tissue culture-based Agrobacterium-mediated methods are the most commonly used method for CRISPR / Cas9 system transformation,but the transformation cycle is long,chimeras are easy to produce,and tissue culture of some varieties is difficult,which seriously affects the genetic transformation process of rice.From the perspective of genetic engineering and molecular breeding,the ideal method is to transform germ cells.Pollen is an ideal target for introducing foreign genes into germ cell lines because of its abundant resources and easy handling.The surface of rice pollen has a germination holes,and foreign materials can be absorbed into the pollen through the germination hole.However,due to the large molecular weight and poor stability of the CRISPR/Cas9 system,it is easily degraded or bound quickly.Therefore,there is a need to develop new technologies that can avoid the tissue culture process and can safely and efficiently deliver the CRISPR/Cas9 system to rice germ cells.With the development of nano-biotechnology,the size of nano-materials(MNP)is nano-scale,and it is safe,low-toxic,and has good cell compatibility,which provides favorable conditions for nano-materials as gene carriers to directly transform rice germ cells.In this study,MNP and rice variety Nipponbare were used as experimental materials.The following four aspects were studied: the binding efficiency of MNP and CRISPR/Cas9 plasmids,and the MNP-CRISPR/Cas9 complex magnetically transfected rice pollen.Process,editing efficiency of offspring by magnetic transfection with CRISPR/Cas9 plasmid,and genetic stability of offspring.Exploring The feasibility of magnetic nanocarrier gene transduction system in genetic transformation of rice breeding.The research results show that:(1)Using MNP as a gene vector to load CRISPR / Cas9 plasmids to prepare complexes with different mass ratios.To analyze the ability of MNP to load and protect the CRISPR / Cas9 plasmid,and study its particle size distribution,zeta potential,and binding morphology.The results show that MNP compresses,adsorbs,and aggregates the CRISPR /Cas9 plasmid through electrostatic interaction to form nanocarrier-gene complexs.MNP can effectively load CRISPR/Cas9 plasmid equivalent to 16 times its mass and protect the loaded CRISPR / Cas9 plasmid from Enzymatic degradation.(2)Comparing the effect of magnetic transfection of different mass ratio complexes on the germination rate of pollen,in order to analyze whether magnetic transfection can transfer the complex to the interior of pollen,We studied the location of the complex in pollen after magnetic transfection.The results show that the pollen density can meet the experimental requirements when the pollination density is 2 flowers / 30ul;the pollen that is magnetically transferred by the compound with different mass ratios can germinate normally.When the compound mass ratio is 1: 4,1: 8,1:16,Germination has a promoting effect,and when the mass ratio is 1: 1,the compound has a suppressing effect;Comparing with the control,magnetically transfected pollens had many white bright spots inside it,and spectroscopic analysis detected that the pollen contained Fe element,which proved that the complex could reach the interior of the pollen through the germination hole.(3)CRISPR/Cas9 plasmid targeting the rice fgr gene was constructed,and used MNPloaded CRISPR/Cas9 plasmids to magnetically transfect mature Japanese pollen,followed by artificial pollination and harvesting of seeds for T1 generation.We investigated the agronomic traits of T1 generation plants,analyzed the effects of magnetic transfection on agronomic traits of plants,and screened T1 generation transgenic plants,Then transgenic plants were subjected to target sequencing and mutant individual phenotypic analysis.The results showed that the T1 plants after magnetic transfection had shorter plant height and lower seed setting rate,and there were no significant changes in other aspects.Through hygromycin and Cas9 gene detection,23 positive plants were detected in 176 T1 generation plants,and the transformation rate was about 13.07%.Sequencing analysis showed that only two strains had mutations at the target site,which were biallelic and heterozygous mutations,respectively.Compared with the control,the mutant plants had shorter dwarfs,shorter flag leaf length,and lower seed setting rate,there were no significant changes in other aspects.(4)The T1 generation transgenic plants were planted according to the strains.The genetic stability of the magnetically transfected offspring was studied by screening T2 generation positive plants and target sequencing analysis.The results showed that a total of155 positive plants were detected in the T2 generation.The hygromycin gene isolation basically accorded with Mendelian genetic laws,indicating that the The foreign gene transferred by the staining method can be passed on to the offspring.The T2 generation of the two lines that had mutations in the T1 generation were separated;some of the lines that did not have mutations in the T1 generation had mutations in the T2 generation,including 2homozygous mutations and 7 biallelic mutations.Indicating that the Cas9 gene can continue to edit gene expression in T2 generation,but the editing efficiency is low.This study shows that MNP can be used as a gene vector to payload and protect the CRISPR / Cas9 plasmid.The MNP-CRISPR / Cas9 complex can be transferred into rice pollen by magnetic transfection,and target gene editing plants can be generated after pollination;some Cas9-positive T1 plants have not been edited for target genes,but can continue to play an editing role through selfing.Compared with genetic transformation methods that rely on tissue culture,this research method is simple and time-consuming,and proves the feasibility of magnetic nanocarrier gene transduction system in genetic transformation of rice breeding,and provides a new method for the cultivation of excellent rice varieties.