| Protoplast means large numbers,close cell states,and highly homologous genotypes.These can bring stable experimental results to study the function and regulation mechanism of special genes in plants.For some valuable plant characteristics,people have been able to skillfully use protoplast as experimental materials in different plants to study the mechanism of the job characteristics.The characters of plants were studied by different experimental methods such as subcellular localization,bimolecular fluorescence complementation and agrobacterium infection.And with the development of technology,m ore and more newer and more efficient techniques will be applied to the study of plant genetic transformation.For example,Crispr/Cas9 technology has been able to be skillfully applied in protoplasts of corn,rice,arabidopsis,tobacco,etc.for targeted gene editing research.This will provide a powerful way to explore deeper genetic traits in plants.Some of the most striking features of the bamboo are well known.For example,the bamboo has rapid growth,uncertain flowering time,and death after flowering.These are all very attractive features to explore.However,as far as the current research progress is concerned,the genetic transformation system has not been established,so the mutant plants of the bamboo can not be obtained.There are few applications of the protoplast of the bamboo,and there is no mature,stable and repetitive conversion system of the protoplast of the bamboo.All these have greatly hindered the further research on the bamboo.This paper will start from the exploration of the preparation of the protoplasts of the bamboo,improve the preparation process of the protoplasts of the bamboo to obtain efficient methods,and further explore how to better use the protoplasts of the bamboo as experimental materials for the establishment and application of the experimental research system.It provides a variety of experimental means for your research on the bamboo,and a powerful help for your experiment.In the process of exploring the establishment and application of instantaneous transformation related system,this paper mainly draws the following important conclusions:(1)The optimal enzymatic hydrolysis solution prepared by phytoplasm of phyllostachys was:cellulose enzyme(RS)2.5%+segregation enzyme(R-10)1.25%(2)The optimum osmotic potential concentration of the protoplast enzymatic hydrolysis solution was 9%(3)The best protoplast enzymolysis time of the protoplast of bamboo was 7h(4)The transient vectors CNX-RFP(endoplasmic reticulum),MAN1-RFP(golgi body)and NLS-MCHERRY(nucleus)were successfully located in the protoplast of phyllostachys pubescens(5)pK7FWG2 binary expression vector was successfully transformed into the protoplast of bamboo,and the pK7FWG2-V11 binary expression vector connected with functional genes was also successfully verified(6)The successful transfer of the RFP instantaneous expression vector and the GFP instantaneous expression vector into the same hydatid hydatids verifies that the hydatid hydatids of hydatid hydatids can co-transform with different carriers(7)The bimolecular fluorescence complementarity(BiFC)system was successfully constructed in the protoplasts of phyllostachys edulis,and two vectors,pB4CYGW-IAA38 and pB4NYGW-ARF40 transformed into protoplasts of phyllostachys edulis(8)The vectors of pK7EGF2-IAA38 and pGWB615-ARF40 were successfully transformed into the protoplast of the bamboo,and the co-precipitation(CO-IP)system was applied to verify the gene interaction(9)It was successfully verified that the plasmid Crispr/Cas9 system could be applied to the protoplast of bamboo,and the vector Crispr/Cas9-bamboo successfully restored the invalid mGFP gene into the GFP gene with fluorescence function... |
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