| The upgrading of crop breeding technology is the basic guarantee of modern agriculture.However,due to the long cycle and high investment of traditional breeding technology,it is difficult to quickly and efficiently solve the new problems emerged during the modern agricultural production.Gene editing technology developed in recent years,especially the CRISPR gene-editing technology,does not rely on the natural genetic variation resources,is able to change a single or combined traits of crops so that significantly improves the efficiency and specificity of breeding,eventually reaches the goal of “design breeding”,thereby meeting the needs of crop breeding in the economic development and environmental changes.The CRISPR-Cas9 system can lead to loss-of-function mutations.Therefore,in order to rapidly evolve new gene resources with important agronomic value,this study utilized plant base-editing and guided editing systems to develop editing technologies for high-density mutation target fragments and important site saturation mutation libraries.In this way,high-throughput replacement mutation resources for important gene functional domains or key amino acid sites in rice can be created,which not only breaks the bottleneck of single editing target,but also innovates the method of in situ directed evolution of crop genes.Taking advantage of these breakthroughs,several novel alleles of herbicide-resistant genes were excavated in this study,which are urgently needed for direct seeding rice breeding,and provide important genetic resources for light and simplified rice production.The work shown here can be divided into four parts.1.Development of efficient crop base editing toolsTo start with,cytosine deaminase(APOBEC),n Sa Cas9(D10A)and uracil glycosylase inhibitor(UGI)optimized by rice codon were used to construct the CBE vector SPCAS9-BE3.In order to improve the editing efficiency of SPCAS9-BE3,three copy UGI were fused on its basis to form SPCAS9-e BE3.The efficiency test results confirmed that the editing efficiency and clean single base substitution efficiency of SPCAS9-e BE3 were greatly improved.Similarly,another CBE vector sp CAS9-CDA was constructed by using n Sp Cas9-D10 A and fusion with rice codon-optimized Petromyzon marinus cytidine deaminase(Pm CDA)and uracil glycosylase inhibitor(UGI).Here,the Sp Cas9-e CD expression cassette coupled with the esg RNA expression cassette or the t RNA-sg RNA expression system was used to improve the editing efficiency and accuracy of Sp Cas9-CDA.The results of the target gene showed that the mutation form of the non-ideal type was greatly reduced,especially the C to T single base replacement efficiency of Sp Cas9-e CDA in the t RNA-sg RNA expression system was increased to 75%,and no other mutation forms were included.2.Development of high-density base editing library technology to direct evolution of new herbicide-resistant alleles of rice Os ACC1 gene.First,for the 1653 bp coding region of the Os ACC1 carboxyltransferase domain(CT domain)in the rice genome,which interacts with aryloxy phenoxy propionic acid herbicides,141 sg RNA were designed based on all NGG PAM feature sequences recognized by Sp Cas9 to form the sg RNA library.Then,e ABE vectors responsible for Ato-G editing and e BE3 and e CDA vectors responsible for C-to-T editing were used to construct three base editing libraries that could target the 141 sg RNA regions.These libraries were introduced into rice callus to achieve high-density mutation of important rice gene fragments based on base editing libraries.Through herbicide resistance screening,it was found that Os ACC1-I1879 V,-C2186 R and-W2125 s could improve the resistance of plants to arylphenoxypropiolic acid herbicide.Finally,genetic and phenotypic analysis showed that the Os ACC1-I1879 V mutation increased resistance without affecting the main agronomic characteristics.Therefore,the Os ACC1-I1879 V gene resources derived from directed evolution of high-density base editing library technology may have important production value in rice resistance improvement.3.Development of saturation guided editing library technology to tap the herbicide resistance potential of Os ACC1 gene.Crop genes,especially those with important functions,are often regulated mainly by a few key amino acids.In order to deeply explore the resistance potential of Os ACC1 key location,this study designed guided-editing libraries that mutated specific amino acid coders into random NNN coders at 6 key location that may be involved in the determination of herbicide resistance.Here,the guided editing tools that can accurately achieve any type of nucleotide substitution are used to achieve saturated mutations that replace one amino acid with another 19 amino acids at these key location.By importing 6guide-editing libraries,this study obtained 16 different types of herbicide-resistant monoamino acid variants of Os ACC1,and 5 of them were newly reported in natural plants containing wild resistant weeds,providing new germplasm resources for the development of rice herbicide-resistant varieties.4.Technical verification of directional evolution of rice editing librariesBy using the efficient PE editing tool in the laboratory,this study carried out multiple types of amino acid substitutions for the new herbicide resistance location 1879 in rice that has not been reported,and made targeted mutation of the herbicide Os ACC1 suppressor gene.Mutations in different amino acids at the same resistance site may lead to differences in drug resistance.In this study,19 key amino acid mutation types of drug resistance were obtained,and the substitution amino acid types with high efficiency and excellent agronomic traits were identified. |
