Establishment And Optimization Of Screening System For CKX1 Gene Mutant In Wheat TILLIING Mutant Library

TILLING technology is a reverse genetics technique for studying gene function by using a chemical mutagen EMS to induce point mutations and performing high-throughput screening techniques for mutation site scanning.This technology established a technical platform for the large-scale screening of mutants at the molecular level,and provided a new direction for studying the function of plant genes.Based on the preliminary work of the laboratory,this study sequenced the unsequencing part of wheat TaCKX1 gene,improved the EMS induced TILLING population of wheat variety Jimai22,established and optimized the mutant detection system based on HRM technology.The target gene TaCKX1 was screened for mutants.The main results are as following:1.Supplemental sequencing of wheat TaCKX1 gene sequenceIn order to better study the wheat TaCKX1 gene and obtain the complete genomic information of the three partial homologous genes of this gene,this study used Primer 5by referring to the existing wheat TaCKX1 gene sequence in NCBI and the CKX1 gene sequence information of similar species such as rice and maize.The software designed the sequencing fragment of the TaCKX1-B subgroup and the TaCKX1-D fragment,and cloned the unsequenced subgenomics with the cultivated variety Jimai 22 genomic DNA as a template.After sequence alignment,the sequence was determined to be the untested sequence of wheat TaCKX1 gene.The results laid the foundation for the subsequent exploration of this gene.2.Establishment and optimization of HRM mutant screening systemBased on the laboratory-made Q-PCR system,the unsaturated fluorescent dye SYBR Green I was changed to Eva Green,and the dye concentration,final concentration of Mg²⁺,final primer concentration and template dosage were adjusted.The system was optimized to establish a HRM mutant screening system.By constructing a template optimized for the HRM system with four single-base difference vectors containing only one base difference,the system dosage of the four aspects was optimized to achieve the goal that the established HRM system can distinguish single-base mutations.After optimizing the system,it is determined that when the amount of dye added in the reaction system is 0.1875?L,the concentration of Mg²⁺is 4.5 mmol/L,the amount of template is100-200 ng,and the final concentration of primer is 5?M,the system can distinguish single base difference.The SNP was typed to meet the requirements of screening single-base mutation screening targets.3.Establishment of a technology platform for wheat mutant scanning detectionIn order to establish a screening system of the wheat TILLING library based on HRM technology,the sequence information of the wheat TaCKX1 gene,which is closely related to the control of grain size and yield,was determined in the laboratory.However,due to the high specificity of the HRM technology for screening primers,it is necessary to further correct the genetic information of the three partial homologous subgroups of the wheat TaCKX1 gene.The three subgroups of this gene were resequenced by redesigning specific primers to correct for differences in the presence of previous sequencing.In this study,a total of 51 pairs of primers were designed and screened in three subgroups using the corrected TaCKX1 gene sequence,and further HRM reactions were carried out on primers in which a single bright band and no primer dimer could be specifically amplified.Through two rounds of PCR amplification and HRM analysis of606 EMS-induced mutant genomic DNA,sub-subgroup mutants were screened in three subgroups of TaCKX1 gene,and G base to A base were successfully screened.Base mutation.The pre-screening of mutant exon regions of TaCKX1 gene verified the operation of the platform,and a technology platform for scanning and detecting mutants of any target gene was established.