Study On The Function Of Wheat TaCKX1 Gene

Cytokinin（cytokinin,CK）is a class of plant hormones with multiple functions,which can promote cell division and differentiation,improve grain yield and regulate abiotic stress,etc.Cytokinin oxidation/dehydrogenase（CKX）is the only enzyme that irreversibly degrades cytokinin.Triticum aestivum L.belongs to the Gramineae family and is one of the three major food crops in the world,providing about 20%of human protein and calories.Therefore,it is of great significance to study the function of Ta CKX gene to develop high quality and high yield wheat varieties.In this study,the Ta CKX1 mutant lines in the M2 generation of Jimai 22（JM22）wheat were identified,the phenotypic effects of Ta CKX1 gene mutation were preliminarily studied by analyzing the plant height,thousand grain weight and the relative expression levels of Ta CKX1 gene in different periods of the Ta CKX1 mutant lines.The activity of CRISPR/Cas9 gene editing vectors targeting Ta CKX1 constructed in the previous laboratory was detected by optimizing the protoplasm preparation system and transient transformation system of wheat.Based on the optimization of wheat genetic transformation system,p Ta CKX1-g1 transgenic positive plants were obtained,which laid a material foundation for further study of Ta CKX1 gene function.The main results are as follows:1.Identification of Ta CKX1 mutant lines and analysis of their physiological functions and gene expressionIn order to study the function of Ta CKX1 gene,a total of 6 M2 generation Ta CKX1mutant lines（JM5、JM23、JM52、JM98、JM138 and JM274）were obtained.Cloning and sequencing showed that all of them had C→T or G→A single base mutation induced by EMS.The analysis of plant height,thousand grain weight and Ta CKX1 gene expression in the identified mutant lines showed that the plant height and thousand grain weight of JM274 were significantly higher than those of other lines,and the expression of Ta CKX1 gene at flowering and maturity was lower than that of other lines,indicating that Ta CKX1 gene is a negative regulator of cytokinin and is closely related to wheat yield.2.Preparation of protoplasm system and optimization of transient expression system of Jimai 22 and activity detection of Ta CKX1 gene editing vectorsIn order to detect the activity of Ta CKX1 gene editing vectors,the preparation and transformation conditions of wheat protoplasts were optimized.In this study,the optimum conditions for the preparation of wheat protoplasm Jimai 22 were determined by orthogonal experiments.The results showed that with the cellulase concentration of1.5%,macerozyme concentration of 0.75%,mannitol concentration of 0.8 mol/L,the protoplast damage was the least,the highest protoplast yield of 6.97x10⁶was obtained,with little protoplast damage and activity of 93.99%,which was higher than that of other combinations.Under the conditions of protoplast concentration of 2x10⁵cells/m L,PEG concentration of 25%,plasmid concentration of 1μg/μL and transformation time of 20min,the recombinant plasmid p AN580 was transformed into protoplasts by PEG-Ca²⁺mediated method,and the transformation efficiency reached to 65.22%.After transferring the Ta CKX1 gene editing vectors into protoplasts,one of them was found to have editing activity and was named p Ta CKX1-g1.3.Optimization of wheat genetic transformation system and identification of p Ta CKX1-g1 transgenic positive plantsThe p Ta CKX1-g1 vector was transferred into young wheat embryos by agrobacterium mediated method,and 38 transgenic plants were obtained through induction and differentiation of callus,and then the plant DNA was extracted,PCR amplification,enzyme digestion,specific primer amplification,enzyme digestion and other experimental steps,two p Ta CKX1-g1 transgenic positive plants were identified in the A sub genome and one p Ta CKX1-g1 transgenic positive plant was identified in the B sub genome,which laid a material foundation for further study of the function of the Ta CKX1 gene.