Construction Of TaCKX1 Gene CRISPR/Cas9 Targeted Editing Vector And Establishment Of Genetic Transformation System

Common wheat(Triticum aestivum L.,2n = 42,AABBDD)is an allohexaploid,which is one of the most important food sources in China and many countries in the world,and provides about 20% of energy for human beings.Due to its large genome,complex genetic structure and multiple copies of most genes,the traditional forward or reverse genetics methods are unable to meet the analysis of the function of the wheat genome and the genetic improvement,resulting in the wheat genome research far behind other food crops such as maize and rice.With the completion of sequencing,assembly and annotation of the whole wheat genome,establishing an efficient reverse genetics approach becomes the key to wheat genomics research and genetic improvement.Gene editing technology,especially the Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9(CRISPR/Cas9)system,is currently the most promising technical system for mutation or gene modification.It has the characteristics of good specificity,high efficiency and high throughput.At present,this technology has been widely applied in animal and plant research,which provided new techniques and ideas for plant breeding.In order to improve the wheat cultivar Jimai 22,this study successfully constructed a TaCKX1 gene targeted editing vector and established an in vitro culture and regeneration system using immature embryos on the basis of the previous work in the laboratory.At the same time,in order to detect the editing efficiency of vectors,a set of wheat mesophyll cell protoplasm preparation and transient expression technology operating procedures were established.Further,some whole exon capture sequenced Jimei 22 TILLING mutant lines were analysed for TaCKX1 mutation sites.The main results are as follows:1.Establishment of wheat immature embryo in vitro regeneration systemsIn order to set up an effective TaCKX1 gene transformation platform,a wheat immature embryo regeneration system was established in this work.The results showed that addition of 2,4-D(2.0 mg/L)and proline amide(500 mg/L),glutamine(500 mg/L),and hydrolyzed casein(300 mg/L)to the MS basal medium yielded the average callus of92.2%.Embryonic callus were produced with pale yellow color and nodules on the surface.The effect of both asparagine and hydrolyzed casein in the medium was better than that of hydrolyzed casein alone.On the medium without any hormone,the efficiency of callus differentiation of wheat immature embryos was significantly higher than that of the medium with hormones.2.Construction of TaCKX1 gene targeted editing vectorSix pairs of sgRNAs within the first exon of TaCKX1 were designed and synthesized,and inserted into the pTaU6-sgRNA carrier skeleton.Then,the target fragment with the target sequence and gRNA amplify using specific primers with homology arms was inserted into the vector backbone pYAO-Cas9 through homologous recombination.The Sanger sequencing result showed that a TaCKX1 gene CRISPR/Cas9 recombinant expression vector was successfully constructed,which provided prerequisites for subsequent functional analysis and genetic improvement of this gene.3.Transient expression system of wheat protoplastsIn order to test the editing efficiency of the TaCKX1 gene editing vector,factors affecting the preparation process of wheat mesophyll protoplasts were analyzed through systematic orthogonal and random design experiments.The optimal combination of parameters including the age and leaf section of the seedlings,the osmotic pressure of the enzymatic hydrolysate,the enzyme concentration,and the enzymatic hydrolysis time were determined.A set of efficient protoplast preparation and transient expression procedures for wheat mesophyll cells was established.Comprehensive analysis of the orthogonal test and the completely random design showed that the largest number and the most complete protoplasts were obtained through the combination of the first true leaf of wheat with a seedling age of 7 d,the cellulase concentration(M/V)of 1.5%,the isolating enzyme concentration(M/V)of 0.75 %,the mannitol concentration of 0.8 mol/L,and the digestion time of 5 h,.The transient expression of GFP plasmid AN580 in protoplasts was achieved through PEG-mediated system,resulting a transformation efficiency of of about40% for Jimai 22 wheat protoplasts.4.Detection of TaCKX1 gene mutation siteExamination of some whole exon captured and sequenced TILLING mutant lines in our laboratory revealed that,2 TaCKX1 mutants in the A subgenome,4 mutants in the B subgenome,and 5 mutants in the D subgenome were identified among the 13 tested mutant lines.The detection of the TaCKX1 gmutation sites laid the foundation for the subsequent research on the functional studies of this gene and the genetic improvement of the variety Jimai 22.