Construction Of Expression Vectors Of Knocking Out BoPDS And SRK15 Gene In Brassica Oleracea L. By CRISPR/Cas9 System And Genetic Transformation

Brassica oleracea L.is not only an important vegetable crop in China,but also one of the leafy vegetable crops widely cultivated all over the world.The cultivation area can reach900,000 hm² every year in China.Brassica oleracea L.plays an important role in the annual balanced supply of vegetables.Brassica oleracea L.is a typical cross-pollination plant,which shows obvious heterosis.At present,breeders mainly prepare the first hybrid generation by self-incompatibility lines,but the self-incompatibility characteristics that the Brassica oleracea L.had makes its reproduction needs artificial pollination in bud stage.Artificial pollination not only requires low efficiency,time-consuming and high cost,but will lead to the low rate of production,all above the factors seriously restrict the improvement of the agronomic traits of Brassica oleracea L..In recent years,CRISPR/Cas9?clustered regularly interspaced short palindromic repeats/Cas9?system has been widely used.As a new gene editing tool,CRISPR/Cas9 has advantages of simple operation,low cost and short cycle,showing great potential in site-directed editing of genes in different plants.The CRISPR/Cas9system has been successfully applied in rice,wheat,arabidopsis,tobacco and other plants.In recent years,CRISPR system has also been successfully applied to Brassica oleracea L.,but it is not yet mature.In this study,we used CRISPR/Cas9 technology to knock out the PDS gene and SRK gene of Brassica oleracea L.and obtained transgenic plants,laying a solid foundation for the establishment of a simple and efficient gene editing system by CRISPR/Cas9 system in Brassica oleracea L.and the application of new germplasm resources.The main results and conclusions are as follows:1.Optimization of protoplasts preparation system and establishment of transient transformation system in Brassica oleracea L..The results of optimization of protoplasts preparation system showed that the optimal enzyme solution for protoplasts isolation was 1%CelluseR-10+0.5%MacerozymeR-10+0.6 mol/L mannitol+10 mmol/L MES+1 mmol/L CaCl₂+0.1%BSA+5 mmol/L?-mercaptoethanol.The explants of Brassica oleracea were incubated in enzyme solution for6 h at 26?in a dark rotary shaker at 60 rpm.The isolated protoplasts were centrifuged at500×g for 5 minutes.The protoplasts yield amounted to 3.0×10⁶/g fresh weight and the vitality was about 90.9%.The PYBA 1132 vector with green fluorescent protein can be successfully transformed into the purified protoplasts from hypocotyls,cotyledons and head leaves of Brassica oleracea L.by 20%PEG4000,and the transformation efficiency was up to43%,35%and 19%respectively.The protoplasts isolated from hypocotyls were the best suitable for transient transformation.The mitochondrion and plastid localization vectors COX and 969 were also successfully expressed in the protoplasts from hypocotyls.These results indicated that the transient transformation system has been established.2.The targeting ability of sgRNA was detected by protoplasts transient transformation system of Brassica oleracea L..The results of this study showed that there were base substitutions and insertions in the sequences near target 1 and target 2 of BoPDS gene,and the substitution occurred at the seventh base after the PAM sequence of target 1,changing from T base to G base.There were two types of base insertion in the vicinity of target 2.The first type was a T base insertion in front of the PAM sequence,and the second type was a two-base insertion at the third position and the sixth position in front of the PAM sequence.The editing efficiency was about 12.5%.There were base deletions and insertions in the sequences near target 1 of SRK gene.The CRISPR/Cas9 system did not edit the target 2 of SRK gene after sequencing the PCR products.The first editing type was the loss from the fourth base to the seventh base in front of the PAM sequence of target 1.The second editing type was the insertion of a C base at the fourth position in front of the PAM sequence of target 1.The editing efficiency was about 10%.3.Establishment of CRISPR/Cas9 gene knockout system in Brassica oleracea L..The PBSE401-BoPDS-CRISPR and PKSE401-SRK15-CRISPR expression vectors were constructed to transform cotyledons of Brassica oleracea L.by Agrobacterium mediated transformation.The PCR products of target sites from transgenic plants were sequenced and the results showed that there were no mutations at the target sites of BoPDS gene.According PCR sequencing results of SRK gene from 35 PKSE401-SRK15-CRISPR transgenic plants,we found knockdown occurred at the target site 1 in 3 plants,and 0 plant at target site 2,the editing efficiency for SRK gene reached 10%.Further sequencing analysis of PCR fragments cloning showed that the knockdown efficiency was 75%at the target site 1.