Establishment Of Mutant Materials Of BNACIPK23 And BNACIPK24 In Brassica Napus L. And Study On Salt Tolerance Function

As an important oil-bearing crop in China,Brassica napus L.has many important economic effects.However,abiotic stress affects the production of Brassica napus L.,and salt stress is a serious hazard to Brassica napus L.CIPK is a plant-specific protein kinase that can respond to salt stress injuries in plants.In this study,Bna CIPK24 and Bna CIPK23,two members of the CIPK family in Brassica napus L.,were identified by bioinformatics analysis,and the functional deletion mutants of Bna CIPK24 and Bna CIPK23 were created by CRISPR/Cas9 technology,to preliminarily explore the functions of CIPK24 and CIPK23 in Brassica napus L..The main results are as follows:(1)In the ‘Westar’ genome of Brassica napus L.,Bna CIPK24 has two homologous copies,which are similar to those of Brassica rapa,Brassica oleracea and Arabidopsis thaliana and contain 12–13 exons.The evolutionary tree shows that Bna A04.CIPK24 and Bra A04.CIPK24 belong to the same branch,while Bna C04.CIPK24 and Bol C04.CIPK24 belong to the same branch.Protein sequence alignment showed that the two homologous copies of Bna CIPK24 had high similarity and high conservation with the amino acid sequences of Bra A04.CIPK24,Bol C04.CIPK24 and At CIPK24,and both contained NAF motif specific to CIPK protein kinase.(2)The expression patterns of the two copies of Bna CIPK24 in different tissues were similar,both showing high expression in flowers and siliques,and low expression in roots,stems and leaves.The expression levels of two homologous copies of Bna CIPK24 were significantly up-regulated after 1 h of treatment with 200mmol/L NaCl,suggesting that Bna CIPK24 might respond to salt stress at the transcriptional level.(3)For the two homologous copies of Bna CIPK24,a two-target vector that could target the two copies was designed using CRISPR/Cas9 technology,and the hybrid mutant was obtained by genetic transformation using Brassica napus L.‘Westar’ as the receptor material.Compared with the wild type,the mutant showed lower germination rate and weaker growth vigor after salt treatment.It indicated that Bna CIPK24 positively regulated salt stress.(4)Arabidopsis thaliana T-DNA mutant atcipk23 showed salt sensitivity under salt stress.In order to explore whether Bna CIPK23 in Brassica napus L.had the same function,protein sequence analysis and expression pattern analysis were performed on Bna CIPK23.There were six homologous copies of Bna CIPK23 in the genome of ‘Westar’ and the evolutionary tree showed that Bna A07.CIPK23 and Bna C07.CIPK23 belonged to the same branch as At CIPK23.The gene structures of Bna A07.CIPK23 and Bna C07.CIPK23 and At CIPK23 were also similar.The protein sequence analysis showed that the sequence similarities of Bna A07.CIPK23 and Bna C07.CIPK23 and At CIPK23 were high,while the homology of other homologous copies was low.The tissue expression pattern showed that Bna A07.CIPK23 was highest expressed in roots,and Bna C07.CIPK23 was highest expressed in flowers.The expression levels of the two copies in stems were both low.After 1 h of salt stress treatment,the expression of Bna C07.CIPK23 was significantly increased,indicating that Bna C07.CIPK23 might specifically respond to salt stress in Brassica napus L.(5)‘Westar’ as the receptor material,a functional deletion mutant of Bna CIPK23 was created by CRISPR technology,which created the conditions for the subsequent verification of the function of Bna CIPK23 in salt stress in Brassica napus L.