Cloning Of BnaABCG14.A09 Gene And Its Function In Salt Tolerance In Brassica Napus

Rapeseed is one of the main oil crops in China and plays a decisive part in the supply of edible oil in China.However,China’s total rapeseed output cannot meet the national consumption demand,and it still needs to make up for the shortage of domestic supply through a large number of imports.In recent years,China has vigorously promoted the cultivation of oil crops,but abiotic stresses such as land salinization and drought have seriously affected the growth and yield of plants.As a signal molecule in the plant cell,plant hormones play an active role in response to abiotic stresses.Among them,cytokinin plays a very important role in response to abiotic stresses.For example,plants can improve their adaptability to stress by adjusting the spatial distribution of cytokinin.Previous studies have shown that At ABCG14,act as a long-distance transporter of cytokinin,which can mediate the polar transport of cytokinin and transport cytokinin from root to shoot.Thus,participating in plant morphogenesis and playing a notable role in abiotic stress response.In this study,we cloned the BnaABCG14.A09 gene in Brassica napus by homologous sequence analysis,and preliminarily explored the function of the gene by bioinformatics analysis,subcellular localization,overexpression,and gene editing.The main results are as follows:（1）Fifteen genes similar to At ABCG14 were identified in the Bn PIR database.Phylogenetic tree analysis showed that Bna A09T0398000ZS and Bna C05T0276100ZS were on the same branch with At ABCG14.However,only the Bna A09T0398000ZS gene was amplified from the cDNA of Brassica napus,indicating that Bna A09T0398000ZS played an essential role in Brassica napus.Therefore,the gene was used as a candidate gene for At ABCG14 in Brassica.napus and named as BnaABCG14.A09.（2）The expression of the BnaABCG14 gene in different tissues of Brassica napus was analyzed by qRT-PCR.The result showed that the expression of the BnaABCG14gene in the root was the highest,which was consistent with that in Arabidopsis thaliana,indicating that BnaABCG14 protein may have similar functions as At ABCG14 in Arabidopsis thaliana.（3）Bioinformatics analysis showed that BnaABCG14.A09 protein belongs to the ABC transporter family,which is composed of 649 amino acids and contains six transmembrane helix regions.BnaABCG14.A09 may be a relatively unstable hydrophilic protein.There are 74 phosphorylation sites in BnaABCG14.A09,accounting for 11.4%of the total sequence,without glycosylation sites and signal peptides.（4）Bn ABCG14.A09-eGFP vector was constructed and transformed into Brassica napus.Fluorescence analysis of the root protoplast showed that Bn ABCG14.A09 was located in the cell membrane,which is consistent with the results in Arabidopsis thaliana.（5）BnaABCG14.A09 overexpression vector was constructed and transformed into Brassica napus.A total of 12 overexpression plants were obtained.Primary roots of overexpressed plants were significantly longer than those of wild type plants.（6）The CRISPR/Cas9 knockout vector of the BnaABCG14 gene was constructed and transformed into Brassica napus.Three T₀ generation gene editing plants were obtained.Gene editing mutants showed primary root elongation compared with wild type.（7）Under 200 m M NaCl treatment,the BnaABCG14 gene editing mutant was more tolerant to salt stress than the wild type.In addition,the DAB staining result showed that the BnaABCG14 gene editing mutant could inhibit the accumulation of hydrogen peroxide（H₂O₂）under salt stress.Moreover,the antioxidant enzyme gene expression analysis showed that the antioxidant enzyme gene expression in the root of the gene-editing mutant were significantly higher than that of the control group.To sum up,the function of ABCG14 transporter in Brassica napus was preliminarily studied by cloning At ABCG14 homologous genes.The BnaABCG14.A09protein is located on the cell membrane and belongs to the membrane transporter.The BnaABCG14 gene editing mutant was obtained by CRISPR/Cas9 gene-editing technology,which confirmed that BnaABCG14 gene knockout can make plants more salt tolerant.These results indicated that ABCG14 as a cytokinin transporter played an important role in the process of plant resistance to salt stress,and provided a preliminary reference for further research on salt tolerance and drought resistance breeding of Brassica napus.