Functional Analysis Of Salt-tolerant Gene BnBBX22.A07 In Brassica Napus L.

There are many saline-alkali lands in China,and breeding salt-tolerant varieties is an effective way to develop and utilize saline-alkali lands.Brassica napus L.is one of the most important oil crops in the worldwide,and the rapeseed planting areas show a declining trend in China.Therefore,it is particularly important to cultivate salt-tolerant varieties of B.napus and make full use of these saline-alkali lands to increase the cultivation area,and alleviate the shortage of oil in China.Mining the salt-tolerant genes of B.napus and analyzing the molecular mechanism of salt tolerance are the key to guiding the breeding of salt-tolerant varieties of B.napus.B-box(BBX)proteins are a kind of zinc finger structural transcription factor,and play a crucial role in plant growth and development regulation.In recent years,there have been more and more studies on the function of BBX protein in response to abiotic stress.However,the role of BBX proteins in salt stress responses in B.napus is not well understood.In this study,the BBX gene family members of B.napus were identified,and the key candidate gene BnBBX22.A07 for salt tolerance was identified based on public transcriptome data and real-time quantitative PCR(RT-qPCR).We screened interaction protein and downstream regulatory gene of BnBBX22.A07.Combined with the phenotype,transcriptome,physiology and expression analysis of related genes in over-expressing lines,the molecular regulatory mechanism of BnBBX22.A07 in response to salt stress was explored.The main findings are as follows:1.The identify of key candidate gene BnBBX22.A07 for salt tolerance:68 BnBBX genes were identified in B.napus,and were divided into five groups based on the phylogenetic tree and conserved domain analysis.There were multiple tandem duplication and segmental duplication events in BnBBX genes.The promoter regions of BnBBX genes contained multiple stress response elements.Public transcriptome data showed that the expression of group IV subfamily was induced under salt stress in Arabidopsis,including AtBBX18,AtBBX22 and AtBBX23.We examined the expression of all homologous alleles of the BnBBX18,BnBBX22 and BnBBX23 under salt stress by RT-qPCR,and found that only the BnBBX22 was up-regulated under salt stress.In all homologous copies of the BnBBX22,only the expression level of BnBBX22.A07 was always higher under salt stress compared to control,and also up-regulated by PEG6000.So,we use BnBBX22.A07 as a key candidate gene for salt tolerance.2.BnBBX22.A07 was expressed in all organs examined,and the highest expression was detected in roots.The BnBBX22.A07 coding sequence encodes a protein of 290 amino acid including two typical B-box zinc finger domains.Subcellular localization and yeast transcriptional activation activity analysis showed that BnBBX22.A07 was a transcriptional activator localized to the nucleus.3.Overexpression of BnBBX22.A07 enhances salt and drought tolerance in transgenic Arabidopsis and B.napus.Under both normal and salt stress conditions,the over-expressing Arabidopsis plants showed longer primary roots compared with the wild type(WT).Similarly,the root length and fresh weight in the over-expressing B.napus plants was higher than that in the control plants.In addition,the salt-treated over-expressing B.napus plants exhibited significantly higher soluble protein and chlorophyll content(SPAD)contents,lower malondialdehyde(MDA)contents,and higher peroxidase(POD)activity compared to the control plants.Under drought stress,the over-expressing Arabidopsis plants exhibited significantly lower MDA contents,higher soluble protein contents,and higher POD activity compared with the WT.After one week of recovery,the over-expressing B.napus plants basically returned to normal growth,showing a relative water content(RWC)of 81.11%,while the control plants had a RWC of only 25.38%.Moreover,the over-expressing B.napus plants exhibited shorter hypocotyl length at the budding stage,more leaf numbers at the seedling stage,multiple main stems from the bottom and more branch numbers at mature stage compared with the control plants under normal conditions.4.BnBBX22.A07 enhances the salt tolerance of B.napus by reducing the production of reactive oxygen species(ROS)under salt stress.We evaluated the transcriptomic dynamics between the over-expressing B.napus and the control plants under normal and salt conditions by RNA sequencing,and performed GO and KEGG enrichment analysis of the differentially expressed genes(DEGs)in salt-treated over-expressing plants versus control plants.The results showed that the up-regulated DEGs were mainly related to metabolism pathways,especially for linoleic acid metabolism,alpha-linolenic acid metabolism,flavonoid biosynthesis,flavone and flavonol biosynthesis,phenylpropanoid biosynthesis,and MAPK signaling pathway.The down-regulated DEGs were mainly found in energy metabolism pathways such as carbon fixation in photosynthetic organisms,glyoxylate and dicarboxylate metabolism,nitrogen metabolism,photosynthesis,carotenoid biosynthesis,glycosaminoglycan degradation and ribosome.In addition,ROS scavenging-related genes,including superoxide dismutase(SOD),peroxidase(POD),glutathione transferase,and flavonoid biosynthesis-related genes,were significantly up-regulated in over-expressing plants compared with the control plants under salt conditions.DAB and NBT staining results showed that the hydrogen peroxide(H2O2)content was at a lower level in the over-expressing plants compared with that in the WT plants under low salt stress(100 m M NaCl),while the superoxide(O2.~-)content was at a lower level under high salt stress(200m M NaCl).Consistent with the ROS levels,the catalase(CAT)and POD activities were also significantly increased in over-expressing plants compared with the control plants under low salt stress,and the SOD activity was significantly increased under high salt stress.5.BnBBX22.A07 interacted with BnHY5.C09.Yeast two-hybrid,bimolecular fluorescence complementation and split luciferase assays showed that BnBBX22.A07could interact with BnHY5.C09.BnHY5.C09 was up-regulated under salt stress.The overexpression of BnHY5.C09 enhances salt tolerance in transgenic Arabidopsis.The BnHY5.C09 over-expressing seedlings displayed better growth with higher root length and fresh weight values,higher soluble protein contents,lower MDA contents,higher CAT,SOD and POD activity,less H2O2 and O2.~-contents of leave compared to WT seedlings under salt stress conditions.In addition,AtSOD1,AtCAT2,AtPerx34 were upregulated in the BnHY5.C09 over-expressing Arabidopsis plants compared with the WT plants under salt stress.6.BnHY5.C09 interacted with BnWRKY33.C03.Yeast one-hybrid and dual-luciferase transient expression assay showed that BnHY5.C09 could bind the G-box or C-box motifs in the BnWRKY33.C03 promoter and activate the expression of BnWRKY33.C03.BnWRKY33.C03 was up-regulated under salt stress.The BnWRKY33.C03 over-expressing seedlings displayed better growth with higher root length and fresh weight values,higher soluble protein contents,lower MDA contents,higher CAT,SOD and POD activity,less H2O2 and O2.~-contents of leave compared to WT seedlings under salt stress conditions.In addition,AtSOD1,AtCAT2,and AtPerx34 were upregulated in the BnWRKY33.C03over-expressing Arabidopsis plants compared with the WT plants under salt stress.7.AtWRKY33 functions as a downstream of the BnBBX22-BnHY5 cascade,mediating the salt tolerance of plants.Transcriptome sequencing of BnBBX22.A07over-expressing B.napus plants showed that BnBBX22.A07 regulated the expression of BnWRKY33 under salt stress.The RT-qPCR results showed that the expression of AtWRKY33 in BnHY5.C09 over-expressing Arabidopsis plants was significantly higher than that in the WT plants under normal and salt stress.We edited AtWRKY33 in BnBBX22.A07 over-expressing Arabidopsis plants(BnBBX22-OE)using CRISPR/Cas9system to generate the cWRKY33 BnBBX22-OE plants.Salt sensitivity assay showed that the BnBBX22-OE plants exhibited better growth with higher root length and fresh weight than WT plants under salt stress.However,the cWRKY33 BnBBX22-OE plants showed a salt-sensitive phenotype with root length and fresh weight comparable to that of WT plants.The wrky33 mutant was more sensitive to salt stress than the WT,and root length of the wrky33 plants was significantly lower than that of the WT plants under salt stress.In summary,this study identified the BBX genes family of B.napus,and revealed the molecular mechanism of BnBBX22.A07 regulating salt tolerance of plants,of which the BnBBX22-BnHY5-BnWRKY33 module coregulates WRKY33-mediated ROS homeostasis in response to salt stress.Our findings provide a theoretical basis for breeding salt-resistant transgenic crops.