Identification Of WRKY Transcription Factor Family Of Camelina Sativa And Its Function In Mediating Salt Stress Response

Camelina sativa,a special oil crop with seeds rich in ɑ-linolenic acid(18:ω3),is the excellent resources for production of high-value functional oils and fats.And it has excellent agronomic characteristics such as short growth period(80-100 days),low input of water and fertilizer,less infection of pests and weeds,and easy for simple cultivation.Camelina is being developed as an ideal crop characterized with “low-consumption,high-efficiency and environment-friendly” for sustainable agricultural production system in the world.In particular,compared with Brassica rapa and soybean,C.sativa has strong resistance to low temperature,drought and salt stress,indicating that camelina is enriched in superior genes responsible for high stress resistance being exploited.Plant-specific WRKY transcription factor protein family is involved in plant growth,development and stress resistance and other life activities.However,there are few reports on the mechanism underlying high resistance and the evolution and function of WRKY transcription factors in C.sativa.This study focused on the identification of key genes such as WRKY transcription factors that mediate the salt stress resistance mechanism of C.sativa.In this study,C.sativa variety SC-N1 was used as test material to systematically detect its salt stress response characterization.The transcriptome analysis of C.sativa salt stress response was conducted by RNA-seq to screen the transcription factors involved in the salt stress response of C.sativa.Genome-wide identification of C.sativa WRKY family members and expression profiles,phylogenetic evolution and functional variation of WRKY family were analyzed to identify candidate WRKY transcription factors that mediate salt stress resistance of C.sativa.The genetic transformation system of C.reinhardtii,a single cell photoautotrophic model plant,was used to test the CsWRKYs of the candidate C.sativa.The target CsWRKY transgenic strains with overexpression and CRISPR-Cas9 knockout were cultivated to analyze the physiological and biochemical phenotypes of the transgenic strains and identify the CsWRKY and its biological functions that mediate the salt stress response of C.sativa.This study will provide a scientific basis for mining the salt tolerance related genes of C.sativa and analyzing the molecular regulation mechanism of C.sativa in response to salt stress.The main research results are as follows:1.Characterization of C.sativa seedlings response to salt stressUnder the condition of hydroponics,6 true leaf seedlings were selected and treated with salt stress to detect the morphological,physiological and biochemical parameters of seedlings.Compared with the control group(1/2 Hoagland nutrient solution),leaf wilting and growth retarded were found in the seedlings subjected to salt stress for 3 days,and the dose-response was observed.Three different doses(100,150 and 200 m M)Na Cl treatment of camelina plant height was reduced by 25.26%,37.59% and 44.58% respectively,the aboveground fresh weight was reduced by 3.22%,13.44% and 21.51% respectively,the underground part fresh weight was reduced by 41.18%,47.06% and 50.0%,respectively,chlorophyll a(Chla)content was reduced by 10.29%,27.94% and 32.35%,respectively,content of chlorophyll b(Chlb)was reduced by 28.26%,52.17% and 56.52% respectively.The chlorophyll fluorescence parameters(Fm,Fv/Fm,Y(II),q P and ETR)and photosynthesis of seedlings were decreased under salt stress.On the contrary,salt stress(200 m M)significantly increased the anti-osmotic substances,and the contents of carotenoids,proline,soluble sugar and increased by 40.0%,10.6-fold and 47.0%,respectively,compared with the control.Compared with the control,the activities of SOD(superoxide dismutase)and POD(peroxidase)and T-AOC(total antioxidant capacity)were increased by 51.86%,156.89% and 50.18%.In addition,catalase(CAT)activity of salt-stressed seedlings was about 4 times higher than that of the control.It is obvious that C.sativa can resist salt stress by increasing the synthesis and accumulation of anti-osmotic substances,enhancing the activity of antioxidant enzymes and scavenging ROS.2.Transcriptome analysis of C.sativa seedlings and screening of transcription factors in response to salt stressIn order to explore the salt tolerance genes of C.sativa and reveal the regulation mechanism of salt stress response,the transcriptome analysis of C.sativa seedlings and their control materials treated with 175 m M Na Cl stress for 1 day was conducted by RNA-seq technique.The results showed that a total of 42.47 Gb of C.sativa transcriptome data were obtained,and 99402 Unigenes and 5902 differentially expressed genes(DEG)(2917up-regulated and 2985 down-regulated)with functional annotation were assembled.GO enrichment analysis showed that a large number of DEGs were mainly concentrated in molecular functions(33.60%),cellular components(13.60%)and biological processes(52.79%).KEGG pathway analysis annotated 1442 DEGs into 44 metabolic pathways,among which up-regulated DEGs were significantly enriched in ascorbic acid and aldehyde acid metabolism,arginine proline metabolism,oxidative phosphorylation and phenylpropane-like synthesis pathways,while down-regulated DEGs were mainly involved in photosynthesis.Further comprehensive analysis of differentially expressed genes screened out more important transcription factor families,including WRKY,MYB,BHLH,AP2/ERF and BZIP.Among them,WRKY transcription factor genes were up-regulated with high number and level,indicating that WRKY transcription factor played a more important role in salt stress response of C.sativa seedlings.3.Genome-wide identification and phylogenetic analysis of C.sativa CsWRKY transcription factorsA total of 242 CsWRKY protein members were identified from C.sativa genome using omics analysis tools,which were coded by 224 CsWRKY genes distributed unevenly on 20 chromosomes,among which 15 CsWRKY genes produced 33 WRKY alternative splicosomes.According to WRKY and zinc finger motifs,CsWRKY family members can be divided into three major groups(I,II and III),of which the second major group contains 149 WRKYs and is further divided into five subgroups(IIa,IIb,IIc,IId and IIe).CsWRKY protein contains 10 conserved motifs,among which WRKYGQK heptapeptide is the most conserved.However,variations in WRKYGXK,WRKYGKK and WRKYGEK were detected in CsWRKYS of multiple IIc subclasses.These mutations may lead to functional diversification of CsWRKY proteins.The evolution of CsWRKY gene family has undergone strong purifying selection,and there are 137 segmental duplication events not found,which are the key evolutionary drivers for the expansion and functional variation of CsWRKY gene family.In addition,166 and 173 CsWRKY genes were detected corresponding to 65 At WRKY and 111 Br WRKY(Brassica rapa)homologous genes,respectively,suggesting that the expansion of C.sativa WRKY gene family may occur after the separation of Arabidopsis and Brassica rapa.4.Temporal and spatial expression profile analysis of CsWRKY gene and CsWRKY screening in response to salt stress in C.sativaUsing transcriptome data from 12 different tissues and organs of C.sativa,including root,stem,leaf,flower and seed,CsWRKY gene expression profile analysis showed that more than half of the CsWRKY genes were expressed in at least one tissue and organ,and 89.11%,80.69%,78.71%,88.61% and 76.73% of the genes were expressed in root,stem,mature leaf,flower and early development seed,respectively.Seventy CsWRKYs were expressed in 12 tissues and organs,among which 24 were highly expressed genes.Many CsWRKY genes have tissue-specific expression patterns,for example,expressed in two tissues/organs,CsWRKY25 and 34 in inflorescence and late developing seeds,CsWRKY174 in germinating seeds and roots,and CsWRKY73 and 203 in flowers and mature leaves.CsWRKY111,208 and 226 in root tissues,CsWRKY182 and 202 in flower tissues,and CsWRKY204 in early developing seeds were expressed only in one tissue/organ.These results suggest that CsWRKY plays an important role in the development of C.sativa tissues/organs.Some CsWRKYs may be involved in the regulation of basic cell life activities,while the other CsWRKYs may play a differential regulatory role in different tissues/organs.The transcriptome data of C.sativa seedlings under salt stress and quantitative PCR were used to detect the expression profiles of CsWRKY genes.Nine CsWRKY8,9,10,43,48,49,50,162 and 242 genes were screened out as CsWRKYs which mediated salt stress response.The expression levels of CsWRKY9,CsWRKY43 and CsWRKY162 were more than 5 times higher than those of the control shoot tissues.CsWRKY9,43 and 162 were also up-regulated in root tissues under salt stress,and their expression levels were more than 6 times higher than those in control tissues.However,CsWRKY8 and 10 were significantly down-regulated(p<0.05),and the other 4 CsWRKYs were not up-regulated to a significant level(p<0.05).5.The functions of CsWRKY9,43 and 162 genes in C.sativa were identified by genetic transformation system of C.reinhardtiiThe analysis of WRKY transcription factor family in lower and higher plants revealed that there was only one WRKY gene in the genome of C.reinhardtii,a single cell photoautotrophic model.Overexpression of heterogenous WRKY genes in C.reinhardtii could effectively eliminate the interference of multiple endogenous WRKY genes in host.At the same time,we obtained the WRKY mutant LMJ605 and its wild-type strain CC5325.These results provided a unique test host germplasm for identifying the CsWRKY functions of C.sativa selected in this study in response to salt stress.The most important CsWRKY9,43 and 162 of the 9 candidate CsWRKYs above were selected as target genes and cloned into the polyclonal site of p HR13 vector suitable for expression in C.reinhardtii cells,respectively,to construct the overexpression vectors p HR13-CsWRKY9,p HR13-CsWRKY43 and p HR13-CsWRKY162.The glass bead method was used to transfer the expression vectors into the easily transgenic C.reinhardtii CC849,and the transgenic strains with stable and effective expression of the target genes were obtained by molecular detection.The growth phenotypes of untransformed control strains(CC849)and transgenic strains under normal culture conditions and salt stress were detected.The results showed that the biomass and photosynthesis of transgenic strains were slightly higher than that of control strains under normal culture conditions,but not significant(p<0.05).After culture for 4 days under 150 m M Na Cl salt stress,the biomass of control strains CC849 and transformed strains CsWRKY9,43 and 162 decreased by 33.57%,25.85%,14.47% and 8.63%,respectively,compared with the normal culture conditions.After culture for 4 days under 175 m M Na Cl salt stress,the biomass of control strains CC849 and transformant CsWRKY9,43 and 162 decreased by 59.40%,27.22%,18.79% and 16.31%,respectively,compared with the normal culture conditions.Chlorophyll content and chlorophyll fluorescence parameters showed that salt stress inhibited photosynthesis in algal cells,but the damage degree of transgenic algal strains was significantly lower than that of control CC849(p<0.05).The phenotypes of CC849,CC5325 and its WRKY mutant LMJ605 and transformant CsWRKY162 under normal culture conditions and salt stress showed that the function loss of endogenous WRKY gene(LMJ605)did not significantly inhibit the growth and photosynthesis of algal cells under normal culture conditions.However,the growth of the mutant LMJ605 was severely inhibited under salt stress.Under 150 m M Na Cl salt stress,the biomass of control strains CC849,CC5325,LMJ605 and transformant CsWRKY162 decreased by33.26%,10.50%,58.04% and 11.54%,respectively,compared with that under normal conditions.Under 175 m M Na Cl salt stress,the biomass of CC849,CC5325,LMJ605 and transformant CsWRKY162 decreased by 56.69%,25.68%,67.54% and 17.21%,respectively,compared with that of normal culture.When the salt concentration reached 200 m M Na Cl,the growth of algal strains CC849,CC5325,LMJ605 and CsWRKY162 were severely arrested and died with the extension of culture time.LMJ605 suffered the most damage,while CsWRKY162 suffered less damage,and the survival time of transformed algal strain CC849 was more than 2 times longer than that of control strain(CC849).The results of these genetic transformation experiments showed that CsWRKY9,CsWRKY43 and CsWRKY162 could significantly improve the salt resistance/tolerance of host cells,and CsWRKY162 had the strongest effect.6.Overexpression of CsWRKY162 and construction and phenotypic analysis of CRISPR/Cas9 knockout mutant in C.sativaThe overexpressed strains and knockout strain of CsWRKY162 gene were selected to construct respectively,and the mechanism of CsWRKY162 mediating salt stress response in C.sativa was further analyzed.CSWRKY162 gene overexpression vector p CAMBIA1303-CsWRKY162 and CRISPR-CsWRKY162_ta/b and CRISPR-CsWRKY162_tc/d were successfully constructed.The constructed vectors were introduced into C.sativa by Agrobacterium mediated impregnation,and the seeds of T0 generation were obtained.After screening and identification,the homozygous CsWRKY162 transgenic plants and CsWRKY162 gene knockout plants(including the knockout vector CRISPR-CsWRKY162_ta/b)were obtained,and the CsWRKY162 gene sequence was deleted and replaced by base.Phenotypic analysis of transgenic plants showed that the growth and development of CsWRKY162 overexpressed plants were not significantly different from that of control plants under normal growth conditions.Under 175 m M Na Cl salt stress,the growth of control plants was inhibited.The plant height and fresh weight of CsWRKY162 overexpressed plants were about 1.5-fold and 1.3-fold,and the CsWRKY162 overexpressed plants showed no symptoms of injury.Under normal conditions,the biomass of CsWRKY162 gene knockout plants did not significantly change compared with the control plants.Under 175 m M Na Cl salt stress,CsWRKY162 gene knockout plants were dwarfized,and their victim symptoms were significantly higher than those of the control plants.Compared with the control plants,the height of CsWRKY162 gene knockout plants decreased by about 20%,and the fresh weight per plant decreased by about 40%.In response to salt stress,the expression of stress related genes NHX2,HKT and NCED3 were up-regulated and down-regulated in CsWRKY162 overexpressed plants and knockout plants,respectively.The genetic transformation of C.sativa demonstrated that CsWRKY162 is an important transcription factor mediating the response to salt stress.Transcriptome sequencing and correlation analysis of these C.sativa are in progress,in order to further clarify the expression mechanism and regulatory network of C.sativa salt stress resistance mediated by CsWRKY162.In summary,the characterization of salt stress response of C.sativa was discussed in this paper.Genome wide identification of WRKY transcription factors and their expression profiles revealed the evolutionary characteristics of CsWRKY family members.Nine CsWRKY transcription factors were screened to mediate the response of C.sativa to salt stress.The functions of CsWRKY9,CsWRKY43 and CsWRKY162 in C.sativa were evaluated using a unique single-cell model plant genetic transformation system.The overexpressed and knockout mutant of CsWRKY162 in C.sativa was successfully constructed,demonstrating that CsWRKY162 is an important transcription factor positively regulating the resistance/tolerance mechanism under salt stress.This study provides new knowledge,target genes and related research basis for comprehensively analyzing the functions of CsWRKY family members and the molecular mechanism of resistance to salt stress and other stresses,and breeding cultivars with excellent stress resistance...