Response Characteristics Of Different Genotype Chinese Cabbages To Salt And Alkali Stress

Chinese cabbage is widely grown in China,but soil salinization is a serious threat to its growth,development and yield quality.In this paper,based on the identification of salt tolerance of different genotype Chinese cabbages,two Chinese cabbage cultivars with different salt tolerance were used as test materials to investigate the growth condition,osmolytes,light energy utilization characteristics and reactive oxygen metabolism under salt and alkali stress,and to screen the salt tolerance genes by transcriptome analysis for the investigating of the response characteristics of Chinese cabbage to salt and alkali stress.The main findings of the study are as follows:1.The salt damage index increased significantly with the increasing days of salt stress（0.5%Na Cl）in all different genotype Chinese cabbages,but there were significant differences between different genotypes.A mathematical model of the salt damage index and related parameters in Chinese cabbage seedlings was established by stepwise regression analysis,and two factors,seedling relative fresh weight and leaf relative electrolyte permeability,were selected with coefficients of determination as high as 0.9815（P<0.001）.The results of the cluster analysis using the selected two factors as statistical parameters were consistent with those of the salt damage index,indicating that it could be used as a convenient indicator for the identification of salt tolerance in Chinese cabbage.The two selected cultivars,Qinghua（salt tolerant）and Biyu（salt sensitive）,were selected for subsequent experiments.2.Using the screened salt-tolerant cultivar Qinghua and the salt-sensitive cultivar Biyu to conduct salt-alkali interaction stress treatments,we found that Qinghua was salt-tolerant but alkali-sensitive,while Biyu was salt-sensitive but highly alkali tolerant.This was evidenced by the fact that the growth of Qinghua was not significantly inhibited by the salt treatment,and the proline,organic acid and carbohydrate contents were significantly increased,and the content of Na⁺increased less,but under alkali treatment,the electrolyte permeability increased significantly and the content of osmoregulatory substances did not change significantly,with significant decreases in K⁺,Ca²⁺and Mg²⁺.Biyu was significantly damaged by salt stress,with a 298%-364%increase in Na⁺significantly higher than in Qinghua,an increase in osmoregulatory substances under alkali treatment,Mg²⁺content remaining at a high level and the increase of malic acid content in root was significant.3.The Qinghua leaves did not appeared significant inhibition of net photosynthetic rate（Pn）,stomatal conductance（Gs）,water use efficiency（WUE）,maximum photochemical quantum yield of PSII（Fv/Fm）and non-photochemical quenching（NPQ）under salt stress,but its net photosynthetic rate was reduced by 10-29%under alkali stress.Light energy utilisation was significantly inhibited in Biyu under salt stress,with net photosynthetic rate decreasing by18%-26%and Gs,WUE and Fv/Fm also decreased significantly.The net photosynthetic rate of leaves was not significantly impaired under alkali treatment and WUE maintained a high level,indicating that high-tolerance Chinese cabbage has high light energy utilisation.4.The activities of SOD,POD,CAT,AAO,APX,DHAR and MDHAR enzymes increased to 150-200%in Qinghua and the gene expression increased to 2.5-3.5 folds that of the control under salt stress,but under alkali stress,the activities and relative expression of antioxidant enzymes did not change and the contents of H₂O₂ and MDA increased by 143%-190%and234%-294%compared with the control,respectively.Under salt treatment,Biyu significantly increased its H₂O₂ and MDA content to 152%-208%and 240%-263%,respectively,but its H₂O₂and MDA content did not increase under alkali stress,while the activity of antioxidant enzymes and gene expression both increased significantly.5.A total of 2859 differentially expressed genes（DEGs）were identified by transcriptome sequencing analysis of Qinghua and Biyu treated with salt stress for 15 d.The number of DEGs was significantly lower in Qinghua than in Biyu.Cation/H⁺antiporter17,V-H⁺-ATPase and VDAC were more highly expressed in Qinghua than in Biyu,and played a positive role in the exchange of Na⁺and H⁺in Qinghua,kept Na⁺in the vesicles and also maintaining the Ca²⁺content in Qinghua.In addition,the expression of the GOLS related genes was higher in Qinghua than Biyu,which alleviated the osmotic stress caused by salt stress and had a positive effect on the salt tolerance of Qinghua.6.By further analysis of the transcriptome data,11 key genes were screened and the expression of 11 genes in the roots and leaves of Qinghua and Biyu was determined,and the expression levels of 4 of these genes were found to be highly positively correlated with salt tolerance in Chinese cabbage.To select the key genes more precisely,we also determined the expression of these 4 genes in 39 Chinese cabbage cultivars and found that the expression of the Br GOLS2 gene in each cultivar correlated the highest with the salt tolerance.7.Salt stress induces the expression of Br GOLS2 gene in Chinese cabbage.Bioinformatics analysis,gene cloning and construction of an overexpression vector for the Br GOLS2 gene revealed that the open reading frame（ORF）of Br GOLS2 is 993 bp in length and encodes a protein containing 330 amino acids.Analysis of the Br GOLS2 protein predicted a molecular weight of approximately 37.86 k D and no transmembrane structural region.The Br GOLS2protein sequence is related to GOLS2 gene in Arabidopsis thaliana and salt mustard,with comparative similarities of 99%and 87.83%,respectively.The Br GOLS2 gene was successfully cloned and transferred into Agrobacterium tumefaciens for subcellular localization in tobacco and was found to be specifically expressed in the cell membrane.