Cloning And Functional Analysis Of SaENO2 Gene From Sophora Alopecuroides

Plants are subject to various environmental stresses during their growth and development.Salt-alkali stress is one of the stresses that restricts plant growth and development.Due to the aggravation of soil salt and alkali stress,the cultivated land area is decreasing day by day,and the yield and quality of crops are further affected.Sophora alopecuroides is a perennial plant of Sophora in Leguminosae,which has the characteristics of drought resistance and salt tolerance.It is widely distributed in arid and semi-arid areas,and it is a gene resource bank rich in resistance genes.In this study,Na₂CO₃ and NaHCO₃ with mole ratio of 1:1 were used to simulate alkali stress to screen the expression library of cDNA yeast of Sophora alopecuroides,and the enolase gene SaENO2 was obtained.The function of SaENO2 gene was verified by the gene reintroduction into yeast BY4743 and Arabidopsis overexpression of SaENO2.This experiment will help to discover and identify the stress resistance genes of bitter beans,enrich the understanding of the biological functions of plant enolase,and help deepen the understanding of the molecular mechanism of plant growth and development and stress response.The main results of this study are as follows:1.Na₂CO₃ and NaHCO₃ with a molar ratio of 0.0026 mol/L of 1:1 were used to screen the expression library of Sophora alopecuroides cDNA yeast,and a total of 9alkali-resistant clones were screened,and the 9 genes were sequenced and sequence analyzed.2.After bioinformatics analysis,a candidate gene,SaENO2,was selected to be an alkali resistant gene.The total length of the vector of SaENO2 gene is 1909 bp,1835 bp long after the vector sequence was removed,and ORF is 1335 bp,encoding444 amino acid residues.The molecular weight of the protein encoded by SaENO2gene is 47.74 kDa,and the PI of the isoelectric point is 5.70.The similarity between amino acid sequence and soybean homologous gene was 95.72%,while the similarity with Arabidopsis was 88.06%.The phylogenetic tree analysis showed that SaENO2had the closest relationship with the homologous genes of Vigna radiata var.Radiata and Vigna angularis.3.Real-time quantitative PCR was used to detect the changes of SaENO2expression in the root,stem and leaf of Sophora alopecuroides after 72 h under control,1.2%NaHCO₃,1.2%NaCl and 8%PEG treatment.The results showed that the mRNA expression of SaENO2 gene was the highest in the stem,and the highest in the stem under 1.2%NaHCO₃ treatment.Compared with the control,the expression of SaENO2 gene increased in 1.2%NaHCO₃,1.2%NaCl and 8%PEG,with the highest increase in 1.2%NaHCO₃,followed by 8%PEG.So SaENO2 gene can respond to stress to some extent.4.In this experiment,seamless plant cloning technology was used to successfully construct the plant recombinant vector pCAMBIA3301-2*flag-GFP-SaENO2 and subcellular localization analysis using tobacco.The result of subcellular localization was found that SaENO2 gene was expressed in both cytoplasm and nucleus.5.The SaENO2 gene of Sophora alopecuroides was transformed into yeast BY4743.After diluting wild-type and transgenic yeast 10⁰,10¹,10²,10³and 10⁴times respectively,yeast was cultivated on YPDA solid medium containing 0 M,0.001 M,0.002 M,0.0026 M,0.003 M,0.005 M,0.006 M and 0.007 M NaHCO₃ and 0 M,0.5M,0.68 M,1 M,1.25 M and 1.5 M NaCl.The experiment uses wild type as a control to observe the growth status of yeast.The results showed that under NaCl and NaHCO₃ treatment,the growth of transgenic yeast was significantly better than that of the control,and under NaHCO₃ treatment the effect was more significant.It shows that SaENO2 can improve the tolerance of plants to salt and alkali to a certain extent.6.The gene SaENO2 was overexpressed in Arabidopsis,and Arabidopsis thaliana strains were subjected to stress treatment in soil and medium respectively.The results showed that under the stress of mannitol and NaHCO₃ in culture medium and NaHCO₃ in soil,the tolerance of transgenic Arabidopsis seeds was significantly higher than that of wild type Arabidopsis.The tolerance of transgenic Arabidopsis thaliana seedling plants to NaHCO₃ stress was also significantly stronger than that of wild type and mutant Arabidopsis thaliana.It is suggested that SaENO2 gene can improve the alkali resistance and drought resistance of plants to a certain extent.