Research On Key Soybean Isoflavones Synthesis Enzyme Genes GmIFSl And GmCHS7 Involving In Plant Responses To Salt Stress

At present,salinization of soil in world became progressively worse and has been one of important factors limiting the growth and production of crop in agriculture.Isoflavones possessed outstanding antioxidant capacities in vitro,was considered as one part of antioxidant system in plant,and would play important role while plant faced with environmental stress factors.Isoflavones synthase(IFS)and chalcone synthase(CHS)were the critical synthases,which control synthesis of isoflavones in soybean.Hence,it was significative to research on expression and regulation of GmIFS and GmCHS genes,which may involve in plant response to salt tolerance.In this paper,soybean cultivar N23674 was cultivated as experimental material,in solar greenhouse condition.The effect of differential salt stress on growth of soybean and isoflavones content in seed was estimated.Resulted showed that plant height,leaf area and fresh weight of soybean seedlings decreased after 80 mM or 100 mM NaCl treatments.The salt treatment also significantly increased isoflavones accumulation in the seed(P<0.05).Apparently,salt stress reduced the soybean plants growth and promoted the isoflavones accumulation in the seeds.In greenhouse condition,soybean seedlings planted by sand-cultured method were treated by 80 mM NaCl solution.At time point of 0 h,2 h and 48 h NaCl treatment,the expression of GmIFSl and GmIFS2 in mature leaves on top of plants and roots collected were anlysised by RT-PCR method.After two weeks,mature leaves on top of soybean plants were collected and used for isoflavones content determination.The results showed that,80 mM NaCl treatment could enhance expression of GmIFS1 and GmIFS1 genes and isoflavones content in leaf of soybean,and promoted isoflavones accumulation in leaf.In root,up-regulate expression of GmIFS1 and GmIFS2 were not significant,while isoflavone content in roots of soybean did not significantly increase.As to expression of GmCHS7 gene in leaves and roots of soybean was increased by salt stress,this meaned the expression of GmCHS7 may contribute to isoflavones accumulation.The expression vector with GmCHS7 gene was constructed and used for transformation of soybean cotyledon hairy roots.The transgenic hairy roots were detected by Semi-quantitative PCR.However,the result showed that expression of GmCHS7 gene in cotyledon hairy roots could not significantly promoted isoflavonoids accumulation in transgenic hairy roots.GmIFSl gene from soybean cv.N23674 was sub-cloned into expression vector pCambia1300 and used for Agrobacterium rhizogenes-mediated transformation of soybean cotyledon hairy roots.The transgenic cotyledon hairy roots were detected by PCR,and the positive cotyledon transgenic hairy roots were used for salt treatment.Transgenic cotyledon hairy roots were treated by 100 mM NaCl stress for one week.Isoflavones content,fresh weight,relative water content and main root length of hairy roots were measured.The results showed that transgenic hairy roots accumulated more isoflavones than vector hairy roots under salt treatment condition.The salt tolerance of transgenic cotyledon hairy roots was also improved.Apparently,in salt stress condition,expression of GmIFSl gene promoted isoflavones accumulation and improved the salt tolerance of soybean hairy roots.With Agrobacterium tumefaciens-mediated leaf disk method,expression vector pCambia 1300 anchored with GmIFSl gene were also transformed into tobacco(Nicotiana tabacum).After DNA molecular determination,copy number estimation and expression analysis of transgenic IFS1 gene,the salt tolerance and antioxidant capacity of leaf tissue of low-copy transgenic tobacco was determined under different salt stress condition.The results showed that,plant height and fresh weight and antioxidant capacity of leaf tissue of transgenic tobacco was higher than wild tobacco under 85mM NaCl treatment.This may indicate that expression of GmIFSl gene in tobacco increased antioxidant capacity of tobacco and improved salt tolerance of transgenic tobacco.The CDS of CHS7 was also isolated by screening N23674 leaf cDNA library and sub-cloned into expression vector pCambia1300 used for sequence analysis and transforming tobacco as above,the CDS of CHS was similar to GmCHS7 of wild soybean.PCR determination,copy number and expression of transgenic GmCHS7 gene were analyzed,the low-copy transgenic tobacco were used for determination of salt tolerance and antioxidant capacity and flavonoids content of transgenic and wild tobacco.The results showed expression of GmCHS7 has promoted flavonoids accumulation and antioxidant capacity in leaves of transgenic tobacco.For plant height and fresh weight of leaf tissue of transgenic tobacco were higher than wild tobacco in 85mM NaCl treatment,this may mean expression of GmCHS7 could promote flavonoids content,and improve salt tolerance of tobacco.In summary,salt stress could promote expression of GmCHS7 and GmIFS1 and GmIFS2 genes and isoflavones accumulation in soybean.Over-expression of GmIFSl or GmCHS7 genes could increase isoflavones content or flavones in cotyledon hairy root or tobacco and improve salt tolerance of cotyledon hairy roots and tobacco.This work can suggest that the expression of key isoflavones synthesis enzyme genes GmCHS7 and GmIFS1 is involved in salt stress tolerance of legumes and other plants.