Evaluation Of Salt Tolerance Of Five Hybrid Mulberry Germplasms And Creation Of Salt Tolerance Germplasms

Mulberry is not only the feed tree species of silkworm,but also an important ecological tree species for desertification control,soil and water conservation,and saline-alkali land management.Exploring salt tolerance genes of mulberry and cultivating salt-tolerant varieties of mulberry are not only conducive to the management of saline-alkali land,but also of great significance for the realization of ecological and economic values of mulberry.In this study,the salt tolerances of five hybrid mulberry germplasms were evaluated.At the same time,the effect of GABA on salt tolerance of mulberry was analyzed.The two key enzymes of GABA metabolic pathway in mulberry,glutamic acid decarboxylase（Mul-GAD）andγ-aminobutyric acid transaminase genes（Mul-GABA-T）were cloned,and their expression characteristics were analyzed.The roles of Mul-GAD and Mul-GABA-T in the response to salt stress in mulberry were preliminarily explored.The transgenic Mul-GAD or Mul-GABA-T gene hairy root mulberry was obtained using transgenic hairy roots technology,and the salt tolerance of the transgenic mulberry plants was identified.The information provided will provide genetic resources and new germplasm for mulberry salt-tolerant breeding;at the same time,will lay the foundation for further research of the functions and mechanisms of Mul-GABA-T and Mul-GAD genes.Our data provided will be great significance to achieve the ecological value of mulberry and promote the development of mulberry industry.The main results are as follows:（1）Evaluation of salt tolerance of five hybrid mulberry germplasmsFive widely cultivated hybrid mulberry germplasms,Jisang 3,Luza 1,Zheza 1,Guisangyou 12 and Guisangyou 62,were used as experimental materials of salt tolerance evaluation.The physiologica indexes of seedling height,plant biomass,leaf chlorophyll content,net photosynthetic rate and stomatal conductance of five germplasms under different concentrations of NaCl stress were comprehensively analyzed,and the salt tolerances of five hybrid mulberry germplasms were evaluated.The results showed that Guisangyou 62 had the strongest salt tolerance.（2）Effects of GABA on salt tolerance of mulberry seedilgsIn order to explore the effect of GABA on salt tolerance of mulberry,the content of GABA in mulberry seedlings before and after NaCl treatment was measured.It was found that the accumulation of GABA in mulberry leaves was induced by NaCl treatment.By applying exogenous GABA to mulberry seedlings under NaCl stress,it was found that the inhibitory effect of NaCl on the growth of mulberry seedlings and the damage on leaves.were significantly alleviated by exogenous GABA.At the same time,it was found that the accumulation of O^2-and H₂O₂ in root tips and leaves of mulberry seedlings were reduced,and the production of ROS was inhibited.Therefore,the synthesis and metabolism of GABA may be associated with the salt tolerance of mulberry.（3）Cloning ofγ-aminobutyric acid transaminase gene of mulberry and its effect on salt tolerance of mulberryTheγ-aminobutyric acid transaminase gene（Mul-GABA-T）of mulberry was cloned from Husang 32.The full-length coding region of the gene was 1536 bp,encoding 511 amino acids.The theoretical isoelectric point of the encoded protein was 7.6,and the molecular weight of the protein was 56.63 KDa.The protein is rich in random coil andα-helix and no transmembrane structure.It has multiple potential phosphorylation sites,one O-glycosylation site and three N-glycosylation sites.The protein has high homology with theγ-aminobutyric acid transaminases from other species,and has the closest relationship withγ-aminobutyric acid transaminase of Morus notabilis.The Mul-GABA-T gene has no specific expression in mulberry,but its expression abundance was significant different among different tissues or organs,and it exhibited the highest expression in leaves and the lowest expression in flowers and fruits.Mul-GABA-T gene was successfully transformed into Arabidopsis thaliana and the transgenic plants were obtained.It was found that NaCl stress significantly inhibited the root growth and seed germination of Mul-GABA-T transgenic A.thaliana.Compared with the wild type,transgenic plants showed salt-sensitive phenotypes.However,exogenous GABA could reduce the O^2-accumulation in transgenic plants under salt stress and effectively alleviate the inhibition of salt stress on plant growth.The transgenic GABA-T gene hairy root mulberry plants were also obtained by root transformation system.The results showed that the expression of GABA-T gene in hairy roots also reduced the salt tolerance of transgenic mulberry.Therefore,GABA-T gene may be involved in the response of plants to salt stress by regulating the metabolism of GABA,and negatively regulate the resistance of plants to salt stress.（4）Cloning,expression characteristics and salt tolerance analysis of glutamate decarboxylase gene of mulberryMul-GAD gene of mulberry was cloned from Husang 32,and the full-length coding region of the gene was 1509 bp,encoding 502 amino acids.The theoretical isoelectric point of the coding protein was 5.88,and the molecular weight of the protein was 57.00 KDa.The coding protein was rich in random coil and helix,and it had no transmembrane structure but had multiple potential phosphorylation sites and three N-glycosylation sites.The protein has high homology with glutamic acid decarboxylase from other species,and has the closest relationship with glutamic acid decarboxylase of M.notabilis.Mul-GAD gene had no expression specificity in mulberry,but its expression abundance was significantly different among different tissues or organs.It exhibited the highest expression in roots and lower expression in fruits.The promoter of Mul-GAD gene（pMul-GAD）was cloned,and it was found that the promoter contained multiple plant hormone responsive elements such as abscisic acid（ABA）,salicylic acid（SA）and methyl jasmonate（Me JA）,as well as multiple light responsive elements and low temperature responsive elements.The plant expression vector of pMul-GAD was successfully constructed.Through transient infection of tobacco and GUS histochemical staining test,it was found that pMul-GAD had light,ABA,SA,MEJA and NaCl-induced expression activity.The transgenic Mul-GAD hairy root mulberry plants were successfully obtained by root transformation system.Salt stress test showed that overexpression of Mul-GAD gene in hairy roots of transgenic mulberry plants confers the transgenic plants more tolerance to salt stress.