Cloning And Functional Analysis Of A SlGAMYBL2 Gene In Abiotic Stress

Tomato is one of the most important model plants for plant science research and also is an important vegetable crop in China.Global climate warming,shortage of water resources,as well as abiotic stress factors,such as high salinity,low temperature,high temperature and drought have affected tomato growth and productivity.The function of genes which participate in plant stress resistance has become an important basis for improving plant stress resistance.GAMYB was found as a transcription factor in the GA signal response pathway,but GAMYB was found to be regulated by the stress hormone ABA(abscisic acid),indicating the potential roles of GAMYB in stress resistance.However,whether GAMYB can increase the stress resistance in tomato is rarely reported.In this study,the SlGAMYBL2,a homologous GAMYB in tomato,was cloned and overexpressed in tobacco plants.The phenotype was observed,and physiological along with biochemical analysis were conducted to study the function of tomato SIGAMYBL2 under abiotic stress.The results are as follows:1.SlGAMYBL2 has the typical R2R3 DNA domain of GAMYB,and has two special conserved regions(BOX1,BOX2)at the C-terminus.The evolutionary tree constructed by MEGA software indicated that the SlGAMYBL2 encoded protein belongs to the GAMYB family,and share high affinity with HvGAMYB.By analyzing its upstream sequence,in addition to the discovery of a typical promoter core sequence,there are also cis-acting elements that can respond to abiotic stress.2.The 35S promoter-driven overexpression vector for SlGAMYBL2 gene expression was successfully constructed.3.Transfer SlGAMYBL2 overexpression vector to tobacco by Agrobacterium-mediated method.After the kanamycin resistance screening,PCR and GUS staining identification,the transgenic tobacco that overexpressed SlGAMYBL2 was verified.Compared to the wild-type plants,the edges of the transgenic tobacco leaves were saw-toothed,and the leaves showed a pronounced wave-like shrunken with shorter petiole.Through microscope observation,the number of stomata in the leaves of transgenic plants was significantly greater than that of wild-type plants.4.Wild-type(WT)and over-expressed SlGAMYBL2 transgenic tobacco seeds were separately grown in MS medium containing 0 mmol/L,50 mmol/L,100 mmol/L,150 mmol/L,and 200 mmol/L mannitol.The germination rate of both experimental samples was as high as 95%in 0 mmol/L mannitol.With the increase of mannitol concentration,the germination rate gradually decreased in both experimental samples.However,the germination rate of transgenic tobacco was significantly higher than that of WT.In the media with 150 mmol/L mannitol,the germination rate of 53.6%was significantly higher than the wild type germination rate of 41.2%(please add the Standard error here).In 200 mmol/L mannitol,the transgenic germination rate was 17.40%(±SD),which was significantly higher than the wild-type germination rate 0.8%.The germination of wild-type seeds was significantly affected and the germination rate was low.Compared to wild-type seeds,transgenic tobacco seeds exhibited higher germination rates under stress.5.7 days old seedlings of WT and SlGAMYBL2 transgenic tobacco plants were future treated with stresses,the results showed that the root lengths of transgenic tobacco seedlings and WT seedlings in the control group had no significant difference,and the root length of the transgenic plants was extremely significant longer under treatment with 400 mmol/L mannitol,0.2 mmol/L GA,0.1 mmol/L ABA and 4? than that of WT under the same treatments.Under 40? treatment,the root length of transgenic seedlings was significantly longer than that of wild seedlings;however,under 200 mmol/L NaCl treatment,the root length of WT seedlings(1.63 cm(±SD))was significantly longer than that of transgenic seedlings(0.50 cm(±SD)).The above results showed that the transgenic plants had strong resistance to mannitol,GA,low temperature,high temperature and other abiotic stresses,but decreased resistance to high salt stress.The results showed that the transgenic plants showed higher superoxide dismutation(SOD)and over-oxidation than the wild-type plants under the low-temperature stress of 0.1 mmol/L ABA and 300mmol/L mannitol at 4?.Catalase(CAT),catalase(POD)activity and lower relative conductivity,malondialdehyde(MDA)content.However,under the stress treatment of sodium chloride,it showed the opposite trend.In summary,overexpression of SlGAMYBL2 enhanced the resistance of transgenic tobacco to ABA,mannitol,and low temperature stress.