Study The Expression Of IN2-2Promoter Induced By Acetanilide And2-Chlorobenzenesulfonamide In Plants

Ethanol-induced system, tetracycline-inducible system, copper-inducible system and dexamethasone-inducible system are popular in chemical inducible gene expression research in plant. Limited by change field condition, stability of inducer and the cost of inducer, these chemical inducible gene expression system are widespread used in laboratory,rarely chemical inducible gene expression system is used in practice production.The safener inducible system have many advantages compared with these chemical inducible system. First, safener inducible system may induced gene expression directly does not require transcription factors, so the safener inducible system is simple and easy to use. Secondly,the inducer of herbicide safener has been used widely in agriculture. Therefore, the safener inducible system is feasible to use in field.IN2-2, IN2-1and IN5-2promoter from maize can be induced expression by analogues of acetanilide and herbicide safener. Currently, the researches about herbicide safener inducible promoters performed mainly in cereal crops and Arabidopsis.In this study,the GUS gene was cloned at downstream of IN2-2promoter to transform tobacco, mustard and tomato respectively.Then, the transgenic plants were detected the GUS expression after treated with acetanilide and2-chlorobenzenesulfonamide respectively. And the expression stability of IN2-2promoter in tomato also was investigated after42℃high temperature stress.The results are as follows:(1) Generation transgenic mustard plantsPCABarIN2-2/GUS and PCABar35S/GUS control vectors were transformed into mustard plants by Agrobacterium-mediated method, total6IN2-2promoter transgenic mustard plants and four35S promoter transgenic mustard plants were produced.(2) Generation transgenic tomato plantsPCAKanIN2-2/GUS vector was transformed into tomato plants by Agrobacterium-mediated transformation,12independent IN2-2promoter transgenic tomato plants were produced finally. T1generation was obtained by self-pollination.(3) Generation transgenic tobacco plants PCAKanIN2-2/GUS and PCAKan35S/GUS control vectors were transformed into tobacco by Agrobacterium-mediated transformation, total6IN2-2promoter transgenic tobacco plants and three35S promoter transgenic tobacco plants were generated.(4) GUS expression detection in transgenic plants after acetanilide inducingGUS expression could be detected in the leaves of PCAKanIN2-2/GUS transgenic tobacco plants after acetanilide inducing. No obvious GUS exprssion difference were observed after50mg/L and100mg/L acetanilide inducing, the expression intensity were weaker than the PCAKan35S/GUS transgenic control plants, but a stronger expression was observed compared with200mg/L inducingAcetanilide could induce GUS expression in the cotyledons and hypocotyls of the germinated transgenic mustard seed. A more GUS expression was observed after treated the germinated transgenic mustard seeds with100mg/L acetanilide, and the expression level is near the PCABar35S/GUS control.The expression intensity were weaker induced with50mg/L and200mg/L acetanilide compared with200mg/L treatment. Moreover,1-2days later seed germination time after acetanilide treatment than untreated control was observed,the results indicated a minor inhibition effect of acetanilide on seed germination.Acetanilide could induce GUS gene expression in roots, stems, leaves and flowers of PCAKanIN2-2/GUS transgenic tomato. GUS expression was observed at the third day after inducing, and the expression reached maximum at the fifth day. More strong expression in root and flower than in stems and leaves were observed after inducing.50mg/L or100mg/L were appropriate concentration to induce the expression, the highest GUS activity of92.7nmol/min/mg was detected in the leaves at the fifth day treated with100mg/L acetanilide.(5) GUS expression detection in transgenic plants after2-chlorobenzenesulfonamide inducing2-chlorobenzenesulfonamide strongly inhibited mustard seeds germination, hypocotyl almost could not extend beyond seed coat. Strong GUS expression were observed in cotyledons and hypocotyls, and expression level were near the expression of35S promoter control.However,no GUS expression was detected in the seed coat.Like the acetanilide,2-chlorobenzenesulfonamide treatment also could induce GUS gene expression in roots, stems, leaves and flowers of PCAKanIN2-2/GUS transgenic tomato The GUS expression reached the maximum at4-5days after inducing. GUS activity assay showed the highest activity reached75.17nmol/min/mg at the third day treated with100mg/L.In addition, severe phytotoxicity was observed after treated with200mg/L concentration.(6)The high temperature stress of PCAKanIN2-2/GUS transgenic tomato plantTo imitate the high temperature stress in field environment,the PCAKanIN2-2/GUS transgenic tomato plants were stressed under42℃for24h, no GUS expression was observed in leaves, but trace GUS activity could be found in root after12h stress.