| Special and efficient expression of plant disease-resistance genes can enhance crop resistance to disease; however, the yield of transgenic crops can reduce because overexpression of exogenous gene. So the questions "how to regulate gene expression and which gene expression to regulate" must be resolved in plant transgene for disease resistance. In this study, a desired synthetic pathogen-inducible promoter (SPIP) was obtained using the dipolymers such as2×F-box,2×S-box,2×GST1-box and2×W-box and35S minimal promoter. Then SPIP was used to regulate nonexpressor of pathogenesis-related genes1(npr1) expression in pepper, which is a key regulatory gene from Arabidopsis thaliana and can make the plant tissue obtain resistance to disease. In this way, transgenic pepper with broad-spectrum resistance can be obtained, and this study can provide reference for realization of accurate expression of target gene.Dipolymers of cis-acting elements such as2×F-box,2×S-box,2×GST1-box and2×W-box were constructed by linking two same cis-acting elements using one copy sequence (ACTAGA). Then the artificial promoter fragments were formed by linking the different dipolymers using one copy random sequence (GAAGATAATC) as the interval sequemce unit (ISU). The artificial promoter fragments were connected with mini35S by three copy ISU. By this way, we constructed the8SPIPs is FSGW-mini35S, FSWG-mini35S, GWFS-mini35S, GWSF-mini35S, SFGW-mini35, SFWG-mini35S, WGFS-mini35S and WGSF-mini35S respectively. The pBI121as the basic framework was digestion by Hind Ⅲ, Bam H Ⅰ and T4ligase at16℃for one night, and SPIPs replaced the CaMV35S promoter which could drive gus expression. The recombinant plasmid(pBI121-SPIPs) was transferred into Agrobacterium tumefaciens GV3101, the positive clones of the single colonies were determined by PCR.The results showed that the vector pBI121-SPIPs was successfully constructed and transferred into GV3101.The inducibility of the eight synthetic pathogen-inducible promoters in pepper leaf was evaluated by Agrobacterium tumefaciens-transient genetic transformation assye. Real-time quantitative PCR (QPCR) was used to screen the SPIPs with low basal expression and high inducible expression. The results showed that the gus transcript level under the control of WGFS-mini35S increased respectively7.88fold and29.48fold, compared with35S after inoculation with SA and Ralstonia solanacearum. However, the gus basal transcript level was0.0604fold of35S. WGFS-mini35S promoter had the advantages such as low basal activity and high expression activity. In this study, a pair of primers were designed according to the base sequences of npr1. npr1was obtained by PCR amplification using cDNA as template which was obtained by reverse transcription of RNA of Arabidopsis thaliana. npr1was cloned into the pUC19by digestion with Bam I and Sac I and ligation, then the pUC19-npr1were transferred into E. coli DH5a. Then the recombined vector were selected by Amp resistant selection and PCR.The pUC19-npr1and pBI121-WGFS-mini35S were digested with BamH I and Sac I, the object DNA fragments were recycled and ligated, and the recombined vectors were transferred into the E. coli DH5a. Then the pBI-WGFS-mini35S-nprl was selected by Kan resistant selection and PCR. Followed, the correctly recombined vector were transferred into Agrobacterium tumefacie GV3101. WGFS-mini35S and nprl fragment were detected by PCR. The result showed that the vector pBI-WGFS-mini35S-nprl was successfully constructed.To establish efficient regeneration system is the first step of crop disease-resistant gene engineering. Cotyledons of pepper (Capsicum annuum L) as explant were cultured in medium containing MB+30g/L of sucrose+8g/L of agar+0.5μmol/L of PBU+1.425μmol/L of IAA+1.14μmol/L of ABA+75μmol/L of Spd+100mg/L of Vc+29.41μmol/L of AgNO3(pH5.8～6.0) to induce buds. The results showed that the rate of adventitious bud induction reached to100%, the induced adventitious bud were distinct, green and good, and the highest rate of superior buds reached to89.36%. The adventitious bud elongation and rooting of peppers can be improved by elongation medium of adventitious bud(PBU0.5μmol/L+IAA5.71μmol/L+GA32.89μmol/L) and rooting medium(1/2MS+IAA5.71μmol/L+NAA2.69μmol/L). The rates of adventitious bud elongation and rooting reached to86.67%and100%, respectively. The pepper cotyledon was infected in the GV3101containing pBI121-WGFS-mini35S-nprl for7min and was co-cultivated in dark place for4days. Then explant were transferred to high efficient bud induction medium with cefotaxime sodium250mg/L and kanamycin100mg/L. PCR amplification of transgenic pepper DNA showed that the nprl and WGFS-mini35S gene was successfully integrated into the pepper genome. |
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