Plant material
Z. japonica used in this study was collected from Fangchenggang, Guangxi, China.
DNA extraction and primer design
Leaves of Z. japonica were used as materials to extract genomic DNA from young leaves that had grown well. A MiniBEST Plant Genomic DNA Extraction Kit (TaKaRa, 9768) was used to extract genomic DNA from the leaves of Z. japonica following the manufacturer’s instructions. Based on the full-length cDNA sequence of ZjFes1 obtained by RACE18, three identical and high annealing temperature specific primers (SP Primer) were designed, and four specifically designed degenerate primers, AP1, AP2, AP3 and AP4, were used for thermal asymmetric interlaced PCR (TAIL-PCR). Typically, at least one of these degenerate primers can react with specific primers by TAIL-PCR based on the difference of annealing temperature, and the flanking sequence of known sequence can be obtained by three nested PCR reactions. Because the length obtained in one experiment cannot meet the experimental requirements, we continue to acquire the flanking sequence according to the sequence information obtained in the first genome walking. Four genome walkings were conducted. Twelve SP Primers were designed. DNAMAN software was used to combine the four fragments described above into a consensus sequence by combining overlapping fragments. Specific primers were designed to amplify 2 kb sequences according to the results (Table 1), and the experimental results were verified.
Cloning and construction of the plant expression vector and sequence analysis of promoter
The full-length promoter sequence was amplified using high fidelity polymerase 2 × TransStart FastPfu PCR SuperMix (-dye) (TRANSGEN BIOTECH, AS221-01) using the DNA of Z. japonica as a template following the manufacturer’s instructions. The PCR products were detected using 1% gel electrophoresis. The results showed that the size of the bands was the same as that of the target fragments, and the PCR products were recovered using a MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (TaKaRa, 9762). The pCXGUS-P plasmid is a vector designed to detect the activity of plant promoters. The promoter activity is detected by the dyeing intensity of GUS. We used XcmI to digest the empty vector to obtain T vector. After recovery, the product was recombined with T vector, and then the recombinant vector was transformed into E. coli DH5α Competent Cells (TaKaRa, 9057) following the manufacturer’s instructions. The positive samples identified by PCR were verified by sequencing at the Guangzhou Sequencing Department of Invitrogen. The sequencing results were compared using DNAMAN software. The plasmid was extracted from the correct bacterial solution and designated pZjFes1::GUS. The sequence analysis of cis-acting elements that could possibly be found in the promoter was performed using the plant-CARE online prediction database (plant cis-acting regulatory element, https://bioinformatics.psb.ugent.be/webtools/plantcare/html/)20.
Agrobacterium-mediated genetic transformation of pZjFes1::GUS into Arabidopsis thaliana
The fusion vector pZjFes1::GUS was transformed into Agrobacterium Rhizobium strain GV3101 chemically competent cells (Biomed, BC304) using the freeze–thaw method following the manufacturer’s instructions. Transgenic plants of A. thaliana were obtained by floral dipping. Plants in nutrient soil were cultured to form a large number of immature flower clusters. The monoclone of A. tumefaciens GV3101 was selected and inoculated in liquid LB medium containing kanamycin and rifampicin (50 µg/mL). The monoclone was cultured overnight at 200 rpm and 28 °C. A volume of 2 mL bacterial solution was transferred to a 500 mL flask culture (containing 200 mL liquid LB with 50 µg/mL kanamycin and rifampicin added) and was cultured overnight at 200 rpm and 28 °C. The next day, the OD600 of Agrobacterium solution was 1.8–2.0. The solution was centrifuged at 5000 rpm for 15 min at 4 °C. The supernatant was discarded, and the precipitate of A. tumefaciens was resuspended in 1/2 volume (100 mL) osmotic medium (1/2 Murashige-Skoog, 5% sucrose, 0.5 g/L MES, 10 µg/mL 6-BA, 200 µl/L Silwet L-77, and 150 µM acetyleugenone, pH 5.7), resulting in an OD600 of approximately 1.6. The bacterial solution was adsorbed on the transformed plants using the floral dip method (5 min), wrapped with film to keep it fresh, and cultured overnight, followed by the removal of the film. The plants were cultured until the seeds were ripe, and they were harvested. A mixed disinfectant consisting of 70% ethanol and 30% bleaching water was used to soak the seeds for 3 min, suspend them continuously, and wash them three times with anhydrous ethanol. The dried seeds were evenly dispersed on the surface of solid screening medium containing hygromycin (25 µg/mL). After stratification at 4 °C for 2 days, the seeds were germinated in a light incubator and cultured for 2 weeks at 21 °C and 16 h light/8 h darkness. The development of seedlings and length of roots were used to determine whether they were transformants.
GUS dyeing and activity analysis
The expression of GUS reporter gene in Arabidopsis tissues was determined using a GUS staining kit (Solarbio, G3060) following the manufacturer’s instructions. The seedlings, leaves, flowers and siliques to be dyed were immersed in GUS dye solution and incubated overnight at 37 °C. The chlorophyll was removed with 75% ethanol until the background color disappeared completely. The results were documented by photography using a Canon 60d camera.
The material needed to determine Gus enzyme activity was frozen rapidly with liquid nitrogen, and then ground into powder by ball mill. The extraction buffer solution (50 mM NaH2PO4 (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1 (w / v) sodium dodecyl sulfonate, 10 mM β-mercaptoethanol) were added to extract protein. After centrifugation at 4 °C, 12,000 r/min for 10 min, the supernatant was taken as protein extract. The protein concentration was determined by Bradford method. 4-MUG, the substrate of GUS reaction, was added and reacted at 37 °C for 30 min. Fluorescence measurement was carried out under the condition of 365 nm excitation light and 455 nm emission light. Three independent biological repeats were conducted. Finally, the GUS enzyme activity value was calculated according to the relative change of product in unit time.
Source: Ecology - nature.com
