Tripure

For tissue-specific expression patterns of TaHsfC3-4, tissues of wheat at different growth and development stages were collected for RNA sample isolation. In order to examine the expression levels of TaHsfC3-4 during the seed maturation stage, the total RNA was extracted from the tissue of wheat at different days after anthesis. In addition, to determine whether the expression of TaHsfC3-4 was induced by 200 µM ABA and 20% PEG6000 treatments in wheat, the roots and the second-expanded leaves of young wheat seedlings were harvested separately after various times of treatment for the extraction of RNA. All the above RNA were extracted by using the TRIpure (Aidlab, Beijing, China), according to the manufacturer’s protocol, and its concentration was measured using NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA). Then, 1 µg of purified RNA was used as template to reverse-transcribe to cDNA. In addition, to detect TaHsfC3-4 in transgenic plants, total RNA was extracted from three-week-old transgenic Arabidopsis plants, and semi-quantitative RT-PCR was performed with specific primers designed by using Primer 5.0 software. The specific primers are listed in Table S1.

RNA was extracted from liver samples using TRIpure (Aidlab, Biotechnologies Ltd, Beijing, China) isolation reagent based on the instruction outlined in the manufacturer's guide. The level of expression of some metabolic genes was assessed according to a previously described method [36 (link)]. Briefly, Easyscript one-step RT-PCR supermix kit (TransGen Biotech Co., Ltd., Beijing, China) was used for the semiquantitative process based on the manufacturer's instruction. cDNA was first synthesized by incubating the RNA template (500 ng) at 45^oC for 30 minutes. A thermal cycler (C100 Touch thermal cycler, Bio-rad Laboratories) was used to carry out the amplification process using gene-specific primers as listed in Table 2. The PCR conditions included an initial denaturation at 94°C for 5 minutes, followed by 45 cycles of 94°C for 30 seconds, another 30 seconds at an annealing temperature of gene-specific primers and 1 min at 72°C. The PCR products were run on an ethidium bromide-stained agarose gel (1.5%) in Tris Borate EDTA buffer and viewed under UV light (UVP BioDoc-It™ Imaging system (Upland, CA, USA). The intensity of the bands was analyzed using Image J software [43 ]. Results are expressed as the mean ratio of the intensity of each gene to that of two reference genes (GAPDH and β-actin).