Experimental materials and design
Two malting barley cultivars, ZU9 and Hua30, which are widely planted locally and differ in the responses of malt quality traits to heat stress according to the previous studies [12], were used in this experiment. The seeds of both ZD9 and Hua30 were obtained from authors’ laboratory, Zhejiang University, China. The seeds were imbibed in water for 3 h and sown into 10 L pots filled with 2.5 kg mixed soil (peat soil : vermiculite = 1:1) in early Nov, 2018 and the pots were placed in a greenhouse of Zijingang campus, Zhejiang University (Hangzhou, China, 120.2°E, 30.5°N). Compound fertilizer of 10 g (N : P2O5 : K2O = 13:7:5) per pot was mixed uniformly with soil before sowing and 5 seedlings were remained in each pot through deleting extra seedlings at 2-leaf stage.
At heading stage, all pots with 5 plants per pot were moved into three growth chambers, referred to C1, C2 and C3, respectively thereafter. C1 was used as control, which was 18/24℃ (night/day, 12/12hours) of temperature during the whole maturity stage. C2 and C3 were the heat stress treatments, which remained the same temperature as the control during no heat treatment, and they were 26/32℃ (night/day, 12/12hours) from 7th and 14th day after heading and lasted for 9 days, respectively. All plants were well irrigated and the relative humidity was maintained at 75% and the light was supplied from 6:00 to 18:00 at a light intensity of 50000 Lux.
Grain samples were taken randomly from the two barley cultivars of both control and treatments on the day just before treatment, and 1st, 3rd, 5th, 7th and 9th day after treatments. The sampled grains were immediately placed into liquid nitrogen and transferred to a refrigerator at -80℃ for use of RNA extraction.
Total RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted from the sampled grains with a TaKaRa MiniBEST Plant RNA Extraction Kit (TAKARA BIO INC, Beijing, China) according to manufacturer’s instructions, and then RNA was treated by RNase-free DNase I (TAKARA BIO INC, Beijing, China) for digesting template DNA. The extracted RNA quality was identified by gel electrophoresis. The concentration of RNA solution was measured by Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific™, US) and then accurately diluted to 200 ng/µl. The 20 µl RT reaction solution system, consisting of 4 µl PrimeScipt RT Master Mix (TAKARA BIO INC, Beijing, China), 5 µl total RNA solution and RNase free H2O, were used to prepare first-strand cDNA synthesis by reverse transcription (37℃, 10 min). The single-strand cDNAs were amplified using TB Green Premix Ex Taq (TAKARA BIO INC, Beijing, China) with gene-specific forward and reverse primers of HvCslF6, HvCslF9, HvBmy1 and limit dextrinase mRNA (Table 1), while actin gene (HvACT) was amplified with specific primers (forward:5'-GACTCTGGTGATGGTGTCAGC-3';reverse:5'-GGCTGGAAGAGGACCTCAGG-3') as an internal control for the reactive amount of RNA. The whole real-time PCR reaction was carried on the background of LightCycler480 System (Roche Diagnostics, Mannheim, Germany) with a de-naturation step of 30 seconds, followed by 40 cycles of 95℃ for 5 s, 60℃ for 30 s and melting curve analysis at 65℃ for 15 seconds. Three technical replicates were performed for both control and treatments. Meanwhile, the expression of malt quality genes was normalized by HvActin expression values. LightCycler480 Software (Roche, version 1.5.0) was used to analyze the raw data.