Hiscriptr 3 rt supermix for qpcr gdna wiper

According to the manufacturer’s protocol, the total RNA was extracted from the leaves and roots of pecan using a Universal Plant Total RNA Extraction Kit (Bioteke, Beijing, China) and stored at -80°C until further use. The purity and integrity of the isolated total RNA was analyzed by agarose gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA). First-strand cDNA was synthesized using a cDNA Synthesis Kit (HiScript ^®RIII RT SuperMix for qPCR +gDNA wiper, Vazyme, Nanjing, China). The qRT-PCR was performed on a 7500 Real-Time PCR system (Applied BiosystemsTM, Foster City, CA, USA) using a Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The specific primers were synthesized by Tsingke Biotechnology Ltd (Nanjing, China), the details of the primers were provided in Table 1, melting curve analyses were used to verify the efficiency of the primers (Supplementary Figure S1). The Actin gene was used as an internal reference gene (Zhu et al., 2022 (link)), and the relative expression levels of pecan Nia, NiR, Gln, GOGAT, and GDH genes were determined using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)). Values represent mean calculated from three biological replicates and three technological repeats. The pecan N assimilate genes were renamed according to the Arabidopsis gene name and the NCBI pblast results.

Quantitative PCR (qPCR) was performed to verify the results of mRNA-seq analysis. The cDNA template was synthesized by reverse transcription using a kit (HiScript^R III RT SuperMix for qPCR (+gDNA wiper), Vazyme Biotechnology Co., LTD., Nanjing, China). The qPCR was performed using SYBR green (AceQ Universal SYBR qPCR Master Mix, Vazyme Biotechnology Co., LTD., Nanjing, China) on a 7500 Fast Real-time PCR System (Applied Biosystems, Foster, CA, USA). The primers were designed according to the gene sequences from M. aeruginosa PCC 7806SL complete genome, with 16S rRNA [26 (link)] as the reference gene and synthesized by GENEWIZ (Suzhou, China) (the primers were shown in Supplementary Table S1).

Infected tea plant leaves were harvested and immediately frozen in liquid nitrogen. RNA was extracted using a HiPure Plant RNA Mini Kit b (Magen, China). The RNA was reverse-transcribed into cDNA using a HiScriptRIII RT Super Mix for qPCR (+gDNAwiper) (Vazyme, China) according to the manufacturer’s instructions. qRT-PCR was carried out according to our previous method [32 (link)]. Actin was used as a reference gene, and the relative expression levels were calculated using the 2^−ΔΔCT method. The primers used are listed in Table 1.