4.8. Expression Analysis of Resistance-Related Genes by qRT-PCR

The relative transcript levels of resistance-related genes were determined with a quantitative reverse transcription PCR (qRT-PCR) method. The root and hypocotyl tissues of cotton were harvested at 6, 12, 24, 48, and 96 hpt, as mentioned above. Total RNA was extracted from cotton Lumianyan 21 using an RNAprep Pure Plant Kit (Tiangen, Beijing, China) and reversely transcribed into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Japan). The G. hirsutum Ubiquitin gene was used as an endogenous reference for the normalization of cotton mRNA [56]. qRT-PCR was performed to quantify the transcript levels of the following genes: three key genes in the lignin metabolism pathway (peroxidase, POD; polyphenol oxidase, PPO; and phenylalanine ammonia lyase, PAL), three pathogenesis-related (PR) genes (disease-related protein gene, PR10; basic chitinase, CHI; and cadinene synthase, CAD); two core genes in the phenylpropanoid metabolic pathway (4-coumaric acid-CoA ligase, 4CL, and cinnamic acid hydroxylase, C4H1) and an important gene responding to NO signaling pathway (nitric oxide associated 1, NOA1) with specific primers (Table 4).