3.5 Transcriptome sequencing analysis of WT andMdASMT9-OE lines under heat stress
To gain a deeper understanding of the impacts of overexpressingMdASMT9 on the heat tolerance of apple plants, transcriptional sequencing was conducted on the leaves exposed to 48°C for 1h. Three cDNA libraries were constructed in triplicate, and after quality control, 95.56 Gb of clean data were obtained, with a minimum of 94.02% Q30 bases per sample. The clean data from each sample were separately mapped to the apple reference genome, resulting in mapping rates between 90.85% and 91.95%. The sequencing data were proved suitable for transcriptome analysis based on these results.
Analysis of differentially expressed genes (DEGs) between WT and transgenic plants was conducted based on fragments per kilobase per million mapped fragments (FPKM) with a fold change (FC) of at least 2 and a false discovery rate (FDR) of less than 0.01. A total of 370 DEGs were identified between WT and OE-3, with 205 being up-regulated and 165 being down-regulated. In the comparison between WT and OE-4, there were 453 DEGs identified in total, including 361 up-regulated genes and 92 down-regulated (Figure 5A and 5B). Among these DEGs, we identified 48 DEGs are expressed simultaneously in both OE-3 and OE-4 (Figure 5C and 5D; Table S2). There are 9 transcription factors (TFs), including 5 heat stress transcription factors (HSFs). The relative transcripts of 5 HSFs were induced by heat stress in both WT and OE lines; however,HSFA1d , HSFA2-like and HSFA9b abundance was higher in OE lines than in WT (Figure 5E). In contrast, HSFB1 andHSFB2b abundance was lower in OE lines than in WT.