Ultrapure rna kit

Total RNA was extracted with an ultrapure RNA Kit (Tiangen, Beijing, China) and template cDNA synthesis was performed using the PrimeScript™ RT Master Mix (Tiangen, Beijing, China) following the manufacturer’s instructions. A part of the template cDNA was used for RNA-Seq. Meanwhile, the template cDNAs amplified with a 20 μL of reaction solution by using TOROGreen^® qPCR Master Mix (Toroivd, Shanghai, China). The qRT-PCR program consisted of a preliminary step of 60 s at 95 °C, followed by 40 cycles at 95 °C for 10 s, and 60 °C for 30 s. The Actin7 from melon was used as an internal control [55 (link)]. The relative gene expression level was calculated via the 2^−ΔΔCt method. Three biological and three technical replicates were used for each sample. All the primers used in this study are listed in Table S2. In addition, the raw transcriptome sequencing data was used to analyze the tissue specificity of NF-Y members and their roles in fruit ripening, including five melon plant tissues, including roots, leaves, male flowers, female flowers, and fruits (PRJNA383830) [62 (link)], as well as fruit development of green-fleshed (Dulce) and orange-fleshed (Tam-Dew) at 10, 20, and 30 days after anthesis (DAA) and maturity, respectively (PRJNA286120) [37 (link)].

Total RNA was extracted from the pericontusional area using the Ultrapure RNA Kit (TIANGEN, Beijing, China), following the manufacturer’s instructions. RNA was reverse-transcribed to complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, MA, USA). Primers for each transcript were synthesized by Invitrogen (Shanghai, China) and are listed in Table 2. GAPDH was used as endogenous controls for GLUT-1, GLUT-3, and HIF-1α. The SYBR®Green Real-time PCR Kit (Takara, Liaoning, China) was used for qRT-PCR assays using the ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA). Relative expression was normalized to that of endogenous controls using the comparative cycle threshold method, and the fold change in gene expression was calculated using the 2^-∆∆Ct method.Table 2

cDNA of HIF-1α, GLUT-1, GLUT-3, and GAPDH

Gene	Primer sequences(5′-3′)	Product
HIF-1α	Sense: AGAGTCAAGCCCAGAGTCAC	116 bp
	Antisense: TGGGACTGTTAGGCTCAGGT
GLUT-1	Sense: TTATTGCCCAGGTGTTCGGC	93 bp
	Antisense: GTAGCAGGGCTGGGATGAAG
GLUT-3	Sense: TGTGGCTCAGGTCTTTGGTT	99 bp
	Antisense: GCGCTCTGTAGGATAGCTGG
GAPDH	Sense: GGGCTCTCTGCTCCTCCCTGT	107 bp
	Antisense: ACGGCCAAATCCGTTCACACC

PCR analysis was used to detect the expression of pathogenic genes in the bacteria. The bacterial culture used to obtain the samples was the same as that described above. The bacteria were collected from the samples and subjected to centrifugation. RNA was obtained through mucopolysaccharide hydrolysis, cracking thalli, deproteinisation, and centrifugation according to the Ultrapure RNA Kit (TIANGEN, China) manufacturer instructions. The obtained RNA was reverse-transcribed into cDNA using the Takara Kit (Takara, USA), and RT-PCR was implemented (Applied Biosystems 7500 Fast Real-Time PCR System, Foster City, CA, USA) with SYBR Premix Ex Taq™ (Takara, USA), using the primers and cDNA templates. As a housekeeping gene, 16S rRNA was used for calibration. The primers (5ʹ-3ʹ) used in this study are listed in Table 1. The primers were used in a volume of 20 µL per tube. The PCR thermal profile was 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The comparative CT (2 − ΔΔCT) method was used to evaluate the gene expression differences between groups.Table 1

Primer sequences for RT-PCR of oral pathogenic genes

Genes	Primer Sequences (F: forward; R: reverse)
16S rRNA	F:CCTACGGGAGGCAGCAGCAGTAG
	R:CAACAGAGCTTTACGATCCGAAA
Fim	F:CTGAACGAACTGCGACGCTATATGCA
	R:GTTTTTTAGTCGTTTGACGGGTCGAT
Gtf	F:AGCAATGCAGCCAATCTACAAAT
	R:ACGAACTTTGCCGTTATTGTCA