Selection Of Reference Genes For QRT-PCR In Pumpkin And The Expression Analysis Of Catalase Family Genes

Pumpkin (Cucurbita L.) is an important cucurbit vegetable in the world due to its abundant nutrition. In order to detect gene expression of pumpkin we should select the genes which stably expressed (designated as internal genes or reference genes) under different stress conditions. To achieve this, we firstly establish the qRT-PCR technique to select the internal genes based on the transcriptomics data of pumpkin obtained previously in our lab. Thus, the expression level of genes in CAT family was characterized and the different expression level of these genes was further investigated under environmental stress. Our results set up the foundation for the study of gene expression of pumpkin1. Selection pumpkin reference genes. Ten candidate reference genes were screened from the transcriptomics data of pumpkin according to the bioinformatics analysis. Using qRT-PCR, we thus analyzed these ten candidates as well as two well-studied reference genes (CAC, EF-1A) in different tissues of two pumpkin cultivars, and abiotic stresses (salt, cold, drought), biotic (bacterial fruit blotch, watermelon Fusarium Wilt, melon Fusarium wilt, root-rot) and hormones (GA3, ABA, NAA, ETH). The expression pattern of 60 samples in total were measured, and the stability of the 12 candidate genes were evaluated by two softwares geNorm and NormFinder. The results showed that the best reference genes were Unigenel6959 and CL5002.Contig7 by geNorm software, while the best one is CL5547.Contig2 by NormFinder software. The stability of the single or combination reference genes was validated by the expression level of catalase gene (CAT) under drought stress, and the traditional internal gene EF-1A as control. The results showed that the combinations of two reference genes (Unigene16959 and CL5002.Contig7) or three reference genes (Unigene16959, CL5002.Contig7, CL5547.Contig2) were more accurate than the traditional reference gene EF-1A.2. Expression analysis of the CAT gene family members in pumpkin under different stress treatments. The H2O2 content in the leaves as well as the expression of CAT1, CAT2 and CAT3 at transcriptional level were investigated under abiotic stress, biotic stress and hormone treatments. The results showed that the expression of CAT gene was stimulated by above stresses, however, the expression varied with different treatments. Among CAT gene family members, the expression of CAT1 and CAT2 changed obviously, whereas the expression of CAT3 had no significant changes. The expression of both CAT1 and CAT2 were upregulated with the increasing H2O2 contents, which indicated that these two genes played important role involving in the antioxidant systems among the CAT gene family. Additionally, the H2O2 can regulate the expression of CAT family genes at transcriptional level as a single molecule, which then strengthened the activity of catalase and decomposed the extra H2O2 to maintain a stable redox state in pumpkins.