Table 1.
Semi-quantitative PCR and real time PCR primers.
Table 2.
The information of CAD genes in melon.
Figure 1.
Intron-exon structures of CAD genes from melon.
Exons and introns are indicated by open boxes and lines respectively. Numbers above boxes indicate the exon sizes. The intron sizes are not to scale. The names of CAD genes and intron-exon structure are indicated at the left and right sides respectively.
Figure 2.
Phylogenetic relationship among CmCADs and CADs from other plant species.
The amino acid sequences were aligned by the Clustal W2 program, and the neighborjoining tree was drawn with TreeView. The corresponding GenBank and the melon genome (https://melonomics.net/) were noted in the phylogenetic tree and the accession number in the melon genome were CmCAD1 (MELO3C019548P1), CmCAD2 (MELO3C018492P1), CmACD3 (MELO3C003735P2), CmCAD4 (MELO3C005809P1) and CmCAD5 (MELO3C023272P1). The number for each interior branch was the percentage of bootstraps value (1000 replicates). Black circle denoted five CmCADs. CADs belong to the following plant species: Arabidopsis thaliana: AtCAD1 (AY288079), AtCAD2 (AY302077), AtCAD3 (AY302078), AtCAD4 (AY302081), AtCAD5 (AY302082), AtCAD6 (AY302075), AtCAD7 (AY302079), AtCAD8 (AY302080), and AtCAD9 (AY302076); Oryza sativa: OsCAD1 (AAN09864), OsCAD2 (DQ234272), OsCAD3 (AAP53892), OsCAD4 (BK003970), OsCAD5 (BK003971), OsCAD6 (CAD39907), OsCAD7 (CAE05206), OsCAD8A to D (BK003972), and OsCAD9 (AAN05338); Sorghum bicolor: SbCAD2 (Sb04g005950), SbCAD4-2 (Sb10g006300), SbCAD4-3 (Sb10g006290), SbCAD4-4 (Sb10g006280), SbCAD4-5 (Sb10g006270), SbCAD5 (Sb07g006090), SbCAD6 (Sb06g001430), SbCAD7 (Sb06g028240), SbCAD8-1 (Sb02g024220), SbCAD8-2 (Sb02g024210), and SbCAD8-4 (Sb02g024190); Triticum aestivum: TaCAD1 (GU563724), TaCAD2 (TC143210), TaCAD3 (TC143265), TaCAD4 (TC144004), TaCAD5 (TC149391), TaCAD6 (TC149393), TaCAD7 (TC170425), TaCAD8 (TC170426), TaCAD9 (TC170429), TaCAD10 (TC172690), and TaCAD11 (TC179401); Aralia cordata: AcCAD1 (D13991); Eucalyptus globulus: EgCAD1 (AF038561); Festuca arundinacea: FaCAD1a (AF188292); Lolium perenne: LpCAD1 (AF472591), LpCAD2 (AF472592), and LpCAD3 (AF010290); Medicago sativa: MsCAD1 (AF083333) and MsCAD2 (AF083332); Nicotiana tabacum: NtCAD1 (X62343) and NtCAD2 (X62344); Picea abies: PaCAD1 (X72675); Populus tremuloides: PtCAD1 (AF217957) and PtSAD (AF273256); Pinus taeda: PtaCAD1 (Z37992); Saccharum officinarum: SoCAD1 (AJ231135); Zea mays: ZmCAD1 (AJ005702) and ZmCAD2 (Y13733); Vitis vinifera: VvCAD (CBI34634.3), VvCAD6 (XP002269356.1), VvCAD9 (XP002279832.1). Cucumis sativus: CsCAD6 (XP004136373.1), CsCAD9 (XP004150677.1), CsCAD11 (XP004140716.1), CsCAD12 (XP004137094.1), CsCAD13 (XP004145884.1), CsCAD14 (XP004162965.1), Hordeum vulgare: HvCAD1A (BAJ84795.1), HvCAD1B (BAJ98188.1), HvCAD6 (BAK01962.1); Glycine max:GmCAD1 (XP003543132.1); Cicer arietinum: CaCAD1 (XP004485621.1); Gossypium hirsutum: GhCAD3 (ACQ59091.1); Ricinus communis: RcCAD (XP_002510582.1); Theobroma cacao: TcCAD9 (EOY15101.1); Fragaria vesca: FvCAD6 (XP004291336.1).
Figure 3.
Alignment of amino acid sequences of CmCADs.
Conserved important regions identified previously are marked as follows: white arrows denotes catalytic zinc ion coordinating residue, black arrows denotes structural Zn ion coordinating residue, the black circle denotes key residues for substrate specificity. The black square denotes key Phe299/Gly(300) residues for substrate specificity. The white square denotes key Trp199 and Asp123 residues for substrate binding. Locations of the Zn1, Zn2, and NADPH binding domains are shown in boxes. The alignment was performed with the ClustalW2 software program.
Figure 4.
Transcript levels of these five CmCAD in different melon organs.
The gene expressions of CmCAD in different organs in melon plants were determined by qRT-PCR in root, developing leaves, mature leaves, young stems, pistillate flower petals and staminate flower petals in melon plants. 18s were used as internal control. The expression level of the genes in mature leaves was set as “1.0”. Data represent the means±SD (n = 3) of three biological samples. The experiments were repeated 3 times with similar results.
Figure 5.
CmCADs relative expression in developing stages of melon fruit after pollination were determined by qRT-PCR.
18s were used as internal control. The expression level of CmCADs in melon fruit at 15days after pollination was set as “1.0”. Data represent the means±SD (n = 3) of three biological samples. The experiments were carried out in triplicate.
Figure 6.
The expression of CmCAD1, 2, 3, 4 and 5 in melon fruit after different hormonal treatments.
IAA and ABA (100 µM) treatments were given for 3 h as described in materials and methods section. Expression analysis was carried out by real time qPCR. For each gene, the relative abundance of mRNA was normalized against the 18S in the corresponding samples. The expression level of the genes in untreated melon fruit by IAA and ABA was set as “1.0”. Data represent the means±SD (n = 3) of three biological samples. The experiments were repeated 3 times with similar results.
Figure 7.
The expression levels of CmCADs after treatment with ethylene and 1-MCP.
The transcript levels of CsCADs were measured by real time qPCR in melon fruit treated, and 18S were used as internal control. The expression of the genes in untreated melon fruit after 1 day of storage was set to 1.0. Data represent the means±SD (n = 3) of three biological samples. The experiments were repeated 3 times with similar results.