Cloning, molecular characterisation, and expression of putative invertase inhibitor genes and their potential relationship with the activity of vacuolar acid invertase in peach fruit

Figures & data

Table 1. INH Primers for PCR amplification.

Gene name	Forward primer sequence (5′–3′)	Reverse primer sequence (5′–3′)	Product size (bp)
PpINH1	AGCCAATGCAGGGTTTACTTG	GGAGAGCTGCTTGAAAAACC	389
PpINH2	ATGGGTGCCAATCTCATCGAC	TCAAAGCATTGTCCTCGCAAT	441
PpINH3	GTTTCCACACCAGGAGTTTAGAG	TACATCAAAGCAATGTCCTGGC	622
PpINH4	GCCTGCAAGACGAGTGAAC	TTTCATTGGCTGAAACTGCTGC	907
PpINH5	AAGGAAGGAAAAGATGAGGCGT	TGCCGAGCTTATGTTGGCTT	633

Table 2. Primers of peach invertase gene and its inhibitor genes used for real-time fluorescence quantitative PCR.

Gene name	Forward primer sequence (5′–3′)	Reverse primer sequence (5′–3′	Product size (bp)
PpINH1	ATGTCCCACAAGGCAGTCAA	CAGCCGCAACATCAAGAAGAG	165
PpINH2	CTCAGATCCCCGAAGCTACAC	ACGATCGGTCTCCTGGGCTC	134
PpINH3	ACCGAACTATGCTCTCTGCG	TCAGAGTGTCGGTCGACTTG	122
PpINH4	TTCCCTTAGATGAAGGCGGTG	ATGTGGGCTAGTCCTGGAAC	130
PpINH5	GCCACAAACATCAACTCCACC	GTAGGGTAGGAATGGCGTCAG	118
PpVIN2	ACAAGGTCTTCCGTGGCAAA	AGCAGCCCCATAAATTGCCT	186
TEF2	TGAAGGAGAGGGAAGGTGAAAG	GGTGTGACGATGAAGAGTGATG	129

Table 3. Invertase inhibitor genes and their deduced amino acid in peaches.

Gene name	Protein length (AA)	Chr	pI	MW (KDa)	SP
PpINH1	145	G5	4.34	15.07	NO
PpINH2	146	G3	4.51	15.29	NO
PpINH3	176	G1	5.78	18.63	Yes
PpINH4	206	G1	5.40	22.11	NO
PpINH5	183	G5	6.72	20.10	Yes

Figure 1. Alignment of deduced amino acid sequences of inhibitor genes in peaches. Regions with amino acid identity are marked with different colours. The four conserved cysteine residues are indicated by crosses at the top of the alignment.

Figure 2. Evolutionary relationship between 18 genes from the PMEI-RP family. The tree was constructed using sequences from 7 different species, including peach. The two main groups are indicated using a dashed line, and the subgroups are identified using Roman numerals. Asterisks (*) indicate invertase inhibitors that have been characterised experimentally. The genes from peach (PpINH1–5) are shown in boxes. The other InvInh gene sequences are cDNAs from potato (StInvInh1, ADM49012; StInvInh2A, ADM49015; StInvInh2B, ADM49016; StInvInh3, ADM49013), tobacco (NtInvInh1, CAA73333; NtInvInh2, CAA73334), tomato (SlInvInh1, CAA09420), Arabidopsis thaliana (AtInvInh1, NP_564516; AtInvInh2, NP_201267; AtPMEI1, NP_175236; AtPMEI2, NP_188348), Zea mays (ZmINH1, ATT24363), and Kiwi (AdPMEI1, BAC54964).

Figure 3. Relative expression of INH genes in peach fruit (‘Yulu’) stored at four different temperatures. (A: 0°C, B: 5°C, C: 10°C, D: 20°C). Values are the means ± SE of triplicate assays. Different letters indicate a significant difference (P < 0.05) between 0°C, 5°C, and 10°C using Duncan's multiple range test at each time point.

Figure 4. Relative expression of INH genes in peach fruit (‘Yulu’ and ‘Hujingmilu’) stored at 5°C. (A: PpINH1, B: PpINH2, C: PpINH3, D: PpINH4, E: PpINH5). Values are the means ± SE of triplicate assays. Different letters indicate a significant difference (P < 0.05) using Duncan's multiple range test.

Figure 5. VIN activities and PpVIN2 expression in peach fruit (‘Yulu’ and ‘Hujingmilu’) stored at 5°C. Values are the means ± SE of triplicate assays. Different letters indicate a significant difference (P < 0.05) using Duncan's multiple range test.

Table 4. Pearson's Correlation between the PpINHs expression and VIN activity of ‘Yulu’ and ‘Hujingmilu’ peaches stored at 5°C.

	VIN activity (Yulu)	VIN activity (Hujingmilu)
PpINH1	−0.9743*	−0.8912*
PpINH2	−0.7054	−0.6149
PpINH3	−0.4183	0.3557
PpINH4	−0.3766	0.1315
PpINH5	0.1161	0.2983

*Indicates a significant correlation at P = 0.05.