Cloning and characterisation of a pepper aquaporin, CaAQP, which reduces chilling stress in transgenic tobacco plants
Yan-Xu Yin 1, Wei-Li Guo 1, Ying-Li Zhang 1, Jiao-Jiao Ji 1, Huai-Juan Xiao 1, Fei Yan1, Yan-Yan Zhao1, Wen-Chao Zhu 1,2, Ru-Gang Chen 1, Wei-Guo Chai3, Zhen-Hui Gong 1*
1College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China
2Guizhou Academy of Agricultural Sciences, Guiyang, Guizhou 550006, P. R. China
3Institute of Vegetables, Hangzhou Academy of Agricultural Sciences, Hangzhou, Zhejiang 311104, P. R. China
*To whom correspondence should be addressed. E-mail:
Supplementary materials
Supplemental Figure 1 Phylogenetic tree of proteins homologous to CaTIP1-1 (CaAQP) and TIPs from Arabidopsis
Relationship tree generated using MEGA5.0 program. The rooted gene tree (majority-ruleconsensus from 1000 bootstrap replicates) resulted from heuristic searching in Mega5.0. The tree is based on the deduced amino acid sequences of CaTIP1-1 and 10 TIP genes from the Arabidopsis genome database. GenBank accession numbers are in parentheses after each species and gene name. The asterisks indicated the CaTIP1-1 (CaAQP).
Supplemental Figure 2 Phylogenetic relationship of CaTIP1-1(CaAQP) with other TIPs.
Multiple sequences alignment was performed using MEGA5.0. The asterisks indicated the CaTIP1-1. The phylogenetic tree was constructed using the Neighbor-Joining method with amino acid sequences of each TIP protein. The length of each branch is proportional to the divergence of the protein sequence from other members.
Supplemental Figure 3 Multiple amino acid sequence alignment of CaAQP, PhAQP, NtTIP, NgMIP3 and NtAQP1.
Identical, conserved and semi-conserved amino acid residues are denoted by different color. Two conserved NPA motif repeats are boxed. The intron positions, marked by black arrow, the larger intron about 1664bp exhibited typical GT/AG splice junctions. GenBank accession numbers of the corresponding proteins are as follows: CaAQP(ACY74345.1), PhAQP (AAL49753.1| AF452015_1), NtTIP (BAF95576.1 ) and NgMIP3 (AF290619_1 ). The six transmembrane helices (TMH1–TMH6) are determined by a Kyte and Doolittle hydropathy plot of the amino acid sequence(http://gcat.davidson.edu/DGPB/kd/kyte-doolittle.htm) and TMHMM ( signature sequence of aquaporins is signed by green underline.
Supplemental Figure 4 Effect of overexpressing CaAQP on the transcript levels of endogenous AQPs in transgenic tobacco plants.
The expression of CaAQP, NtTIP, NtAQP1 and NgMIP3 was measured by Semi-quantitative PCR with NtL25 as internal control under standard culture conditions. PCR was performed for 28-32 cycles for different genes. WT means wild-type; T2-1, T2-2 and T2-3 are independent transgenic lines.
Supplemental Figure 5 Analysis of expression levels of stress-responsive genes in the WT and the transgenic lines.
The plants were exposed to low night temperature for treatments as described in Materials and methods. The expression of NtNCED1, NtDREB2, NtDREB3 and NgERD10C was measured by qRT-PCR analysis with NtL25 as internal control at day 3 (CH3) of chilling stage and at day 1 (R1) of recovery stage. Bars with different lower case letters in each group were significantly different by Duncan’s multiple range test (p<0.05). Three biological experiments were performed, which produced similar results. WT indicates wild-type; T2-1 and T2-2 are independent transgenic lines. CK0 indicates standard culture conditions.
Supplemental Table 1 Homology analysis of the transcript-derived fragments (TDFs) to gene sequences in the database using BLAST
TDF / TDF length[bp] / Homolog accession / E-value / Organism and putative homolog annotationKH70-1 / 372 / XM_003624327.1 / 1e-56 / Medicago truncatula Ser/Thr protein kinase
KH70-2 / 193 / XM_002300260.1 / 1e-31 / Populus trichocarpa predicted protein
KH70-3 / 115 / AF442387.1 / 2e-49 / Capsicum annuum ascorbate peroxidase mRNA
KH70-4 / 226 / AY496104.1 / 6e-87 / Capsicum annuum thioredoxin mRNA
KH70-5 / 145 / DQ114394.1 / 0.0 / Capsicum annuum cytochrome P450 mRNA
KH70-6 / 445 / AF290619.1 / 3e-145 / Nicotiana glauca putative gamma TIP (MIP3) mRNA
KH70-7 / 134 / DQ114394.1 / 4e-15 / Capsicum annuum cytochrome P450 mRNA
Supplemental Table 2 Primers used in this investigation.
Primersa / Primer sequence (5′→3′)b / ExplanationBD SMARTⅡTM A Oligonucleotide / AAGCAGTGGTATCAACGCAGAGTACGCGGG / First strand cDNA synthesis
3’-CDS / AAGCAGTGGTATCAACGCAGAGTAC(T)30VN(N=A, C, G, or T; V=A, G, or C) / 3′-RACE
5’-CDS / (T)25VN(N=A,C,G, or T; V=A,G, or C) / 5′-RACE
RA32 / CTGATCTAGAGGTACCGGATCC / 3′-RACE
UPM1 / CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT / 5′-RACE
UPM2 / CTAATACGACTCACTATAGGGC / 5′-RACE
NUP / AAGCAGTGGTATCAACGCAGAGT / 5′-RACE
GSP1 / CCTGTGCCCTCCTTGTATTC / 3′-RACE
GSP2 / TTACTCCCAAATCTCCCTTCT / 5′-RACE
CaAQPF1 / TGTCCAGTTTTTTCCCCTCT / Reverse PCR
CaAQPR1 / AAAGATCAAGACATAATGGATGC / Reverse PCR
CaAQPF2 / CGATGGCGTCACTACTCCTC / RT-PCR
CaAQPR2 / CACCAACGAAAGCACCGA / RT-PCR
CaAQPF3 / CGGGGTACCATGCCGATCCGCCAAATTG / Vector construct
CaAQPR3 / GCTCTAGATTAAAAATCTCCTCCACTTGGGATT / Vector construct
CaAQPF4 / ATGCCGATCCGCCAAATTG / Transformation confirmed
CaAQPR4 / TTAAAAATCTCCTCCACTTGGGATT / Transformation confirmed
CaAQPF5 / GCTCTAGACTCTTCAGTTTGGTTGTAGGC / Vector construct
CaAQPR5 / CGGGGTACCGCACCGAAAGTAACAGCAG / Vector construct
NtMIP3F / CTGTATCCTTTGGGCCAGCTTT / RT-PCR
NtMIP3R / AGCAGAGGTTGAAATTTCGTGA / RT-PCR
NtTIPF / CTGTCTCATTTGGTCCTGCTTT / RT-PCR
NtTIPR / GCAACAGAGGTTGAAATTTGAAT / RT-PCR
NtAQP1F / TGGGGCCTTTACTGGAGCTTC / RT-PCR
NtAQP1R / TGAAAGCAACAGATAGAGGTT / RT-PCR
CaAQPR6 / TGATGTACAGAAGTCCCCTG / RT-PCR
NtERD10C-F / AAGGTTGAAGAGGGTAGCGC / RT-PCR
NtERD10C-R / CTTGGGCATGAGTTGGCTTT / RT-PCR
NtNCED1F / ACACGAACTCCAACACCCTT / RT-PCR
NtNCED1R / TTCGGGTATTTTTCCGTTGA / RT-PCR
NtDREB2F / CCGACGCTAAGGATATTCAG / RT-PCR
NtDREB2R / CTTTCTGGCGTTTCAGGAGT / RT-PCR
NtDREB3F / ACAGGAGAAGATTCAAAGGA / RT-PCR
NtDREB3R / GAACACTGAGGTGGAGGTAA / RT-PCR
NtL25F / CCTAAAGTATCCCCTCACCACAG / RT-PCR
NtL25R / CTTTCTTCGTCCCATCAGGC / RT-PCR
CaUbi3-F / TGTCCATCTGCTCTCTGTTG / RT-PCR
CaUbi3-R / CACCCCAAGCACAATAAGAC / RT-PCR
aThe UPM primer used in RACE contains 0.4M UPM1 and 2M UPM2.
bKpnⅠenzyme site was boxed in primers of CaAQPF3 and CaAQPR5, and XbaⅠenzyme site was boxed in primers of CaAQPF5 and CaAQPR3 separately.
Supplemental Table 3 Comparison of intron lengths and numbers of TIP1 in Arabidopsis and Solanaceae
Gene name / Accession number / Number of intron / Length of intron[bp] / Donor and acceptor splice sites.
AtTIP1-1 / NM_129238.3 / 1 / 91 / TA/GG
AtTIP1-2 / NM_113559.3 / 1 / 172 / GT/AG
AtTIP1-3 / NM_116377.1 / 0 / 0 / -
aSlTIP1-1 / AB845616.1 / 1 / 1262 / GT/AG
aSlTIP1-2 / AB845617 / 2 / 87/79 / （GG/CA)/(GT/AG)
aSlTIP1-3 / Not Found
StTIP1-1 / bPGSC0003DMG400028182 / 1 / 665 / GG/CA
StTIP1-2 / bPGSC0003DMG400030308 / 2 / 95/69 / (GG/CA)/(GG/CA)
StTIP1-3 / bPGSC0003DMG400007134 / 1 / 1338 / GT/AG
cCaTIP1-1(CaAQP) / GU116569 / 1 / 1664 / GT/AG
a,The amino acid sequence length was either confirmed by cDNA sequencing or predicted using SL2.40 gene models
b, Potato genome sequencing consortium (PGSC) nomenclature
c, Intron was confirmed by sequencing and blasted using the database of pepper genome (