Cloning and Characterisation of a Pepper Aquaporin, Caaqp, Which Reduces Chilling Stress

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

Supplemental Table 2 Primers used in this investigation.

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

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 (