Isolation and expression pattern of COR15b and KIN1 genes in watermelon and pumpkin

COR15b and KIN1 (COR 6.5) genes encode polypeptides of 15 KDa and 6.5 KDa, respectively. They are involved in the dehydration tolerance mechanisms and play important role under cold stress. cDNA sequences of COR15b and KIN1 genes were first isolated from leaves of watermelon ( Citrullus lanatus ) and pumpkin ( Cucurbita moschata ). Sequencing results indicated that the open reading fragments (ORF) of COR15b in watermelon ( ClCOR15b ) and COR15b in pumpkin ( CmCOR15b ) were 348 and 426 bp, which encoded 116 and 141 amino acids, respectively. The putative amino acids of CmCOR15 b shared 98.58% identities to COR15b in Arabidopsis ( AtCOR15b ), but ClCOR15b had only 80.85% identities to AtCOR15b because there appeared two mutations at the positions of 220 (C/T) and 418 (T/A) in ClCOR15b and T/A mutation produced a transcript end codon (TAA), which led to a lack of 26 amino acids. Similar with KIN1 in Arabidopsis ( AtKIN1 ), ORFs of both KIN1 in watermelon ( ClKIN1 ) and KIN1 in pumpkin ( CmKIN1 ) were 198 bp, encoding two short polypeptides of 65 amino acids. The putative amino acids of ClKIN1 and CmKIN1 shared 98.48 and 90.51% identities to AtKIN1 respectively, although they also contained some mutation sites. Real-time quantitative PCR results indicated that, during cold stress condition, transcripts of CmCOR15b , ClKIN1 and CmKIN1 significantly increased, suggesting that they could take part in the cold tolerance. However, ClCOR15b kept stable during cold stress, implying that its role during cold stress could be changed because of the lacked sequence.


INTRODUCTION
Plants have evolved diverse adaptive mechanisms that enable them to tolerate abiotic stresses, such as low temperature. It has been reported that cold acclimation could increase the cold tolerance in response to low, nonfreezing temperatures (Thomashow, 2001). And many studies indicates that the enhancement of cold tolerance that occurs during cold acclimation is due, in part, to the action of cold-regulated genes and CBF/DREB transcription factors are key regulators for expression of many cold-regulated genes (Stockinger et al., 1997;Jaglo-Ottosen et al., 1998;Liu et al., 1998;Nakayama et al., 2007). Some of cold-regulated genes have been assigned to known classes of proteins. For example, many coldinduced hydrophilic polypeptides belong to the hydrophilin *Corresponding author. E-mail: zkangg@163.com. family, which includes late embryogenesis abundant (LEA) proteins. These hydrophilins may protect enzymes against the effects of water limitation in vitro (Reyes et al., 2005). In Arabidopsis, two small hydrophilic polypeptide, designated as COR15 and COR 6.5, have been widely elucidated.
COR15a encodes a 15 KDa protein with substantial similarities in its amino acid sequence to those encoded by LEA genes. It is located in the stromal compartments of chloroplasts and is involved in the dehydration tolerance mechanisms of cold-stressed plants. Over-expression of the COR15a gene can reduce the propensity of membranes to form hexagonal-phase lipids during freezing stress and enhance the cold tolerance (Lin and Thomashow, 1992;Artus et al., 1996;Steponkus et al., 1998;Zhou et al., 2009). A homolog of the COR 15a gene (COR15b) (with 82% amino acid similarity to COR15a) has also been discovered in Arabidops thaliana. And transcripts for both CORl5b and CORl5a increase dramatically in response to low temperature (Wilhelm and Thomashow, 1993).
KIN1 is an up-regulated gene during cold acclimation and KIN1 from Arabidopsis thaliana is particularly interesting because it codes for a 6.5 KDa polypeptide that bears some compositional similarity to the fish Ala-rich antifreeze proteins. This similarity as well as its increased expression during cold acclimation has led to the speculation that KIN1 might be involved in cold tolerance in plants (Wang et al., 1994).
So far, studies on COR15b and KIN1 genes have mainly been focused on Arabidopsis plants. Here, cDNA sequences of COR15b and KIN1 genes were cloned from watermelon and pumpkin plants and their transcript levels during cold stress period were further explored. This is the first report on COR15b and KIN1 genes in watermelon and pumpkin plants.

Plant materials and chilling stress
Seedlings of watermelon (Citrullus lanatus cv. Xiaolan) and pumpkin (Cucurbita moschata cv. Hongmiben) were grown in pots in soil and sand mixture (8:1), which were placed in plastic growth chambers (30/22°C day/night, 75% of RH，12-h photoperiod with a PPFD of 250 µmol m -2 s -1 ). Three leaf stage of uniform and healthy watermelon and pumpkin seedlings (about 14 cm high) were selected for experiments.
Some watermelon and pumpkin seedling were transferred to a climatic chamber (a PPFD of 150 µmol m -2 s -1 , a 12 h photoperiod and a relative humidity of 70%) to subject cold stress for 3 d at 8°C.

RNA extraction and molecular cloning
Total RNAs of the last fully developed leaves after 1 day of cold stress were isolated using Trizol (Sigma). The primers for cloning cDNA sequences containing ORFs for COR15b and KIN1 were designed according to COR15b (NM-129814) and KIN1 (NM_121601) in Arabidopsis, respectively. The specific primers used for COR15b amplification were: ATCTCACTTTCTCCATCT (forward primer) and GGTTGAATCAGGACTTTG (reverse primer) and cycle parameters were 95°C for 3 min, 35 cycles of 95°C 30 s, 56°C for 30 s, 72°C for 1 min and an extension of 72°C for 8 min. Primers used for KIN1 were TCTCATCATCACTAACCAAAAC (forward primer) GACCCGAATCGCTACTTG (reverse primer) and cycle parameters were 95°C for 8 min, 45 cycles of 95°C 40 s, 58°C for 40 s, 72°C for 1 min and an extension of 72°C for 10 min.
The amplified fragments were isolated from gels, purified using Geneclean (Takara) and cloned into pMD 20-T vector (Takara). Each product was completely sequenced using the Applied Biosystems 3710 DNA capillary sequence for three times.

Bioinformatics analysis
Similarity search was done with the BLASTX program (http://www. ncbi.nlm.nih.gov/blast /Blast.cgi). Multiple sequence alignments of COR15b and KIN1 with other COR15b and KIN1 proteins were conducted using the CLUSTAL X (version 1.81) program. The phylogenetic trees were constructed using the MEGA software 4.0.
Sequences used here for phylogenetic analysis were selected Guozhang et al. 5667 according to their reported functions.

Quantification of the transcripts of COR15b and KIN1 genes by real-time quantification PCR
The last fully developed leaves from cold-stressed and control (at 30/22°C) leaves of watermelon and pumpkin plants were harvested directly into liquid nitrogen and stored at -80°C. Three separate samples of cold stress treatment and control were used for realtime PCR. First strand cDNAs of the above samples were synthesized from 2 µg of total RNA using the first cDNA synthesis kit (Takara). A serial dilution of 100, 50, 5, 0.2 and 0.04 ng of first strand cDNA was used for all transcripts to generate a standard curve by plotting the threshold cycle (Ct) values against log (ng cDNA) and to ensure that the efficiencies of the individual transcripts were equal. The log10 value of the dilution was plotted against the CT (threshold cycle) values obtained. For each sample, the amount of the COR 15b and KIN1 transcript was expressed relative to the amount of Actin transcript. The copy number of COR15b, KIN1 and Actin genes were calculated according to its molecular weight and then converted into the copy number based on Avogadro's number by the formula: number of copies = (amount (ng) × 6.022 ×10 23 )/ (length (bp) × 1 × 10 9 × 650). The pairs of specific primers were used to amplify the Actin in watermelon and pumpkin plants were TGGACTCTGGTGATGGTGTTA (forward primer) and ATGAG GGATGGCTGGAAAA3 (reverse primer).The pairs of specific primers used to measure the transcript levels of COR15b were TTTCGTGACGGATAAGA (forward primer) and TTCCTCAGTC GCAGTTT (reverse primer). And the pairs of primers were to measure the transcript levels of KIN1 were TGTTCTGC TGGA CAAGG (forward primer) and ACCCGAATCGCTACTTG (reverse primer), respectively. Using these two pairs of primers, 103 and 143 bp cDNA fragments were amplified, respectively.

Cloning of COR15b and KIN1 genes in watermelon and pumpkin plants and sequence analysis
Based on the reported AtCOR15b (NM-129814) and AtKIN1 (NM-121601) gene, respectively, the predicated cDNA fragments of COR15b and KIN1 were amplified from watermelon and pumpkin plants (Figure 1). The sequencing showed that the amplified fragments of COR15b from both watermelon (ClCOR15b) and pumpkin plants (CmCOR15b) were 505 bp. The fragments of KIN1 from watermelon (ClKIN1) and pumpkin (CmKIN1) plants were 276 and 275 bp, respectively. ORF of ClCOR 15b and CmCOR15 b were 348 and 426 bp, respectively. ORFs of both ClKIN1 and CmKIN1 were 198 bp ( Figures  2 and 3).
Compared with AtCOR15b, ClCOR15b cDNA sequence had two mutations at positions of 220 bp (C/T) and 418 bp (T/A). C/T mutation only resulted in an amino acid change (H/Y) at position of 50. However, T/A mutation formed a transcript end codon (TAA). Accordingly, putative protein sequence of ClCOR15b had only 115 amino acids, 26 amino acids fewer than those of AtCOR 15b (141 amino acids) (Figures 2 and 4). Accordingly, putative amino acids of ClCOR15b cDNA sequence had 5668 Afr. J. Biotechnol.
Phylogenetic tree from the amino acid sequences of some plant KIN genes also showed their division into three main classes (Figure 7). Both ClKIN1 and CmKIN1 belonged to Class I and they were clustered together AtKIN (NM-121601), AtKIN (NM-121602) and AtKIN (X151474). ClKIN1 had higher identity to NM-121601 and X151474, but CmKIN1 had higher homology to NM-121602.

Expression Patterns of COR15b and KIN1 genes in leaves of in watermelon and pumpkin plants during cold stress
Real-time quantification PCR was undertaken to investigate the transcript levels of COR15b and KIN1 genes in watermelon and pumpkin plants under 8°C cold stress condition. During cold stress periods, the transcripts of CmCOR15b, ClKIN1 and CmKIN1 genes in watermelon and pumpkin plants increased rapidly and significantly higher than those at 30/22 7°C conditions (Figure 8). During cold stress periods, however, ClCOR15b transcripts increased slightly and kept stable.

DISCUSSION
In this paper, cDNA sequences of COR15b and KIN1 genes were first isolated from watermelon and pumpkin plants (Figures 1, 2 and 3). Sequence analysis indicated that putative amino acids of CmCOR15b, ClKIN1 and Cm KIN1 shared high identities to AtCOR15b and AtKIN1, respectively (Figures 4, 5, 6 and 7), although there were some base mutations in their sequences. It has been reported that transcript levels of AtCOR15b and AtKIN1 increased quickly in response to low temperature and were speculated to take part in the cold tolerance (Wilhelm and Thomashow, 1993;Wang et al., 1994). In this paper, during cold stress, transcript levels of CmCOR 15b, ClKIN1 and CmKIN1 genes also increased dramatically (Figure 8), suggesting they could take part in cold tolerance in these plants.
Compared with AtCOR15b, however, putative amino acid sequence of ClCOR15b lacked a fragment of 26 amino acids because of a mutation (T/A), which brought about a transcript end codon (TAA) (Figure 2). Accordingly, putative amino acids of ClCOR15b protein shared low identity to those of AtCOR15b (80.85%) (Figure 4). Its transcript expression patterns differed temporally from CmCOR15b ( Figure 8) and AtCOR15b (Weretilnyk et al., 1993). This inferred that its role during cold stress could be changed because of the lacked sequence and this needed to be further explored.