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Cloning and functional characterization of SAD genes in potato.

Li F, Bian CS, Xu JF, Pang WF, Liu J, Duan SG, Lei ZG, Jiwan P, Jin LP - PLoS ONE (2015)

Bottom Line: Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD.A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv.This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China; Institute of Potato in Guizhou Province, Guiyang, China.

ABSTRACT
Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

No MeSH data available.


Related in: MedlinePlus

Process of buds induction from potato tuber dish A: Nov.26th, 2012; B: Dec.1th, 2012; C: Dec.5th, 2012; D: Dec.10th, 2012.
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pone.0122036.g003: Process of buds induction from potato tuber dish A: Nov.26th, 2012; B: Dec.1th, 2012; C: Dec.5th, 2012; D: Dec.10th, 2012.

Mentions: The full-length ORF (1182 bp) of the ScoSAD was sub-cloned into T-vector and verified by DNA sequencing. ScoSAD was further digested by Xba I and Sma I and then inserted it into the pBI121 (AF485783) binary vector through using Xba I and Sma I restriction enzyme site. The destination vector showed that ScoSAD was driven by CaMV35S promoter and followed by the selectable neomycin phosphotransferase II marker gene for selection marker in agrobacteria transformation. Then, the resulting construct carrying the ScoSAD coding sequence was introduced into Agrobacterium tumefaciens by electroporation method. Mini-tubers (Solanum tuberosum cv. Zhongshu8) were inoculated with A. tumefaciens strain EHA 105 containing the 35S:ScoSAD plasmid on MS medium (4 mg/l ZT and 0.2 mg/l indole-3-acetic acid [IAA]) and incubated at 28°C in the dark for 2 d. Then, the explants were transferred to MS medium containing 4 mg/l ZT, 0.2 mg/L IAA, and 250 mg/l TIM for 7 d (Fig 3). The rooted transgenic plantlets were selected by means of kanamycin resistance, and further screened by PCR for the SAD genes. Of the 35 regenerated plants, 15 (42.8%) were PCR positive. A ~500-bp fragment was amplified from the positive control pBI121:ScoSAD plasmid and some of the transgenic plants, but not the non-transgenic plants. These plants were transferred to an illuminating incubator under the condition that temperature is 22 ± 1°C, illumination intensity is 400 μmol m–2 s–1, photoperiod is 14 h/10 h, and relative humidity is 70 ± 5%.


Cloning and functional characterization of SAD genes in potato.

Li F, Bian CS, Xu JF, Pang WF, Liu J, Duan SG, Lei ZG, Jiwan P, Jin LP - PLoS ONE (2015)

Process of buds induction from potato tuber dish A: Nov.26th, 2012; B: Dec.1th, 2012; C: Dec.5th, 2012; D: Dec.10th, 2012.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380360&req=5

pone.0122036.g003: Process of buds induction from potato tuber dish A: Nov.26th, 2012; B: Dec.1th, 2012; C: Dec.5th, 2012; D: Dec.10th, 2012.
Mentions: The full-length ORF (1182 bp) of the ScoSAD was sub-cloned into T-vector and verified by DNA sequencing. ScoSAD was further digested by Xba I and Sma I and then inserted it into the pBI121 (AF485783) binary vector through using Xba I and Sma I restriction enzyme site. The destination vector showed that ScoSAD was driven by CaMV35S promoter and followed by the selectable neomycin phosphotransferase II marker gene for selection marker in agrobacteria transformation. Then, the resulting construct carrying the ScoSAD coding sequence was introduced into Agrobacterium tumefaciens by electroporation method. Mini-tubers (Solanum tuberosum cv. Zhongshu8) were inoculated with A. tumefaciens strain EHA 105 containing the 35S:ScoSAD plasmid on MS medium (4 mg/l ZT and 0.2 mg/l indole-3-acetic acid [IAA]) and incubated at 28°C in the dark for 2 d. Then, the explants were transferred to MS medium containing 4 mg/l ZT, 0.2 mg/L IAA, and 250 mg/l TIM for 7 d (Fig 3). The rooted transgenic plantlets were selected by means of kanamycin resistance, and further screened by PCR for the SAD genes. Of the 35 regenerated plants, 15 (42.8%) were PCR positive. A ~500-bp fragment was amplified from the positive control pBI121:ScoSAD plasmid and some of the transgenic plants, but not the non-transgenic plants. These plants were transferred to an illuminating incubator under the condition that temperature is 22 ± 1°C, illumination intensity is 400 μmol m–2 s–1, photoperiod is 14 h/10 h, and relative humidity is 70 ± 5%.

Bottom Line: Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD.A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv.This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China; Institute of Potato in Guizhou Province, Guiyang, China.

ABSTRACT
Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

No MeSH data available.


Related in: MedlinePlus