Presence and expression of NAD+-dependent sorbitol dehydrogenase and sorbitol-6-phosphate dehydrogenase genes in strawberry
Presence and expression of NAD+-dependent sorbitol dehydrogenase and sorbitol-6-phosphate dehydrogenase genes in strawberry
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DOI：
10.1080/14620316.2007.11512219
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发表时间：
2007-03-01
影响因子：
1.9
通讯作者：
Yamaki, S.
中科院分区：
文献类型：
--
作者：
Duangsrisai, S.;Yamada, K.;Yamaki, S.
以草莓（Fragaria X ananassa Duch.）变种“Nyoho”）不清楚，尽管草莓属于蔷薇科。因此，研究了草莓中NAD（+）依赖性山梨醇脱氢酶（NAD-SDH）和山梨醇-6-磷酸脱氢酶（S6 PDH）基因的存在或不存在，以及它们与生长相关的表达水平。利用RACE技术获得了草莓NAD-SDH基因的全长cDNA（FaSDH）。全长克隆由1，399 bp组成，并含有编码361的1，083 bp开放阅读框。计算分子量为38.8 kDa的氨基酸，预测等电点（pI）为6.08。该基因与其它植物NAD-SDH基因的同源性为79 - 85%。其氨基酸序列含有锌和NAD结合位点，证实该序列属于NAD-SDH基因家族。Southern杂交结果表明，FaSDH基因组拷贝数低于苹果。草莓果实中FaSDH转录本的表达量在幼嫩期和成熟期高于其他时期。成熟叶中的转录水平高于茎尖，折叠叶和幼叶中的转录水平也高于茎尖。草莓果实和叶片中均检测到NAD-SDH活性，且果实中的活性与其它果实相当。根据野草莓S6 PDH基因设计特异引物，进行RT-PCR扩增，结果表明，在草莓叶片和果实中均能扩增出一条清晰的单一条带。序列分析表明，该片段与其它植物的S6 PDH基因序列具有较高的同源性，表明S6 PDH基因存在于草莓基因组中，其mRNA在叶片和果实中表达。然而，无论是蛋白质或其酶活性检测叶片或果实。
The sorbitol metabolic pathway in strawberry (Fragaria X ananassa Duch. var.'Nyoho') is unclear, despite strawberry belonging to the family Rosaceae. Therefore, the presence or absence of genes for NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH) and sorbitol-6-phosphate dehydrogenase (S6PDH) in strawberry, and their levels of expression related to growth were investigated. A full-length cDNA encoding the NAD-SDH gene in strawberry (FaSDH) was obtained using rapid amplification of cDNA ends (RACE). The full-length clone consisted of 1,399 bp and contained a 1,083 bp open reading frame encoding 361. amino acids with a calculated molecular mass of 38.8 kDa, and a predicted isoelectric point (pI) of 6.08. This nucleotide sequence showed 79 - 85% similarity with the NAD-SDH genes of other plants. Its amino acid sequence contained both zinc- and NAD-binding sites, confirming that this sequence belonged to the NAD-SDH gene family. Southern blotting suggested that the genomic copy number of FaSDH was less than in apple. The expression of FaSDH transcripts in strawberry fruit was higher at the young and mature stages than at other stages. The transcript level in mature leaves was higher than in the shoot tip, and in folded and young leaves. NAD-SDH activity was detected in strawberry fruits and leaves and the activity in fruit was comparable to that in other fruits. Reverse transcriptase (RT)-PCR using specific primers designed based on the S6PDH gene of Fragaria vesca, showed a clear single band in leaves and fruits of strawberry. Sequence analysis showed this RT-PCR fragment, named FaS6PDH, to have high homology to the S6PDH gene sequence of other plants, suggesting that the S6PDH gene exists in the strawberry genome and that its mRNA is expressed in leaves and fruits. However, neither the protein nor its enzyme activity were detected in leaves or fruit.