[Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii].
Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD260/280, OD260/230, RNA integrity (RIN) and yield of the total RNA with this method were 2.01, 2.02, 9.50 and 260 mirog.g-1, respectively. The open reading frame (ORF) of dehydroascorbate reductase (DHAR), named as SgDHAR, was cloned by rapid amplification of cDNA ends (RACE) and RT-PCR method from S. grosvenorii. The GenBank accession number for this gene is KC907731. The SgDHAR gene contains a full-length cDNA of 1,252 bp including ORF of 819 bp and encodes a predicted protein of 272 amino acids. The molecular mass is 30.217 7 kD and the isoelectric point is 8.76. Homology comparison showed that it shared 87% nucleotide sequence homology with Cucumis sativus. Expression patterns using qRT-PCR analysis showed that SgDHAR was mainly expressed in fruit and stem, followed by flower, and was lowest in root, while the expression level was 6.83 times in triploid. T than that in diploid. Therefore, SgDHAR gene may be involved in abortion of triploid seedless S. grosvenorii.
Rong-Chang
RC
Wei
Huan
H
Zhao
Xiao-Jun
XJ
Ma
Ke
K
Mi
Chang-Ming
CM
Mo
Li-Mei
LM
Pan
Long-Hua
LH
Bai
Qi
Q
Tang
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D019662:Cucurbitaceae / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D035264:Flowers / Q000737:chemistry / Q000235:genetics; D005638:Fruit / Q000737:chemistry / Q000235:genetics; D008968:Molecular Conformation; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D018517:Plant Roots / Q000737:chemistry / Q000235:genetics; D018547:Plant Stems / Q000737:chemistry / Q000235:genetics; D010946:Plants, Medicinal / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D018749:RNA, Plant
D018076:DNA, Complementary; D018749:RNA, Plant; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate)