Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii.
- Author:
Rong-Chang WEI
;
Huan ZHAO
;
Xiao-Jun MA
;
Ke MI
;
Chang-Ming MO
;
Li-Mei PAN
;
Long-Hua BAI
;
Qi TANG
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Base Sequence;
Cloning, Molecular;
Cucurbitaceae;
chemistry;
genetics;
DNA, Complementary;
genetics;
Flowers;
chemistry;
genetics;
Fruit;
chemistry;
genetics;
Molecular Conformation;
Open Reading Frames;
Oxidoreductases;
genetics;
metabolism;
Phylogeny;
Plant Roots;
chemistry;
genetics;
Plant Stems;
chemistry;
genetics;
Plants, Medicinal;
chemistry;
genetics;
Protein Structure, Secondary;
RNA, Plant
- From:
Acta Pharmaceutica Sinica
2014;49(1):115-123
- CountryChina
- Language:Chinese
-
Abstract:
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.
