[Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii]

Yao Xue Xue Bao. 2014 Jan;49(1):115-23.
[Article in 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Cucurbitaceae / chemistry*
  • Cucurbitaceae / genetics
  • DNA, Complementary / genetics
  • Flowers / chemistry
  • Flowers / genetics
  • Fruit / chemistry
  • Fruit / genetics
  • Molecular Conformation
  • Open Reading Frames
  • Oxidoreductases* / genetics
  • Oxidoreductases* / metabolism
  • Phylogeny
  • Plant Roots / chemistry
  • Plant Roots / genetics
  • Plant Stems / chemistry
  • Plant Stems / genetics
  • Plants, Medicinal / chemistry
  • Plants, Medicinal / genetics
  • Protein Structure, Secondary
  • RNA, Plant*

Substances

  • DNA, Complementary
  • RNA, Plant
  • Oxidoreductases
  • glutathione dehydrogenase (ascorbate)