RESUMO
Siraitia grosvenorii is a traditional Chinese medicine also as edible food. This study selected six candidate reference genes by real-time quantitative PCR, the expression stability of the candidate reference genes in the different samples was analyzed by using the software and methods of geNorm, NormFinder, BestKeeper, Delta CT method and RefFinder, reference genes for S. grosvenorii were selected for the first time. The results showed that 18SrRNA expressed most stable in all samples, was the best reference gene in the genetic analysis. The study has a guiding role for the analysis of gene expression using qRT-PCR methods, providing a suitable reference genes to ensure the results in the study on differential expressed gene in synthesis and biological pathways, also other genes of S. grosvenorii.
Assuntos
Cucurbitaceae , Genética , RNA Ribossômico 18S , Genética , Reação em Cadeia da Polimerase em Tempo Real , Métodos , Padrões de ReferênciaRESUMO
To evaluate the genetic background of triploid female and diploid male strains of Siraitia grosvenorii and provide the biological reference for good varieties breeding of seedless S. grosvenorii. Inter simple sequence repeats (ISSR) marker was developed to analyze the genetic background among 28 samples of S. grosvenorii, and cluster analysis and double principal coordinate analysis were revealed by the NTSYS-pc software and GenAIEx software, respectively. Out of 100 ISSR primers selected, 13 primers were used for amplification and a total of 131 unambiguous bands were obtained, among which 99 (PPB = 75.57%) were polymorphic. The results of cluster analysis and double principal coordinate analysis showed that there was a certain rich of genetic background in triploid female and diploid male strains of S. grosvenorii. But the genetic similarity coefficients of majority were bigger and the genetic distance was closer. The complexity of the genetic background in triploid female and diploid male strains of S. grosvenorii is lower and germplasm innovation strategies should be carried out to enrich the genetic background of the parents of seedless S. grosvenorii.
RESUMO
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.