Reference gene selection for transcriptional profiling by RT-qPCR in the 28-spotted larger potato ladybird

Henosepilachna vigintioctomaculata is one of the most serious defoliates attacking potatoes. However, studies on functional genes have greatly been limited due to the insufficiency of effective and stable endogenous references to normalize RT-qPCR data. In this report, nine housekeeping genes (RPL4, RPL6, RPL13, RPL32, RPS18, ACT, EF1α, GAPDH and α-TUB) involved in different biological processes were selected. Their expression levels under diverse experimental conditions including developmental stages, tissues, temperatures and host plants were determined using RT-qPCR technology. The tested candidate genes were comprehensively ranked based on five alternative stability analysis methods (Ct value, geNorm, NormFinder, BestKeeper and ReFinder). The results revealed that the optimal internal reference genes varied under different experimental conditions. Any gene pair among the five candidates (RPL4, RPL13, RPL32, RPS18 and EF1α) was a suitable reference gene set under different temperatures and on different host plants. A combination of RPL6 and RPL13 was recommended as the best reference gene set across different developmental stages. A pair of RPS18 and EF1α was ranked as the optimal reference gene combination within different tissues. The most suitable reference genes were RPS18 and RPL13 under four different experimental conditions. Our findings not only establish an accurate and reliable normalization of RT-qPCR data, but also lay a solid foundation for further functional gene researches in H. vigintioctomaculata.     

Bt毒素诱导下小菜蛾实时定量PCR 内参基因的筛选

【目的】筛选出Bt毒素诱导后的小菜蛾Plutella xylostella (L.)的实时定量PCR最适内参基因。【方法】选取核糖体18S rRNA (18S rRNA)、 肌动蛋白（ACTB）、 延伸因子（EF1）、3-磷酸甘油醛脱氢酶（GAPDH）、 核糖体蛋白L32 (RPL32)、 核糖体蛋白S13 （RPS13）、 核糖体蛋白S20 （RPS20）和β-微管蛋白(TUB)基因作为候选内参基因, 以geNorm、 Normfinder和BestKeeper软件分析这8个基因在Bt毒素诱导后的小菜蛾不同品系中肠组织中的表达稳定性。并应用筛选出来的内参基因分析小菜蛾氨肽酶2（aminopeptidase N2, APN2）基因的表达水平。【结果】geNorm软件以RPS13和EF1为最稳定内参基因, NormFinder和BestKeeper软件均以RPS13和RPL32为最稳定基因。使用3种不同内参基因分析Bt毒素诱导后的小菜蛾Bt抗性和敏感品系中ANP2表达水平时, 新的内参基因EF1和传统内参基因RPL32表现了良好的稳定性, 二者作为标准化因子, ANP2表达量结果基本一致, 而使用18S rRNA作为内参基因, 却导致部分表达量分析结果有所误差。【结论】筛选出PRS13,RPL32和EF1可以作为小菜蛾某些试验条件下的内参基因, 对小菜蛾基因表达研究奠定了一定基础, 也对其他昆虫内参基因的筛选具有参考价值。     

Validation of housekeeping genes for gene expression studies in an ice alga <Emphasis Type="Italic">Chlamydomonas</Emphasis> during freezing acclimation

Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.     

牧草盲蝽实时定量PCR内参基因的筛选

【目的】筛选出适合牧草盲蝽Lygus pratensis在不同条件下稳定表达的内参基因。【方法】选取编码牧草盲蝽α-微管蛋白、β-微管蛋白、肌动蛋白、延伸因子、琥珀酸脱氢酶复合体A、泛素、转录起始因子TFIID亚基、谷胱甘肽S-转移酶、核糖体蛋白L32及TATA盒结合蛋白的10条基因为候选内参基因,采用qRT-PCR技术测定其在牧草盲蝽不同性别成虫、成虫不同组织、不同龄期、对功夫菊酯不同抗性成虫、不同温度及不同杀虫剂分别处理后的成虫共6类样品中的表达量;采用BestKeeper, geNorm, NormFinder及RefFinder 4种计算程序对各候选基因的表达稳定性进行评价。【结果】依据获得的有效qRT-PCR数据前提,利用不同计算方法综合分析得出：在牧草盲蝽成虫不同组织中和对功夫菊酯不同抗性成虫中最稳定表达的内参基因均为RPL32;不同温度处理、不同性别成虫中、不同杀虫剂处理和不同龄期牧草盲蝽中最稳定表达的内参基因分别为UBQ, SDHA, β-tubulin和TAF。通过geNorm软件判断配对变异数确定的内参基因最佳数目,结合RefFinder对基因表达稳定性综合排序的结果可得：牧草盲蝽不同成虫组织中、不同性别成虫中、不同龄期、对功夫菊酯不同抗性成虫中、不同温度及杀虫剂分别处理的成虫等各组的最优内参基因组合分别为RPL32+TAF, SDHA+GST, TAF+GST+UBQ, RPL32+GST, UBQ+ACT+β-tubulin和β-tubulin+TAF+RPL32。【结论】本研究获得的牧草盲蝽在不同条件下表达最稳定及最适内参基因组合,都可用于后续的基因定量表达研究。     

Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca)

Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 μg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.     

Evaluating a set of reference genes for expression normalization in multiple tissues and skeletal muscle at different development stages in pigs using quantitative real-time polymerase chain reaction

Gene expression analysis requires the use of reference genes consistently expressed under various conditions. In many cases, however, the commonly used reference genes are not uniformly expressed independently of tissues or environmental conditions. To provide a set of reliable reference genes in pigs, we used quantitative polymerase chain reaction to examine expression of six common reference genes (GAPDH, ACTB, H3F3A, HPRT1, RPL32, and RPS18) in adult tissues and prenatal skeletal muscles at 33, 65, and 90 days postcopulation from Tongcheng (obese-type) and Landrace (lean-type) pigs. The expression stability of these reference genes was evaluated by NormFinder, BestKeeper, and geNorm methods. Our data suggest that the reference genes were expressed variably in different tissues, developmental stages and breeds. RPS18, PRL32, and H3F3A could be used as internal controls to normalize gene expression in pig tissues and developmental skeletal muscle. The combination of internal control genes was necessary for accurate expression normalization. During skeletal muscle development, H3F3A and RPS18 would be the most appropriate combination to normalize gene expression in Tongcheng pigs, whereas the combination of PRL32 and RPS18 would be more suitable in Landrace pigs. In different tissues, the expression of PRL32 and RPS18 was the most consistent, and the combination of three genes (RPL32, RPS18, and H3F3A) is the most suitable for accurate normalization.     

Loss of the rpl32 gene from the chloroplast genome and subsequent acquisition of a preexisting transit peptide within the nuclear gene in Populus

Gene transfer events from organelle genomes (mitochondria and chloroplasts in plants) to the nuclear genome are important processes in the evolution of the eukaryotic cell. It is highly likely that the gene transfer event is still an ongoing process in higher plant mitochondria and chloroplasts. The number and order of genes encoded in the chloroplast genome of higher plants are highly conserved. Recently, several exceptional cases of gene loss from the chloroplast genome have been discovered as the number of complete chloroplast genome sequences has increased. The Populus chloroplast genome has lost the rpl32 gene, while the corresponding the chloroplast rpl32 (cp rpl32) gene has been identified in the nuclear genome. Nuclear genes transferred from the chloroplast genome need to gain a sequence that encodes a transit peptide. Here, we revealed that the nuclear cp rpl32 gene has acquired the exon sequence, which is highly homologous to a transit peptide derived from the chloroplast Cu-Zn superoxide dismutase (cp sod-1) gene. The cp rpl32 gene has acquired the sequence that encodes not only for the transit peptide, but also for the conserved N-terminal portion of the mature SOD protein from the cp sod-1 gene, suggesting the occurrence of DNA sequence duplication. Unlike cp SOD-1, cp RPL32 did not show biased localization in the chloroplasts. This difference may be caused by mutations accumulated in the sequence of the SOD domain on the cp rpl32 gene. We provide new insight into the fate of the inherent sequence derived from a transit peptide.     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Evaluation of reference genes for quantitative real-time PCR in the brain,pituitary, and gonads of songbirds

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.     

The First Intron of Human c-fms Proto-oncogene Contains a Processed Pseudogene (RPL7P) for Ribosomal Protein L7

During sequence analysis of the first intron of the human c-fms oncogene, we identified an open reading frame encoding the ribosomal protein L7 (RPL7). The presence of this sequence within intron 1 of the c-_fms gene was confirmed by Southern blot hybridization and by sequence analysis of two independent cosmid clones (cos2-e and cos1-22) that span the human genomic c-_fms locus. The RPL7 sequence was detected in a region of sequence overlapped by the cos2-e and cos1-22 cosmid clones but oriented opposite to the c-fms gene. We demonstrated that the sequence is identical to the full-length RPL7 cDNA sequence, but lacks any recognizable introns, has a 30-bp poly(A) tail, and is bracketed by two perfect direct repeats of 14 bp. We also showed that despite the fact that the 5′ flanking region of the RPL7 sequence contains a potential TATA box upstream of an intact open reading frame, this pseudogene (RPL7P) is not actively transcribed.