Standard addition quantitative real-time PCR (SAQPCR): a novel approach for determination of transgene copy number avoiding PCR efficiency estimation.

Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for analysis was established. Samples from six individual T(0) tomato (Solanum lycopersicum) plants were analyzed by SAQPCR, and the results confirmed by Southern blot analysis. The approach produced accurate results and required only small amounts of plant tissue. It can be generally applied to analysis of different plants and transgenes. In addition, it can also be applied to zygosity analysis

Phenolic Composition and Antioxidant Properties of Different Peach [Prunus persica (L.) Batsch] Cultivars in China

China is an important centre of diversity for Prunus persica. In the present study, 17 Chinese peach cultivars were evaluated for phenolic content and antioxidant activity. Neochlorogenic acid (NCHA), chlorogenic acid (CHA), procyanidin B1 (B1), catechin (CAT), cyanidin-3-O-glucoside (C3G), quercetin-3-O-galactoside (Q3GAL), quercetin-3-O-glucoside (Q3GLU), quercetin-3-O-rutinoside (Q3R), and kaempferol-3-O-rutinoside (K3R) were identified and quantified. CHA and CAT were the predominant components in both the peel and pulp of this fruit. In general, peel extracts showed higher antioxidant activities than the pulp counterparts, consistent with the observed higher phenolic content. The melting peach cultivar “Xinyu” showed the highest antioxidant potency composite (APC) index. The principal component analysis (PCA) of peel phenolics showed a clear distinction between the melting peach and nectarine. Overall, peach cultivars rich in hydroxycinnamates and flavan-3-ols showed relatively higher antioxidant activities and might be excellent sources of phytochemicals and natural antioxidants

Transcriptome analysis provides insights into the regulation of metabolic processes during postharvest cold storage of loquat (Eriobotrya japonica) fruit

Loquat fruit: Investigating spoilage during cold storage Next-generation sequencing of loquat fruit stored in different ways reveals the genes and proteins that help reduce fruit spoil. Loquat fruit become rigid and woody during cold storage, thanks to the build-up of lignin polymers in the fruit flesh. Lignification can be prevented with exposure to heat or preconditioning to cool temperatures before cold storage. Zhangjin Fei at Cornell University, New York, US, Qingbiao Wu at Zhejiang University in Hangzhou, China, and co-workers used comparative RNA sequencing to explore the molecular mechanisms inherent in lignification. The team placed three loquat groups in post-harvest cold storage for 8 days: one pre-treated with heat, one conditioned at cool temperatures, and one with no pre-treatment. They identified key genetic differences between the groups and several protein families that may regulate lignification

EjODO1, a MYB transcription factor, regulating lignin biosynthesis in developing loquat (Eriobotrya japonica) fruit

Lignin is important for plant secondary cell wall formation and participates in resistance to various biotic and abiotic stresses. Loquat undergoes lignification not only in vegetative tissues but also in flesh of postharvest fruit, which adversely affects consumer acceptance. Thus, researches on lignin biosynthesis and regulation are important to understand loquat fruit lignification. In loquat, a gene encoding an enzyme in the lignin biosynthesis pathway, Ej4CL1, was reported to be regulated by transcription factors, including EjMYB1, EjMYB2, EjMYB8 and EjAP2-1, knowledge of this process is still limited. With the aim of identifying novel transcriptional factors controlling lignin biosynthesis in loquat, the promoter of Ej4CL1 was utilized to screen a cDNA library by yeast one hybrid assay. A novel R2R3 MYB, named EjODO1, was identified. Real-time PCR analyses indicated that EjODO1 is highly expressed in lignified stems and roots. During fruit development, expression of EjODO1 decreased along with the reduction of lignin content and became undetectable in mature ripe fruit. Thus, EjODO1 is likely to be involved in lignification of vegetative organs and early fruit development but not in mature fruit or postharvest lignification. Dual-luciferase assay indicated that EjODO1 could trans-activate promoters of lignin biosynthesis genes, such as EjPAL1, Ej4CL1 and Ej4CL5 and transient overexpression of EjODO1 triggered lignin biosynthesis. These results indicate a role for EjODO1 in regulating lignin biosynthesis in loquat which is different from the previously characterized transcription factors

Determination of transgene copy number of six transgenic tomato plants by standard addition qPCR (SAQPCR).

<p>A quantified amount of 10.20 ng of tomato genomic DNA, which contains around 10,000 molecules of <i>ELIP</i>, was included in each PCR reaction.</p>a<p>Refers to the copy number per diploid genome.</p

Effects of pHE DNA addition on PCR efficiency.

a<p>A two-copy transgenic T₀ tomato plant was used;</p>b<p>The concentration was 10.20 ng µl⁻¹, containing 10,000 <i>ELIP</i> and 10,000 <i>HPT</i> molecules µl⁻¹;</p>c<p>The concentration was 0.051 pg µl⁻¹, containing 10,000 <i>ELIP</i> and 10,000 <i>HPT</i> molecules µl⁻¹.</p

Construction of recombinant plasmid pHE.

<p>pELIP: plasmid harboring tomato <i>ELIP</i> gene; pHPT: plasmid harboring <i>Escherichia coli HPT</i> gene; pHE: plasmid harboring both tomato <i>ELIP</i> and <i>E. coli HPT</i> gene.</p

Southern blot analysis of six transgenic tomato T₀ lines.

<p>A) <i>Hind</i> III digestion; B) <i>BamH</i> I digestion. M, λDNA/<i>Hind</i> III marker; L1-L6, transgenic tomato T₀ lines, were the same as those appeared in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053489#pone-0053489-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053489#pone-0053489-t003" target="_blank">Table 3</a>; P, positive control (plasmid); WT, negative control (wild type).</p