Zhaolei Liu

Reference Gene Selection for Quantitative Real-Time PCR in Chrysanthemum Subjected to Biotic and Abiotic Stress

Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1α (elongation factor 1α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.

Heterologous Expression of a Nelumbo nucifera Phytochelatin Synthase Gene Enhances Cadmium Tolerance in Arabidopsis thaliana

Phytochelatin synthase (PCS) is a key enzyme involved in the synthesis of phytochelatins, which are thought to play important roles in heavy metal detoxification. The sacred lotus (Nelumbo nucifera), one of the most popular ornamental species, has been shown to be a potential phytoremediator of heavy metal polluted water. However, the phytochelatin synthase gene in N. nucifera has not been identified yet. Here, we report the isolation and function characterization of a N. nucifera homologue of phytochelatin synthase. The sequence obtained shares a high degree of similarity with PCSs from other plant species and was named as Nelumbo nucifera phytochelatin synthase1 (NnPCS1). By using quantitative RT-PCR, we found that the expression of NnPCS1 in leaves of N. nucifera was dramatically increased in response to Cadmium (Cd) treatment. We further showed that, when exposed to Cd stress, Arabidopsis transgenic plants heterologous expressing NnPCS1 accumulated more Cd when compared with wild type. These results suggest that NnPCS1 involved in the response of N. nucifera to Cd stress and may represent a useful target gene for the phytoremediation of Cd-polluted water.

Candidate reference genes for gene expression studies in water lily

The selection of an appropriate reference gene(s) is a prerequisite for the proper interpretation of quantitative Real-Time polymerase chain reaction data. We report the evaluation of eight candidate reference genes across various tissues and treatments in the water lily by the two software packages geNorm and NormFinder. Across all samples, clathrin adaptor complexes medium subunit (AP47) and actin 11 (ACT11) emerged as the most suitable reference genes. Across different tissues, ACT11 and elongation factor 1-alpha (EF1alpha) exhibited a stable expression pattern. ACT11 and AP47 also stably expressed in roots subjected to various treatments, but in the leaves of the same plants the most stably expressed genes were ubiquitin-conjugating enzyme 16 (UBC16) and ACT11.

The effect of exogenously applied nitric oxide on photosynthesis and antioxidant activity in heat stressed chrysanthemum

The effect of exogenously applied nitric oxide on the heat tolerance of Chrysanthemum morifolium was investigated by applying the NO donor sodium nitroprusside (SNP). We found that the SNP partially alleviated the heat stress by slowing down the reduction of photosynthetic pigment content and net photosynthetic rate. SNP treatment also lowered the increase in the non-photochemical quenching of fluorescence and malondialdehyde content and maintained higher activities of superoxide dismutase, peroxidase, catalase and ascorbate peroxidase.

Identification and expression of an APETALA2-like gene from Nelumbo nucifera.

Arabidopsis transcription factor APETALA2 (AP2) controls multiple aspects of plant growth and development, including seed development, stem cell maintenance, and specification of floral organ identity. Based on sequence similar of Arabidopsis AP2 and its homologues genes from other plant species, degenerate RT-PCR and rapid amplification of cDNA ends assay were used to clone AP2 genes from lotus (Nelumbo nucifera). A 2,048-bp cDNA fragment was obtained, which contains a 1,536-bp open reading frame encoding a protein of 511 amino acids. The protein contains two AP2 domains that are conserved in AP2 proteins from other plant species, thus was named as N. nucifera APETALA2 (NnAP2). Quantitative RT-PCR revealed that NnAP2 gene was expressed in flowers, roots, leaves, and stems of N. nucifera, with flowers which have the highest transcript levels. Further analysis showed that in all five lotus cultivars examined, including “Zhongguogudailian,” “Yaoniangyujiao,” “Jinxia,” “Hongtailian,” and “Yiliangqianban,” petals always have the highest expression levels when compared with the other four flower organs, though the number of petals in these cultivars ranged from simple to thousands. However, NnAP2 expression level in four nonsimple petal flower cultivars was higher than that in the simple petal flower cultivar Zhongguogudailian, indicating that NnAP2 may play a role in specification of petal identity during the evolutionary process of the ancient species N. nucifera.

PCR primers targeting cis-acting promoter elements in plants

The cis-acting elements present in gene promoters are important for promoter function. Thermal Asymmetric Interlaced PCR (TAIL-PCR) provides a simple means for isolating promoter sequences, but the necessary primers can be troublesome to design. Here, we describe an approach, which targets cis-acting elements for TAIL-PCR. The method combines the advantages of TAIL-PCR and SiteFinding-PCR. The new method proved successful for isolating a number of plant promoter sequences.

Iris lactea var. chinensis (Fisch.) cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana

IlCDT1, a cysteine-rich protein, was isolated from Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis). Its transcription was up-regulated by the exogenous application of Cd. The truncated IlCDT1 (25-54) containing 14 Cys residues confers Cd tolerance to yeast as the intact IlCDT1, indicating that Cys residues are required for Cd tolerance presumably by chelating Cd. When the gene was constitutively expressed in A. thaliana, root length of transgenic lines was longer than that of wild-type under 100 μM or 200 μM Cd stress. However, Cd absorption in wild-type was more than in two trangenic lines under 100 μM Cd exposure. IlCDT1 may directly bind Cd, through chelating Cd and avoiding the Cd uptake into the cells. Together, IlCDT1 may be a promising gene for the Cd tolerance improvement. SUMMARY Cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.