&lt;p&gt;Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells&lt;/p&gt;

et al. 2021

Preprint

Rapidly growing interest in nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into plant cells. However, little is known about how the size and shape of nanoparticles influences transport in plants and use of their cargoes, limiting development and deployment of nanotechnology in plant systems. Here, we employ non-biolistically delivered DNA-modified gold nanoparticles (AuNP) spanning various sizes (5 – 20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana (Nb) leaves. Generally, smaller AuNPs demonstrate more rapid, higher, and longer-lasting levels of association with plant cell walls compared to larger AuNPs. We observe internalization of rod-shaped but not spherical AuNPs into plant cells, yet surprisingly, 10 nm spherical AuNP functionalized with small-interfering RNA (siRNA) are most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery, and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious bio-cargo delivery for future developments in plant nanobiotechnology.

Section: Methodsmentioning

confidence: 99%

Nanoparticle Cellular Internalization is Not Required for RNA Delivery to Mature Plant Leaves

Zhang

Goh

et al. 2021

Preprint

“…Some genes necessary for maintaining basic cell functions have been found in plants, called housekeeping genes, whose expression levels are less affected by environmental conditions and developmental stages [5,6]. The housekeeping genes or other genes used as reference genes cannot be stably expressed in all transcriptional conditions, and the expression stability of genes is usually biased for speci c conditions [7][8][9]. Therefore, it is necessary to select the reference genes for speci c conditions to ensure the accuracy in qRT-PCR experiments.…”

Section: Introductionmentioning

confidence: 99%

“…In order to detect whether there is any difference in the expression stability of actin family members when C. sinensis is exposed to various light conditions, six pairs of distinctive primers (corresponding to CsACT1, CsACT2, CsACT(3-4), CsACT(5-6), CsACT (7)(8), and CsACT(9-10) genes from C. sinensis genome) were investigated in this study. In addition, six common reference genes of C. sinensis from other families, including CsUBC1, CsGAPDH, CsTBP, CseIF-4α, clathrin adaptor complex subunit (CsCLATHRIN1), and tap42-interacting protein of 41 kDa (CsTIP41) were evaluated together [28,30].…”

Section: Introductionmentioning

confidence: 99%

Evaluation and Selection of Suitable qRT-PCR Reference Genes for Light Responses in Tea Plant (Camellia Sinensis)

Tang

Sun

et al. 2020

Preprint

Background: Tea plant (Camellia sinensis) is an important woody economic crop used for processing leaf-type beverages. Tea has been proved to be beneficial to human health because it is rich in tea polyphenols and other active ingredients. Numerous studies have shown that light is a necessary environmental condition to control the growth and metabolism of C. sinensis. Gene expression experiments are always performed to explore the transcriptional regulation mechanism of plants widely based on the technique of quantitative real time polymerase chain reaction (qRT-PCR). The screening and application of reference genes are necessary for the normalization of gene expression under specific conditions. However, the reference genes for systematic analysis of light-induced transcription mechanisms are still not available in C. sinensis.Results: In this research, we identified actin family genes that are always used as reference genes with high frequency and without distinction for various expression experiments in C. sinensis. Six pairs of distinctive primers (corresponding to CsACT1, CsACT2, CsACT(3-4), CsACT(5-6), CsACT(7-8), and CsACT(9-10) genes) were designed to evaluate their expression stability in response to light quality (LQ), light intensity (LI), and photoperiod (PD). Simultaneously, six other family members (CsUBC1, CsCLATHRIN1, CsGAPDH, CsTBP, CsTIP41, and CseIF-4α) of C. sinensis commonly used as reference genes were also investigated. The stability rankings of gene expression were calculated by the statistical algorithms of geNorm, BestKeeper, NormFinder, and RefFinder softwares. Conclusions: CsACT(5-6), CsTIP41, and CsACT(3-4) were the most stable genes for light quality (LQ), light intensity (LI), and photoperiod (PD) treatments, respectively. This study provides a basis for the selection of reference genes for future research on the transcription mechanism of light response in C. sinensis. Moreover, the analysis of actin family members of C. sinensis will help to understand the individual transcription mechanism of housekeeping family.

mentioning

confidence: 99%

mentioning

confidence: 99%

Evaluation and selection of suitable qRT-PCR reference genes for light responses in tea plant (Camellia sinensis)

Liu

Tang

et al. 2021

Scientia Horticulturae

Background: Tea plant (Camellia sinensis) is an important woody economic crop used for processing leaf-type beverages. Tea has been proved to be bene cial to human health because it is rich in tea polyphenols and other active ingredients. Numerous studies have shown that light is a necessary environmental condition to control the growth and metabolism of C. sinensis. Gene expression experiments are always performed to explore the transcriptional regulation mechanism of plants widely based on the technique of quantitative real time polymerase chain reaction (qRT-PCR). The screening and application of reference genes are necessary for the normalization of gene expression under speci c conditions. However, the reference genes for systematic analysis of light-induced transcription mechanisms are still not available in C. sinensis.Results: In this research, we identi ed actin family genes that are always used as reference genes with high frequency and without distinction for various expression experiments in C. sinensis. Six pairs of distinctive primers (corresponding to CsACT1, CsACT2, CsACT(3-4), CsACT(5-6), CsACT(7-8), and CsACT(9-10) genes) were designed to evaluate their expression stability in response to light quality (LQ), light intensity (LI), and photoperiod (PD). Simultaneously, six other family members (CsUBC1, CsCLATHRIN1, CsGAPDH, CsTBP, CsTIP41, and CseIF-4α) of C. sinensis commonly used as reference genes were also investigated. The stability rankings of gene expression were calculated by the statistical algorithms of geNorm, BestKeeper, NormFinder, and RefFinder softwares.Conclusions: CsACT(5-6), CsTIP41, and CsACT(3-4) were the most stable genes for light quality (LQ), light intensity (LI), and photoperiod (PD) treatments, respectively. This study provides a basis for the selection of reference genes for future research on the transcription mechanism of light response in C. sinensis. Moreover, the analysis of actin family members of C. sinensis will help to understand the individual transcription mechanism of housekeeping family. BackgroundQuantitative real time polymerase chain reaction (qRT-PCR) is a universal technique to analyze gene expression because of its high sensitivity, good repeatability, and fast speed [1,2]. In the process of using qRT-PCR to analyze gene expression, the purity and concentration of the sample cannot be absolutely guaranteed that may cause experimental errors [2,3]. In order to make the results more accurate, it is necessary to use the reference gene as a reference to standardize the expression level of the target gene and correct the discrepancy of transcription e ciency and cDNA usage [2,4]. Stable expression is the basis for selection of reference genes because of the selection of unstable reference genes will lead to test deviation. Some genes necessary for maintaining basic cell functions have been found in plants, called housekeeping genes, whose expression levels are less affected by environmental conditions and developmental stages [5,6]. The housekeeping genes o...