rna interference in the asian longhorned beetle:identification of key rnai genes and reference genes for rt-qpcr

Asian long horn beetle (ALB)
Light Shoulder star disease is a serious forest invasion pest in the United States, Canada and Europe.
RNA interference (RNAi)
This technology is being developed as a new pest management approach.
Here, we identified Dicer, Argonaute, and double-stranded RNA-
Binding protein (dsRBP)
And proteins involved in dsRNA transport and systemic rna interference.
We also compare the expression of six potential reference genes that can be used to normalize gene expression and select gapdh and rpl32 as the most reliable genes between different tissues and stages
Double notestranded RNA (dsRNA)
Targeted gene encoding of apoptosis inhibitors (IAP)
Causing this gene to be significantly knocked down, resulting in 90% of larvae and 100% of adult deaths.
There is no mortality in larvae and adults of dsRNA targeting genes injected with green fluorescent protein (
As negative control)was observed.
These data show that there is a functional rna interference mechanism in the BSA and potential rna interference
A method of controlling this insect can be developed.
Asian long horn beetle (ALB), (
Department of moths)
It is a boring insect native to China and South Korea, and has invaded several countries in the United States, Canada and Europe.
The beetle has been recorded in more than 100 different trees and is considered an important invasive species worldwide.
For example, if the population found is not controlled by the current eradication plan, the potential economic impact of 2016 people in the United States will reach $889 billion.
Early detection, isolation, and eradication efforts remain the main strategies for the current targeting of albumin.
However, the cost and environmental issues of removing or chemical treatment of large host trees in or near newly infected areas require the development of new methods to effectively control such invasive pests.
The recently completed genome and transcription group sequencing provides valuable tools for the development of new molecular methods to control albumin.
RNA interference (RNAi)
Is the mechanism of gene silence triggered by double-stranded RNA (dsRNA).
By targeting and silencing the basic genes involved in insect survival, rna interference is being developed as a new pest management approach.
Small interfering rna (
Small interfering RNA, miRNA (microRNA)and piRNA (PIWI-associated RNA)
Pathways have been identified in insects.
These pathways are different in many ways, such as RNA precursor molecules and specific enzymes involved in their processing.
Although piRNAs is independent of Dicer (
RNase III enzyme)
Activity, mirna needs Drosha (RNase III-family enzyme)
Handling stem-
The cyclic dsRNA structure of its precursor molecule.
Dicer 1 and 2 enzymes also require the processing of long rna into mirna or siRNAs, respectively.
Treated small rna is loaded onto the silent complex with the help of dsRNA bindingproteins (dsRBP).
In insects, these dsRBPs are unique to each Dicer; Loquacious (Loqs)
For Dicer 1, r2 2 of Dicer 2 and Pasha of Drosha. Argonautes (Ago)
It is the main protein of the silent complex consisting of two domains, the PAZ domain involved in dsRNA binding and the PIWI domain responsible for RNase activity.
In all three pathways, specific Ago mediated target recognition and silence of miRNA (Ago-1), siRNA (Ago-2), and piRNA (
Eggplant, OBB and ago3).
The presence or absence of core rna interference genes does not seem to be the only reason for the different efficiency of rna interference observed in different insects.
Several other factors, such as the digestion of dsRNA by dsRNase enzymes present in the intestinal cavity and blood lymph, the transport of dsRNA to within and within cells and their systemic transmission, even the level of expression of rna interference genes has been shown to affect the efficiency of rna interference between the insects under test.
In order to determine the knock-down efficiency of dsRNA, reverse transcription quantitative PCR (RT-qPCR)is often used. Although RT-
QPCR is a robust method that combines high specificity, accuracy, and sensitivity detection, which is influenced by several factors, such as the stability of reference genes, the number and purity of RNA used, and primer efficiency.
A key step may compensate for most of the variability at RNA and PCR levels, that is, to select a reference gene for constituent expression in samples and treatments in the study.
Recent studies have shown that there are no universal reference genes suitable for normalizing gene expression in different organisms and conditions.
To facilitate the validation of reference genes, four models based on different statistical algorithms, Genorm, NormFinder, BestKeeper, and delta-
Ct is integrated into a free network tool, RefFinder.
RefFinder provides an overall final ranking based on the calculation of the geometric mean of each program to estimate the stability of candidate reference genes.
Current studies are aimed at identifying and verifying rna interference mechanisms, as well as identifying reliable reference genes that are standardized in gene expression studies.
According to the homology with the sequence of rna interference genes identified in other insects, the analysis was performed to identify the genes involved in the rna interference pathway.
Then verify the reliable reference gene and (
Apoptosis inhibitor
Gene expression.
Rna interference biological assay was carried out using dsRNA-targeted genes for HB larvae and adults.
Targeted injection of DsRNA (dsIAP)
After entering the larvae and adults, the target gene was knocked down and both larvae and adults died.
This is the first study to provide evidence for interfering reactions and to validate reference genes for expression analysis of albumin.
These data contribute to future research on gene expression and the development of rna interference technology.
Methods based on this and other invasive Woodboring insects.