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Assessment of the multi-gene silencing effect on insect growth and development
In the previous experiments, I have observed that the RNAi effect was greatly varied with different target genes; therefore, simultaneous targeting of multiple genes could be one of futuristic strategy for enhancing of RNAi effect in the management of insect pests. But, in insects very few reports were available regarding this strategy (Miller et al., 2012; Wang et al., 2013) while, in case of H. armigera, this information is lacking. Therefore, I want to know what could be the possible consequences if I silence more than one gene simultaneously. With this objective, I set an experiment for a comparative study using RNAi to target single and multiple genes simultaneously. In this regard, I silenced both CYP6AE14 and CYP6B7 genes simultaneously and separately to compare their effects on larval growth and development using Ex-vivo insect bioassay.
Ex-vivo insect Bioassay using detached cotton leaf disc
To assess the effect of multi-gene silencing on insect growth and survival, insect bioassay was performed with a detached cotton leaf. Fresh cotton leaves were cut into 19.16 cm2 diameter circle. Leaf discs were washed with nuclease-free water, then placed on petri dish containing pre-mounted (1%) agar. Edges of leaf discs were covered with a thin layer of molten agar in order to absorb phenolic compounds present at cutting edges and to maintain moisture in the leaf discs. Then, the surface tension of leaf discs were reduced by treating with 5 µl of 0.01% Titron-X. To silence CYP6AE14 and CYP6B7 genes individually and simultaneously, I have used previously synthesized concatemeric dsRNA of CYP6AE14 and CYP6B7. In vitro transcribed CYP6AE14 and CYP6B7 dsRNA at 1 µg/cm2 of leaf disc was applied individually with a pre-wetted soft paint brush and allowed for drying. Meantime, CYP6AE14 and CYP6B7 dsRNAs were mixed to give 1 µg/cm2 of each, then this mixed dsRNAs were applied on the surface of the leaf disc. For non-target control, dreb1A dsRNA at 1 µg/cm2 concentration was applied on the leaf disc and allowed for drying. On each leaf disc, five-day old single larva was released and there were 20 replicates per each treatment and controls. On every alternate day, leaf discs were changed and fresh dsRNA was applied in order to ensure continuous availability of dsRNA on leaf disc and this was continued until pupation. Effect of dsRNA on target genes expression was assessed on day six of the experiment by extracting the total RNA from two larvae of each treatment and controls. Total RNA isolation, cDNA synthesis and RT-qPCR assays were performed as described earlier (Chapter 3, section). To assess the effect of above dsRNAs on larval growth and development, larval weight was recorded on day three and day five of the dsRNA treatment. From day one onwards larval mortality rates were recorded until pupation.
In this study, I have experienced difficulties in simultaneous silencing of multiple genes using RNAi. Therefore, to explore possible causes for less efficient RNAi in multi-gene silencing, I have investigated two main factors such as dsRNA uptake and dilution effect and limitation of core RNAi components to process multiple dsRNAs.
Regarding first factor, I hypothesized that mixing of different dsRNAs may lead to decreased quantities of each dsRNA uptake. To test this hypothesis, I diluted cognate (CYP6AE14) dsRNA with non-target dreb1A dsRNA in 1:1 and 1:2 ratios. Insect bioassay was performed using this diluted dsRNAs similarly as described earlier in this section. Effect of above diluted dsRNAs on target gene (CYP6AE14) expression was assessed on sixth day of the experiment by extracting the total RNA from two larvae of each treatment and control. Effect of diluted dsRNA on larval growth and development was assessed by recording the larval weight on day three and day five of the dsRNA treatment. From day one onwards larval mortality rates were recorded until pupation.
Second, having a mixture of dsRNA can compete for core RNAi machinery thus core components (Dicer-2 and Ago2) of RNAi are limiting in processing of multiple dsRNAs that result in reduced RNAi effect. In this regard, I have assessed the expression levels of Dicer-2 and Ago2 in both CYP6AE14 and CYP6B7 dsRNAs administered larval samples and also in CYP6AE14 dsRNA diluted with dsRNA of non-target dreb1A treated larval samples. For this analysis, I have used the total RNA extracted from above experiments. RNA isolation, DNAse I treatment, cDNA synthesis and RT-qPCR assays were performed as described earlier (Chapter 3, section).
3.5 Gene pyramiding to silence multiple genes
To overcome the dsRNA dilution effect due to multiple genes dsRNAs mixing, I envisage that genes pyramiding in a single construct that ought to overcome the above hurdles.
Pyramiding of CYP6AE14 and CYP6B7 genes
To make gene pyramiding, earlier cloned concatemers of CYP6AE14 and CYP6B7 were used for gene pyramiding. Previously, both CYP6AE14 and CYP6B7 have been cloned in HindIII site of pBluescript II KS(+) plasmid. The pBluescript II KS(+) plasmid was digested using restriction enzymes to release both inserts and then the inserts were sequentially cloned into pTZ57R/T vector to make genes pyramiding.
First, CYP6B7 (concatemer) was released from the pBluescript plasmid by restriction digestion with BamHI and SalI. Similarly, pTZ57R/T plasmid was digested with BamHI and SalI. Then both digested plasmids were resolved on 1.5% agarose gel along with uncut plasmid. Once after resolving, the linearized pTZ57R/T vector and released CYP6B7 concatemer insert were excised from the gel and eluted using NucleoSpin® Extract II kit (Macherey-Nagel). Then, both vector and insert were ligated using T4 DNA ligase and transformed into ultra-competent E. coli DH5α cells. The presence of the CYP6B7 insert in pTZ57R/T vector was confirmed by PCR amplification using M13 universal primers. The confirmed colonies were inoculated into 5ml of LB broth having 100 µg/ ml of ampicillin antibiotic and incubated at 37°C for overnight with 220 rpm shaking. Restriction digestion, gel elution, ligation and transformation, PCR amplification using M13 primers and plasmid isolation were performed similarly as described earlier (Chapter 3, section).
After confirmation of the presence of the CYP6B7 insert in pTZ57R/T vector, this was digested with EcoRI and KpnI to clone CYP6AE14. Similarly, CYP6AE14 was released from the pBluescript plasmid by restriction digestion with EcoRI and KpnI. Then both digested vector and insert were resolved on 1.5% agarose gel and linearized vector and inserts were gel eluted. Then vector and insert were ligated and transformed similarly as described earlier. The presence of CYP6AE14 (concatemer) insert in pTZ57R/T vector was confirmed by PCR amplification using M13 universal primers. The confirmed colonies were inoculated into 5ml of LB broth having 100 µg/ ml of ampicillin antibiotic and grown overnight at 37°C for overnight with 220 rpm shaking. Plasmids were extracted from the overnight grown culture and resolved on 1.2% agarose gel along with control plasmid (without insert).
Finally, restriction digestion was performed to confirm the presence of both inserts in pTZ57R/T vector. Restriction digestion was performed with following enzymes to yield following products. Restriction enzymes, EcoRI and KpnI were used to release CYP6AE14; BamHI and SalI were used to release CYP6B7 and EcoRI and PstI were used to release both inserts. The restriction digestion was performed similarly as described earlier (Chapter 3, section). The digested products were resolved on 1.5% agarose gel to confirm the release of inserts.