we construced a vector with two promoters are constructed in one plasmid but their directions are reversed, double-stranded RNA will be produced during the transcription period.
2. hpRNA construct
We found that the hairpin RNA actually possess higher stability and higher efficiency in gene silencing in living organisms including higher plants like Arabidopsis thaliana. In general, the hairpin RNA harbor similar effect with double-stranded RNA, what is different is that there is a loop inserted between the sense arm and anti-sense arm.
① Hairpin RNA with normal loop
② Hairpin RNA with intron loop
As the intron sequence will be sliced during the pre-mRNA processing and the stability is much higher than the previous two methods according to a relevant paper, it will be a better option for us to silence the target sequene.
In Our experiment，we selected 120 base-pair-long noncoding sequence from Chloramphenicol as normal loop.
Strain select
We use the M-JM109LacY (dicer deficit type, can’t degrade dsRNA), as a manufactory to produce dsRNA.
Reference
[1] Total silencing by intronspliced hairpin RNAs.
[2] Young Sik Lee and Richard W. Carthew, Making a better RNAi vector for Drosophila: use of intron spacers
[3] Molecular cloning（the 4th edition
Target organism
Plant
At first, we aimed at setaria virids, a kind of maliganant weed resistant to pesticide glyphosate.
But we met an huge obstacle : select vital gene fragments with high specificity in purpose of decreasing RNAi off-taget rate. But the truth is existing genome data of weeds is deficient right now and there are rare gene notes for sequencing species. Also,many malignant weeds are homologous from the victim crops they do harm to, the gene discrepancies is too less.
We felt hopeless after doing a lot of work then we paid attention to an easier one—Arabidopsis , which has clear genetic background. It is an ideal candidate for tandem RNAi verification.
Pest
In consideration，we chose aphids as experiment materials. First, It is a kind of worldwide pests, creating great impact in agriculture fields. Second, studies towards on aphids are relatively mature. And the materials is easy to get form nature.
Target gene
Arabidopsis
We find a target gene—trxz, whose deficiency will result in complete albino phenotype , it’s very easy to observe.
TRX z is a branch of plastidial thioredoxin (TRX), lying between x- and y-type TRXs thus was named TRX z, which is a likely candidate for mediating thiol-modifications in chloroplasts.
Recently, several independent studies could demonstrate that TRX z constitutes a subunit of the plastid-encoded RNA polymerase (PEP). Meanwhile the generation of the full PEP complement is an essential early step during chloroplast bio-genesis and its disturbance cannot be reversed if a certain time point in the program has been passed.
The Arabidopsis knockout mutant lines of TRX z exhibit a complete albino phenotype and was inhibited in chloroplast development.
What’s more, we noticed that a analysis of promoter-GUS indicated that TRX z is expressed in young tissues with decreasing expression during leaf maturation.
So our verification need to be at the young stage of the otherwise the phenotype maybe not so obvious.
Aphid
Theoretically, any genes necessary for insects survival, growth, development, reproduction or feeding can be used as RNAi targets(Vilcinskas 2011). The ecdysone receptor (ECR) is a such kind of gene, which could up-regulate the vital genes expression through the interaction with ultra-stable protein (USP) under the existence of ecdysteroid hormone.
Therefore, Ecr gene silencing can effectively increasing the insects mortality and reducing the reproduction rate. Studies have shown when fed aphids with 7.5 ng / ul dsRNA Ecr, the mortality rate up to 70% after 8 days.
According to the literature, the similarity of Ecr gene sequence between pea aphid and grain aphid was 98.4% and with more than 90% similarity in other kind of aphids, so we chose ECR as the target sequence of RNAi. So Ecr is an Ideal RNAi target site to kill aphids with a broad spectrum destruction effect.
