We design a locust bio-pesticide by applying with RNAi technology targeting to different stage of locust in order to inhibit the growing rate of locust. We employ the synthetic biology method to Saccharomyce cerevisiae and Metarhizium acridum which can affect locust by interfering critical genes in locust metabolism and growth.
RNAi techonolgyRNAi is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. During the process, double strand RNA would be cleaved into siRNA by DICER. siRNA will assemble with multiple proteins and form RNA-induced silencing complex (RISC). RNAi can specifically identify target gene and suppress its expression. This technology is precise, efficient and stable. Before we designed dsRNA sequence, we first blast the target gene to human, crops and other species to prevent the affect of RNAi to them. We chose sequence which has low-identity to those species so that our dsRNA wouldn’t affect to them. Before we started our experiment, we searched and read huge amount of papers about RNAi design on locust. Finally, we chose CHS (chitin synthase), TSP (trypsin-like serine protease) and ATPase (vacuolar ATPase subunit H) gene as our target gene. By using specific program, we attain our dsRNA sequence and then we decided to measure its effect by using transferred yeast and metarhizium.
Phase of Locust Target gene Mortality Ⅲ instar LmHR3 100% Ⅳ instar Lm-TSP 100% LmHR3 100% Ⅴ instar LmSPC1 86.7% Lm-TSP 46% LmHR3 100% Lm-Vhasfd 96.7% LmE75 100% Adult LmSPC1 80%Before we started our experiment, we searched and read huge amount of papers about RNAi design on locust. Finally, we chose CHS (chitin synthase), TSP (trypsin-like serine protease) and ATPase (vacuolar ATPase subunit H) gene as our target gene.By using specific program, we attain our dsRNA sequence and then we decided to measure its effect by using transferred yeast and metarhizium.
CarH Light-Control systemLight control system has many advantages comparing to chemical induction.1.fast and efficient rate of inducing2.easy to manipulate by computer3.low toxicity to cellTo select an appropriate light control system for our project, there are three factors we should consider:1.showing high induction2.no extra chemical reagent3.light has no influence to strainConsequently, we choose CarH light control system.
Light control system Light organism Induction fold othre Phy/PIF3interactio11 R/FR Yeast 1000 Exogenously added PCB CRY2/CIB1 with TALE DNA operon12 blue mammal Up to 30 TALE DNA operon should be constructed CRY2/CIB1 and CRISPR/Cas9system10 blue mammal 500 UVR8/COP1interaction5 UVB mammal 800 CarH system2 Green Mammal and plant 350 EL2226 blue E.coli and yeast ＜5 OptoINVRT and OptoEXP6 blue yeast ＜5 Yeast optogenetic control system3 blue yeast 3CarH protein is from Thermus thermophilus. This protein has cobalamine (Vitamin B12), which has a photosensitive Co-C bond, as a prosthetic group. In the dark, CarH is a tetramer that binds strongly to CarO DNA operon and represses downstream protein translation2. When excited by green light, the photosensitive Co-C bond breaks, this disrupts the tetramer structure, and then gene expression can start. From the research we know CarH maximum absorption spectrum is 525nm, we decided to apply green light control system to manipulate yeast dsRNA synthesis. When the yeast is incubated in dark condition, CarH will bind to dsRNA synthesis part which will block the dsRNA synthesis. When exposing to 525nm light environment, CarH tetramer photolysis which resulted in dsRNA synthesis expression.
dsRNA measurementsiRNA is unstable in cell and could only function several days. We decided to employ dsRNA in our experiment. dsRNA is composed of an antisense and sense RNA sequence of targeted gene and a single strand RNA loop structure. dsRNA would be combined to plasmid and deliver into host cell, converted into siRNA and finally interfered gene expression. To measure the concentration of dsRNA, we decided to use pepper RNA. Pepper RNA is a fluorescent RNA, an aptamer that binds and activate fluorescent dyes. Pepper RNA can bind to (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC) and emit stable fluorescence8. We then measure the concentration of dsRNA by fluorescence strength and compare its value to standard curve.
Mechanism of Metarhizium insecticidesMetarhizium has a high rate of pathogenic to 3rd instar locust and 4th, about mortality rate from 70%~80%. On the contrast, adult locust has a related low mortality of 60%13.The process of Metarhizium virulence can be divided into three steps:1.spore attachment：penetration through the body wall；2.colonization in vivo： evade the immune system of insect hosts；3.killing the host：produce compounds such as beauvericin and destruxin and re-sporulation.In order to enhance Metarhizium virulence ability to locust, we employ RNAi technics, which shows that high infection to Metarhizium when transformed dsRNA sequence targeted to locust gene.
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- RNAi techonolgy
- CarH Light-Control system
- dsRNA measurement