1. The effectiveness of our engineered system
Our engineered system worked, which is demonstrated by the facts that phyllotreta striolata were successfully killed by application of shRNAs, the products of our engineered system.
Adult P. striolata were obtained from Shenzhen University field station, and kept in glass bottles. The tissue culture seedlings of Chinese cabbage, Brassica chinensis leaves were placed into the above bottles (Fig. 5.1).
Fig. 5.1 Adult P. striolata and Brassica chinensis leaves were placed into the glass bottles for RNAi efficiency test. Each siRNA/shRNA sample has two repeats.
The solutions of shRNA (10 ng/mL) were separately sprayed onto the leaves of Chinese cabbage every third day, each solution has two repeats. Around twenty adult beetles of P. striolata were tested per siRNA/shRNA sample.
The survival rates of adult beetles, were recorded at different days after shRNA treatment (Table 5-1).
Fig. 0 Death number of the beetles after different days of treatment were recorded
Table 5.1 RNAi efficiencies of siRNA/shRNA
Different days of shRNA treatment were displayed in X axis, the survival rates of the beetles were displayed in Y axis. Results show that, except for the negative control: water and ALR-shRNA-1 sample, all the other samples tested could trigger RNAi mechanism, which was demonstrated by the significant survival rate decrease after treatment (Fig.5-2).
Fig. 5.2 The survival rate of Phyllotreta striolata at different days after siRNA/ shRNA treatment.
The topical spray application of shRNA to a target insect is an effective, simple, safe and a relatively inexpensive technology for insect control.
2. The effectuveness of Phyllotreta striolata attractants
We intend to reduce the application cost by using Phyllotreta striolata attractants. The idea is to add attractants to shRNA solution to trap Phyllotreta striolata. In this way we do not have to spray our product on all the vegetables in the fields. Instead, we could use attactant included shRNA solution, or use vegetables that have been sprayed with attactant included shRNA solution to trap Phyllotreta striolata. and kill the beetles there.
We chose two potential attractants to test, which are lemon yellow and sucrose. Both are safe and relatively cheap. Chinese flowering cabbages were placed in nylon net cages, one set of the cabbages were sprayed with attractants, another set of cabbages were sprayed with water were and placed in the same cage as control (Fig. 1).
Fig. 1 Chinese flowering cabbages that were sprayed with water or lemon yellow were placed in nylon net cages
Each attractant is tested in a separate cage. Around 30 adult beetles were released into each cage, let the beetles stayed in the cages for 2 days (Fig. 2).
Fig. 2 Lemon yellow and lemon yellow plus sucrose were tested in a separate cage
After 2 days, we found more beetles were attracted to the cabbages that were sprayed with lemon yellow compared to the cabbages that were sprayed with water, which was demonstrated by the ratio of biting holes on the leaves (Fig.3). There were 125 holes on the leaves that were sprayed with lemon yellow, while 60 holes on the leaves that were sprayed with water. The ratio of holes is 2.08 (Table 1).
Fig. 3 Lemon yellow has the function of attracting Phyllotreta striolata.
When we added sucrose to the lemon yellow, trapping effect was even better, more beetles were attracted by the attractants, which again was indicated by the increase of the ratio of the biting holes (Fig.4). there were 227 holes on the leaves that were sprayed with lemon yellow plus sucrose, while 53 holes on the leaves that were sprayed with water. The ratio of holes is 4.28 point zero eight (Table 1)
Fig. 4 Lemon yellow plus sucrose has better effect in attracting Phyllotreta striolata.
Table 1 Comparison of the trapping effects of lemon yellow and lemon yellow plus sucrose.
Based on the data from our experiments, solution of sucrose plus lemon yellow have the best trapping effect on Phyllotreta striolata.
3. Comparison of the cost and safety for using chemical pesticide and shRNA
For large scale production of our product by in vitro transcription system, the main cost includes: primers, NTPs and T7 polymerase. By calculating, the production cost for shRNA is $1.5/mg.
Basis for the calculation:
1) Primers for shRNA template synthesis
We need to order 2 primers, P1 38 (17+21) bases, and Primer 2, 29 (8+21) bases, the total primer bases is 67. The price for primer synthesis is one dollar for 14 bases (100 ul, 100pM), with 1ul primer, we get 20ul template, 1ul template we get 10ug shRNA. Cost for one primer ordering is ~ $5 (67/14), for one ordering, we get 20 mg shRNA (100 x 20 x 10 ug = 20000 ug). So, to produce 1 mg shRNA, the cost for primers is $ 0.25 ($5/20mg)
2) NTPs for template synthesis and in vitro transcription
The price for NTP is 20 Dollar for 1ml, 40mM. For 20 dollars NTP, we get around 20 mg NTP (0.0001L x 40mM x 500 mg/mM = 20 mg), only a very small portion of NTP is used for template production. So, to produce 1 mg shRNA, the cost for NTP is $ 1 ($20/20mg)
3) T7 polymerase
We will use E coli expression system to produce T7 polymerase, the cost is very low, no more than 0.25 dollars cost for making 1 mg of shRNA. Thus, the total cost for producing 1 mg of shRNA is $1.5 (0.25 + 1 + 0.25)
Table 2 Comparison of the cost and safety for using chemical pesticide and shRNA