Team:FAFU-CHINA/Project

侧边栏



Background


No one knows since when did humans begin to attempt to domesticate honeybees, just like no one knows exactly how important these little creatures can be to human civilization and world environment.
Honeybees may be most famous for the stored honey they serve up, but there are a few more useful things they do as well. They produce beeswax, which gives us the manufactures of candles and many other sorts of products. They produce royal jelly, which is extremely famous for preserving people’s health. Moreover, they pollen plants, which is the most fundamental ecological process and makes everything in nature possible.
Today, in China, apiculture is a vital economic generator for the country, producing average 245,000 metric tons and estimating at 238,000 metric tons of consumption in honey per year. In America, it creates 210,000 direct jobs for people, and it is a crucial element of U.S agriculture, which relies on apiculture for pollination. it is impossible to define the economic value of apiculture. According to a data released by Times, almost 1/3 of food of the world is dependent on honeybee’s pollination. If all the honeybees die out, thousands of plants will follow, which could leads to millions of people starving in the following years.
Einstein once said that’if all the bees die out, humans will follow a few years later.’ It is unsettling to inform you that, honeybees have started to disappear. In recent years，beekeepers have witnessed an annual 30% losses in their colonies. For the Europe alone, bees are greatly declining, from 5 million hives in 1988 to 2.5 million today. Since 2006, a phenomenon called Colony Collapse Disorder has spread to every corner of the world. We don’t exactly know what is causing it. The only thing we know is that it is pretty serious, and measures must be taken immediately.


Sacbrood Virus
Sacbrood Virus has been a scourge to apiculture over the world for almost half of a century. Sacbrood Virus is a typical RNA virus. It causes failure of larval pupation, and results in larval death. With SBV accumulating beneath larval skin, Infected larva changes in color from pearly white to pale yellow, forming a brown gondola-shaped scale after death. Different Sacbrood virus samples originate from various geographic regions. In China, it is classified as Chinese Sacbrood Virus (CSBV). According to a research from Jilin University, in some provinces of China, 60 percent of bee tribes carries CSBV, and the death rate of larvae is almost 100 percent during outbreaks.
CSBV is one of the main causes of the significant decrease of Chinese honeybee populations in the past 40 years. So far, no effective control method exists for this bee disease, besides the prevention strategy, the only one beekeepers adopting currently. FAFU-China team aims to investigate the possibility of controlling this disease through silence of CSBV’s RdRp (RNA-dependent RNA polymerase) gene by using RNA-interference technology.


RNA-interference
The RNA-interference naturally exists in many eukaryotes, including honeybees. It plays an crucial role in defending somatic cells against viruses. The biological process is known for it’s ability to inhibit gene expression by causing the destruction or silence of specific messenger-RNA molecule.
Double-stranded ribonucleic acid (dsRNA) molecules is essential to RNA-interference. In the very beginning, the Dicer enzyme initiates the biological process by cleaving dsRNA into small interfering RNA (siRNA). Next, the small interfering RNAs are soon separated into two single strands. The positive-strand which is also known as guide-RNA is then integrated into an active RNA-induced silencing complex, RISC. RISC can bind to it’s target mRNA by base-pairing and cleave it, preventing the mRNA from producing a protein.


Theory
Replication of CSBV
As a typical RNA virus, CSBV’s replication process is initiated once the messenger RNAs of CSBV, which is CSBV’s original genetic information, are injected into somatic cells. However, a essential complex for the replication of genetic information of CSBV is lacked in the somatic cells of honeybees, RNA dependent RNA polymerase, RdRp in short. RdRp can be synthesized directly by ribosome of honeybees using mRNA of RdRp as temperate. With the existence of RdRp, the replication of genetic information of CSBV can be completed and CSBV will soon spread out infect the whole organism.
This year，FAFU-China aims to investigate the possibility of controlling this disease by inhibiting the expression of RdRp gene through RNA-interference.


CSBV.Silencer 1.0
We designed a engineered bacteria capable of producing double-stranded RNA of RNA dependent RNA polymerase （RdRp）to initiate the process of RNA-interference. We inserted the RdRp gene into plasmid L4440, and inserted T7 promoters in the upstreams of both strands of the plasmid. two contemporary messenger-RNAs of RdRp can be synthesized simultaneously after T7 polymerases detect the promoters in both strands and start the transcription from 5 prime to 3 prime.
The contemporary messenger RNAs will then bind together into double stranded RNA by base-pairing. Long dsRNA of RdRp is then cleaved by Dicer enzyme and short small interfering RNA is formed. small interfering RNA will soon separate into two single strands. One is called passenger RNA, which is soon degraded. And the other strand called guide RNA will then integrated into an RNA Induced Silencing Complex, RISC in short. RISC then binds to the specific sequence of mRNA of RdRp and destruct it, thereby preventing it from being used as translation template to produce RdRp.
We transferred plasmid L4440 into E.coli so that dsRNA of RdRp can be largely produced. Then we mixed our product, CSBV.Silencer, into forage of honeybees, and feed it to infected hives. After the infected honeybees take in CSBV.Silencer, the replication process of CSBV in will be destructed in the somatic cells of honeybees, for the essential RdRp complex can not be formed.


CSBV.Silencer 2.0
On the basis of CSBV.Silencer 1.0, we decided to improve our product,out of two concerns.
In the field of food industry, safety concern is always a sensitive problem. E.coli is always taken as potential pathogen by consumers unconsciously，and because of that, we decided to replace E.coli by Yeast which originally exists in honey.
If we want to transfer CSBV.Silencer into honeybees, we need to consider the influence imposed by immune system and digest system. We need to find a way to prevent our product from being degraded by immune cells and digestive solution. The need to enable the engineered bacteria getting through the digest system without being harmed also requires us to replace the E.coli by using eukaryote as carrier.
However, if we want to use eukaryotic system to produce the dsRdRp, there is still a problem needed to be solved. In eukaryotic system, there is no T7 polymerase, which causes failure in transcription of L4440, leading dsRdRp unable to be synthesized. To solve this, we have another plasmid capable of producing T7 polymerase, PyES2.
After galactose induces Gal1 promoter, activating RNA polymerase to transcript the gene of T7 RNA polymerase, mRNA of T7 RNA polymerase can be expressed and T7 polymerase is produced. the produced T7 polymerase can go back to the T7 promoter in the upstream, and continue making more T7 polymerases. The circulation of making T7 polymerase ensures the abundance of T7 polymerases, which actually accelerates the process of producing dsRdRp.
Our next product, CSBV·Silencer 2.0, will be the engineered yeast containing plasmid PyES2 and plasmid L4440. However, in this stage, we have only come up with the idea and are still trying to recombine the plasmid PyES2. As long as we have the recombined plasmid PyES2 ready, we will then transfer PyES2 and L4440 into yeast, and have our product applied to practice.
Meanwhile,we noticed that IPTG could induce the expression of dsRdRp in HT115, and galactose could improve the efficiency of GAL1 in eukaryotic system


Easier to spread
Eusocial insects like ants, honeybees, bumble, termites have the habit of cooperative care of young. The larvae in the family are unable to forage and feed themselves, and because of that, the food delivered by workers are essential. this habit enables CSBV.Silencer flow through the whole insect family rapidly.Through the mechanism, CSBV.Silencer can be diffused easily and the effect can be detected rapidly.
Similarly, the mechanism can be adopted in the prevention of other virus disease of eusocial insects or biological control. We are planning to develop a medicine of termite control through silence of certain gene. We predict that the medicine can be delivered with food throughout the whole colony, and achieve the purpose of pest control.