Team:BNU-CHINA/Introduction

Team:BNU-CHINA - 2015.igem.org

Project Safety

Background

Nematode has been up to over 5000 of 200 genus[1]. Among them, over 100 species of nematodes are damaging the agricultural, forestal and economic crops of China who widely parasitize the roots, stems, leaves, flowers, buds and seeds of manifold plants, harming the development of agriculture and forestry seriously.

A newest research by USA indicates that the plant-parasitic nematodes damage leads to about 8 billion dollars’ losses to their croppers, which takes 12% of the whole value of the crop output. Meanwhile, according to incomplete statistics, the losses caused by paratrophy nematodes can reach 100 billion every year worldwide[2]. China’s researchers have done some relevant surveys as well, and according to incomplete statistics, 17 provinces such as Anhui, Hainan, Hubei, Gansu, Zhejiang and Fujian have reported root knot nematode disease once, among which the morbidity of some severe regions in Shandong province can up to 2/3[3]. Therefore we can note that plant-parasitic nematodes have brought out severe lost to global agriculture and forestry already.

Loss the Fig
Figure 1.hahahahah

The size of plant-parasitic nematodes is usually too small for naked eyes to catch sight of. In addition, the present survey about the agricultural damage of nematodes are so limited that people often fail to notice and prevent it in time. The plant-parasitic nematodes are furnished with scalpellus functioning to stab into the plant cells to obtain nutrients after seeking out the host with the amphids on their forehead, which as a result, could damage the host as well as bring in pathogenic fungus and therefore causing complex harm to them. Up to now, the pathogenesis of plant-parasitic nematodes can be concluded in theory as follow:

  1. The nematodes give raise to severe mechanical injury to the host plants when feeding on them with the scalpellus.
  2. Other pathogen accompanied with the nematodes induce plant disease.
  3. Some nematode secretion is toxic to the plants which as a consequence damages them.

In most canses, the mechanical damage by nematodes to host is a drop in the bucket, therefore the latter two theories are relatively common[4].

The majority of harmful nematodes in agriculture and forestry belong to Tylenchida while a little belongs to Dorylaimida. Meloisogyne spp. Heterodera spp. Aphelenchoides composticola, Ditylenchus dipsaci are nematodes that gravely damage plants around the world. Among them, Meloisogyne spp. mainly destroy the roots of plants by forming root-knot to hinder their development, which in turns, causes the roots rot. According to statistics, a slight Meloisogyne spp. disease can lead to a 20%~30% output reduction, and 50%~70% even a total loss if it’s severe. Heterodera spp. also target the roots of plants primarily, weakening and decomposing the roots. Ditylenchus dipsaci damage the underground part such as the tuber, tuberous root, bulb of the plants which results in their rot and malformation while some local part over ground would also be influenced to turn to malformation, ,Aphelenchoides composticola such as Bursaphelenchus xylophilus and Aphelenchoides besseyi generally aim at the overground part of plants who infect the leaves and causing lesion and leaf tip drying, and more than that, infect the trunks to kill the whole plant rapidly.

Hence one can see that plant-parasitic nematode cause serious losses to a variety of agricultural crops worldwide. Since the traditional methods based on the use of nematocides and antihelminthic drugs are associated with major environmental and health concerns, the development of biocontrol agents to control nematodes is of major importance[5]. In this case, our project is designed to control the losses caused by nematodes effectively and timely with biocontrol agents. And after the bait-kill system successfully established, we are looking forward to further expansion to other agricultural pests for the sake of achieving our ultimate goal – establishing a database containing attractant base and toxic protein base in allusion to all sorts of agricultural pests.

As to our project this year, we designed two models separately to attract and poison nematodes. We made E.coli synthesize limonene to bait plant-parasitic nematodes and then kill them with two kinds of toxic proteins Bace-16 and Mpl afterwards. Furthermore, we introduced a photo-regulated bi-direction transcription system to the whole system, by which means we would be able to regulate the .expression of attractant and toxic proteins through the control of light. That’s to say, the E.coli would express attractant to bait nematodes when exposed to light whereas express toxic proteins to kill them as a result of the promoter’s reverse in the dark.


Module 1: Bait

Limonene is a monoterpenoid, which is a raw material widely used in medicine and chemicals. There are two conformations in the nature. One is D configuration, the other is L configuration. Limonene can attract root knot nematodes of citrus or other plant parasitic nematodes[6]. Our design based on the following two limonene synthase sequences which are named Citrus unshiu CitMTSE1 for d-limonene synthase and Mentha spicata 4S-limonene synthase. At the same time, we also designed another synthase sequence which can synthesize the isoprene GPP, the precusor substance of synthesizing the limonene. We transformed these two parts into E.coli to realize the expression of limonene in prokaryotic cell.


Module 2：Killer

Bace16 and MpL, these two protein have a good toxicity toward the nematodes. Bace16 is a kind of serine proteases from a bacterial parasite of the nematode named Bacillus sp. B16. After the expression of Bace16 has completed, the protein can be secreted out of the cell. This proteases could decompose the intestine protein of the nematodes thereby the nematodes will be killed[7]. MpL is a novel lectin isolated from parasol mushroom. It is a kind of intracellular expression protein. MPL could bind with glycan of the nematodes specifically. So it can stop the growth of the nematodes from L1 phase to adults[8].


Module3：Suicide

The system of attracting and killing above would carry out a series of functions. But at the same time, our recombination E.coli could suicide after a certain time. The suicide system is controlled by a regulatory switch. With the continual proliferation of the cells, the thallus can accumulate an organic named AHL. AHL can move across the cytomembrane freely. When it accumulates to a certain degree, it will combine with the intracellular proteins to induce the activation of the promoter of the suicide gene. Later the suicide system starts and the E.coli will die.

In order to attract and kill the nematodes, we also built a bidirectional photoinduced system. In this system, the expression of the attractive organics and the toxin are under different conditions. We have already known the promoter PompR is regulated by red light. When there is no light, the promoter combines with the ompR protein to function. We added the int gene behind the promoter, so the promoter would control the expression of the int protein. Under the effect of the int protein, the reversed promoter can reverse to activate the expression of the other side. In a word, we linked the genes of attractive organics and the toxin on each side of the Pcon respectively. And we controlled the different module by light.


Modeling

Our modeling can be divided into four parts. Firstly, in order to enable our project to be applied in life, we designed a device placed in soil, which can attract and kill nematodes by modified engineering bacteria inside. Secondly, assuming there is a farmland, we took advantage of nematodes’ movement analogue simulation to find the best position where the device should be placed. Thirdly, according to this concentration, we obtained the best size of the device by calculating. At last, we established a database to enlarge applied range of our method to kill other pests. We would appreciate that new synthetic biological and environment-friendly methods can be shared and improved with the science researchers all over the world.


Human Practice

During our project, we constantly did the communication and practice. We improved our plan by doing the human practice. At the same time, we also helped people understand the meaning of the synthetic biology and showed them the charm of the synthetic biology through our project. What’s more, we did a survey to publicise the damage of the plant parasitic nematodes and told the details to the farmers about the solutions. We interviewed the related experts, dropped in the countryside, analyzed the soil sample, communicated with other teams, did assistance and so on. The goal we would like to achieve is to help more people with the development of ourselves.


  1. 陈立杰, 段玉玺. 植物寄生线虫虫种资源的分类鉴定[C]. 2006年全国寄生虫种质资源共享与利用学术交流会2006:29-33.
  2. Chitwood, David J. Research on plant‐parasitic nematode biology conducted by the United States Department of Agriculture–Agricultural Research Service[J]. Pest Management Science, 2003, 59(6-7):748-753.
  3. 付令国. 植物病原线虫的发生与防治技术初探[J]. 农业与技术 2015, Vol.35, No.09
  4. 董娜, 张路平, 康云晖. 植物线虫寄生策略及致病机理[J]. 河北师范大学学报：自然科学版, 2003, 27:298-301. DOI:doi:10.3969/j.issn.1000-5854.2003.03.024.
  5. Duncon LW (1991) Current options for nematode management. Annu Rev Phytopathol 29:469–490CrossRef
  6. Ali J G, Alborn H T, Stelinski L L. Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes[J]. Journal of Ecology, 2011, 99(1):26–35.
  7. Niu Qiuhong ,, Huang Xiaowei ,, Tian Baoyu ,, et al. Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor[J]. Applied Microbiology & Biotechnology, 2006, 69(6):722-730.
  8. 05urga S, Pohleven J, Renko M, et al. A novel β-trefoil lectin from the parasol mushroom (Macrolepiota02procera) is nematotoxic.[J]. Febs Journal, 2014, 281(15):3489–3506.