Team:ROADS SY/Poster

Poster: ROADS_SY



RAAR: Rice Arsenate Accumulation Reduction

Authors and Affiliations

  1. Cao, Shihong from Northeast Yucai Foreign Language School
  2. Hong, Suning from Northeast Yucai Foreign Language School
  3. Li, Mengzhe from Perkiomen School
  4. Li, Xinbo from Northeast Yucai Foreign Language School
  5. Sun, Qiancheng from Northeast Yucai Foreign Language School

Abstract

Thanks to hybridization and genetic modification, rice sustains a huge proportion of the world population. However, arsenate accumulation poses a severe threat to the rice-consuming population, who are generally unaware of the threat and unable to deal with it. We decided to lower the arsenate accumulation of rice by identifying and modifying the genetic sequence related to the arsenate level. Specially, we adopted both the conventional method and the bioinformatic method to identify the related genetic sequence. By doing so, we intended to promote the fusion of bioinformatics and bioengineering. The assumption is that Ospt4 is responsible for the arsenate accumulation of many crop species, including rice. We planned to knock out the genetic sequence and perform contrast experiments to examine whether Ospt4 is related to arsenate accumulation

Introduction

In this poster your will see:

Why we chose the topic of our project in the Inspiration, Problem, and Idea sections.

How we designed our experiments in the Engineering section and the three sections below it.

What our results will look like in the Results section.

Inspiration

Because of covid-19, all high school student in China stayed home and took online courses. Several of our team members, while staying home, gradually learned to cook. In China, the primary staple food is rice. While washing rice, Yudan Wang (one of our team leaders) wondered whether rice contains harmful chemicals that cannot be removed by flowing water. After looking up the internet for some information, she found out about the problem of arsenic accumulation within rice. She then shared her finding with the team and conducted a survey on people’s knowledge about arsenic pollution. To learn more about the survey, check out our human practice section!

Problem: Arsenic Accumulation

Arsenic (As), the 33rd element in the Periodic Table of Elements, is metalloid that has the similar properties as Phosphorous, but unlike P, As is very dangerous. Elemental arsenic and arsenic sulfate and trioxide compounds are classified as "toxic" and "dangerous for the environment" in the European Union under directive 67/548/EEC. The International Agency for Research on Cancer (IARC) recognizes arsenic and inorganic arsenic compounds as group 1 carcinogens. In addition, most of its compounds are potential poisons, for example, white arsenic (As trioxide). As mentioned above, As is extremely dangerous to the human body, so dangerous that the regular amount of As in a human body is 0. If As accumulates over dangerous levels in a human body, the human is exposed to diseases like skin cancer, cardiovascular diseases, diabetes and cognitive development problems.

Arsenic in Humans

The element Arsenic can be found in human bodies in 4 different ways.

  1. iAs: Short for As ions, pentavalent (Arsenate — AsV) or trivalent (Arsenite — AsIII). Both AsV and AsIII are toxic to humans but by different mechanism. AsV, a phosphate analogue, interferes with energy and phosphate metabolism, and AsIII interferes with nucleic acid synthesis.
  2. MMAs: Monomethylarsonous acid (MMA(III)), which target mitochondria in cells, cutting out the power supply for the cells, and monomethylarsonic acid (MMA(V)), along with dimethylarsinic acid (DMA) induce single-strand DNA breaks, suggesting that they can damage cells.
  3. DMA: dimethylarsinic acid
  4. TMA: trimethylarsinic acid, because TMA is very rarely found in human bodies, little research has been done on this The iAs’ in human bodies is converted to MMAs’ then DMAs in a process called arsenic methylation.

How Arsenic Gets to Humans

Unfortunately, As deposits have been found in almost every human. This deposit of As is usually introduced to the human body in two ways. a) Drinking water contaminated with iAs or b) Eating plants that have As deposits within them. Most of the plants that have As deposits are rice plants, and the plants have worried scientists for a long time, due to the health hazards within the deposits they can possess.

Idea: Biochem vs Bioinfo

Ospt4 & AsV

Plants including rice take up arsenate (AsV) by phosphate transporters. In plants, AsV can be reduced to AsIII inside plant cells, and AsIII is accumulated in the vacuoles. From precedent research, we have found that some of the genes of the OsPht1 family of genes are responsible for the AsV uptake. In addition, other groups that are researching the reason for the AsV uptake pointed out that OsTP4, another gene in the OsPht1 family is probably responsible for the AsV uptake.

Machine Learning & Genes

In late July, we were informed that the laboratory we had contacted could not open in August because the reopening of all Chinese universities was postponed. Unable to carry out the experiments, we wondered whether there was another approach to verify Ospt4 as the genetic sequence that could affect the arsenic level of rice. Through a conversation with a graduate who majored in bioinformatics, we learned that frontier researchers are now using machine learning to analyze the relationship between human genes and diseases. This idea inspired us. If we regarded the accumulation of arsenic as a plant disease, we could use the same method to explore the connection between Ospt4 and the arsenic accumulation within rice. Eventually, we developed a plan of dual verification.

Engineering

First, we will gather data from real rice samples and use our algorithm to determine whether Ospt4 controls the arsenate level with in rice. After confirming with the bioinformatic method that OsPT4 is the most important factor of AsV uptake, we will do experiments make sure that OsPT4 was the most important factor in AsV uptake, using the biochemical method. A process that could be simplified as Plant —> Measure —> Record —> Repeat.

Plant

To speed up the process, we will use hydroponic experiments to grow the rice.

First, we surface sterilize the rice seeds for 30 min with diluted Sodium Hypochlorite (NaClO), followed by thorough rinsing for 30 min with deionized water. Seeds were germinated in darkness at 25 °C for 3 days.

Then we transfer the germinated seeds to the hydroponic solution: 1.25 mM NH4NO3, 200 μM KH2PO4, 0.35 mM K2SO4, 1 mM CaCl2, 1 mM MgSO4, 20 μM Fe-EDTA, 20 μM H3BO3, 9 μM MnCl2, 0.32 μM CuSO4, 0.77 μM ZnSO4, and 0.39 μM Na2MoO4. This solution will give the rice plants the nutrients and acidic environment it needs to grow. The hydroponic experiments were conducted in a greenhouse with a 16-h-light (30 °C)/8-h-dark (22 °C) photoperiod and ∼70% relative humidity.

In our hydroponic solution mentioned above, we made some modifications to control the expression of OsPT4, 20 μM AsV with 0 or 300 μM KH2PO4 were used as P-deficient As treatment (−P +As) and P-sufficient As treatment (+P +As), respectively.

For the details about "Measure" and "Record," please visit the third minor section and the Results section respectively.

Human Practice: Survey

Q1: Do you pay special attention to chemical residues when washing rice?

Q2: How many times do you usually wash rice?

Q3: Do you know that rice contains arsenic?

Q4: Do you know the health risks of long-term exposure to inorganic arsenic?

Q5: What is your age?

Q6: What is your diploma?

Q7: what region of china do you live in now?

In the survey, we found that about 70% of people washed rice 2-3 times, indicating that people attach importance to food safety. However, we also found that people's understanding of inorganic arsenic is not comprehensive. 60% of people do not pay attention to chemical residues, and almost one fifth of the participants assumed that arsenic intake causes no long-term risk. What it more, since the average educational level of the participants are higher than that of the whole nation, the real situation is probably worse that reflected in the survey. Because there is no appropriate approach for people to remove arsenic from rice at home, genetic modification will be a thorough solution to the problem.

Algorithm
Measurement

P-Concentrations: ∼0.05 g of crushed dry sample was digested with H2SO4−H2O2 at 300 °C. After cooling, the digested samples were diluted to 100 mL in distilled water. P concentration was analyzed by the molybdenum blue method based on dry weight. The determination of phosphorus, arsenic, silicon and germanium are examples of the use of heteropoly-molybdenum blue in analytical chemistry. The following example describes the determination of phosphorus. A sample containing the phosphate is mixed with an acid solution of MoVI, to produce [PMo12O3]-40. This anion is then reduced to form the blue colored ion, [PMo12O7]-40.The amount of the blue colored ion produced is proportional to the amount of phosphate present and the absorption can be measured using a colorimeter to determine the amount of phosphorus (See diagram above). [3] As-Concentration: To determine AsV amounts, 17-d old rice plants will be transferred to growth media containing various AsV concentrations (1, 5, 10, 25, and 50 μM) in the absence and presence of 100 mM P. After 30 min, the roots will be rinsed briefly with deionized water, and the shoots and roots separated and dried, which we will ground and digest with HNO3 to determine As concentration using ICP-MS or AFS. However, the advanced machinery used for testing As concentration may even be too hard for us to get, so we may need to find some different ways to test the concentration of AsV, the more common ways are ICP-MS or AFS.

Results

Below is the results we expect from our experiments. The data below represents an average number, instead of data from a single experiment. When we are actually conducting our experiments, we will have many groups at the same time, and we omit the highest and lowest concentrations for every group, and plot the averages.

Conclusions and Future Research

Our research will have a dual impact. It will a) demonstrate that bioinformatics can improve the efficiency and safety of genetic engineering research and b) examine whether the knockout of the (OsPT4) gene could result in a significant reduction in arsenic deposition and contribute to food safety and public health.

After our research on this topic has finished, we can continue experimenting with the other genes in the Pht1 family, like the possible factors of AsV uptake, OsPT1 and OsPT8, and the less studied ones such as OsPT2,3 or 5. This would help lower the AsV uptake even more, lowering the As levels so that the amount of Arsenic in human bodies coming from rice would not harm humans.

References

[1] “Arsenic”,WHO https://www.who.int/zh/news-room/fact-sheets/detail/arsenic

[2] “Be careful of arsenic in rice change the cooking method”,BBC china, 2,8,2017 https://www.bbc.com/zhongwen/simp/science-38913626

[3] “arsenic”, https://en.wikipedia.org/wiki/Arsenic#Inorganic_compounds

[4] “OsPT4 Contributes to Arsenate Uptake and Transport in Rice” 22 December 2017 https://www.frontiersin.org/articles/10.3389/fpls.2017.02197/full#B35

[5] “social experiments” https://www.wjx.cn/mobile/statnew.aspx?activity=81573768&reportid=#1

[6] “arsenic”, https://www.niehs.nih.gov/health/topics/agents/arsenic/index.cfm