C4 genes promoter reporter assay

INTRODUCTION
Almost half of the world's population (3 billion people) feeds on rice which makes it the most important food crop in the world (McLean, Hardy, & Hettel, 2013). It provides 21 % of global human per capita energy and 15 % of per capita protein. It also provides minerals, vitamins and fiber (Dawe, Pandey, & Nelson, 2010). Aside from nutritional importance, it has also a great role in Asia's economy as well as the world's. About 144 million rice farms with an area of 158 million hectares are harvested every year (McLean et al., 2013). Millions of small time farmers consider it as their main source of income and more than 90% of these rice farms are found in Asia ("South East Asia & The Importance of Rice _ South East Asia Backpacker Magazine," 2010).
On the local perspective, Philippines considers rice as its staple food. A recent survey from Food and Agriculture Organization (2014) shows that within a year the average rice consumption of a Filipino is 123 kilograms. This ranks as sixth in the world. Also, the Consumer Price Index (CPI) of the Philippines for rice is very high (8.9%) which comes fourth in the world ranking next to Vietnam, Sri Lanka and Indonesia (Cruz,2014).
Considering the importance of rice, it would be a very big problem if production of it would be insufficient. Now, an estimate of 925 million people suffer from chronic hunger and 14,400 children die every day because of hunger-related causes and this is expected to worsen in the future because by 2050 the world's population is anticipated to reach 9 billion. In order to meet this demand, rice production in Asia needs to increase by 60%. That is for every hectare of rice that currently provides for 27 people will be needing to provide food for 47 people in the future (Kajala et al., 2011). Aside from this, an unpredictable weather condition, in consequence to climate change, threatens stable food production. In addition to that, competition in land use is also a problem, most farmlands are now being converted into lands used for industrial and bio-fuel purposes. (Karki, Rizal, & Quick, 2013).
A solution being considered is to improve the photosynthesis of rice by introducing the C4 pathway (Kajala et al., 2011). In doing this, genes responsible for leaf anatomy and biochemical processes should be inserted in rice and expressed and appropriately. In making sure that the expression of these certain genes are expressed appropriately, strong and cell-specific promoters should be used.
According to Matsuoka, M. et al (1994), promoters of PEPC (Phosphoenolpyruvate carboxylase) and RuBPC (Ribulose bisphosphate carboxylase) from maize direct cell-specific expression when inserted in rice but this research alone is not updated and a study made by Nomura and his colleagues (2005) suggests that expression driven by ZmMe does not only direct expression in bundle sheath cells but also in mesophyll cell and vascular bundles. To further assimilate these claims, an updated research must be done. In addition to that, tissue specificity of these promoters must be studied to know the efficiency of these promoters. This study focuses on assessing the two promoters that promises cell-specific expression: ZjPCK which directs expression in bundle sheath cells and ZmPEPC which directs expression in mesophyll cells.
Objectives of the Study
The study's goal is to analyze the activity of ZjPCK promoter and ZmPEPC promoter.
More specifically this study aims to:
Observe the tissue specificity of ZjPCK promoter and ZmPEPC promoter by using 5-day old seedlings in GUS (β-glucorunidase gene) assay;
Show the cellular localization of expression of GUS driven by ZjPCK promoter and ZmPEPC promoter using leaf blade, leaf sheath and roots;
Quantify the expression of GUS driven by ZjPCK promoter and ZmPEPC promoter and compare these to the expression promoter of constitutive gene ZmUbi; and,
Know the number of inserts (copy number analysis) that happened in the transgenic lines used in the experiment.


Significance of the Study
This study will provide grounds for future experimentations regarding the creation of C4 rice specifically for the scientists at International Rice Research Institute (IRRI). The promoters in this study provide cell-specific expression for the genes found in the C4 pathway enabling an accurate transport of enzymes. These promoters can also overexpress genes in a cell-specific manner. Fusing these promoters with the genes in C4 pathway will greatly improve the progress of the developing the C4 rice.
The creation of C4 rice will solve the problem in rice production. In consequence, hunger will be lessened throughout the world especially in the rice producing countries. Also, economy of rice producing countries will increase because of the added yield which will also lead to poverty alleviation.

Scope and Limitations
The study will be testing the promoters with different specificities in expression. Their specificity will be determined by GUS assay while their strength of expression will be measured by doing Reverse Transcription Polymerase Chain Reaction (RT-PCR). On the other hand, copy of gene insertions in the genome of the transformed rice will also be screened by Southern blotting. The test of expression will be up to the transcriptional level but the translational level of expression will not be a part of the screening. Northern blot and western blot will not be done because RT-PCR and GUS assay alone are enough to quantify expression of the GUS.
Promoter testing is the aim of the study so the procedures will be limited in screening the promoters. The expression of the enzyme (PEPC and Rubp) in the transformed rice will not be included in the scope of procedures. The duration of the study is for six months which will start on June 2015 and will end on November 2015. All materials including the laboratory where the experiment will be conducted will be provided by International Rice Research Institution (IRRI).





References:
Cruz, C. J. F. (2014). Surveillance of the Philippine Rice Market. Retrieved from http://www.bsp.gov.ph/downloads /EcoNews/EN14-02.pdf

Kajala, K., Covshoff, S., Karki, S., Woodfield, H., Tolley, B. J., Dionora, M. J. A., … Quick, W. P. (2011). Strategies for engineering a two-celled C 4 photosynthetic pathway into rice, 1–10. http://doi.org/10.1093/jxb/err022
Karki, S., Rizal, G., & Quick, W. P. (2013). Improvement of photosynthesis in rice (Oryza sativa L.) by inserting the C4 pathway. Rice (New York, N.Y.), 6(1), 28. http://doi.org/10.1186/1939-8433-6-28

Matsuoko M., Kyozuka J., Shimamoto K.,& Kano-Murakami Y., (1994). The Promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice).The Plant Journal 6(3), 311-319.
McLean, J., Hardy, B., & Hettel, G. (2013). Rice Almanac, 4th edition. IRRI, Los Baños, Philippines. http://doi.org/10.1093/aob/mcg189
South East Asia & The Importance of Rice _ South East Asia Backpacker Magazine. (n.d.).