Summary of the Proposal The goal of this project is to identify differentially expressed genes and proteins in susceptible and

resistant maize inbred lines. Aflatoxin contamination is a big problem in the production of maize crops. Specially, in southern Unites States due to drought and high temperature conditions. Aflatoxin is a secondary metabolite produced by fungi Aspergillus flavus and Aspergillus parasiticus. It can infect maize crops both in the fields and in the storage area. Several pre and post- harvest strategies have been applied to reduce aflatoxin contamination but none of them are very effective. The most effective method of aflatoxin reduction is the development of resistant maize inbred lines. These resistant maize inbred lines can be developed by identifying differentially expressed proteins which can further lead to identification of genes involved in the resistance to aflatoxins by employing PCR technique. Traditionally two dimensional (2DE) gel electrophoresis was a preferred method for identifying differentially expressed proteins. 2DE can resolve thousands of proteins in a single gel and provides valuable information related to separated proteins. However, reproducibility and gel to gel variation is a major limitations related to this technique. In the proposed work, to overcome the limitations with traditional 2DE, two dimensional difference gel electrophoresis (DIGE) technique will be used to identify differentially expressed proteins in maize inbred lines. Two resistant maize inbred lines (Mp715 and Mp718) and two susceptible maize inbred lines (Mp04:87 and Va35) will be selected for this study. Protein spots obtained from 2D DIGE, will be further analyzed with MALDI-TOF-MS technique by peptide mass fingerprinting (PMF). Protein sequences obtained from MALDI-TOFMS will be further used to identify gene sequences through sequencing reaction. It is a novel analysis procedure for profiling and comparative investigations of differentially expressed proteins to facilitate the development of DNA markers for maize resistance breeding. Specific objectives: 1) Identify differentially expressed protein in maize susceptible and resistant inbred lines by employing 2D DIGE technique 2) Incorporation of MALDI-TOF-MS for further analysis of protein spots obtained from 2D DIGE Relevant Background Need for the identification of differentially expressed proteins in susceptible and resistant maize inbred lines Aflatoxin contamination is a world -wide problem affecting large number of commodities including maize (Zea mays L.). Aflatoxin is a potent secondary metabolite and carcinogen found in nature which is produced primarily by fungi Aspergillus flavus and Aspergillus parasiticus [14]. Aflatoxin contamination can pose a great economic impact on maize growers annually due to contaminated maize grains. Contaminated maize grains may result in the health problems for both humans and live stocks if it enters the food supply [5, 6]. Dietary exposure of aflatoxin is

responsible for hepatocellular carcinoma which is fifth most common cancer in the world [7]. Chronic exposure of aflatoxin in maize grains can cause growth retardation in children and even death when consumed in large quantities [8, 9]. It is a serious problem in developed and developing countries. More than fifty countries have set a rule for controlling aflatoxin in food and feed [10]. United States Food (USA) and Drug administration set the limits for the sale of grains with aflatoxin level exceeding 20 ppb [11]. The best strategy to reduce aflatoxin contamination in maize inbred lines is to reduce aflatoxin accumulation in corn grains. Cultural practices, irrigation and use of fertilizers are some of the current practices to reduce aflatoxin accumulation in corn grains [5]. None of the above mentioned practices are as effective as to control aflatoxin contamination when environmental conditions are favorable for the growth of fungus[12]. The most effective method of aflatoxin reduction is the development of resistant maize inbred lines[13, 14]. These resistant lines can be developed by identifying differentially expressed proteins which can further lead to identification of genes involved in the resistance to aflatoxin. Traditionally two dimensional (2DE) gel electrophoresis was a preferred method for identifying differentially expressed proteins[15]. In 2D gel electrophoresis method, proteins first undergo isoelectric focusing (IEF) step which separates protein according to their isoelectric point (pI) and in the second dimensional electrophoresis proteins separate orthogonally according to their molecular weight. [16, 17]. This method can resolve thousands of proteins in a single gel and provide valuable information related to proteins. However, reproducibility and gel to gel variation is a major limitations related to this technique[18]. Our Proposed Approach In order to overcome the limitations with traditional 2DE, we are proposing two dimensional difference gel electrophoresis (DIGE) technique to identify differentially expressed proteins in maize inbred lines. Preliminary Results Quantification of 2D protein gels for differentially expressed proteins associated with resistance to aflatoxin accumulation in maize inbred lines:

Two dimension gel electrophoresis (2DE) technique was employed to obtain protein gel images from resistant and susceptible maize inbred lines. Resistant (Mp719) and susceptible (Mp04:87) maize inbred lines has been chosen to identify differentially expressed regions of protein spots. 1D gel images were shown in the figure 1 A) and in 1 C). 3-D view (mesh plot) has been shown by 600 600 coordinates in 1B and 1D. In these figures the red and yellow region is highly expressed in compare to blue region. This represents that some of the common proteins are present in both resistant (Mp719) and Figure 1. (A). 2D gel image of resistant Mp719 showing proteins expressed at 14 days after A. flavus inoculation. (B) 3D view of the Mp719 protein gel image by Matlab image toolbox. susceptible (C) 2D gel image of susceptible maize inbred line Mp04:87 showing total proteins expressed maize at 14 days after A. flavus inoculation. (D) 3D view of the Mp04:87 protein gel image by inbred line Matlab image toolbox. (E) 3-D view of differentially expressed proteins between Mp719 and Mp04:87. The red and yellow peaks represent proteins highly expressed in Mp719 (F)A plot (Va35). Two showing differentially expressed proteins along an x =300 coordinate section from an maize alignment of 2D gel imges (Mp719 and Mp04:87). inbred lines are aligned in Figure 1E). The red and yellow peaks shows proteins are highly expressed in Mp719 in compare to Mp04:87. These results visualize differentially expressed protein regions. In this method, each protein spot is uplifted from the background by uplifted edges and shows different pixel intensities of the protein spot. Protein spots which are highly expressed have high pixel intensity and they are brighter in compare to background. Protein spots which are less expressed have lower pixel intensity and are less bright in the gel. In the Figure 1F) two protein gel images were aligned at coordinate (x= 300). The plot showing differentially expressed proteins in the peak form. The red peak shows resistant maize inbred line Mp719 and blue peak

shows susceptible maize inbred line Mp04:87. These results, clearly shows that proteins are differentially expressed in susceptible and resistant maize inbred lines. Plan of Work Resistant and susceptible maize kernels will be collected on summer (June- July). Protein will be extracted using using TCA\acetone precipitation in combination of a phenol extraction step. Three CyDye (Cy2, Cy3, and Cy5) will be used for labeling 50 g of protein extracted from each sample. Protein extract obtained from maize inbred lines will be labeled with 400 pmol of Cy3 and Cy5 fluorescent dyes, while Cy2 dye will be used to label the internal standard, which consists of a Figure 2. Flow chart of Cydye labeling in 2D-DIGE. Protein pooled sample comprising equal sample of susceptible maize inbred lines Va35 and mp04:87 and amounts of all samples to be resistant lines Mp715 and Mp718 will be labeled with Cy3 and Cy5. compared. After protein labeling Internal standard is the pool of all samples and will be labeled with the three samples will then be run Cy2. Gel will be imaged with Typhoon TRIO Variable Mode Imager in PROTEAN II XL cell (Bio(GE Healthcare). Rad) on 12 % SDS-page gel and run at 16mA for 30 minutes followed by 24 mA for 5 hour in the same gel to reduce gel to gel variation. Protein gel will be imaged with Typhoon TRIO Variable Mode Imager (GE Healthcare). Gel analysis will be performed with using Matlab image processing toolbox. Differentially expressed protein spots will be further analyzed using MALDI-TOF-MS technique by peptide mass fingerprinting (PMF). In this procedure, 2D gel will be first destain, and spot will be cut by protein spot cutter. Disulfide bonds present in proteins will be first reduced and alkylated for tryptic digestion. MALDI-TOF-MS spectrum will be used to determine the mass of protein and database search for PMF will identify proteins. These protein sequences will be converted in to gene sequences and through primer design, PCR (polymerase chain reaction) will be performed. Anticipated Outcome 2D DIGE technique will provide more number of protein spots in a single gel and with the use of MALDI-TOF technique, large number of differentially expressed proteins will be identified

in different maize inbred lines [16, 19]. Protein spots will be more visible in two different gels by using Cydyes [20]. The spots present between two different gels will be easily identifiable and spot repeatability in a gel will be in an acceptable range [21]. Poor protein spot resolution which is present due to high background noise in a gel will be enhanced due to less gel to gel variation[22]. Differentially expressed proteins will provide information towards differentially expressed genes related to aflatoxin resistance by employing PCR technique[23, 24]. It is a novel analysis procedure for profiling and comparative investigations of differentially expressed proteins to facilitate the development of DNA markers for maize resistance breeding Potential Scientific Impact Traditional 2D gel electrophoresis technique has some limitation such as gel to gel variation and time consuming. This technique is highly sensitive and has straightforward protocols which make it relatively easy to use. The 2D-DIGE technique will dramatically improve the reproducibility, sensitivity, and accuracy of protein quantitation. With the use of this technique we can easily identify differentially expressed proteins in different maize inbred lines. Literature Cited
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