Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Agricultural and Environmental Sciences
Non Technical Summary
Tomatoes (Solanum lycopersicum) are the second most consumed vegetable in the U.S, behind potatoes. Tomatoes grow best when the daytime temperature is between 65 and 85 degrees Fahrenheit (18.3oC-29.4 oC). When temperature reaches above 95 degrees (35 oC) Fahrenheit, fruit-set is severely disrupted, and red tomatoes won't turn color as the pigment (lycopene) cannot be synthesized properly. Tomatoespreferwell-drained soilwith apHof around 6 to 6.8. Plants experience toxicity to excess levels of aluminum (Al), iron and manganese, and deficiency ofcalcium, magnesium, and phosphorus in acidic soils (pHbelow 5.5). We have taken the first step to understand the underlying molecular mechanisms of tomato plant responses to these stress factors. Using genomic and proteomics analysis of stress treated plants, a list of stress-responsive genes have been identified. Among these genes are transcription factors (TFs) which regulate perception of stress signals, and activate/repress the transcriptional rate of single or multiple stress-responsive genes. More importantly, some TFs expressed changes at protein and/or transcript levels in response to the stress treatment. Reported studies have shown that alteration in the expression of these TFs has the potential to produce stress tolerant phenotypes. In this project, we will focus on the TFs as primary targets of CRISPR/Cas9 gene editing with a final goal to develop mutant plants to be used in breeding or cultivar development. The project will provide training to graduate and undergraduate students with competent skills in this important Biotechnology field.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Goals / Objectives
The goals of this project are to develop CRISPR/Cas9 gene editing system of tomato for research and student training, and to produce tomato lines that would help understanding the function of stress-responsive genes and to be used as novel genetic materials in tomato breeding.
Project Methods
Developing the CRISPR/Cas9-mediated gene editing system of tomatoesTwo stress-responsive transcription factor (TFs) geneswill be selected based on the data obtained from proteomics/ transcriptome analysis of tomatoes under aluminum (Al) stress treatedconditions. The tomato gene sequences will be retrieved in the annotatedtomato genome on Sol Genomics Network (https://solgenomics.net/). The tissue-specific expression of the target gene in tomato plants will be searched using an online database: web-based tools for visualizing functional genomics and other data (http://bar.utoronto.ca/efp_tomato/cgi-bin/efpWeb.cgi). In this platform, once the target gene is pasted in the Browser, the database will show where it is expressed. If the gene is mainly expressed in roots and mature fruits, changing the expression of this gene would not affect leaf development and other traits, in other words, the ectopic effect is rather small. So this type of genes will be selected for genome editing to study root tolerance to Al toxicity.To design the guide RNA (gRNA) sequences, the downloaded gene sequence will be translated into protein to determine the orientation. If it is an antisense sequence, it will be reverse- transcribed using online program (http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/revcomp.html ). cDNA sequence will be loaded onto CRISPR P v2.0 to design guide RNA (gRNA) sequence (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR). gRNA sequences will be synthesizedandcloned onto p201N:Cas9 vector. The plasmid will be transferred into E.coli competitive cells. Plasmid will be extracted from positive clones and inserts validated by sanger sequencing. Selected plasmids will be used to transform Agrobacterium tumefaciens strain AGL1. A. tumefaciens harboring the CRIPSR/Cas 9 construct will be used to inoculate tomato 'Money Maker' using the cotyledon-inoculation method.Plantlets will be regenerated on selective media. Putative transformation events will be validated usingPCR analysis witha pair of primers covering the insert region. The postive T0plants will be rooted and transplanted to potting mix to estabolish into individual plants in greenhouse.For genotyping and mutation analysis in T0 plants, DNA will be extracted from tomato leaves, PCR will be performed using primer sequences covering the predicted mutation sites, and the amplified DNA will be Sanger sequenced.To determine the heritable genome editing effects, the T0 plant flowers will be bagged for strict self-pollination to obtain homogenous lines in T1-T2 plants. Genotyping of the target sites as well as off-target sites will be confirmed using Sanger sequencing of PCR products covering the targeted gene region. Expression level of the target gene will be determined using quantitative PCR.Objective 2. Developing the CRISPR/Cas9-mediated gene edited tomato lines for the selection of stress tolerance lines In this objective, we will select a number of genes associated with tomato plant responses to Al and other abiotic stress factors including salt and heat. These genes will be targeted for generating CRISPR-edited plants following the procedures described above. The genome-edited plants will be evaluted for phenotypic changes based on properties ofroot, shoot,flowering, and fruitsunder stress treated conditions. Lines showing improved tolerance to stress factors will be selected, and the use of the elitelines in tomato breeding or developing intocultivars will be evaluated.