Function verification of soybean GmACP1 alleles by TALENs开题报告

1. 研究目的与意义、国内外研究现状（文献综述）

Transcription activator-like effector nucleases (TALENs) are a distinct class of proteins secreted by plant pathogenic bacteria from the genus Xanthomonas. These TALENs are injected via the type III secretion pathway into plant cells (Bogdanove et al.,2010) where they are translocated to cellular nucleus binding to gene promoters specific to TAL effectors that results in the activation of expressing downstream genes that cause bacterial colonization and spread that eventually leads to onset of disease ( Cermak et al.,2011).These TALENs have in the past been documented to have led to development of disease in most notably cotton and rice (Bogdanove et al., 2010; Boch and Bonas, 2010) with devastating results that led to reduced productivity of the crops and great economic losses to farmers.TALENs apart from having their DNA binding domain they also posses a FokI cleavage domain .FokI is a type IIS endonuclease from the bacteria species Flavobacterium okeanokoites. This enzyme comprises of two regions, an N-terminal that acts as a DNA binding domain and a C-terminal that acts as a DNA cleavage domain (Sun et al., 2013). In order to form a functional complex, two FokI molecules are required to bind to the target region of DNA strands in a tail-to-tail orientation ensuring that proper spacer length between the binding sites exists after which they should then dimerize in a way that the two nuclease cleavage domains couple to form a functional endonuclease that cleaves DNA at the downstream site of the recognition sequence (Scholze and Boch 2010; Hafez and Hausner,2012)Phosphorus is essential mineral element utilized by all living cells and organisms however there is a great need to improve the efficiency of this finite and non-renewable resource and also reasonable use of P resources in agriculture. In maize, wheat and barley, P has been shown to increase dry matter content (Rai et al.,2013) In soybean, low phosphorus levels have a detrimental impact to growth and development of the plant hence there is a need to understand the mechanism and target chromosomes as well as genes to fully comprehend soybean phosphorus efficiency. A study by Zhang et al., (2014) led to the discovery of the chromosome 8 located gene GmACP1 was responsible for Phosphorus efficiency in soybean. References1. Bogdanove, A. J., et al. (2010). "TAL effectors: finding plant genes for disease and defense." Curr Opin Plant Biol 13(4): 394-401.2. Cermak, T., et al. (2011). "Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting." Nucleic Acids Res 39(12): e82.3. Sun, N. and H. Zhao (2013). "Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing." Biotechnol Bioeng 110(7): 1811-1821.4. Zhang, D., et al. (2014). "The acid phosphatase-encoding gene GmACP1 contributes to soybean tolerance to low-phosphorus stress." PLoS Genet 10(1): e1004061.5. Scholze, H. and J. Boch (2011). "TAL effectors are remote controls for gene activation." Current Opinion in Microbiology 14(1): 47-53.6. Avinash Rai, Sumit Rai, Amitava Rakshit Mycorrhiza-mediated phosphorus use efficiency in plants Environmental and Experimental Biology (2013) 11: 1071177. Boch J, Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol. 2010; 48:419436. [PubMed: 19400638]8. Mohamed Hafez, Georg Hausner Homing endonucleases: DNA scissors on a mission Genome, 2012, 55(8): 553-569, 10.1139/g2012-049

2. 研究的基本内容和问题

A study by Zhang et al., (2014) led to the discovery of the chromosome 8 located gene GmACP1 was responsible for Phosphorus efficiency and also compared two haplotype proteins from within the gene where they found that proteins from H9 haplotype had a more favourable activity than those from the H4 haplotype. The objective of the study is to use TALENs to induce a modification in H4 and H9 haplotype to change the nucleotide base at position 581 to T and A respectively and to assess the resulting protein activity in soybean plants with regard to phosphate efficiency.

3. 研究的方法与方案

The first step is to use the TALEN Targeter software to design pairs of custom TAL effectors for use as TAL effector nucleases (TALENs) by identifying good candidate binding sites of the proper length and with the proper spacing and orientation between the two sites. The TALEN Targeter software tutorial can be found in the website https://tale-nt.cac.cornell.edu/tutorials/talentargeterupdated. The procedure to design Tal effectors is as follows;1. Open the software and paste the desired DNA sequence.2. Select the architecture or customize spacer and array lengths to desired proportions.3. Determine themost suitable filter for your results from those available in the kit4. Select upstream base that is preceded with a T for ones desired TAL effectors5. Submit the sequence for query to search for suitable TAL sites6. Retrieve and finally interpret results of TAL sequences found within the desired sequences.After finding the suitable TAL sites, the TALEN box will be constructed by using the FAST TALE?TALEN kit following manufactures?instructions and sequenced to it was correctly constructed.

4. 研究创新点

The innovative aspect of the study is the intended use TALENs to induce a modification in H4 and H9 haplotype to change the nucleotide base at position 581 to T and A respectively and to assess the resulting protein activity in soybean plants with regard to phosphate efficiency. The insights on this study may help in enhancing the productivity of soybean in a global scale for years to come.

5. 研究计划与进展

The first area is creating the TALENs for the Hap4 and Hap 9 sequences The expected outcome of the experiment is to get suitable sequences that can be used for cloning and subsequent transformation in the soybean plant.Status of Progress?The experiment will be carried out using the FAST TALE?TALEN kit. The plasmid will then be extracted and sent for sequencing to ensure that the cloning was successful.