Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 72-78 http://cropscipublisher.com/index.php/tgg 73 2 Structure and Expression Characteristics of TaGW2 2.1 Gene structure and conserved sequence analysis TaGW2 is a gene related to grain size. It encodes a protein called "E3 ubiquitin ligase". This protein has a special "ring finger" structure, which is critical to its function. In hexaploid wheat, this gene has three versions, namely TaGW2-6A, 6B and 6D. Its coding sequence is 1 275 bases, which can produce a protein containing 424 amino acids. Studies have found that the coding part of this gene is not much different in different wheat varieties, that is, it is very conservative. But its promoter region is more different, especially in the 6A and 6B versions. These differences affect grain size and weight and are the reason for the formation of different haplotypes (Su et al., 2010; Qin et al., 2014). The C-terminus of the protein, that is, the LXLX region near the end (amino acids 376 to 424), is particularly important for identifying the target protein. This segment helps TaGW2 bind to other proteins involved in grain development, thereby exerting its effects (Lü et al., 2022). 2.2 Expression patterns in different tissues and developmental stages TaGW2 is always expressed in wheat. All three versions (6A, 6B, and 6D) are expressed, but with different intensities. Generally speaking, 6A is expressed the most, 6B is in the middle, and 6D is the least, especially at the critical stage of grain development (Qin et al., 2014). This gene is not only expressed in grains, but can also be seen in leaves, stems and other parts, indicating that it plays a wide range of roles in the growth and development of wheat (Figure 1) (Su et al., 2010; Zhang et al., 2022). Figure 1 Subcellular localization and spatiotemporal expression of TaGW2L and TaGW2 in wheat (Adopted from Zhang et al., 2022) Image caption: (A) Subcellular localization of TaGW2L and TaGW2 in wheat protoplasts. Scale bars, 10 µm. (B) Expression pattern analysis of TaGW2L and TaGW2 in wheat. SR, seedling roots; SS, seedling stems; SL, seedling leaves; ER, roots at elongation stage; ES, stems at elongation stage; EL, leaves at elongation stage; HR, roots at heading stage; HS, stems at heading stage; HL, leaves at heading stage; 1YS-7YS, 1 cm-7 cm young spikes; 5D-25D, grains at 5-25 days after pollination. Values are presented as mean ± SD (Adopted from Zhang et al., 2022) 2.3 Spatiotemporal expression during key grain developmental stages During grain development, the expression of TaGW2 is particularly active during the rapid cell division period and the late filling period. This suggests that it may play a regulatory role in endosperm cell growth and nutrient accumulation (Bednarek et al., 2012; Qin et al., 2014). Studies have found that the expression of this gene is related to the width of the grain. The lower the expression, the larger the grain and the higher the thousand-grain weight (TGW) (Su et al., 2010). The variation in its promoter region will further affect the expression level of this gene at different stages, thereby affecting the final grain size. 3 CRISPR/Cas9 System Design and Editing Strategy 3.1 Target site selection and gRNA construction To edit TaGW2, the first step is to find a suitable target. Generally, a more conservative region of this gene is selected, which makes it easier to interrupt its function. gRNA (guide RNA) is specially designed to
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