Plant Gene and Traits 2024, Vol.15, No.3, 108-117 http://genbreedpublisher.com/index.php/pgt 111 3.2 Key genes and alleles: description of major genes involved and their known variants A total of 45 genes have been identified as participants in the starch biosynthesis pathway in cassava. These include ADPG pyrophosphorylase (AGPase), granule bound starch synthase (GBSS), starch synthase (SS), starch branching enzyme (SBE), de-branching enzyme (DBE), and glucan, water dikinase (GWD) (Tappiban et al., 2019). Among these, GBSS is particularly noteworthy as it is directly involved in amylose biosynthesis. CRISPR-Cas9 mediated targeted mutagenesis of GBSS, as well as PROTEIN TARGETING TO STARCH (PTST1), has been shown to reduce or eliminate amylose content, significantly altering the physicochemical properties of starch (Figure 2) (Bull et al., 2018). These findings highlight the potential of gene editing techniques in developing cassava varieties with modified starch characteristics for specific applications. Figure 2 Genome editing of MeGBSS andMePTST1(Adopted from Bull et al., 2018) Image caption: (A) Model for association between GBSS, PTST1, and starch granules as proposed by Seung et al. (2015). Image recreated with permission. (B) Binary expression construct containing hptII (hygromycin B resistance) for plant selection; Cas9 codon optimized for cassava usage (pcoCas9) and fused to the eGFP reporter gene and directed to the nucleus via a nucleoplasmin nuclear localization signal (NLS); transcription terminated by sequence from a heat shock protein (tHSP) (Nagaya et al., 2010). A synthetic pU6 promoter [(psynU6; (Nekrasov et al., 2013)] used to drive expression of the protospacer sequence [single guide RNA (sgRNA)] and synthetic scaffold (Jinek et al., 2012) to generate the desired sgRNA. AtFT for early flowering is constitutively expressed by the CaMV35S promoter and terminated by nopaline synthase sequence (tNOS). Binary expression constructs named pCas9-sgGBSS-FT and pCas9-sgPTST-FT. Left and right borders (LB and RB) are shown. Diagram not to scale. (C) Gene maps of MeGBSS and MePTST1. Exons are depicted as blocks, and regions encoding functional domains are shaded. The target sites for the sgRNAs are indicated. Scale bars are shown (Adopted from Bull et al., 2018) Figure 2 presents the genome editing strategy used to modify the MeGBSS and MePTST1 genes in cassava. The illustration details a model of how GBSS and PTST1 associate with starch granules, as well as the construction of binary expression constructs used for plant transformation. This approach utilizes CRISPR/Cas9 technology, optimized for cassava, to target specific gene sequences responsible for starch biosynthesis and transport, which are crucial for understanding and manipulating starch accumulation and properties in cassava roots. By employing this targeted editing, the study aims to elucidate the roles of these genes in starch granule formation and potentially improve cassava's starch yield and quality. 3.3 Gene expression patterns: how genetic factors influence the expression and functionality of enzymes in different cassava varieties The expression of starch biosynthesis genes is regulated during root development, which determines the functionality of the enzymes and ultimately the starch properties of the cassava roots (Tappiban et al., 2019). Comparative genomic analyses have revealed that genes involved in starch accumulation have been positively selected in domesticated cassava varieties (Wang et al., 2014). Additionally, transgenic approaches have shown that increasing the expression of genes like AGPase in cassava roots can lead to a substantial increase in starch production (Ihemere et al., 2006). The expression profiles of genes such as those in the UGPase family also vary across different tissues, which may influence the distribution and quantity of starch synthesis within the plant (Ihemere et al., 2006). Understanding these expression patterns is crucial for the development of cassava varieties with desired starch qualities through breeding and genetic modification.
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