International Journal of Marine Science, 2025, Vol.15, No.3, 167-178 http://www.aquapublisher.com/index.php/ijms 173 reproductive axis. Studies have shown that the expression of IGFBP-5 in the abalone gonad maturation period is significantly increased, and it is speculated that it affects gametegenesis and maturation by regulating IGF accessibility (Bai et al., 2019). 6 Gene Editing and Functional Verification Technology 6.1 RNA interference and CRISPR technology attempts in abalone To gain an in-depth understanding of the functional role of key genes on abalone traits, it is necessary to use functional genomic technology to manipulate and verify the target gene. Currently in abalone, RNA interference (RNAi) and CRISPR/Cas9 gene editing are the main attempts. RNA interference uses specific double-stranded RNA to induce gene silencing and has been successfully used in the study of abalone gene function. Zhang et al. (2020) efficiently silenced the HdHSF1 gene of Ezo abalone by injection of dsRNA, and observed significant changes in heat shock-related gene expression and heat-resistant phenotype, thereby verifying the function of HSF1 in abalone's anti-therm. The same method has been applied to the study of abalone AKT genes. After interference, the downstream immune genes of AKT are suppressed, confirming the key role of AKT in the immune pathway (Yao et al., 2025). The advantage of RNAi is that it is easy to operate and does not require genome change, but has limited maintenance time and is generally used for developmental stages or short-term trait analysis. Recently, the scientific research team of the Institute of Oceanography of the Chinese Academy of Sciences has made a breakthrough. The team established an improved abalone embryo semi-dry exposure microinjection technology, injecting Cas9 protein and gRNA into the Ezo abalone abalone fertilized egg, successfully knocking out the specific gene of the abalone and obtaining edited individuals that develop to the larval stage. They reportedly published this result in The CRISPR Journal, achieving the first accurate editing of the genome level of abalone species (Li et al., 2024). Although editing efficiency still needs to be improved, this attempt demonstrates that CRISPR technology can be used in abalone functional genome research. 6.2 Functional gene knockout/overexpression experimental cases 6.2.1 Knockout verification of genes related to growth regulation Through genetic function research, several key genes that regulate abalone's growth have been identified and functionally verified. The mTOR pathway gene mentioned above is one of the hot topics of growth regulation. The study used RNAi to knock down the SLC38A9 transporter gene on the abalone mTOR pathway, which resulted in a significant reduction in the phosphorylation level of RPS6KP protein downstream of the TOR signal, and the abalone anabolic slowed down and the growth was inhibited. This functionally demonstrates that SLC38A9-mediated amino acid signaling is crucial for abalone growth. Similarly, genes on the IGF axis such as IGF1R receptor have also been verified by RNAi: interfering with IGF1R will reduce the growth rate of young abalone by about 15%, indicating that IGF signaling affects abalone growth by promoting cell proliferation (Li et al., 2021). In addition, some growth-related transcription factor genes such as Myostatin (myostatin) have also been studied in preliminary functional areas. Myostatin usually inhibits muscle growth. Researchers use antisense oligonucleotides to inhibit the expression of abalone Myostatin and found that the abalone foot muscle fiber diameter is significantly enlarged, consistent with other species, demonstrating that inhibition of Myostatin can help increase the meat production of abalone (Shiel et al., 2014). These functional validations provide gene targets for the next step of breeding. 6.2.2 Analysis of overexpression function of immune-related genes In the field of immunity and disease resistance, some progress has been made in functional gene verification, but overexpression techniques are more challenging in abalone compared to knockout. Gene injection or transient expression of vectors is usually used to observe gene function. A successful case is the functional study of the abalone antimicrobial peptide NDK gene. The researchers constructed a eukaryotic expression vector of the abalone NDK gene and injected it into the abalone body cavity, and detected an increase in the content of NDK peptides in the abalone hemolymph. After artificial infection with the pathogen, the survival rate of abalone overexpressing NDK was 20% higher than that of the control group, proving that excessive NDK can enhance the antibacterial ability of abalone. Another example is the functional analysis of the heat shock protein HSP24. Some
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