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In order to achieve its nucleotide polymorphism (SNP) accurate typing, this paper establishes a molecular diagnostic technique based on fluorescent quantitative PCR for accurate genotyping of the nucleotide polymorphism (SNP) at the rs755555 locus of the SLC39A13 gene. Firstly, Taqman fluorescent ARMS-PCR detection primers and probes were designed for the rs755555 locus and the internal reference gene peptidylprolyl isomerase A (PPIA). Secondly, positive control plasmids were constructed. Finally, based on genotyping accuracy, the primer and probe combinations were optimized, and the PCR reaction system and conditions for the detection reagents were optimized. The optimal primer and probe combination for the wild-type was: WF1, R1, FP1, PIRF5, PIRR5, PIRP5; the optimal combination for the mutant type was: FMF3, R1, FP1, PIRF5, PIRR5, PIRP5. The optimal reaction system for detecting samples was: 0.1 μL each of upstream and downstream primers and probes for the SLC39A13 gene, 0.1 μL each of upstream and downstream primers and probes for the internal standard, 10 μL PerfectStart Ⅱ Probe qPCR SuperMix UDG, 5.4 μL purified water, and 4 μL sample genome. The feasibility of this detection system was confirmed through reproducibility experiments and the detection of 70 samples. This provides a technical foundation for the development of a detection kit for the polymorphism at the rs755555 locus of the SLC39A13 gene.

In order to solve the problems of low efficiency and cumbersome process of Pichia pastoris genetic manipulation, secretory genetic manipulation tools were constructed and optimized to improve their industrial production and application value. Firstly, the α-amylase was employed as the reporter protein, and the non-integrated expression plasmid was constructed by fusing the self-replicating sequence. Secondly, endogenous signal peptides that best match the target protein were quickly screened and evaluated using universal primers and POE-PCR. Finally, a nutrition-dependent screening marker was constructed with phosphite as the sole phosphorus source. The results indicate that non-integrated plasmids express α-amylase with 2-fold and 1.6-fold higher enzyme activity compared to integrated plasmids, respectively; The use of FLO10, EXG1, and MSB2 signal peptides results in 2.5-fold, 1.94-fold, and 1.75-fold higher α-amylase activity than the conventional signal peptide α-MF, respectively; Arecombinant strain for heterologous expression of α-amylase is successfully screened using the phosphite dehydrogenase gene (ptxD). The process of gene operation is simplified, the genetic operation tools are improved, and green, safe, and low-cost nutrition-dependent screening markers are provided, which lays a foundation for the application and promotion of the Pichia pastoris secretory expression system.