Tussah mid-gut specific gene ApLITAF as well as promoter and application thereof
An intestinal-specific and promoter technology, applied in the field of genetic engineering, can solve problems affecting industries and the people's economy, tussah silkworms are easily affected by diseases, and there are few researches, and achieve good application prospects
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Embodiment 1
[0025] Tissue expression characteristics of embodiment 1 tussah ApLITAF gene
[0026] RNA was extracted from each tissue of tussah silkworm, and cDNA was obtained by reverse transcription. After screening the transcriptome data, the primers were designed as follows:
[0027] ApLITAF-gep-F: 5'-tatgcgataatacgttgcgagtg-3' (SEQ ID NO.1);
[0028] ApLITAF-gep-R: 5'-atggttagctataaaatgttcct-3' (SEQ ID NO. 2);
[0029] Using the cDNA of each tissue of tussah silkworm larvae as a template, using SEQ ID NO.1 and SEQ ID NO.2 as a template, and using APL32 as an internal reference gene, the tissue expression specificity of the ApLITAF gene was verified. The results are as follows: figure 1 The tissue expression profile of tussah silkworm ApLITAF gene shows that 1 and 2 are larval head tissues; 3 and 4 are larval epidermis tissues; 5 and 6 are larval midgut tissues; 7 and 8 are larval silk gland tissues; 9 and 10 are larval tissues Malpighian tubules; 11, 12 are larval hemolymph; 13, 14...
Embodiment 2
[0030] Cloning of embodiment 2 tussah ApLITAF gene
[0031] According to the cloned ApLITAF gene mRNA full-length, design ApLITAF coding sequence primers, as follows:
[0032] ApLITAF-F: 5'-cgccatatgatgaacgttcaagtaatatagacg-3' (SEQ ID NO.3);
[0033] ApLITAF-R: 5'-ccgctcgagttagctataaaatgttcct-3' (SEQ ID NO. 4);
[0034] The cDNA of the midgut tissue of tussah silkworm larvae was used as a template, and the PCR amplification was carried out with SEQ ID NO.3 and SEQ ID NO.4 as primers. The amplification conditions were: pre-denaturation at 98°C for 40 seconds; denaturation at 98°C for 15 seconds, annealing at 54.8°C for 30 seconds, extension at 65°C for 35 seconds, a total of 30 cycles; final extension at 65°C for 5 minutes. Electrophoresis of PCR amplification products figure 2 As shown, the fragment size of the tussah midgut-specific expression gene ApLITAF is about 250bp. The amplified product was recovered and sequenced to obtain the ApLITAF coding region sequence (SEQ ...
Embodiment 3
[0035] Example 3 Immune-related verification of tussah ApLITAF gene
[0036] According to the full length of the cloned ApLITAF gene mRNA, design real-time fluorescent quantitative PCR primers:
[0037] ApLITAF-RT-F: 5'-atgcgataatacgttgcgagtg-3' (SEQ ID NO. 6);
[0038] ApLITAF-RT-R: 5'-agggcagatcatagcggaggg-3' (SEQ ID NO.7);
[0039] RNA was extracted from the midgut tissue of healthy and infected silkworms infected with different pathogens, and the corresponding cDNA was obtained by reverse transcription. The relative quantitative experiment of ApLITAF gene expression was carried out with SEQ ID NO.6 and SEQ ID NO.7 as primers, and the tussah silkworm actin gene was selected as an internal reference Gene, the expression level of tussah ApLITAF gene in normal tussah silkworm and tussah silkworm infected with different pathogens image 3 shown. image 3 , A is the quantitative result of respectively infecting Gram-negative bacteria (Escherichia coli E.coli), Gram-positive b...
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