Cloning,Expression And Function Research Of Vitellgenins Family In Haemaphysalis Flava

Based on the salivary gland proteome and salivary gland transcriptome of H.flava,the primers were designed to amplify the full length of the vitellogenins family gene,and quantitative PCR primers were designed to analyze the expression levels of four Hf-vitellogenins genes in different life phases,feeding stages and in different tissues.The distribution in the egg lays the foundation for exploring new strategies for reducing nutrients,interfering with embryonic development,and achieving control goals.The 3' and 5' ends of Vg-1,Vg-2,Vg-3 and Vg-B were amplified by RACE,and the full length of four genes encoding Vg-1,Vg-2,Vg-3 and Vg-B were obtained.Vg-1 gene was 5453 bp,ORF was 5079 bp,encoding 1693 amino acids;Vg-2 gene was 4953 bp,ORF was 4671 bp,encoding 1557 amino acids;Vg-3 gene was 4811 bp,ORF was 4602 bp,encoding 1534 amino acids The Vg-B gene was 9754 bp and has an ORF of 9576 bp,encoding 3192 amino acids.The results of qPCR analysis indicated that the expression levels of four vitellogenins in different life phases were in the following order(low to hige)larvae < Nymphs < female adult H.flava.In different feeding stages,the expression level of four vitellogenins were in the following order(low to hige)1/4-engorged adult female H.flava < 1/2-engorged adult female H.flava < 3/4-engorged adult female H.flava < full-engorged adult female H.flava.In different tissues,the expression levels of the four genes in the salivary glands were up-regulated with the increase of blood meal,and the expression levels of the four genes in the ovary were down-regulated with the increase of blood meal.In the same tissue of H.flava,the expression level of the four genes were in the follow order Vg-2 > Vg-B > Vg-1 > Vg-3.LC-MS/MS was used to analyze the fresh eggs from H.flava.A total of 221 unique peptides were detected in the eggs of H.flava,and 53 proteins were identified,among which 12 were highly confident proteins.The functional proteins that can be identified were Actin and Vg-2Ah,and Vg-2Ah was the only vitellogenin in the eggs.Vg-1,Vg-2,Vg-3,and Vg-B were not found in the eggs.The alignment shows that Vg-2Ah has a similarity to Vg-2 of 12.59% and is a different member of the Vg family.