Study On Inhibin Gene Immunization And Ovary Expression Of Gthr Mrna In Qingjiaoma Chicken

The poultry are oviparous animals, which have no estrous cycle, estrous behavior, pregnancy and lactation. They could complete follicular development, ovulation and fertilization in a continuous period. There have no corpus luteum after ovulation. As their specific characteristics, the poultry are widely used as good models in biological study of follicular development. In this paper, techniques such as gene cloning, immune-neutralization and reverse transcription-PCR were applied to investigate the influence of inhibin gene immunization on follicular development and GTHR mRNA expression within follicles of distinct categories in qingjiaoma chicken.1. Effect of inhibin gene immunization on egg production in qingjiaoma chickenTo investigate the effect of inhibin gene immunization on egg production in qingjiaoma chicken, two distinct experiments employing different inhibin vaccines were taken. In experiment1,150adult hens were divided into5groups. The hens were treated with125μg/300μl (T1),75μg/300μl (T2),25μg/300μl (T3) pcISI,25μg pcISI+25μg E. coli DNA/300μl (T4), and300μl saline (T5) as control. Booster immunizations were given with the same dosage at20days’interval. In experiment2,390hens were divided into2groups. The trial hens (T6, n=300) were immunized with1010CFU/300μl attenuated Salmonella of recombinant inhibin plasmid pXAIS, and the controls (T7, n=90) were treated with equal volume of attenuated Salmonella of inhibin-free plasmid pVAX-asd.50days after the primary immunization, hens were sacrificed and ovary samples were collected. Blood and egg samples were collected every10days throughout the experiment to decide the level of antibody against inhibin, also to the egg quality and hatching rate. The results showed that immunization against pcISI could elicit remarkably higher inhibin antibody and promote follicular development; while immunization against the recombinant inhibin bacterica could elicit higher inhibin antibody, but have no effect on egg production performance. Both immunizations had no effects on egg quality and hatching rate, indicating that both vaccines were safe. 2. Cloning of FSHR and LHR genes and the expression of GTHR mRNA within follicles of distinct categories in qingjiaoma chickenAfter inhibin immunization, follicles of distinct categories were collected. Total RNA extracted from follicles and primers designed according to the GTHR mRNA sequences were used for RT-PCR to amplify GTHR mRNA fragment. Positive clones were identified and sequenced. BLAST and DNAman softwares were applied to analyze the homology. β-actin was used as an inner control and an optimal semi-quantitative RT-PCR method was established to analyze GTHR mRNA expression levels in follicles. Sequencing analysis indicated that there was no spliced variant in both FSHR and LHR mRNA. Semi-quantitative RT-PCR results showed that both levels of FSHR and LHR mRNA were higher in SYF than LWF. After inhibin immunization, the level of FSHR mRNA increased in LWF and declined in SYF, while the level of LHR mRNA was opposite.