| Marek’s disease (MD) is an immune suppressive, malignant T lymph proliferative disease to chicken which caused by Marek’s disease virus (MDV). After chicken infected by MDV, it can produce serious interference to the protective effect of vaccine and vulnerable to secondary infection caused by other viral and bacterial diseases, finally brings huge economic losses to the poultry industry of countries and regions in the world. The Guangxi Three-Yellow chicken is a local variety with excellent production performance and economic value. The Marek’s disease epidemiological investigations to Guangxi Three-Yellow chicken carried out by our laboratory in recent years showed that even if the flocks had been immunized by the vaccine of CVI998/Rispens and814, MD still occurred. Therefore, in addition to taking effective immune prevention and control, as well as bio-security measures to the flocks, breeding for disease resistance measures should be taken out to improve the nature of flocks’ genetic resistance to MD. Our topic had found that BF gene alternative spliced exon7is related with MD resistance.This topic first used the rapid detection of BF gene alternative spliced exon7technology that had been established by our laboratory to filter the cocks’BF gene exon7from parents’ generation, and after that we got three groups of offspring through breeding:K group (the cocks’BF gene with exon7from parents’generation; Y group (the cocks’BF gene without exon7from parents’ generation); P group (the cocks’BF gene exon7from parents’generation unfiltered),100birds per group. The experiment chickens were challenged with MDV-1virulent strain120015via intraperitoneal injection (500PFU,0.2mL each). Experimental chickens raised10weeks by conventional methods after challenged, and then killed all the survival chickens. The mortalities during the experiment and the survivals at the end of experiment were dissected to observe the neoplastic lesions, and then collected lesions to detect MDV by PCR. The results showed that K, Y and P groups of tumor incidence and mortality rates were32.97%.52.75%;53.85%、71.43%;42.39%,63.04%, had significant difference between the groups (p<0.05); the detecting positive rates of BF gene exon7for K、Y and P groups were42.86%(39/91),19.57%(18/92) and30.77%(28/91), combined with tumor incidence for the Chi-square test showed that Chi-square value and P value for each group were9.548%0.002;6.907.a、0.009;4.866a,0.027. This suggested that the tumors whether occur is highly related with exon7for the three experiment groups (p<0.05). The tumor incidence for the entire BF gene with exon7and without exon7samples from three groups of experiment chickens were22.35%(19/85) and52.38%(99/189), had significant difference (p<0.05); Secondly,20samples of BF gene with exon7and10samples of BF gene without exon7were cloned and sequenced. The analysis of sequences alignment and genetic evolution with MD resistant haploid (B2, B5, B12, B21) and susceptible haploid (B19, B15, B13,) showed that we got ten MD resistant samples from20BF gene with exon7and four MD susceptible samples from10BF gene without exon7. Furthermore, the MD resistant and susceptible individuals have a significant difference in the3and87amino acid sites:susceptible is proline and serine, resistant is serine and arginine; thirdly, the plasmid standards and real-time quantitative PCR standard curves were prepared and constructed for reference gene β-actin and BF gene alternative spliced exon7. The BF gene alternative spliced exon7mRNA (cytoplasmic tail containing exon7or without exon7) expression levels were detected at different phases after MD challenged via the established real-time quantitative PCR technology. The statistics analysis showed that the mRNA expression level of BF gene with exon7and without exon7was not significantly different at4DPI (p=0.6377>0.05); significantly different at7DPI (p=0.0443<0.05) and highly significantly different at14,21,28DPI (p<0.01). The BF gene with exon7mNRA expression levels had maintained a high level during7-28days after MDV challenged, but the BF gene without exon7mRNA expression level decreased significantly.The fourth experiment, a new PCR rapid detection of BF gene alternative spliced exon7technology had been established,300chicken BF gene samples were detected by this method, sequencing the samples that did not match with the old Semi-nested PCR detection results, confirmed that the accuracy of the new method higher, as well as, the detection time is much shorter, could be applied in the actual operation of MD resistance breeding.This issue through animal challenge experiment confirmed the Guangxi Three-Yellow chicken BF gene exon7sequence features consistent with previously reported blood type resistance haplotypes and significantly associated with anti-MD tumor; Simple, rapid method of breeding carried out by selecting the BF gene exon7of the cock can obtain offspring that significantly resist to MD; BF gene with exon7and without exon7mRNA expression levels had significant difference after MD challenged further to determine it is MD resistance related gene. |
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