| There are many eggs with different shell color on the market,and the green shell eggs as one favored by the majority of consumers.Because of the packing characters,the breeders have carried out many researches in the use of green shell egg character and regu Lation mechanism.Currently,the formation mechanism of the green shell characters have been elucidated,the SLCO1B3 gene is the key gene to the green shell character regulation.The expression of the gene will cause bile salts to shell gland,the biliverdin was secreted to the eggshell by the shell gland,forming green shell.The egg shell color shades in the production of the same variety of chickens and the same crowd in the different periods.Even if the SLCO1B3 gene was dominant homozygous genotypes in all of the chickens,the change of the egg shell color remained largely.With the increasing of egg production weeks of the hens,especially after 40 weeks,the color of the green shell eggs gradually became shallow in the Lushi green shell chickens,thus greatly affecting the production efficiency of the enterprise.However,we do not know the reason leading the eggshell color to shoal and the way we deal with the problems? These studies are all very important to theoretical research and production practice.Therefore,in order to reveal the molecular mechanism of the eggshell color became light on the green shell eggs,this project was based on the same batch,good health,feeding and management conditions consistented with Lushi green shell chickens of 20 weeks including 200 individuals as the object of study.By analyzing the change of the eggshell color during the egg production period,the expression level of the SLCO1B3 gene and the methylation level,and analyzing the correlation among them.By adding compound vitamin fodder feeding in the experiment to observe the effects of vitamin to eggshell color changes,to find effective strategies or techniques solving the problem that eggshell color became shallowed.The main results of this study were as follows.1.Variation analysis in different egg production period of the green shell egg color.NR colorimeter of eggshell color was used for quantitative determination ofgreen shell eggs laid by the same batch of 200 chickens in 7 different weeks.Each period was measured for 3 continuous days and 3 points of each egg were measured.Results showed that the average values of eggshell color for each week were 53.53,53.86,56.14,56.59,56.95,56.88,58.57,57.79,and 58.40,respectively.Thus,the egg production period of Lushi chickens can be divided into 3 stages: 20~25 weeks for dark green,26~45 weeks for medium green,46~60 weeks for light green.Variance analysis of different weeks showed that the eggshell color of 20 and 25 weeks were significantly deeper than 30,35,40 and 45 weeks(P<0.05),and the eggshell color of50 and 60 weeks were significantly lighter than the ahead 45 weeks(P<0.05).There are two inflection points in the egg production period of Lushi chickens,which are 25 and 45 weeks.25 weeks in the changed from dark green to green,45 weeks from green to light green.Finally,we can speculate that the value of eggshell color in the whole egg production period mainly concentrated in the range of 50~62 through summarizing the distribution of eggshell color according to the measured data.The results indicated that the eggshell color of Lushi green shell chickens became shallowed gradually with the increasing of the egg production weeks.2.Correlation analysis between the expression of the SLCO1B3 gene and the eggshell color in different egg production period.The shell gland tissue samples of 25,30,35,40,45,50,55 and 60 weeks were used,shell gland tissue was taken from five chickens of each week.By the molecular marker technology of the green shell egg to choose the dominant homozygous genotypes,using q RT-PCR technology to measure the relative expression quantity of SLCO1B3 gene.The results showed that the relative expression level of SLCO1B3 gene of Lushi green shell chicken in 25 and 30 weeks were significantly higher than other laying weeks(P<0.05),and the expression level of 35,40 and 45 weeks were significantly higher than 50,55 and 60 weeks.There were no significant difference in the expression level among 35,40 and 45 weeks,as well as among 50,55 and 60weeks(P﹥0.05).The expression of SLCO1B3 gene in different periods and the color of the eggshell in the corresponding period were analyzed by the Pearson correlation analysis,found they were positively correlated,the correlation coefficien between the expression of SLCO1B3 gene and the value of the eggshell color was-0.729(P<0.05).3.Correlation analysis between the methylation level of the SLCO1B3 gene and expression of the gene and the color of the eggshell.3 individuals were choosed respectively according to the dominant homozygous genotypes in different egg producing period.Bisulphite sequencing technique was used to determination the methylation level of SLCO1B3 gene promoter methylation.The results showed that the promoter whole methylation level of SLCO1B3 gene in the shell gland tissue during 25,30,35,40,45,50,55 and 60 weeks of Lushi green eggshell chicken were 83.93 %,84.37 %,85.27 %、87.76 %,90.82 %,91.67 %,91.07 % and 91.33 %.The results indicated that the whole methylation rate of SLCO1B3 gene in Lushi green eggshell chicken was increased gradually as the increasing weeks of egg production.By Pearson correlation analysis,found the promoter whole methylation of the SLCO1B3 gene and its expression in the shell gland tissue were significant negative correlation,the correlation coefficient was-0.889(P<0.01);the promoter whole methylation level of the SLCO1B3 gene and the color of the eggshell were significant negative correlation,the correlation coefficient between the methylation level and the value of eggshell color was 0.807(P<0.05).Analysis the correlation of each island by the Pearson correlation analysis,we found significant negative correlation between the methylation level of Cp G1,Cp G5,Cp G8 islands and the expression of SLCO1B3 gene(P<0.05),the correlation coefficients were-0.708,-0.779 and-0.709.And significant negative correlation between the methylation level of Cp G1,Cp G5,Cp G8 islands and the eggshell color,the correlation coefficients between the methylation level of the three islands and the value of eggshell color were 0.775(P<0.05),0.710(P<0.05),0.871(P<0.01).Bioinformatics analysis found the transcription factor Sp1 and Oct-1A were respectively in the Cp G5 island and Cp G8 island,so we speculate that the methylation of the two islands may block the combination of transcription factors and the promoter,the gene expression decreased in the later stage of laying cycle as the improvement of methylation level,thus the eggshell color became shallow.4.The influence to the eggshell color of multivitamin in Lushi green eggshell chicken.The chicken flocks were randomly divided into two groups at 43 weeks.The experimental group was fed regular diet containing multivitamin,the control group was fed with regular diet.The eggshell color value in two groups were detected respectively in the 45,50,55 and 60 weeks,and the shell gland tissue samples were taken from five chickens of each week.By the molecular marker technology of the green shell egg to choose the dominant homozygous genotypes,using q RT-PCR technology to measure the relative expression quantity of SLCO1B3 gene.The resultsshowed that the eggshell color of experimental group and control group in 45 and 50 weeks had no significant difference,the eggshell color of experimental group significantly deeper than the control group in 55 weeks(P<0.05),the eggshell color of experimental group deeper than the control group in 60 weeks,but had no significant difference between them.The results indicated that the diet was added multivitamin during the later stage of laying cycle could remission the eggshell shallow.In conclusion,the green eggshell color presented lighter along with increasing of egg production weeks in Lushi green eggshell chicken.The expression of the SLCO1B3 gene was decreased,the whole methylation rate of SLCO1B3 gene was increased gradually;Bioinformatics analysis found the transcription factor Sp1 and Oct-1A were respectively in the Cp G5 island and Cp G8 island,so we speculate that the methylation of the two islands may block the combination of transcription factors and the promoter,the gene expression decreased in the later stage of laying cycle as the improvement of methylation level,thus the eggshell color became shallow;the diet was added multivitamin during the later stage of laying cycle could remission the eggshell shallow. |
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