Expression characteristics of ADSL gene in pectoral muscle tissue of Chishui black-bone chicken and the construction of ADSL gene interference vector
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摘要: 【目的】探究赤水乌骨鸡胸肌组织中腺苷琥珀酸裂解酶基因(ADSL)表达特征及其目的基因的干扰载体构建,为深入研究畜禽组织肌肉中肌苷酸(IMP)的表达规律及ADSL基因影响畜禽风味的机制提供理论参考。【方法】以不同月龄(3、4、5和6月龄)的赤水乌骨鸡(公鸡和母鸡各半)为试验材料,运用实时荧光定量PCR检测不同月龄及不同性别赤水乌骨鸡胸肌组织中ADSL基因的表达情况;同时根据GenBank中鸡ADSL基因mRNA序列(NM_205529.2)设计4对短发夹RNA(shRNA)干扰序列(sh1-ADSL、sh2-ADSL、sh3-ADSL和sh4-ADSL)和1对阴性对照序列(sh-NC)与pGPH1/GFP/Neo载体连接后进行测序,将构建成功的ADSL基因干扰载体转染至成肌细胞,检测并筛选出干扰效率最高的干扰载体。【结果】ADSL基因在不同月龄赤水乌骨鸡胸肌组织中的相对表达量排序为5月龄>4月龄>6月龄>3月龄,赤水乌骨母鸡3月龄母鸡的ADSL基因相对表达量极显著高于公鸡(P<0.01,下同),4月龄母鸡ADSL基因的相对表达量显著高于公鸡(P<0.05),6月龄母鸡ADSL基因的相对表达量极显著低于公鸡;ADSL基因质粒的测序结果显示干扰质粒构建成功,质粒转染后干扰组及阴性对照均可见绿色荧光,空白对照不发光,说明各重组质粒成功转染赤水乌骨鸡成肌细胞;ADSL基因沉默结果显示,sh2-ADSL沉默效果最佳,干扰效率为90.30%。【结论】赤水乌骨鸡胸肌中ADSL基因的相对表达量随着月龄增加表现为先上升后下降的变化趋势;3、4和5月龄赤水乌骨母鸡ADSL基因的相对表达量高于公鸡,随着月龄增加ADSL基因的相对表达量增加,公鸡和母鸡间的差异减小。成功分离提取赤水乌骨鸡成肌细胞,并筛选出RNA干扰效果最佳载体sh2-ADSL,为后续研究ADSL基因调控鸡肉肌苷酸及风味的分子机制提供参考。Abstract: 【Objective】This experiment was carried out to investigate the expression characteristics of adenylosuccinate lyase gene(ADSL) in the pectoral muscle tissue of Chishui black-bone chicken and to construct the interference vector of the target gene,.which provided a theoretical reference for an in-depth study of the expression pattern of inosine monophosphate acid(IMP) in the muscles of livestock tissues and the mechanism by which the ADSL gene affected the flavour of livestock and poultry. 【Method】Chishui black-bone chicken(half male and half female) of different ages(3, 4, 5 and 6 months old) were used as experimental materials. The expression of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken at different ages was detected by real-time fluorescence quantitative PCR. Meanwhile,four pairs of shorthairpinRNA(shRNA) interference sequences(sh1-ADSL,sh2-ADSL, sh3-ADSL and sh4-ADSL) and one pair of negative control sequences(sh-NC) were designed according to the mRNA sequence of chicken ADSL gene in GenBank(NM_205529.2). They were sequenced after ligated with pGPH1/GFP/Neo vector. The constructed ADSL gene interfering vectors were transfected into myoblasts, and the most efficient interference vector was detected and screened.【Result】The relative expression level of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken at different months age was in the order of 5 months>4 months>6 months>3 months. The relative expression level of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken was extremely significantly higher in 3-month-old females than in males(P<0.01, the same bellow). The relative expression level of ADSL gene was significantly higher in 4-monthold females than in males(P<0.05). The relative expression level of ADSL gene was extremely significant lower in 6-month-old females than in males. The sequencing results of the ADSL plasmid showed that the interference plasmid was successfully constructed and the green fluoresce was observed in the interference group and the negative control after plasmid transfection,and the blank control showed no fluoresce, indicating that each recombinant plasmid successfully transfected the myoblasts of Chishui black-bone chicken. The ADSL gene silencing results showed that sh2-ADSL silencing effect was the optimal, with an interference efficiency of 90.30%. 【Conclusion】The relative expression level of ADSL gene shows a trend of increasing and then decreasing with the increase of month age. The relative expression level of ADSL gene in the pectoral muscle tissue of females at 3, 4 and 5 months old higher than that of males. The relative expression level of ADSL genes increases with increasing month age, and the differences between males and females decrease.The experiment is successful in isolating and extracting myoblast from Chishui black-bone chicken, and screening out the sh2-ADSL vector with the best RNA interference effect. This study provides a reference for further research on the molecular mechanism of ADSL gene regulation of meat inosinate and flavor.
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Keywords:
- Chishui black-boned chicken /
- ADSL gene /
- tissue expression /
- myoblast /
- interference vectors /
- meat flavor
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曹少奇,张梅,张苗苗,赵宗胜,黄涛,李岩,孙杰,张金生,李坚,廖和荣.2019.巨型玫瑰冠鸡ADSL基因表达及其与IMP的相关分析[J].中国家禽,41(6):46-48.[Cao S Q,Zhang M,Zhang M M,Zhao Z S,Huang T,Li Y,Sun J,Zhang J S,Li J,Liao H R.2019.Expression of ADSL gene in giant rose-crowned chickens and its correlation analysis with IMP[J].China Poultry,41(6):46-48.]doi: 10.16372/j.issn.1004-6364.2019.06.010. 常山,李小成,徐亚欧,杜华锐,曹特,辅宏璞,于春林,蒋小松.2008.优质肉鸡腺苷琥珀酸裂解酶基因第2和第9外显子单核苷酸多态性分析[J].四川畜牧兽医,(6):25-28.[Chang S,Li S C,Xu Y O,Du H R,Cao T,Fu H P,Yu C L,Jiang X S.2008.Single nucleotide polymorphism analysis of exons 2 and 9 of adenosuccinate lyase gene in high-quality broiler chickens[J].Sichuan Animal&Veteri-nary Sciences,(6):25-28.]doi: 10.3969/j.issn.1001-8964.2008.06.017. 豆腾飞,汪善荣,佟荟全,刘丽仙,谷大海,徐志强,李琦华,贾俊静,葛长荣.2017.武定鸡和大围山微型鸡肌苷酸含量及ADSL基因表达差异研究[J].中国家禽,39(14):7-10.[Dou T F,Wang S R,Tong Q H,Liu L X,Gu D H,Xu ZQ,Li Q H,Jia J J,Ge C R.2017.Study on inosinic acid content and ADSL gene expression in Wuding chicken and Daweishan mini chicken[J].China Poultry,39(14):7-10.]doi: 10.16372/j.issn.1004-6364.2017.14.002. 龚琳琳.2011.鸡ADSL和GARS-AIRS-GART基因遗传变异、表达及其与肌苷酸的相关分析[D].扬州:扬州大学.[Gong L L.2011.Genetic variation and expression of ADSL and GARS-AIRS-GART genes and their associations with meat quality in chicken[D].Yangzhou:Yangzhou University.]doi: 10.7666/d.Y2050127. 虎红红,黄增文,母童,蔡正云,冯小芳,马正旭,顾亚玲,辛国省,国鹏,张娟.2020.静原鸡肌苷酸沉积与ADSL基因mRNA表达量相关性分析[J].基因组学与应用生物学,39(12):5442-5448.[Hu H H,Huang Z W,Mu T,Cai ZY,Feng X F,Ma Z X,Gu Y L,Xin G S,Guo P,Zhang J.2020.Correlation analysis between inosine deposition and ADSL gene mRNA expression in Jingyuan chicken[J].Genomics and Applied Biology,39(12):5442-5448.]doi: 10.13417/j.gab.039.005442. 黄正洋,王钱保,黄华云,李春苗,穆春宇,黎寿丰,赵振华.2022.鸡let-7b在不同生长时期及不同组织中的表达及其生物信息学分析与靶基因预测[J].江苏农业学报,38(2):429-437.[Hang Z Y,Wang Q B,Hang H Y,Li CM,Mu C Y,Li S F,Zhao Z H.2022.Expression of let-7b in different growth stages and different tissues of chicken and its bioinformatics analysis and target gene prediction[J].Jiangsu Journal of Agricultural Sciences,38(2):429-437.]doi: 10.3969/j.issn.1000-4440.2022.02.017. 季从亮.2005.鸡肉IMP含量相关候选基因SNP筛查及其用于地方鸡种群体遗传结构分析的研究[D].扬州:扬州大学.[Ji C L.2005.SNP screening in the related candidate gene for IMP contents of chicken and its application in assessment of genetic structure of indigenous chicken breeds[D].Yangzhou:Yangzhou University.]doi: 10.7666/d.Y747795. 金睿,曹满湖,黄泰来,温建崇.2021.鸡肉肌苷酸含量影响因素研究进展[J].中国家禽,43(10):87-93.[Jin R,Cao MH,Huang T L,Wen J C.2021.Research progress on influencing factors of inosinic acid content in chicken[J].China Poultry,43(10):87-93.]doi: 10.16372/j.issn.1004-6364.2021.10.013. 李庆海,范京辉,楼立峰,张伟武,张雷,王欢欢,李祥,章学东.2013.饲粮能量差异对猪ADSL基因mRNA表达的影[J].养猪,(6):57-59.[Li Q H,Fan J H,Lou L F,Zhang W W,Zhang L,Wang H H,Li X,Zhang X D.2013.Effect of dietary energy differences on mRNA expression of ADSL gene in pigs[J].Swine Production,(6):57-59.]doi: 10.13257/j.cnk0i.21-1104/s.2013.06.032. 刘基伟,徐日福,李奎,白丽景,应正宙,唐中林,杨述林.2010.猪ADSL基因克隆及其在部分组织中的mRNA定量表达[J].畜牧与兽医,42(6):22-27.[Liu J W,Xu R F,Li K,Bai L J,Ying Z Z,Tang Z L,Yang S L.2010.Molecular cloning and mRNA expression of ADSL gene in pig tissues[J].Animal Husbandry&Veterinary Medicine,42(6):22-27.] 刘长青,刘帅,包阿东,陆涛峰,吴宏梅,张洪海,唐学玺,关伟军,马月辉.2008.北京油鸡ADSL基因的克隆、表达及其结构与功能分析[J].动物学研究,(4):353-362.[Liu CQ,Liu S,Bao A D,Lu T F,Wu H M,Zhang H H,Tang XX,Guan W J,Ma Y H.2008.Molecular clone,expression,structure and function study of Beijing fatty chicken ADSLgene[J].Zoological Research,(4):353-362.]doi: 10.3321/j.issn:0254-5853.2008.04.001. 路宏朝,张涛,王令.2016.不同日龄略阳乌鸡肌肉中肌苷酸含量的变化规律[J].江苏农业科学,44(12):277-280.[Lu H Z,Zhang T,Wang L.2016.Changes of inosinic acid content in muscle of Luyang’s chicken at different ages[J].Jiangsu Agricultural Science,44(12):277-280.]doi: 10.15889/j.issn.1002-1302.2016.12.085. 栾德琴.2012.鸡肌肉生长相关基因的表达与肌苷酸关键酶基因网络调控的构建[D].扬州:扬州大学.[Luan D Q.2012.Analysis of gene expression profiles and construc-tion of network for key genes associated with inosine monophosphate in chicken muscles[D].Yangzhou:Yang-zhou University.]doi: 10.7666/d.Y2258561. 潘爱銮,吴艳,申杰,皮劲松,杨林,蒲跃进,梁振华,孙静,张昊,杜金平.2015.不同鸡品种肉质性状比较分析[J].湖北农业科学,54(23):5966-5968.[Pan A L,Wu Y,Shen J,Pi J S,Yang L,Pu Y J,Liang Z H,Sun J,Zhang H,Du JP.2015.Comparative analysis on muscle characteristics of different breed chicken[J].Hubei Agricultural Sciences,54(23):5966-5968.]doi: 10.14088/j.cnki.issn0439-8114.2015.23.044. 石浩,徐亚欧,梅寒,王海,刘红丽,左斌,聂晓庆,胡卫民.2015.米易鸡ADSL基因第2和第9外显子多态性与肌苷酸含量的关联[J].江苏农业科学,43(5):29-32.[Shi H,Xu Y O,Mei H,Wang H,Liu H L,Zuo B,Nie X Q,Hu WM.2015.Association between exons 2 and 9 polymor-phisms of ADSL gene and inosinate content in Miyi chicken[J].Jiangsu Agricultural Sciences,43(5):29-32.]doi: 10.15889/j.issn.1002-1302.2015.05.008. 王红娜,张英杰,刘月琴.2016.绵羊成肌细胞的纯化、培养、鉴定及其成肌诱导分化研究[J].河北农业大学学报,39(1):94-98.[Wang H N,Zhang Y J,Liu Y Q.2016.Astudy of purification,culture,identification and differen-tiation of sheep myoblast cells[J].Journal of Hebei Agri-cultural University,39(1):94-98.]doi: 10.13320/j.cnki.jauh.2016.0016. 王红杨.2015.胚胎期至生长早期鸡肌肉发育及肌内脂肪沉积蛋白质组研究[D].北京:中国农业科学院.[Wang HY.2015.The proteomic study of muscle development and intramuscular fat deposition of chickens at embryonic and early growth stages[D].Beijing:Chinese Academy of Agri-cultural Sciences.]doi: 10.7666/d.Y2787516. 王珏,樊艳凤,唐修君,马丽娜,葛庆联,陆俊贤,杨星星,丁红梅,高玉时,龚道清.2020.不同品种肉鸡屠宰性能及肌肉品质的比较分析[J].中国家禽,42(7):13-17.[Wang J,Fan Y F,Tang X J,Ma L N,Ge Q L,Lu J X,Yang X X,Ding H M,Gao Y S,Gong D Q.2020.Comparative analy-sis on slaughter characters and meat quality in different breeds of broilers[J].China Poultry,42(7):13-17.]doi: 10.16372/j.issn.1004-6364.2020.07.003. 吴琼,杨福合,邢秀梅.2012.中国环颈雉与其它禽类肌肉肌内脂肪含量比较分析[J].特产研究,34(4):1-3.[Wu Q,Yang F H,Xing X M.2012.Intramuscular fat content(IMF) analysis in Chinese ring-necked pheasants and other avians species[J].Special Wild Economic Animal and Plant Research,34(4):1-3.]doi: 10.16720/j.cnki.tcyj.2012.04.020. 吴玉麟.2021.和盈黑鸡PHGDH基因对肉用性状和成肌细胞增殖分化的影响[D].扬州:扬州大学.[Wu Y L.2021.Effect of PHGDH gene on meat traits and prolifera-tion and differentiation of myoblasts cells in Heying black chicken[D].Yangzhou:Yangzhou University.]doi: 10.27441/d.cnki.gyzdu.2021.001296. 夏婷婷.2014.巢湖鸭群体遗传多样性和IMP含量与ADSL基因多态关联分析[D].合肥:安徽农业大学.[Xia T T.2014.Analysis of population genetie diversity and the association of IMP contents with ADSL gene polymor-phism in Chaohu duck[D].Hefei:Anhui Agricultural University.] 徐善金.2011.鸭ADSL与PurH基因序列特征及表达与肌肉肌苷酸(IMP)含量的相关性分析[D].南京:南京农业大学.[Xu S J.2011.Analysing seouence characters of ADSL and PurH gene and correlation berweengenes expression and IMP content in duck[D].Nanjing:Nan-jing Agricultural University.] 张梅.2018.玫瑰冠鸡的冠型鉴定及FABP和ADSL基因与肉品质的关联分析[D].石河子:石河子大学.[Zhang M.2018.Association analysis of FABP and ADSL genes and meat quality coronary identification of Rose crowned chickens[D].Shihezi:Shihezi University.] Imaeda A,Tomoike F,Hayakawa M,Nakamoto K,Kimura Y,Abe N,Abe H.2019.N6-methyl adenosine in siRNAevades immune response without reducing RNAi activity[J].Nucleosides,Nucleotides&Nucleic Acids,38(12):972-979.doi: 10.1080/15257770.2019.1641205.
Kmoch S,HartmannováH,StibůrkováB,Krijt J,ZikánováM,Sebesta I.2000.Human adenylosuccinatelyase (ADSL),cloning and characterization of full-length cDNA and its isoform,gene structure and molecular basis for ADSLdeficiency in six patients[J].Human Molecular Genetics,9(10):1501-1513.doi: 10.1093/hmg/9.10.1501.
Mao H G,Cao H Y,Liu H H,Dong X Y,Xu N Y,Yin Z Z.2018.Association of ADSL gene polymorphisms with meat quality and carcass traits in domestic pigeon(Columba livia)[J].British Poultry Science,59(5):604-607.doi: 10.1080/00071668.2018.1493188.
Shu J T,Bao W B,Zhang X Y,Ji C J,Han W,Chen K W.2009.Combined effect of mutations in ADSL and GARS-AIRS-GART genes on IMP content in chickens[J].British Poultry Science,50(6):680-686.doi: 10.1080/00071660903391709.
Tsai M,Koo J,Yip P,Colman R F,Segall M L,Howell P.L.2007.Substrate and product complexes of Escherichia coli adenylosuccinate lyase provide new insights into the enzymatic mechanism[J].Journal of Molecular Biology,370(3):541-554.doi: 10.1016/j.jmb.2007.04.052.
Wang T,Wiater E,Zhang X M,Thomas J B,Montminy M.2021.Crtc modulates fasting programs associated with 1-C metabolism and inhibition of insulin signaling[J].Pro-ceedings of the National Academy of Sciences of the United States of America,118(12):e2024865118.doi: 10.1073/pnas.2024865118.
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