CN103233002A - 一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点及应用 - Google Patents
一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点及应用 Download PDFInfo
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Abstract
一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点及应用，属于遗传学技术领域，本发明提供一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点---脂肪酸去饱和酶基因（fattyaciddesaturase1,FADS1）位点。即含有rs174556单核苷酸多态性的FADS1位点。由于该位点的单核苷酸多态性rs174556碱基Ｃ突变为T，从而与乳汁多不饱和脂肪酸水平密切相关联。FADS1基因rs174556T/T次等位基因纯合子携带者乳母乳汁花生四烯酸及二十碳五烯酸水平显著降低，该位点存在决定乳汁多不饱和脂肪酸水平的单核甘酸多态性。FADS1位点及其编码蛋白对阐明多不饱和脂肪酸代谢机制及建立个体化营养干预，建立针对不同遗传背景而确定适宜的多不饱和脂肪酸膳食推荐摄入量具有重要作用。
Description
技术领域
本发明属于遗传学技术领域，涉及一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点，和检测所述基因位点的方法，以及所述基因位点的的用途。
背景技术
孕妇及母乳的营养是保证胎婴儿健康的关键。母乳中的脂肪酸不仅是婴儿重要的能量来源，而且长链多不饱和脂肪酸（LCPUFA）与婴儿的视功能和脑发育密切相关。n-3系列LCPUFA主要为二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）， n-6系列LCPUFA主要为亚油酸和花生四烯酸（AA）。AA、EPA及DHA是最重要的三种LCPUFA，是人类早期发育的必需脂肪酸。在孕末期和新生儿出生的头几个月，脑细胞数量迅速增加，体积增大，为满足这一时期生长的需要，必须供给充足的营养物质。由于婴儿组织和器官的生长发育旺盛，大部分生理功能快速完善，对多不饱和脂肪酸的需求量也较大，而胚胎期的储备通常较少，新生儿，尤其是早产儿，体内的脂肪酸去饱和酶活性较低，由α-亚麻酸合成EPA 和DHA 的能力低。大部分EPA和DHA都是通过乳汁或奶粉摄入。孕妇及乳母PUFA的膳食推荐摄入量一直是国际研究的热点，但以往的研究大多集中在孕妇及乳母膳食的变化对其血浆及乳汁中PUFA水平的影响，没有考虑到个体的遗传因素差异。FADS1位于人类11号染色体（11q12-11q13.1），由12个外显子和11个内含子构成，△5脂肪酸去饱和酶由FADS1编码，是多不饱和脂肪酸代谢通路上重要的限速酶。去饱和酶的活性直接影响PUFA合成。第一篇报道FADS基因多态性影响血清磷脂脂肪酸水平是在2006年(Schaeffer L, Gohlke H et al.
Hum Mol Genet 2006;15:1745-1756)。Innis Sheila 研究小组于2008年发现 FADS多态性影响孕妇血浆和红细胞膜磷脂以及乳母乳汁中的n-6和n-3多不饱和脂肪酸水平，（Lin Xie, Sheila M. Innis. J Nutr. 2008,
138(11):2222-8）。而母亲乳汁中的脂肪酸水平通过母婴传递途径直接影响婴儿的脂肪酸营养状况。此后的多项研究分别探讨了不同国家不同人群的FADS基因多态性对血液、乳汁及组织中脂肪酸水平的影响。目前的研究主要集中在白种人群，中国孕妇及乳母PUFA水平是否也受FADS基因多态性的影响尚未见报道。
发明内容
本发明要解决的技术问题是公开一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点。
本发明同时公开了检测所述基因位点的方法。
本发明还公开了所述基因位点的用途。
本发明通过NCBI(http://www.ncbi.nlm.nih.gov/mapviewer)数据库，获得FADS1基因位点序列。通过http://www.ncbi.nlm.nih.gov/SNP及http://snp.cshl.org/数据库，在FADS1基因上选择含有限制性酶切位点的单核苷酸多态性位点rs174556。下列所示为这个单核苷酸多态性的核苷酸序列。加黑部分为引物序列，方框内是发生互换的碱基，划线处则是MboI限制性内切酶识别的酶切位点，箭头所示内切酶的切割部位。具体信息如下。
以中国东北地区115个汉族健康乳母为研究对象。乳母均由外周静脉抽取抗凝血5ml，用Wizard®Genomic DNA
Purification Kit试剂盒提取全血基因组DNA。步骤如下：1.5ml Eppendorf离心管中加入细胞裂解液900μl，加入融化的全血300μl吹打混匀，室温反应20min，以13000 rpm离心3min，吸弃上清，加入300μl核裂解液，充分振荡反复吹打至团块溶解，37℃水浴30min，加1.5μl RNase充分混匀，37℃水浴15min，加入100μl蛋白沉淀液，充分混匀，室温反应20min，以13000
rpm离心3min，将上清移至加入300μl异丙醇的离心管中，反复颠倒混匀，室温反应15min，以13000 rpm离心3min，轻轻弃上清，留白色沉淀，用吸水纸吸干，加70%乙醇200μl洗涤1次，离心轻轻弃上清，用吸水纸吸干，室温干燥20min，加100μl DNA溶解液，65℃水浴2h，制成DNA样品，于-20℃冰箱保存。使用美国BECKMAN公司的Du®640紫外分光光度仪，分别取波长260nm、280nm 两个波段，测得其相应DNA的OD260nm及OD280nm值，然后再通过公式，计算出DNA含量，公式为：DNA含量=50mg/ml×OD260nm×稀释倍数，另外通过公式计算出DNA纯度，公式为：DNA纯度=OD260nm/OD280nm。依据相应的碱基序列合成引物。正向引物为：5’ AAGCAGGGACCTCAAGAC 3’，反向引物为：5’ AGCCCACCAAGAATGTAA 3’。PCR反应体系为20μl，其中含上下游引物各0.4μM， 8μl 2×Taq MasterMix ，DNA模板30∼50ng。PCR扩增条件为：①94℃ 5min ②96℃ 30s，62℃ 60s，72℃ 35s 2个循环；③95℃ 30s，60℃60s，72℃ 35s 6个循环；④94℃ 55s，58℃ 60s，72℃ 35s 28个循环；⑤72℃10min。取PCR产物5μl用1.5%琼脂糖凝胶电泳，以DL2000为分子量标准品。所扩增的片段长度为153bp（6886371～6886683）。在PCR扩增后的反应体系中，加入适量的限制性内切酶MboI及其酶切缓冲液，置37℃温箱5小时。经2.5%琼脂糖凝胶电泳，根据酶切图谱判断基因型。存在3种基因型，其中有完全切开的纯合基因型，未切开的纯合基因型，既有切开又有未切开的杂合基因型。
检测乳汁中8种多不饱和脂肪酸水平，步骤如下：向10ml玻璃离心管中加入200μL乳汁、33μL十七烷酸（C17:0）内标物、2ml甲醇-苯（4:1）溶液。振荡使其充分混匀，1min后缓慢加入200μL乙酰氯溶液，边加边摇匀。然后以Teflon膜做内衬，旋紧试管螺旋盖。将试管于100℃油浴中保温1h。待离心管冷却后，缓慢加入5ml 6%的K2CO3溶液，以终止反应，中和混合物。振荡混匀，于3000r/min条件下离心10min。小心吸取上层苯相液于1.5mL
EP管中用于气相色谱分析。应用岛津GC-14C型气相色谱仪和100m×0.25mm×0.20μm色谱柱。初始柱温140℃；体积流速1.76ml/min；线速度为26.8cm/s；分流比30:1。
实验结果发现rs174556的主等位基因是C，次等位基因是T，次等位基因频率为31.3%。该SNP的基因型分布符合Hardy-Weinberg 平衡(见表1)。建立SPSS数据库，利用SPSS16.0软件分析数据。应用方差分析及Kruskal-Walis H检验比较不同基因型的乳母膳食及乳汁中多不饱和脂肪酸水平。结果发现不同基因型的乳母膳食中五种脂肪酸：亚油酸（18:2
n-6），α 亚麻酸（18:3
n-3），花生四烯酸（20:4 n-6），二十碳五烯酸（20:5
n-3）和二十二碳六烯酸（22:6 n-3）水平没有显著性差异（P>0.05），见表2。FADS1基因rs174556 T/T次等位基因纯合子携带者乳母乳汁花生四烯酸水平显著降低（P=0.046），其它基因型携带者乳母乳汁中的n-6系列脂肪酸水平没有显著性差异，见表3。FADS1基因rs174556 T/T次等位基因纯合子携带者乳母乳汁二十碳五烯酸水平显著降低（P=0.039），其它基因型携带者乳母乳汁中的n-3系列脂肪酸水平没有显著性差异，见表4。综合分析以上结果，CC，CT及TT基因型的乳母膳食中花生四烯酸和二十碳五烯酸水平没有差异（P>0.05），而此等位基因纯合子TT携带者乳母乳汁中花生四烯酸及二十碳五烯酸水平显著降低（P=0.046，P=0.039）。说明携带TT基因型的乳母乳汁中这两种多不饱和脂肪酸水平较低，需要从膳食中更多地摄入富含这两种多不饱和脂肪酸的食物来补充乳汁中的花生四烯酸和二十碳五烯酸含量，以满足婴儿的正常发育。
根据本发明，可对健康乳母进行如下所示方法的筛查，rs174556基因型为TT的个体为乳汁中花生四烯酸和二十碳五烯酸水平易缺乏人群。对此类基因型携带者乳母在日常生活中应加大富含花生四烯酸和二十碳五烯酸的食物摄入，如这些乳母的婴儿为早产儿或低体重儿，应密切监测婴儿的喂养方式及多不饱和脂肪酸的摄入水平，以促进婴儿的生长发育和大脑及视力发育。这是本发明的一个重要用途。
表1 FADS1 SNP rs174556 的基本特点和基因分型结果
表2乳母FADS1基因不同基因型膳食n-6 和 n-3脂肪酸水平比较
表3乳母FADS1基因不同基因型乳汁n-6脂肪酸水平比较
表4乳母FADS1基因不同基因型乳汁n-3脂肪酸水平比较
表1：FADS1
SNP 的基本特点和基因分型结果
表2乳母FADS1基因不同基因型膳食n-6 和 n-3脂肪酸水平比较（Mean±SD）
LA：亚油酸，ALA：α 亚麻酸，AA：花生四烯酸， EPA：二十碳五烯酸，
DHA：二十二碳六烯酸，*M±Q
表3乳母FADS1基因不同基因型乳汁n-6脂肪酸水平比较(Mean±SD，mg/ml)
| 乳汁脂肪酸 | C/C | C/T | T/T | P |
| LA | 1.387±0.868 | 1.131±0.581 | 1.122±0.818 | 0.461 |
| GLA | 0.191±0.169 | 0.127±0.066 | 0.131±0.063 | 0.148 |
| DGLA | 0.224±0.140 | 0.187±0.101 | 0.150±0.110 | 0.209 |
| AA | 0.335±0.200 | 0.239±0.143 | 0.227±0.177 | 0.046 |
| C22:4 | 0.076±0.049 | 0.061±0.032 | 0.050±0.034 | 0.135 |
| n-6 | 2.212±1.315 | 1.745±0.864 | 1.680±1.161 | 0.208 |
LA：亚油酸，GLA：γ-亚麻酸，DGLA：二高γ-亚麻酸，AA：花生四烯酸，
C22:4：二十二碳四烯酸， n-6：n-6脂肪酸
表4乳母FADS1基因不同基因型乳汁n-3脂肪酸水平比较(Mean±SD，mg/ml)
| 乳汁脂肪酸 | C/C | C/T | T/T | P |
| ALA | 0.644±0.442 | 0.519±0.366 | 0.431±0.301 | 0.242 |
| EPA | 0.041±0.029 | 0.026±0.016 | 0.036±0.026 | 0.039 |
| DHA | 0.224±0.152 | 0.155±0.086 | 0.157±0.125 | 0.110 |
| EPA+DHA | 0.265±0.176 | 0.181±0.097 | 0.192±0.148 | 0.082 |
| n-3 | 0.909±0.590 | 0.701±0.435 | 0.623±0.383 | 0.238 |
ALA：α 亚麻酸，EPA：二十碳五烯酸， DHA：二十二碳六烯酸， n-3：n-3脂肪酸
根据本发明，对于纯母乳喂养婴儿，基因型为TT的乳母及她们的婴儿需提高花生四烯酸和二十碳五烯酸这两种多不饱和脂肪酸的推荐摄入量，做到有针对性地进行营养干预。正因为FADS1
rs174556位点的碱基突变，可能造成脂肪酸去饱和酶活性降低，从而影响多不饱和脂肪酸代谢。本发明为开展基于遗传学背景的个体化营养干预提供了十分重要的实验依据，可促进婴儿的健康生长，并将带来十分可观的社会与经济效益。
具体实施方式
下面实施例是对本发明的进一步解释。
受试者均由外周静脉抽取抗凝血5ml，用Wizard®Genomic DNA
Purification Kit试剂盒提取全血基因组DNA。步骤如下：1.5ml Eppendorf离心管中加入细胞裂解液900μl，加入融化的全血300μl吹打混匀，室温反应20min，以13000 rpm离心3min，吸弃上清，加入300μl核裂解液，充分振荡反复吹打至团块溶解，37℃水浴30min，加1.5μl RNase充分混匀，37℃水浴15min，加入100μl蛋白沉淀液，充分混匀，室温反应20min，以13000
rpm离心3min，将上清移至加入300μl异丙醇的离心管中，反复颠倒混匀，室温反应15min，以13000 rpm离心3min，轻轻弃上清，留白色沉淀，用吸水纸吸干，加70%乙醇200μl洗涤1次，离心轻轻弃上清，用吸水纸吸干，室温干燥20min，加100μl DNA溶解液，65℃水浴2h，制成DNA样品，于-20℃冰箱保存。使用美国BECKMAN公司的Du®640紫外分光光度仪，分别取波长260nm、280nm 两个波段，测得其相应DNA的OD260nm及OD280nm值，然后再通过公式，计算出DNA含量，公式为：DNA含量=50mg/ml×OD260nm×稀释倍数，另外通过公式计算出DNA纯度，公式为：DNA纯度=OD260nm/OD280nm。
应用引物5’
AAGCAGGGACCTCAAGAC 3’和5’
AGCCCACCAAGAATGTAA 3’ 进行PCR扩增。PCR反应体系为20μl，其中含上下游引物各0.4μM， 8μl 2×Taq MasterMix ，DNA模板30∼50ng。PCR扩增条件为：①94℃ 5min ②96℃ 30s，62℃ 60s，72℃ 35s 2个循环；③95℃ 30s，60℃60s，72℃ 35s 6个循环；④94℃ 55s，58℃ 60s，72℃ 35s 28个循环；⑤72℃10min。取PCR产物5μl用1.5%琼脂糖凝胶电泳，以DL2000为分子量标准品。在PCR扩增后的反应体系中，加入适量限制性内切酶MboI及其酶切缓冲液，置37℃温箱5小时。经2.5%琼脂糖凝胶电泳，根据酶切图谱判断基因型。存在3种基因型，其中有完全切开的纯合基因型（C/C），未切开的纯合基因型（T/T），既有切开又有未切开的杂合基因型（C/T）。rs174556基因型为TT的个体乳汁中花生四烯酸和二十碳五烯酸水平较低，rs174556基因型为CC的个体乳汁中花生四烯酸和二十碳五烯酸水平较高。TT基因型母亲的婴儿如果是纯母乳喂养的话，应该加大膳食花生四烯酸和二十碳五烯酸的摄入以保证乳汁中相应的多不不饱和脂肪酸的充足水平，满足婴儿的生长发育要求。
Claims (3)
1.一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点，其特征在于：核苷酸序列为
加黑部分为引物序列，方框内是发生互换的碱基，划线处则是MboI限制性内切酶识别的酶切位点，箭头所示内切酶的切割部位。
2.根据权利要求1所述的一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点的检测方法，其特征在于：
乳母均由外周静脉抽取抗凝血5ml，用Wizard®Genomic DNA
Purification Kit试剂盒提取全血基因组DNA；步骤如下：1.5ml Eppendorf离心管中加入细胞裂解液900μl，加入融化的全血300μl吹打混匀，室温反应20min，以13000 rpm离心3min，吸弃上清，加入300μl核裂解液，充分振荡反复吹打至团块溶解，37℃水浴30min，加1.5μl RNase充分混匀，37℃水浴15min，加入100μl蛋白沉淀液，充分混匀，室温反应20min，以13000 rpm离心3min，将上清移至加入300μl异丙醇的离心管中，反复颠倒混匀，室温反应15min，以13000 rpm离心3min，轻轻弃上清，留白色沉淀，用吸水纸吸干，加70%乙醇200μl洗涤1次，离心轻轻弃上清，用吸水纸吸干，室温干燥20min，加100μl DNA溶解液，65℃水浴2h，制成DNA样品，于-20℃冰箱保存，使用美国BECKMAN公司的Du®640紫外分光光度仪，分别取波长260nm、280nm 两个波段，测得其相应DNA的OD260nm及OD280nm值，然后再通过公式，计算出DNA含量，公式为：DNA含量=50mg/ml×OD260nm×稀释倍数，另外通过公式计算出DNA纯度，公式为：DNA纯度=OD260nm/OD280nm，依据相应的碱基序列合成引物，正向引物为：5’ AAGCAGGGACCTCAAGAC 3’，反向引物为：5’ AGCCCACCAAGAATGTAA 3’，PCR反应体系为20μl，其中含上下游引物各0.4μM， 8μl 2×Taq MasterMix ，DNA模板30∼50ng，PCR扩增条件为：①94℃ 5min ②96℃ 30s，62℃ 60s，72℃ 35s 2个循环；③95℃ 30s，60℃60s，72℃ 35s 6个循环；④94℃ 55s，58℃ 60s，72℃ 35s 28个循环；⑤72℃10min，取PCR产物5μl用1.5%琼脂糖凝胶电泳，以DL2000为分子量标准品，所扩增的片段长度为153bp（6886371～6886683），在PCR扩增后的反应体系中，加入适量的限制性内切酶MboI及其酶切缓冲液，置37℃温箱5小时，经2.5%琼脂糖凝胶电泳，根据酶切图谱判断基因型，存在3种基因型，其中有完全切开的纯合基因型，未切开的纯合基因型，既有切开又有未切开的杂合基因型。
3.根据权利要求1所述的一个与乳汁多不饱和脂肪酸水平密切相关联的基因位点在指导孕妇及乳母营养摄取量的应用。
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Non-Patent Citations (4)
| Title |
|---|
| EVA LATTKA ET AL.: "Genetic variants in the FADS gene cluster are associated with arachidonic acid concentrations of human breast milk at 1.5 and 6 mo postpartum and influence the course of milk dodecanoic, tetracosenoic, and trans-9-octadecenoic acid concentrations over the", 《AM J CLIN NUTR》, vol. 93, no. 2, 8 December 2010 (2010-12-08), pages 382 - 391 * |
| LIN XIE AND SHEILA M. INNIS: "Genetic variants of the FADS1 FADS2 gene cluster are associated with Altered (n-6) and (n-3) Essential Fatty Acids in Plasma and Erythrocyte Phospholipids inWomen during Pregnancy and in BreastMilk during Lactation", 《THE JOURNAL OF NUTRITION》, vol. 138, no. 11, 30 November 2008 (2008-11-30), pages 2222 - 2228 * |
| NCBI: "Submitted SNP(ss) Details：ss281034535", 《DBSNP》, 16 December 2010 (2010-12-16) * |
| 孙琳 等: "FADS1／FADS2基因多态性与冠心病发病的关联性分析", 《吉林大学学报（医学版）》, vol. 38, no. 1, 31 January 2012 (2012-01-31), pages 115 - 118 * |
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