CN106947779B - 利用乳腺生物反应器提高乳汁中褪黑素含量的方法 - Google Patents
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Abstract
本发明提供一种利用乳腺生物反应器提高乳汁中褪黑素含量的方法，其是将褪黑素关键合成酶AANAT和HIOMT基因的cDNA序列连接酪蛋白启动子，通过高效的CRISPR/Cas9系统，整合到羊的染色体基因中，通过乳腺反应器表达酶AANAT和HIOMT，从而在乳腺中合成天然褪黑素。转基因羊乳腺反应器具有较高的重组褪黑素合成酶表达水平，并且能够提高乳汁中褪黑素含量。
Description
技术领域
本发明涉及基因工程及农业类畜牧兽医领域，具体地说，涉及一种利用乳腺生物反应器提高乳汁中褪黑素含量的方法。
背景技术
褪黑素(N-acetyl-5-methoxytryptamine)是一种生物钟的调节激素，参与神经和外周组织细胞时钟基因的表达调控。褪黑素是两亲分子，容易进入所有的亚细胞结构空间，包括细胞膜膜结构、细胞质、细胞核和线粒体中。在各种各样复杂的生理活动中，褪黑素是一种强效的活性氧(ROS)和活性氮(RNS)的自由基清除剂。乙酰基转移酶(AANAT)和羟基吲哚-O-甲基转移酶(HIOMT)被证明是合成褪黑素的关键限速酶。血液褪黑素主要合成器官在松果体中，5-羟色胺(5-HT)来自血液中的色氨酸的脱羧反应，在AANAT的催化下转变为N-乙酰基-五羟色胺(NAS)；在乙酰血清素-O-甲基转移酶(HIOMT)的催化下，合成褪黑素。松果体中AANAT酶的活力受光的抑制，表现出昼夜节律，其活力与血液及松果体褪黑素水平一致；HIOMT在褪黑素周期中发挥重要作用，其活力与褪黑素的产生呈正相关。研究表明这两种酶是褪黑素合成途径中的重要催化剂，直接影响褪黑素的合成产量。
褪黑素在不同组织中具有多种生物活性。褪黑素的代谢产物N-acetyl-N-formyl-5-methoxykynuramine(AFMK)和N-acetyl-5-methoxykynuramine(AMK)也是高效的抗氧化剂。褪黑素与自由基发生的反应是一种不可逆的氧化反应，不能被循环利用，它在氧化过程中形成的各级中间代谢产物都具有显著的抗氧化能力，有的作用甚至超过褪黑素本身，形成褪黑素级联放大的抗氧化机制。褪黑素被认为是一种抗衰老的激素，它还具有提高睡眠质量和发展免疫的功能。其中，已知褪黑素可以影响胃肠道平滑肌，具有放松平滑肌和催眠的效果。它可能有助于婴儿更好的夜间睡眠，从而促进免疫系统的发展和早期的昼夜节律建立。外源低浓度的褪黑素可以迅速吸收，发挥抗氧化、抗炎症作用从而影响并调节免疫功能。例如，国外已有高褪黑素含量牛奶“Night milk”等上市，有更好的促进睡眠效果和提高免疫力的功能。在绵羊乳腺中高表达褪黑素合成限速酶AANAT和HIOMT，催化5-羟色胺合成高水平褪黑素，使乳汁中褪黑素含量进一步提高，不仅可以使羔羊增强免疫力，提高成活率；同时对于褪黑素功能乳的开发奠定基础，提高东弗利生等乳用羊的产品附加值。
发明内容
本发明的目的是提供一种利用乳腺生物反应器提高乳汁中褪黑素含量的方法。
为了实现本发明目的，本发明首先提供过表达褪黑素关键合成酶AANAT和HIOMT基因的原核胚的构建方法，包括以下步骤：
(1)分别将褪黑素关键合成酶AANAT和HIOMT的cDNA插入表达载体中，构建得到重组表达载体；
(2)Cas9 cDNA及sgRNA的制备：将经过密码子优化后的Cas9序列及sgRNA序列分别构建到包含T7启动子的载体上，然后通过体外转录分别得到Cas9 cDNA及sgRNA产物并纯化；
其中，sgRNA的作用位点位于羊MSTN基因第三外显子区，sgRNA作用位点的核苷酸序列为5’-GACATCTTTGTAGGAGTACAGCAA-3’；
Cas9 cDNA的核苷酸序列如SEQ ID NO:1所示；
(3)将(1)构建的重组表达载体线性化，然后与Cas9 cDNA及sgRNA一起导入哺乳动物的原核期胚胎细胞中，即得。
本发明所述褪黑素关键合成酶AANAT和HIOMT的cDNA来自羊松果体细胞。优选杜泊羊松果体细胞。
前述方法中，步骤(1)所述表达载体为pBC1(携带有酪蛋白启动子)。分别将AANAT和HIOMT的cDNA插入载体pBC1的XhoI多克隆位点之间，得到重组表达载体pBC1-AANAT和pBC1-HIOMT(质粒结构见图9和图10，载体pBC1-AANAT的全序列如SEQ ID NO:2所示，将SEQID NO:2中第8614～9240位碱基替换成SEQ ID NO:3所示序列即为载体pBC1-HIOMT的全序列)。
前述的方法，步骤(3)是将重组表达载体pBC1-AANAT、pBC1-HIOMT分别用Sal I和Not I限性内切酶进行线性化处理，分别回收大片段(长度分别为16391bp和16795bp)，然后与Cas9 cDNA及sgRNA一起通过原核显微注射直接注入羊原核胚的细胞质中。
进行显微注射时，大片段的注射浓度为80ng/μl；Cas9 cDNA的注射浓度为838ng/μl；sgRNA的注射浓度为38.7ng/μl；单枚原核胚注射总体积为10pL。
本发明还提供按照上述方法制备的过表达褪黑素关键合成酶AANAT和HIOMT基因的原核胚。
本发明还提供所述原核胚在制备动物乳腺反应器、转基因动物以及转基因细胞系中的应用。
本发明还提供一种利用乳腺生物反应器提高乳汁中褪黑素含量的方法，其是将所述原核胚移入受体羊的输卵管中，经过妊娠、分娩，获得阳性转基因羊，获得的转基因羊的原代或后代母羊在泌乳期的乳汁中产生褪黑素，从而提高乳汁中褪黑素含量。
其中，用于鉴定阳性转基因羊的特异性PCR引物包括：
A8-F 5’-GTCCAGCACTTCCTGACCCT-3’
MLT-R 5’-CATCAGAAGTTAAACAGCACAGTTAG-3’；
H4-F 5’-ATACGCTGTTTCCTCATCTTCC-3’
MLT-R 5’-CATCAGAAGTTAAACAGCACAGTTAG-3’；
pBC1-F 5’-GATTGACAAGTAATACGCTGTTTCCTC-3’
pBC1-R 5’-CATCAGAAGTTAAACAGCACAGTTAG-3’。
所述泌乳期包括人工诱导泌乳期和自然分泌泌乳期；所述人工诱导泌乳期和自然泌乳期包括泌乳前期、泌乳中期和泌乳后期。
本发明所述羊为绵羊。
本发明进一步提供按照上述方法制备的褪黑素功能乳。
本发明具有以下优点：
(一)本发明利用羊乳腺反应器合成褪黑素限速酶，从而生产天然褪黑素和褪黑素功能乳。在将羊自身的褪黑素合成酶cDNA连接酪蛋白启动子整合到羊基因组中，最大程度地减少了转基因动物自身的外来蛋白的影响。转入的基因中无标记、无抗性基因、也无细菌载体片段，不仅避免了密码子偏好的问题，更符合转基因安全的要求。
(二)通过高效的CRISPR/Cas9系统，制作褪黑素乳腺生物反应器，获得的羊AANAT和HIOMT基因拷贝数增加1个以上，AANAT和HIOMT蛋白表达上升。利用乳腺生产高水平天然褪黑素，不仅可以提高乳腺健康水平、生产高质量的乳汁，增强羔羊免疫力，提高羔羊成活率；还可以解决目前褪黑素主要依赖化学合成，造成微量杂质残留的问题，又可以规避生物提取潜在的病原物质感染的风险。而且羊乳腺反应器和产品均具有重要的研究价值和商业前景。
(三)实验表明，本发明的阳性转基因羊乳腺细胞褪黑素合成酶的表达量显著提高。在催乳素诱导下，阳性乳腺细胞可合成高水平褪黑素，并且阳性羊血液中的褪黑素水平也显著高于普通羊。转基因羊整个泌乳期阶段都具有分泌褪黑素合成酶的能力，并且通过育种选择，能进一步提高转基因羊的褪黑素表达量。
附图说明
图1为本发明实施例1中线性化pBC1-AANAT/HIOMT载体结构示意图。其中，线性化的pBC1-AANAT为16391bp，pBC1-HIOMT为16795bp；Pβ-casein为羊β-Casein启动子；E1为羊β-Casein外显子1；IVS1为羊β-Casein内含子1。
图2为本发明实施例1中转基因后代的AANAT、HIOMT基因Southern检测结果。其中，AANAT基因阳性条带约为5.2kb，1-5为PCR阳性个体，WT为阴性对照，P为表达载体质粒。HIOMT基因阳性条带约为700bp，内源条带约为11kb，1-10为PCR阳性个体，WT为阴性对照。
图3为本发明实施例1中转染乳腺上皮细胞系中AANAT和HIOMT基因相对表达量。其中，*表示差异显著，P<0.05，**表示差异极显著，P<0.01，n＝4。图4为本发明实施例1中转染乳腺上皮细胞匀浆中褪黑素含量。其中，*表示差异显著，P<0.05，n＝4。
图5为本发明实施例1中转基因阳性羊早晚血清中褪黑素浓度。其中，*表示差异显著，P<0.05，**表示差异极显著，P<0.01，n＝14。
图6为本发明实施例1中转染乳腺上皮细胞系的细胞培养基中添加5-HT(10-4mol/L)后，随时间变化的褪黑素合成量。
图7为本发明实施例1中转基因后代乳腺上皮细胞AANAT和HIOMT相对表达量。其中，**表示差异极显著，P<0.01，n＝4。
图8为本发明实施例1中乳腺细胞合成酶表达量及奶中褪黑素的浓度。其中，A中1-4为转基因阳性个体，5-8为野生型个体；B中**表示差异极显著，P<0.01，n＝4。
图9为本发明实施例1中重组表达载体pBC1-AANAT的结构示意图。
图10为本发明实施例1中重组表达载体pBC1-HIOMT的结构示意图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
实施例1利用羊乳腺生物反应器提高乳汁中褪黑素含量的方法
1.基因克隆和表达载体构建
从杜泊羊松果体中提取总RNA(Ovis aries)，合成cDNA第一链。分别扩增出AANAT和HIOMT完整的开放阅读框(ORF)和部分上下游非编码区(引物见表1)。AANAT和HIOMT的PCR产物单酶切后连接到pMDTM19-T载体中测序,基因片段的大小分别为634bp和1048bp。测序验证后的cDNA片段连接到乳腺特异性表达载体pBC1(Invitrogen)上。重组载体命名为pBC1-AANAT和pBC1-HIOMT(SEQ ID NO:1和2，质粒结构见图9和图10)。两者均由β-酪蛋白启动子驱动生产重组蛋白。线性化PBC1-AANAT/HIOMT载体结构见图1。
表1基因克隆引物
2.Cas9 cDNA及sgRNA的制备
将经过密码子优化后的Cas9序列及sgRNA序列分别构建到包含T7启动子的载体pcDNA3.1(+)(Invitrogen)上，然后通过体外转录分别得到Cas9 cDNA及sgRNA产物并纯化。
其中，sgRNA的作用位点位于羊MSTN基因第三外显子区，sgRNA作用位点的核苷酸序列为5’-GACATCTTTGTAGGAGTACAGCAA-3’。
Cas9 cDNA的核苷酸序列如SEQ ID NO:1所示。
3.原核胚细胞质内显微注射生产转基因羊
乳腺表达载体pBC1-AANAT和pBC1-HIOMT进行线性化处理。利用Sal I和Not I限性内切酶可以释放5.9kb的细菌质粒部分与线性DNA大片段，DNA大片段长度分别为16391bp和16795bp。线性DNA为表达构件、包含调控元件和插入的目的基因cDNA。
以杜泊羊为供体进行超数排卵。通过等量混合pBC1-AANAT和pBC1-HIOMT两个表达载体，以及Cas9 cDNA和sgRNA，采用原核显微注射直接注入羊原核胚的细胞质中，生产过表达褪黑素合成基因的绵羊后代。
大片段的注射浓度为80ng/μl；Cas9 cDNA的注射浓度为838ng/μl；sgRNA的注射浓度为38.7ng/μl；单枚原核胚注射总体积为10pL。
从62只供体羊中共获得593枚原核注射胚胎，移植到所有150个受体母羊输卵管内，21天超声检查后77只妊娠，妊娠率为51.33％(表2)。我们共获得98只胚胎移植绵羊后代，其中包括经测序鉴定的34只转基因阳性羔羊，产羔率和转基因PCR测序鉴定阳性率分别为65.33％和34.69％(表2)。其中，有7只携带外源AANAT基因，2只携带外源HIOMT基因，其余25羔羊同时携带AANAT和HIOMT两种基因。
表2原核注射生产转基因绵羊统计
表3转基因后代鉴定引物
*A8-F、H4-F分别鉴定转入的AANAT、HIOMT基因，与MLT-R共同使用；利用pBC1-F/R鉴定转AANAT基因后代，扩增产物大小为765bp，利用pBC1-F/R鉴定转HIOMT基因后代，扩增产物大小为1169bp。
通过Southern印迹杂交法进一步验证PCR测序结果(图2)。使用DIG标记AANAT基因的探针分别与个体基因组及载体质粒杂交。基因组与载体质粒均用HindⅢ单酶切彻底消化，获得阳性条带约为5.2kb。WT为野生型个体，P为表达载体。重复验证个体共5个，标号分别为1-5号阳性个体。
4.细胞系中AANAT和HIOMT基因表达水平
从整合外源AANAT和HIOMT基因的母羊乳腺处取得上皮细胞，经催乳素诱导培养基培养，以便通过β-酪蛋白启动子启动外源基因的表达。Q-PCR检测发现在阳性转染细胞系中，与转染空白载体和对照组细胞系相比，24h至48h范围内AANAT和HIOMT基因在mRNA水平表达差异极显著(N＝4，P＜0.01；图3)。
转染乳腺上皮细胞匀浆中褪黑素含量见图4。
5.转基因后代AANAT和HIOMT基因表达水平
获得转AANAT和HIOMT双基因群体后，分别在早晨4：00和下午16:00对其血清中褪黑素水平进行了测定。转基因组与野生型组羊相比4:00和16:00褪黑素浓度差异不显著(n＝10，P>0.05)(图5)。
取泌乳期转基因和野生型羊乳腺上皮细胞，原代培养下并检测基因表达情况。褪黑素的测定表明，通过培养基添加底物5-HT(10-4mol/L)，2小时后发现野生型和转基因细胞之间差异显著(N＝4，P＜0.01。图6)。Q-PCR显示转基因个体与阴性或是野生型个体相比，mRNA转录水平的差异极显著(N＝4，P＜0.01)，高出约10倍(图7)。Western印记杂交法测定活性受调控的合成限速酶AANAT表达水平。转基因合成限速酶AANAT(23KD)表达水平明显高于野生型(1-4为转基因个体，4-8为野生型个体。内参Actin-β43KD，图8A)。分别在4：00和16:00对转基因组羊奶中褪黑素水平进行了测定。转基因组羊奶中夜间褪黑素含量极显著地高于野生型组(N＝4，P＜0.01，图8B)，提高3倍左右。
本发明利用原核注射技术生产转基因羊，快速引入新的基因——褪黑素合成限速酶基因，产生期望的生产性状——乳中高含量天然褪黑素。并在此基础上建立育种群体，进一步选育表达性状优良的个体，为家畜新品种培育打下良好基础。同时能提高羔羊的健康状况、提高存活率，为羊养殖产业服务。在新品种培育的基础上，该技术用于开发褪黑素功能乳产品，发挥其保健功能的作用以便人类利用，具有积极的社会效益。
羊奶营养价值优良，经济效益高，随着国内消费水平的提高，羊奶的市场前景非常广阔。通过乳腺特异表达载体实现褪黑素合成酶在绵羊乳腺中高效表达，使乳腺分泌高水平褪黑素，改善母羊乳腺功能和羊奶品质，进一步获得具有商业开发价值的褪黑素功能乳转基因羊，可带来巨大的经济效益。
与传统的原核显微注射生产转基因动物的方法(转基因小鼠阳性率约10％，转基因羊阳性率约2～10％)相比，本发明具有很高的阳性率达到34.69％。同时，相比传统的原核注射技术，本方法可实现外源基因的定点整合，可控性和安全性高。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中国农业大学
<120> 利用乳腺生物反应器提高乳汁中褪黑素含量的方法
<130> KHP171110979.6
<160> 9
<170> PatentIn version 3.3
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aagcttgcca ccatggacta taaggaccac gacggcgact acaaggatca cgacattgat 60
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gggctcgaca ttggcaccaa ctccgtgggc tgggctgtga tcacagacga gtacaaagtc 180
cccagcaaga agttcaaggt cctcgggaac accgataggc actccatcaa gaagaatctg 240
attggcgccc tgctctttga cagcggcgag acagccgaag ctacccggct gaaaaggaca 300
gctcggcgga ggtatacaag gcggaagaat cggatttgct acctgcagga aatcttttcc 360
aacgaaatgg ctaaggtgga cgacagcttc ttccacaggc tcgaggagtc cttcctggtg 420
gaagaggata agaagcatga gcggcacccc atctttggca acatcgtcga tgaagtggcc 480
tatcatgaga agtaccccac catctaccac ctccggaaga aactcgtcga cagcaccgac 540
aaggccgatc tgaggctgat ttacctcgct ctggcccaca tgattaagtt tcgggggcat 600
tttctcattg aaggcgacct gaaccccgac aattccgatg tcgacaagct gttcatccag 660
ctggtccaga cctataacca gctcttcgag gagaacccca tcaacgcctc cggggtcgac 720
gccaaagcta tcctgtccgc caggctgagc aagtccaggc ggctcgaaaa cctcattgcc 780
cagctccccg gcgagaagaa gaatggcctg tttgggaacc tgattgctct cagcctcggg 840
ctcaccccta acttcaagtc caacttcgac ctggctgagg acgccaaact ccagctgtcc 900
aaggatacct acgacgatga cctcgataac ctgctggccc agatcggcga ccagtacgcc 960
gatctgtttc tcgctgccaa aaacctgagc gacgccattc tgctgtccga catcctccgg 1020
gtcaacaccg agatcacaaa agcccctctg agcgcctcca tgatcaagcg gtatgatgag 1080
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aaggagatct tctttgatca gtccaagaac ggctacgctg ggtatatcga cggcggggcc 1200
tcccaggagg agttttataa gttcatcaaa cccattctgg aaaagatgga cggcacagaa 1260
gagctgctgg tgaagctcaa cagggaggat ctcctccgga agcaacggac cttcgacaat 1320
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gacttctatc ccttcctgaa ggacaacagg gaaaaaatcg agaagattct gacattccgg 1440
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ctccccaagc actccctcct ctatgagtac ttcaccgtgt ataacgagct gaccaaggtg 1680
aagtatgtca ccgaagggat gcggaaacct gccttcctga gcggggagca gaaaaaagcc 1740
attgtggacc tcctgttcaa gaccaacagg aaagtgaccg tcaagcaact gaaggaagat 1800
tacttcaaaa aaattgaatg ctttgattcc gtggagatct ccggggtgga agatcggttt 1860
aacgctagcc tgggcacata ccacgacctc ctcaagatta tcaaggataa agactttctc 1920
gacaacgaag aaaatgaaga tatcctggag gacattgtcc tgaccctcac actgtttgag 1980
gaccgggaga tgattgaaga gcggctcaag acatacgccc atctcttcga cgacaaggtg 2040
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aacgggatcc gggataagca atccggcaag acaatcctgg acttcctgaa aagcgacggc 2160
ttcgccaaca ggaatttcat gcagctcatt cacgacgata gcctgacctt caaggaagat 2220
atccagaagg cccaggtctc cggccagggg gatagcctgc acgaacatat cgccaacctg 2280
gctggctccc ctgctatcaa gaagggcatc ctccaaacag tgaaggtggt ggatgaactc 2340
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cagaccaccc aaaaaggcca gaagaactcc cgggaaagga tgaaaaggat cgaggaaggc 2460
atcaaagagc tgggcagcca aattctcaag gaacatcccg tcgaaaatac ccagctgcag 2520
aacgagaagc tctacctgta ctatctccag aacggccggg atatgtacgt ggaccaggag 2580
ctggacatta ataggctgtc cgactacgac gtcgatcaca tcgtgcctca gtccttcctc 2640
aaagacgatt ccattgacaa taaggtcctc acccggagcg acaaaaacag ggggaaatcc 2700
gacaacgtcc cttccgaaga ggtggtcaaa aagatgaaaa actattggag gcagctgctc 2760
aacgccaagc tgatcacaca acggaagttc gacaatctca caaaggccga aaggggcggc 2820
ctgtccgagc tggacaaggc tggctttatt aaacggcaac tcgtcgaaac ccggcagatt 2880
accaagcacg tcgctcagat tctggactcc cggatgaaca ccaaatatga cgaaaacgat 2940
aagctcatta gggaagtgaa ggtgatcacc ctgaagagca agctggtctc cgacttcagg 3000
aaagacttcc agttctacaa agtgcgggag atcaacaact atcaccatgc ccatgatgcc 3060
tacctcaatg ccgtggtggg cacagctctc atcaaaaagt accccaagct ggagtccgag 3120
ttcgtctacg gcgattacaa ggtgtacgac gtgcggaaga tgatcgccaa aagcgagcaa 3180
gagattggga aggctaccgc caaatacttc ttctactcca atatcatgaa ctttttcaag 3240
acagagatta ccctcgccaa tggcgaaatc aggaaacggc ccctgatcga aaccaatggg 3300
gagaccggcg agatcgtgtg ggataaaggg agggactttg ctacagtccg gaaagtgctg 3360
tccatgcccc aggtcaacat cgtgaagaaa accgaggtcc aaaccggcgg cttctccaag 3420
gagagcattc tccccaaacg gaacagcgac aagctcatcg cccggaagaa ggactgggac 3480
cccaagaaat atggcgggtt cgattccccc accgtcgctt atagcgtgct ggtcgtggcc 3540
aaggtcgaga aagggaagag caagaagctc aagagcgtca aggagctcct ggggatcacc 3600
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aaggaagtga agaaggatct catcatcaag ctccccaaat attccctctt tgagctggaa 3720
aacggccgga aacggatgct cgcctccgct ggggaactgc aaaaaggcaa tgagctggcc 3780
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tcccccgagg acaacgagca aaagcaactg ttcgtggaac aacacaaaca ctacctggac 3900
gaaatcatcg agcagatttc cgagttttcc aagcgggtca tcctggccga tgccaacctc 3960
gacaaggtcc tgagcgctta caataagcac agggacaagc ccatccggga gcaggctgag 4020
aacattatcc acctcttcac cctgaccaac ctcggggccc ccgctgcctt caagtatttc 4080
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tcgactctag agggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc 60
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cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt 180
cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa 240
agctttaggc tgaaagagag atttagaatg acagaatcat agaacggcct gggttgcaaa 300
ggagcacagt gctcatccag atccaacccc ctgctatgtg cagggtcatc aaccagcagc 360
ccaggctgcc cagagccaca tccagcctgg ccttgaatgc ctgcagggat ggggcatcca 420
cagcctcctt gggcaacctg ttcagtgcgt caccaccctc tgggggaaaa actgcctcct 480
catatccaac ccaaacctcc cctgtctcag tgtaaagcca ttcccccttg tcctatcaag 540
ggggagtttg ctgtgacatt gttggtctgg ggtgacacat gtttgccaat tcagtgcatc 600
acggagaggc agatcttggg gataaggaag tgcaggacag catggacgtg ggacatgcag 660
gtgttgaggg ctctgggaca ctctccaagt cacagcgttc agaacagcct taaggataag 720
aagataggat agaaggacaa agagcaagtt aaaacccagc atggagagga gcacaaaaag 780
gccacagaca ctgctggtcc ctgtgtctga gcctgcatgt ttgatggtgt ctggatgcaa 840
gcagaagggg tggaagagct tgcctggaga gatacagctg ggtcagtagg actgggacag 900
gcagctggag aattgccatg tagatgttca tacaatcgtc aaatcatgaa ggctggaaag 960
cctccaagat ccccaagacc aaccccaacc cacccaccgt gcccactggc catgtccctc 1020
agtgccacat ccccacagtt cttcatcacc tccagggacg gtgacccccc cacctccgtg 1080
ggcagctgtg ccactgcagc accgctcttt ggagaaggta aatcttgcta aatccagccc 1140
gaccctcccc tggcacaacg taaggccatt atctctcatc caactccagg acggagtcag 1200
tgaggatggg gctctagagg gacagccccc ccccaaagcc cccagggatg taattacgtc 1260
cctcccccgc taggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc 1320
ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga 1380
tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg 1440
gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg 1500
ttgcaaagga gcacagtgct catccagatc caaccccctg ctatgtgcag ggtcatcaac 1560
cagcagccca ggctgcccag agccacatcc agcctggcct tgaatgcctg cagggatggg 1620
gcatccacag cctccttggg caacctgttc agtgcgtcac caccctctgg gggaaaaact 1680
gcctcctcat atccaaccca aacctcccct gtctcagtgt aaagccattc ccccttgtcc 1740
tatcaagggg gagtttgctg tgacattgtt ggtctggggt gacacatgtt tgccaattca 1800
gtgcatcacg gagaggcaga tcttggggat aaggaagtgc aggacagcat ggacgtggga 1860
catgcaggtg ttgagggctc tgggacactc tccaagtcac agcgttcaga acagccttaa 1920
ggataagaag ataggataga aggacaaaga gcaagttaaa acccagcatg gagaggagca 1980
caaaaaggcc acagacactg ctggtccctg tgtctgagcc tgcatgtttg atggtgtctg 2040
gatgcaagca gaaggggtgg aagagcttgc ctggagagat acagctgggt cagtaggact 2100
gggacaggca gctggagaat tgccatgtag atgttcatac aatcgtcaaa tcatgaaggc 2160
tggaaagcct ccaagatccc caagaccaac cccaacccac ccaccgtgcc cactggccat 2220
gtccctcagt gccacatccc cacagttctt catcacctcc agggacggtg acccccccac 2280
ctccgtgggc agctgtgcca ctgcagcacc gctctttgga gaaggtaaat cttgctaaat 2340
ccagcccgac cctcccctgg cacaacgtaa ggccattatc tctcatccaa ctccaggaac 2400
ggagtcagtg aggatggggc tctagaggat ccctcgacct gcaggtcaac ggatcacaac 2460
aaactggaaa attcttcaag agaagaatac cagaccaccc tacctgcttc ctgagaaatc 2520
tgtttgctgc tcagaagcaa cagttagaac cagacatgga acaacagact ggttccaaat 2580
caggaaagga gtatgtcaag gctgtatatc gtcaccctga ttatttaact tatatgcata 2640
gtacataata caaaatgcca ggctggatga atcgcaagct ggaatcaaga tttctgggag 2700
aaatatcaat aaacgagata caaagataca ccacacttat ggcagaaaac taagaagaac 2760
taaagagcct cttgatgaaa gtgaaagagg agagtgaaaa agccagctta aaacccaaca 2820
ttcaaaatca agatcatcat ttcatggcaa ataaatgggg aaacaatgga aacagtgaga 2880
gactttattt tcttgggctc caaaatcact gcagattgtg actacagcca tgattaaaag 2940
atgcttgctc cttggaagag aagctattac caaactagaa agcatattaa aaagcagaga 3000
cgttactttg ctgactaagt tctgtctagt caaacctatg gtttttccag tagtcatata 3060
tggatgtgag ttgaactata aagaaagctg agcaccaaag aattgatgct tttgaaattt 3120
ggtgttggag aagtctcttg agagtccctt gaacctgcaa ggagatccaa ccagtccatc 3180
ctaaaggaaa tcagtcctga atattcattg gaaggactga tgctgaaatt gaagattaac 3240
gttttggact cacctaatgc agaagagcca actcactaga aaagacccca tgttggcaaa 3300
aattgaagcc aggaagagaa gtgaatgaca gaggatgaga tggttggatg gcatcgttga 3360
ctgaatggac atgagtctga tcaagttccg ggagacagca aaggacaggg ctgcctggtc 3420
tgctgcagtc catggggttg caaagagtcg gtctcaaatg agtaactaaa caacaaccaa 3480
gcagtagaaa aataaataaa atttgtctct gagatctcag tacctctttc tgtgcatatc 3540
cgtctcctgt tattgtactt tgtcttctgc ttgtaataaa gctgtcctgt tagtaaaatc 3600
tgtttgggtc ctctgaattc ttttagctat caaaaatgga aggtgattat tgtgcaatgt 3660
ccacctctga gtaatataca gagaataaaa gaagggagaa attatgtgca agttctctct 3720
catctcctgc ttctcattta aaagattcta cctcagtggg ggctaaaact ccacatttaa 3780
cagtagcaaa aaccaatatt ccatagcttc ttaggaaacc attttttata ctcttgtatg 3840
taattacatt caagctcaaa agcaaagaag tgattctgcg ttggtgaagg cccaaccata 3900
gaaaagagga agaaaatagg ccacatactg tgcttccccc atagctcagt tggtaaagaa 3960
tctacctaca atgcaggagg cctgggcttg atccctgggt aagggagatc ccctggagaa 4020
ggaaatggta acccactcca gtactcttgc ctgtaaatcc catggacgga ggagcctggc 4080
agctacagcc ttggggtggc aagagttgga catgattaac aactaaacca ctgccaccac 4140
tccacatact gagtgctccc cagtggcact agtggtaaag aaccacctgc cggtgcagaa 4200
gacattaaag acactggctc tatccctgct tgggaagtag ggaagatccc ctagagaggg 4260
aaatagcaac ccactccaga attcttgcct ggaaaatccc atgaatgaag actggcgggc 4320
tgtagtaact ggggtcacaa agagttaaac atgatttagc aactaaacat caccacatta 4380
aaaaaattac caccaaaata gtcatattcc aggctaaggg gaataatagc actagtacct 4440
gagagaactt tctcagattc tctgtcaagt tcttccttct ctcatataac cagtagtcta 4500
gtttacctca tcagatatta actactcatc gattctaaat tatctaatta tggggggggg 4560
cactacattg cattatattt tgtgtccatt gactatcact caatttattt ataaaaaatt 4620
catccatgtt gtttctgtga cagtaactca ttcacattaa ttgtaatatc tcattgcatt 4680
gtatactaca atttatttat acaaaatact attattcaca cttctgttga ttttaatttg 4740
gaacatcaac aataacgtgg ctgagaagct tctttcttta gtatattgtt aaggatttcc 4800
ttgatcaaga ttttacctac ttttctggtc caattggtga gagacagtca taaggaaatg 4860
ctgtgtttat tgcacaatat gtaaagcatc ttcctgagaa aataaaaggg aaatgttgaa 4920
tgggaaggat atgctttctt ttgtattcct tttctgagaa atcagacttt ttcaccttgg 4980
ccttggccac caaaagctaa caaataaagg catatgaagt agccaaggcc ttttctagtt 5040
atatctatga cactgagttc atttcatcat ttattttcct gacttcctcc tgggtccata 5100
tgagcagtct tagaatgaat attagctgaa taatccaaat acatagtaga tgttgatttg 5160
ggttttctaa gcaatccaag acttgtatga cagtaagatg tattaccatc caacacacat 5220
ctcagcatga tataaatgca aggtatattg tgaagaaaaa tttttaatta tgtcaaagtg 5280
cttactttag aaggtcatct atctgtccca aagctgtgaa tatatatatt gaaggtaatg 5340
aatagatgaa gctaaccttg taaaaatgag tagtgtgaaa tacaactaca attatgaaca 5400
tctgtcacta aagaggcaaa gaaacttgaa gattgctttt gcaaatgggc tcctattaat 5460
aaaaagtact tttgaggtct ggctcagact ctattgtagt acttagggta agaccctcct 5520
cctgtatggg ctttcatttt ctttcttgct tccctcattt gcccttccat gaatactagc 5580
tgataaacat tgactataaa agatatgagg ccaaacttga gctgtcccat tttaataaat 5640
ctgtataaat aatatttgtt ctacaaaagt attatctaaa taaatgttac tttctgtctt 5700
aaaatccctc aacaaatccc cactatctag agaataagat tgacattccc tggaatcaca 5760
gcatgctttg tctgccatta tctgacccct ttctctttct ctcttctcac ctccatctac 5820
tcctttttcc ttgcaattca tgacccagat tcactgtttg atttggcttg catgtgtgtg 5880
tgctgagttg cgtctgactg ttatcaaccc catgaatgat agtccaccag gctctactgt 5940
ccatgaaatt ttccagtcaa gaatactgga gtggattgca tttcctactc catttgatta 6000
atttagtgac ttttaaattt ctttttccat attcgggagc ctattcttcc tttttagtct 6060
atactctctt cactcttcag gtctaaggta tcatcgtgtg cttgttagct tgttactttc 6120
tccattatag cttaagcact aacaactgtt caggttggca tgaaattgtg ttctttgtgt 6180
ggcctgtata tttctgttgt gtattagaat ttaccccaag atctcaaaga cccactgaat 6240
actaaagaga cctcattgtg gttacaataa tttggggact gggccaaaac ttccgtgcat 6300
cccagccaag atctgtagct actggacaat ttcatttcct ttatcagatt gtgagttatt 6360
cctgttaaaa tgctccccag aatttctggg gacagaaaaa taggaagaat tcatttccta 6420
atcatgcaga tttctaggaa ttcaaatcca ctgttggttt tatttcaaac cacaaaatta 6480
gcatgccatt aaatactata tataaacagc cactaaatca gatcattatc cattcagctt 6540
ctccttcact tcttctcctc tactttggaa aaaaggtaag aatctcagat ataatttcag 6600
tgtatctgct actcatcttt attttggact aggttaaaat gtagaaagaa cataattgct 6660
taaaatagat cttaaaaata agggtgttta agataaggtt tacactattt tcagcagata 6720
tgttaaaaaa tagaagtgac tataaatact tgataaaaat tatagtgact gcaaatgttt 6780
taggaatata ataagatata ataacagtgg ttgctatttt ctttagcaca agactagtta 6840
acaggctgta ttaaaagatc ttttcttgaa ttaaatattt tcaatttgat taaacctacc 6900
tcagccataa aggcaagcac atttcattta tactatgggg atttgaataa ttattactga 6960
agaagctcta ccaacaaaaa gtttatagag ctatcatatt tagtcaagag ataaagaggg 7020
ttgttaggat atatatgcta tttgaaaggt atttataaaa gaagagtata tttatcaaaa 7080
tttctcaaga acatccaaat ttcaagttta tcatttatct tacaatattt caaaaatatt 7140
aaaatagata catgaaatac agaagtaaat taaagagaaa gtattttact tggtaaaaaa 7200
attctaggtt ggacagagag tgccaggaaa caaaaacaat gaaaaatgtg acctgacagg 7260
aattatagct caaagtatag tagtaagtaa tgaaatggct taaaaattgg tatataaaat 7320
gctagttata aaataaacaa aatgcaataa tatcctccct acatgtaatg aattctaggt 7380
attatgatta tgctcttttt tgaagtcttg acaataaaaa tttttttaga agtttatagg 7440
catcttgaat aaagtgaaac aaattaagaa ttagtatcca tgagaaaaat atagaacaat 7500
tttcctaatt tagtttgaaa atctgggatt gaagatgtgt gtcaagagat gttggtggca 7560
agaacatttt tttttcaaga acttataaaa atgcaacaaa acaaaccatt taatacattt 7620
tggtcaaaat caataatgta ttttatttta tgctccaagg agcataaaat tggggactgg 7680
gcaagagaaa ctgacaccct ggtaaattac caagagataa gtacacagtt actatagtag 7740
aaaataagca tagtgtatga tctctaaaat tatgtgagac aaaggagaga tgacattagg 7800
catgtgggga tgaagactga gtagagaaga aacaatctaa tcagtccaag aaaacatctc 7860
gatcagtgga acaaatagaa gaaatgctaa aatgaaacag aagtcttact ggaaataaaa 7920
gatatgcata agacaaaaat tcatgaaaat cacttagttt agcagagaaa agataaaaat 7980
aaagtatgac cttcttcata tacattgttt gatcatatgc acctcaataa aactgagtct 8040
ccaacagaaa tgaaacatta atattttgtt cactgctcta atcccagaat ctaagcgata 8100
tctggcaata aaaataataa atatatattt tttaataaat gaatcaacca cttaattttt 8160
ctgtaaatat ctgtaacttc tcttctgtct ttccaaaaac actcataagt actgtgaatg 8220
agatgaaaaa gagtgaagta ggatataggc tgttagcaga aaacatctga atggctggca 8280
gtgaaacatt aacttgaaat gtaagattaa tgagtaatag taaattttaa ccttggccat 8340
atgataaaat gttcattaat atttttctag aatacagggc tttttgtttt tgccatgagg 8400
tttgcaggat cttggttccc tgaccaggga tcaaacctgc acaccaggga tcaaacctgc 8460
actcccctgg aagcatggag tcttggacat ttgtattata cactatcttt ggttcctttt 8520
aaagggaagt aattttactt aaataagaaa atagattgac aagtaatacg ctgtttcctc 8580
atcttcccat tcacaggaat cgcggatcct cgaggatgtc cacgccaagc gtccactgcc 8640
tgaaaccctc gcctctgcac ctgccctctg ggatcccagg gtccccaggc cgccagcggc 8700
gccacacgct ccctgccaac gagttccgct gcctcacccc agaggacgct gccggcgtgt 8760
ttgagattga gcgagaggcc ttcatctctg tctccggcaa ctgccccctg aatctggacg 8820
aggtccagca cttcctgacc ctgtgtcccg agctgtccct gggctggttt gtggagggcc 8880
gcctcgtggc cttcatcatc ggctccctgt gggatgagga gagacttact caggagtcgc 8940
tggcactgca caggcccagg ggccacagcg cccacctgca cgcgctggcc gtgcaccgca 9000
gcttccggca gcaaggcaag ggctccgtcc tgctctggcg ctacctgcac cacgtgggcg 9060
cccagccagc cgtgcgccgg gcggtgctta tgtgcgagga cgcgctggtg cccttttacc 9120
agaggtttgg cttccatccc gcgggcccat gtgccatcgt cgtgggctca ctgaccttca 9180
cggagatgca ctgctccctg cggggccacg ccgccctgcg ccggaacagt gaccgctgac 9240
tcgaggatcc ggacccttcc ctattcttgt aagtctaaat ttactaactg tgctgtttaa 9300
cttctgatgt ttgtatgata tttgagtaat taagagccct acaaaaaaat caataatgaa 9360
tggttccaaa ataagcatag ctgagattaa tgattctcag cattagttat aaatagaata 9420
agctggaaaa ccttcacctc ccctccacca ccagatctca atgtctaggc ttacccatgg 9480
agattctgat taactgttct ttctatgtag aagaaactta ttgggaagaa ataatataat 9540
ggactatgat ttaattggtc tgttgagaat ttagatgaag gggattaagt tacaataaag 9600
ccagaattta acttgataat ctcatttggc taagaataac aaacctaaga aggtttgcta 9660
ttttctacaa ttttgaagtt ttccttatgc acaattattt caccacatga ctcatttcac 9720
atcttgtttt tgatatatga gcatatgagg gcaaaatact gaagatgctt atttcaatac 9780
tcagggaaaa ttttcttgcc aaaaggcaag aattgtataa ttcattcact tattttattt 9840
tttttaattt ttaaggtcta agaggatttc aaagtgaatg ccccctcctc acttttggta 9900
agctttagga gattggaggc agactgatca tttttatagt taatatcttt tacatttcat 9960
cttcctggat aagccccaat agtagcaatt tctatcagta taccagcata aagattagtt 10020
ttaaatttat tttcagtgat tgactgttat ttactgacct gaaattatgt atctgttata 10080
tttcaaataa tgcaaaactg tatatatatg gtgttgacag atttgattgg ttttctttca 10140
attgcctata tccttattat tgattgtaat catttataga aaaaacaaaa taatttctta 10200
tacttttatg taaacctgtt agagcttatt ttaaagatca actgcattca catttctaat 10260
ctagtcatta tgagcttcaa ttgttttatc tcacttaaaa tttatatatt gtcttttaat 10320
tcatgagtca aaatacaatc tcacagtcca gatatgggac ttaaaagggg aatagcatat 10380
agttttgata ttcttaaaga tatacatctt tttgtgatca tgattcagca gacattttaa 10440
taaaacaatt ccaagtgagc cgacacttgg tcctagagga atttttataa ccttaagata 10500
aggcacagca tggtgttttt gtaataagat ttcttttatg aaaaagtcac accaaaattg 10560
gaaatggggt gagatgaaga gttataacat ataactaaat ggacatttgt tctctattcc 10620
acagaattga ctgcgactgg aaatatggca acttttcaat ccttgcatca tgctactaag 10680
ataattttta aatgagtata catggaacaa aaaatgaaac tttattcctt tatttatatt 10740
atgctttttc atcttaattt gaatttgagt cataaaccat atactttcaa aatgttaatt 10800
caacattagc ataaaagttc aattttaact tggaaatatc atgaacatat caaattatgt 10860
ataaaaataa tttctggaat tgtgattatt atttctttaa gaatctattt cctaaccagt 10920
catttcaata aattaaccct taggcatatt taagttttct tgtctttatt atatttttaa 10980
aaatgaaatt ggtctcttta ttgttaactt aaatttatct ttgatgttaa aaatagctgt 11040
ggaaaattaa aattgaatag aattctttga attgagttcc aaaggatatc aaaaagtgag 11100
ggaaaagata gggtgagcct atgctgcata tgtccttaga aagtcttggt ttatacctgt 11160
tacctaagtt aaacaattat acttgttcct ttcactctcg aaagtaccca gcattggatg 11220
ttaaatttta tagtcatcct agacaaaaaa aaaaaaaaaa acaaacaacc ctcaaatgtg 11280
atatctgaat cacagctcta cagtgtggta gctaagtggt gctgtgtaag ttagtctcca 11340
agagattcca tttctacatt tataaacagt caatttaagg tgttttattg aagttttaat 11400
gtgaaaagtg cactatatgg tgcatgatag gagttcctgg ttgaatctca tttctgacat 11460
cactgacacc agtgcagcaa ggactagtgt tacaatcaga aggagctgag ttgtgtaatt 11520
ttagccatta atgcccaaga gactagaact tacacaaagc tctaatatcc attgtctctg 11580
tctgtggagt aattatttca ttgccatgaa ttatctgtct gtcatatcct gcatttttat 11640
acatgattca gttcccttca gttcacacaa tgacttgtct aatttcatct ttcctgcatc 11700
ctccatgttt tcctcacttc aggattaagt gaagccgtac ttaggcacaa tatttcttat 11760
ctttaaagaa aaattccatc tttgagagtt gttattgttc agtcactagg tcatgtccaa 11820
ctctttgtga ccccatgcac tgcagcatgc caggcttccc tgcccttcgc tctctcctgg 11880
agtttgctca gactcatgta gattgagtcg gtgatggtat ccaactatct catcaactgt 11940
tgtgcccttc tcctcctacc ctcagtcttt accagcatca gagtctttct cagattcttc 12000
aggttattat ataacaacta tcataaaagg agtatctaaa tggctgtgtc cattatttca 12060
catgttattc tctctttaac ttgctccaat cccaatttta tccctatggg aactgcttta 12120
ttgaagatca ccaacaactt ttattttact aatcgttttg ttttacccaa cctctcagtg 12180
agtgttatga ggtagagttg actatttctt cattttgaaa tattacgctt catttcattt 12240
gatatcctaa agctcataag gtgtggtttt tctcttaact cactagacac ttctttgaag 12300
tctctcttct ggcattttct ccttttccaa aattttaatg gttggagtac cctagatttt 12360
agccttaatt tgtttgatgt tgttcagttc cattctcagc tcagagcttc caactgtatg 12420
tctccaaact tactcgtttt gtaaactcca aactcatgca ctcaactgca ttcttgacct 12480
ccacactgaa ttatctaatt aatgtcctaa atctggcatg accaagcata catttttgtc 12540
tgaatccagt ccccaacttg ctcaaaattt aattaaacgt aattcagtta caaaggcagc 12600
tgatattgta tgcaatagac ctgaatggga acttcacaaa agaagttatc ttaattgtca 12660
ataaaaacat gaaaaatact ctacatcatc aatcttcaga aaaatgcaaa ttaaaggtgc 12720
ctaataatat catgacacaa ccgtcagaat gactgaaatg aaaagaattg taatacagtt 12780
cagttcagtt cagttactca gtcgtctcca actctttgtg accccatgaa ctgcagcatg 12840
acagaccttc ctgtccatca ccaactccca gagtttactc agactatgtc cattgagttg 12900
atgatgccat ccaaccatct catcctctgt cgtccccttc tcctcctgcc ctcagtcttt 12960
cccagcatca gggtcttttc caatgagtca gctcttcgca tcaggtggct aaagtattgg 13020
agtttcagct tcaacatcag tccttctaat taacacccag gactgatctc ttttaggatg 13080
gactagttgg atctccttgc agtccaaggg actctcaaga gtcttctcca acaccacagt 13140
tcaaaagcat caattccttg gcactcagct ttccttatag tccatgtctc acatccacac 13200
atgactattg gaaaaaccat agccttgact aggtggacct ttgttgacaa agtaatgtct 13260
ctgcttttta atatgttgtc tagattggtc ataactttcc ttccaagaag taattgtctt 13320
ttaatttcat ggctgcagtc accatctgca gtgattttgg agccccaaaa tataaagtca 13380
gctgctgttt ccactgttgc cccatctacc ccatctattt gccatgaagt gatgggactg 13440
gatgccacta tcttagtttt ctgaatgttg agctttaagc cagccttttt actctcctct 13500
ttcactttca tcaagaggct ctttagttcc tcttcacttt ctgccataag ggtggtgtca 13560
tctgcatatc tgaggttatt gatatttctc ttggcaattt tgattccagc ctgcacttct 13620
tccagcccag tgtttctcat gatgtactct gcatataaat taaataagca gagtgacaat 13680
atacagcctt gacatactct ttttcctatt tggaaccagt ctgttgttcc atgtccagtt 13740
ctaactgttg tttcctgacc tgcatacagg tttctcaaga ggcaagtcag gtggtctggt 13800
attctcacct gtttcagaat tttccacagt ttattgtgat ccacacagtc aaaggctttg 13860
gcatagccaa taaagcagaa agagatgttt ttctggaact ctcttacttt tttgatgatc 13920
cagtggatgt tggcaatttg atctctggtt cctctgcctt ttctaaaacc agctttaaca 13980
tctggaagtt catggttcac gtaatacaaa atgtaataca aaatgtctgc aaaaacaaag 14040
gaatgaaaag taatgctaaa aaatgttaat atttacagaa atttttatag tagtaaagaa 14100
ttcacctgca atacaggaga accgggttag atccctgggt tggaagacct cctggagaag 14160
gaaatggcta cccaatctag tattcttgtc tggagaaggc aagaatggac agagaagccc 14220
agcgggctat ggtccatcgg gtcacaaaga gtcagaagct accttgcaca cagcaagcac 14280
ggtgcgcgcg cgtgcacaca cacacacaca cacacacaca gacacacaca cactctaaaa 14340
catttaccca agcttgtcca atggaaaatc aaaaagccag caatttaaga tgacatcagg 14400
taccactgtc caggtaagcc tcagaacaca atgaccagta agaagcaaag tgccatatga 14460
gcaactcgaa tttttgcaat gttacctaag agcttccatt tttataatgc aaaagaattt 14520
catatgggga aattgtatta gataaccctg aatgaggagc aagatatagt caaagtaaga 14580
tgctctagta ctatttttta taagcatgat ttgttcagcc aaaggttttt tcccatatgg 14640
ccaatgaact gaaatatgca gtcctgagat ttgcatatat ttctagctga aaccaagtaa 14700
ataatatcct caagaaagaa atcaatagaa aagttggatg aagagtacaa taaagggacc 14760
aaaaatattc agaaataaga actagaggag atattgggaa atccctggtg agtccagttt 14820
aggattttgt actttcactg cagttggcat ggatataatc cctcactggg gaactaagat 14880
cccataagct gtgttggatt gccaaaaaaa taaatattaa gagatatcat tcatagaata 14940
ttttaaagat attttagaga agaggaaatt aaggatgtga gaatttgtat tactttttca 15000
agatactaaa gctatttaga gatagagctg ttactaaaaa cttcagtttc ctaaaaatta 15060
tttgaagcac tgtttaataa attccaaaat atagaggaag gaaaacaaaa tactgaggat 15120
tcatataatg attcagattt agaaacaata taacacagaa ttagtgaatt ctgacaaatt 15180
attaggtagg agtagatagt tcagcattac tcgtatagat ggagtattta atcctttcca 15240
tgagattatc caaatataat aatttcgtat ctatgtgaag tataactatt aagattactt 15300
tataaagtaa atcaagaacc agagaataag aaaaatgttt tgtgaaccag cagatactat 15360
gaacacataa aactcagaac cctgattcct aagacacaca gctaatcctg attattcttc 15420
ctttacatgt gaccatagaa cttcacacaa gttcaagata catttgttga gcacatcagt 15480
atcagttcag tcactcagtc atgtccgaat ctttgtgacc ttgtggactg cagcacgcca 15540
ggctttcctg tccaccacca acccctggag cttactcaaa ctcatgtcca ttgagtcagt 15600
gatcccatcc aaccatctca tcctctgtca tcctcttctc ctgccttcaa tctttcccag 15660
acattggagt cttttccaat gagtcagatc ttcacattag gtggccaaag tataggagtt 15720
tcagcttcag catcaatcct tccaatgaat attccttgat gtaccccttt cgcagtttgg 15780
aaccagtctg ttgttccatg tccagttcta actgctgctt ctggacctgt atacagattt 15840
ctcaggaggc aggtaaagtg gtctggtatt cccatctctt gaagaatttt ccacagttta 15900
ttgtgatcca cacaatcaaa ggctttagcg tagtcaataa agcagatgtt tttctggaac 15960
tctcgtgctt ttttgatgat ccaatggatg ttggcaattt gatctctggt tcctctgcct 16020
tttctaaatc cagcttgaac atctggaagt tcatggtcca cgtactgttg aagcctggct 16080
tggagaattt tgagagttat tttgctagca tgtgagatga gtgcaatcat gtgggtgttt 16140
gaacatactt tgtcattgct tttctttggg attgtggcag tcctgtggcc actgctgagt 16200
tttccaaatt tgctgacata ttgagtgcag cactttcaca gcatcacctt ttaagatttg 16260
aaatagctca actggaattc catcacctcc actagctttg ttcatagtga ggctttctaa 16320
ggccgtttga ctttgcattc cagggtgtct ggctctaggt gagtgatccg ttgacctgca 16380
gcggccgcaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 16440
tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 16500
cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 16560
cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 16620
tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 16680
tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 16740
atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 16800
gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc 16860
aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 16920
aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 16980
gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 17040
cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 17100
atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt 17160
tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 17220
ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 17280
ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 17340
ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 17400
tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 17460
tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 17520
aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct aacgtgagtt 17580
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 17640
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 17700
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 17760
gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt 17820
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 17880
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 17940
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 18000
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 18060
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 18120
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 18180
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 18240
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 18300
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 18360
gaccgagagc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct 18420
ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc 18480
tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct 18540
gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 18600
tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 18660
tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat 18720
tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat 18780
gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcacttga 18840
tgcctccgtg taagggggaa tttctgttca tgggggtaat gataccgatg aaacgagaga 18900
ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg 18960
gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc 19020
agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc 19080
agatctatta atacgatacc tgcgtcataa ttgattattt gacgtggttt gatggcctcc 19140
acgcacgttg tgatatgtag atgataatca ttatcacttt acgggtcctt tccggtgatc 19200
cgacaggtta cggggcggcg acctcgcggg ttttcgctat ttatgaaaat tttccggttt 19260
aaggcgtttc cgttcttctt cgtcataact taatgttttt atttaaaata ccctctgaaa 19320
agaaaggaaa cgacaggtgc tgaaagcgag gctttttggc ctctgtcgtt tcctttctct 19380
gtttttgtcc gtggaatgaa caatggaagt caacaaaaag cagctggctg acattttcgg 19440
tgcgagtatc cgtaccattc agaactggca ggaacaggga atgcccgttc tgcgaggcgg 19500
tggcaagggt aatgaggtgc tttatgactc tgccgccgtc ataaaatggt atgccgaaag 19560
ggatgctgaa attgagaacg aaaagctgcg ccgggaggtt gaagaactgc ggcaggccag 19620
cgaggcagat ccccagccag gaactattga gtacgaacgc catcgactta cgcgtgcgca 19680
ggccgacgca caggaactga agaatgccag agactccgct gaagtggtgg taaccgcatt 19740
ctgtactttc gtgctgtcgc ggatcgcagg tgaaattgcc agtattctcg acgggctccc 19800
cctgtcggtg cagcggcgtt ttccggaact ggaaaaccga catgttgatt tcctgaaacg 19860
ggatatcatc aaagccatga acaaagcagc cgcgctggat gaactgatac cggggttgct 19920
gagtgaatat atcgaacagt caggataaca ggctgcggca ttttgtccgc gccgggcttc 19980
gctcactgtt caggccggag ccacagaccg ccgttgaatg ggcggatgct aattactatc 20040
tcccgaaaga atccgcatac caggaagggc gctgggaaac actgcccttt cagcgggcca 20100
tcatgaatgc gatgggcagc gactacatcc gtgaggtgaa tgtggtgaag tctgcccgtg 20160
tcggttattc caaaatgctg ctgggtgttt atgcctactt tatagagcat aagcagcgca 20220
acacccttat ctggttgccg acggatggtg atgccgagaa ctttatgaaa acccacgttg 20280
agccgactat tcgtgatatt ccgtcgctgc tggcgctggc cccgtggtat ggcaaaaagc 20340
accgggttaa cacgctcacc atgaagcgtt tcactaatgg gcgtggcttc tggtgcctgg 20400
gcggtaaagc ggcaaaaaac taccgtgaaa agtcggtgga tgtggcgggt tatgatgaac 20460
ttgctgcttt tgatgatgat attgaacagg aaggctctcc gacgttcctg ggtgacaagc 20520
gtattgaagg ctcggtctgg ccaaagtcca tccgtggctc cacgccaaaa gtgagaggca 20580
cctgtcagat tgagcgtgca gccagtgaat ccccgcattt tatgcgtttt catgttgcct 20640
gcccgcattg cggggaggag cagtatctta aatttggcga caaagagacg ccgtttggcc 20700
tcaaatggac gccggatgac ccctccagcg tgttttatct ctgcgagcat aatgcatgcg 20760
tcatccgcca gcaggagctg gactttactg atgcccgtta gatctcggcg tatatcaaat 20820
cgcgatcaac aaggccattc atgcaggccg aaagattttt ttaactataa acgctgatgg 20880
aagcgtttat gcggaagagg taaagccctt cccgagtaac aaaaaaacaa cagcataaat 20940
aacccgctct tacacattcc agccctgaaa aagggcatca aattaaacca cacctatggt 21000
gtatgcattt atttgcatac attcaatcaa ttgttatcta aggaaatact tacatatggt 21060
tcgtgcaaac aaacgcaacg aggctctacg aatcgagagt gcgttgctta acaaaatcgc 21120
aatgcttgga actgagaaga cagcggaagc tgtcggcgtt gataagtcgc agatcagcag 21180
gtggaagagg gactggattc caaagttctc aatgctgctt gctgttcttg aatggggggt 21240
cgttgacgac gacatggctc gattggcgcg acaagttgct gcgattctca ccaataaaaa 21300
acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg 21360
gatctatcaa caggagtcat tatgacaaat acagcaaaaa tactcaactt cggcagaggt 21420
aactttgccg gacaggagcg taatgtggca gatcatgcgc acccgtggcc aggacccaac 21480
gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg 21540
ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg 21600
agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc 21660
catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat 21720
gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca 21780
gtgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg 21840
tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg 21900
agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag 21960
cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg 22020
gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac 22080
aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct 22140
gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata 22200
gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcgg 22259
<210> 3
<211> 1045
<212> DNA
<213> 人工序列
<400> 3
gccaccatgt gctcccagga gggtgacgac taccgtctcc tgaaggaata cgccaacggc 60
ttcatggtct cccaggttct ctttgctgcc tgtgagctgg gggtgtttga gctcctggca 120
gaggccctgg agcccttgga ctcagcggca gtgtcttcac gtctgggctc cagcccccgg 180
gggacagagc tgctgctgga cacctgcgtg tccctgaagc tgctgcaagc cgccgtgagg 240
ggaggaaaag ctgtgtatgc caacacggag ctcgccagca cctacctggt gagaggcagc 300
cccaggtccc agcgggacat gctgctgtac tcgggcagga ccgcttatgt ctgctggcgc 360
cacctggccg aggcagtgag ggaagggagg aaccagtatc cgaaagcctt caggatcccg 420
tccgaggagc tcttctctgc catctacagg tcggaggacg aaaggctgcg gttcatggag 480
gacctgcagg acgtgtggag gctggagggg gcgagcgtgc tggccgcctt cgacctgtct 540
ccctccccgc tcatctgcga cctcgggggc ggttccgggg ctctggccag ggcgtgcgtg 600
tccctgtacc cacgatgtcg ggacatcgtc tttgacatcc caggagtggt gcggatggca 660
aagagacatt tctcagcctg ggaggatgag cggatcagct tccatgaagg ggatttcttc 720
aaagaccccc tcccggaggc ggacctgtat atcctggcca gggtcctgca tgactggacc 780
gatgccaagt gctgccacct gctgcagaga ctgtatcggg cctgcaggac aggtggcggc 840
gtgctggtca tcgagagcct cctggacgcg gacgggcggg gccccctgac cacgctgctg 900
tactcgctga acatgctggt gcagaccgag ggccgtgagc gcacccccgc ccagtaccac 960
gcgctcctgg cccccgccgg cttccgagac gtgcagtgcc ggaggacggg gggaacctac 1020
gatgcagtct tgggcagaaa gtgac 1045
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
gtccagcact tcctgaccct 20
<210> 5
<211> 26
<212> DNA
<213> 人工序列
<400> 5
catcagaagt taaacagcac agttag 26
<210> 6
<211> 22
<212> DNA
<213> 人工序列
<400> 6
atacgctgtt tcctcatctt cc 22
<210> 7
<211> 26
<212> DNA
<213> 人工序列
<400> 7
catcagaagt taaacagcac agttag 26
<210> 8
<211> 27
<212> DNA
<213> 人工序列
<400> 8
gattgacaag taatacgctg tttcctc 27
<210> 9
<211> 26
<212> DNA
<213> 人工序列
<400> 9
catcagaagt taaacagcac agttag 26
Claims (3)
1.利用乳腺生物反应器提高羊乳汁中褪黑素含量的方法，其特征在于，构建过表达褪黑素关键合成酶AANAT和HIOMT基因的原核胚，将所建原核胚移入受体羊的输卵管中，经过妊娠、分娩，获得阳性转基因羊，获得的转基因羊的原代或后代母羊在泌乳期的乳汁中产生褪黑素，从而提高乳汁中褪黑素含量；
其中，所述泌乳期包括人工诱导泌乳期和自然分泌泌乳期；所述人工诱导泌乳期和自然泌乳期包括泌乳前期、泌乳中期和泌乳后期；
过表达褪黑素关键合成酶AANAT和HIOMT基因的原核胚的构建方法，包括以下步骤：
(1)分别将褪黑素关键合成酶AANAT和HIOMT的cDNA插入表达载体中，构建得到重组表达载体；
(2)Cas9 cDNA及sgRNA的制备：将经过密码子优化后的Cas9序列及sgRNA序列分别构建到包含T7启动子的载体上，然后通过体外转录分别得到Cas9 cDNA及sgRNA产物并纯化；
其中，sgRNA的作用位点位于羊MSTN基因第三外显子区，sgRNA作用位点的核苷酸序列为5’-GACATCTTTGTAGGAGTACAGCAA-3’；
Cas9 cDNA的核苷酸序列如SEQ ID NO:1所示；
(3)将(1)构建的重组表达载体线性化，然后与Cas9 cDNA及sgRNA一起导入哺乳动物的原核期胚胎细胞中，即得；
步骤(1)所述褪黑素关键合成酶AANAT和HIOMT的cDNA来自羊松果体细胞；所述表达载体为pBC1，分别将AANAT和HIOMT的cDNA插入载体pBC1的XhoI多克隆位点之间，得到重组表达载体pBC1-AANAT和pBC1-HIOMT；所述重组表达载体pBC1-AANAT的全序列如SEQ ID NO:2所示，将SEQ ID NO:2中第8614～9240位碱基替换成SEQ ID NO:3所示序列即为载体pBC1-HIOMT的全序列；
步骤(3)是将重组表达载体pBC1-AANAT、pBC1-HIOMT分别用Sal I和Not I限性内切酶进行线性化处理，分别回收大片段，然后与Cas9 cDNA及sgRNA一起通过原核显微注射直接注入羊原核胚的细胞质中。
2.根据权利要求1所述的方法，其特征在于，步骤(3)进行显微注射时，大片段的注射浓度为80ng/μl；Cas9 cDNA的注射浓度为838ng/μl；sgRNA的注射浓度为38.7ng/μl；单枚原核胚注射总体积为10pL。
3.根据权利要求1所述的方法，其特征在于，用于鉴定阳性转基因羊的特异性PCR引物包括：
A8-F 5’-GTCCAGCACTTCCTGACCCT-3’
MLT-R 5’-CATCAGAAGTTAAACAGCACAGTTAG-3’；
H4-F 5’-ATACGCTGTTTCCTCATCTTCC-3’
MLT-R 5’-CATCAGAAGTTAAACAGCACAGTTAG-3’；
pBC1-F 5’-GATTGACAAGTAATACGCTGTTTCCTC-3’
pBC1-R 5’-CATCAGAAGTTAAACAGCACAGTTAG-3’。
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| CN1944645A (zh) * | 2006-10-17 | 2007-04-11 | 青岛森淼生物技术研究所 | 利用乳腺生物反应器制备重组人的抗凝血酶ⅲ蛋白的方法 |
| CN103484497B (zh) * | 2013-08-08 | 2015-04-29 | 兰诺生物技术无锡有限公司 | 利用家兔乳腺反应器平台生产重组人第七凝血因子的方法 |
| US9790490B2 (en) * | 2015-06-18 | 2017-10-17 | The Broad Institute Inc. | CRISPR enzymes and systems |
| CN105671080B (zh) * | 2016-03-04 | 2020-01-31 | 内蒙古大学 | CRISPR-Cas9系统介导的羊MSTN基因敲除和定点整合外源基因的方法 |
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