CN114410612A - 一种密码子优化的玉足海参海藻糖酶基因及其应用 - Google Patents
一种密码子优化的玉足海参海藻糖酶基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种密码子优化的玉足海参海藻糖酶基因及其应用,属于基因改造与蛋白表达技术领域。本发明通过密码子优化技术对玉足海参的海藻糖酶基因进行改造后,在酵母表达系统中成功表达出具有海藻糖酶活性的蛋白,进而为人工养殖海参提供一种有助于摄食和消化的饲料调节剂,提高海参的摄食和消化吸收效率;同时,将该蛋白应用于大型海藻的精深加工,使海藻的营养得到更充分的释放与利用。
Description
技术领域
本发明涉及基因改造与蛋白表达技术领域,具体涉及一种密码子优化的玉足海参海藻糖酶基因及其应用。
背景技术
玉足海参(Holothuria leucospilota)是我国南海海域的优势海参品种,在富、寡营养环境均能广泛分布。海洋藻类广泛存在于玉足海参的食物中,海藻糖(Trehalose)是海洋藻类重要的细胞骨架和贮藏物质,可以被海藻糖酶(Trehalase,Tre)水解为葡萄糖,为生命活动提供能量物质。玉足海参海藻糖酶(Hl-Tre)是到目前为止,在海洋动物尤其海参中唯一公开报道的海藻糖酶。前期的研究发现,海藻糖酶在玉足海参的食性转换和适应富、寡营养环境过程中,均可能起至关重要的作用。由于海藻糖酶的来源有限,目前没有商用化的海藻糖酶,且通过生化分离得率低、质控难,而化学合成成本高,因此需要通过基因工程手段建立Hl-Tre的高效制备方法。
相对于其它动植物或细菌来源的海藻糖酶,通过海参基因表达的海藻糖酶作为饲料调节剂更有助于提高人工养殖海参的摄食和消化吸收效率;同时,其还能以酶制剂的形式应用于大型海藻的精深加工,使海藻的营养得到更充分的释放与利用。
毕赤酵母是一种常用的甲基营养型酵母,以其为基础构建的毕赤酵母表达系统因遗传背景清楚,表达水平高,且具有良好的工业化性能而被广泛应用于各类生物活性蛋白的高效生产。目前常用的毕赤酵母表达系统主要采用的表达载体为pPIC9K,该载体含有一个甲醇诱导型的启动子AOX1,在发酵过程中需要甲醇做碳源和诱导剂;通过改造该载体的启动子,既能有效表达目的产物,同时可以避免甲醇诱导带来的不利影响。
在众多的基因改造技术中,密码子优化技术是目前应用较广泛的技术。密码子优化是基于氨基酸具有密码子简并性及不同物种密码子使用频率差异原则,通过改变基因序列中的多个适合在毕赤酵母表达系统中表达的密码子,而不改变原有的氨基酸序列,以确保表达的蛋白和内源分泌的蛋白具有相同的理化特性。
发明内容
本发明利用密码子优化技术对玉足海参的海藻糖酶基因进行了改造,改造后的玉足海参海藻糖酶基因在酵母表达系统中成功表达出具有海藻糖酶活性的蛋白。另外,由于甲醇具有对动物体有毒、易挥发、不利于发酵控制和易造成环境污染的缺点,而GAP启动子在葡萄糖等基本碳源存在的条件下,无需诱导即可高效地组成型表达外源基因,且在培养过程中,无需更换碳源,操作步骤更为简便,对于大规模的工业化生产及其友好。因此,本发明采用GAP启动子来构建玉足海参海藻糖酶的毕赤酵母组成型表达系统的载体。
本发明的第一个目的是提供一种密码子优化的玉足海参海藻糖酶基因,其特征在于,其核苷酸序列是以SEQ ID NO.1所示的序列为基础,作出以下(1)~(69)任意一项或多项的改变:
(1)将第2、17、23、42、45、61、71、84、111、114、122、154、203、264、312、365、427、455、490和517位氨基酸密码子GAT替换为GAC;
(2)将第3、102、130、158、361、371、373、396、397、420、494、496、497、504、510和527位氨基酸密码子GGA替换为GGT;
(3)将第4、43、60、299、310、384、439和493位氨基酸密码子CCT替换为CCA;
(4)将第6、147、207、265、300、425、464、532和535位氨基酸密码子CTT替换为TTG;
(5)将第8、54、80、164、208、360和445位氨基酸密码子GCA替换为GCT;
(6)将第9、121、227、330、354、405、444和511位氨基酸密码子GTA替换为GTT;
(7)将第11、97、327、364和385位氨基酸密码子CTC替换为TTG;
(8)将第13、16、51、58、63、74、123、134、196、202、209、232、253、257、260、269、276、321、358、392、407、413、456、478、484和498位氨基酸密码子GAA替换为GAG;
(9)将第14、26、174、513和516位氨基酸密码子CTG替换为TTG;
(10)将第15、56、67、120、137、230、243、274、340、417和519位氨基酸密码子TAT替换为TAC;
(11)将第18位氨基酸密码子TCT替换为TCC;
(12)将第20、25、219、305和399位氨基酸密码子ACA替换为ACC;
(13)将第21、37、52、64、99、125、132、181、189、197、213、220、255、287、334、383、390、401、505和543位氨基酸密码子TTT替换为TTC;
(14)将第22、53、109、156、272、391、426和539位氨基酸密码子GTG替换为GTT;
(15)将第27位氨基酸密码子AAG替换为AAA;
(16)将第29、185、283、289、315、349、525和547位氨基酸密码子AGT替换为TCC;
(17)将第30、233、341、378和471位氨基酸密码子TCA替换为TCC;
(18)将第32、201、479和492位氨基酸密码子ACG替换为ACT;
(19)将第34、292、319和523位氨基酸密码子CTA替换为TTG;
(20)将第35、108、112、258、297和359位氨基酸密码子GAC替换为GAT;
(21)将第36、38、48、91、270、280、346、380、465和466位氨基酸密码子GCC替换为GCT;
(22)将第44、56、226、236、262、362、370、403、522、526和534位氨基酸密码子ACA替换为ACT;
(23)将第49、82、119、143、229、308、316和512位氨基酸密码子ATA替换为ATC;
(24)将第62、148、160、240和406位氨基酸密码子TTA替换为TTG;
(25)将第68、282、295、296和524位氨基酸密码子GAG替换为GAA;
(26)将第69位氨基酸密码子CCA替换为CCT;
(27)将第70、100、113、140、193、239和508位氨基酸密码子ACT替换为ACC;
(28)将第79和118位氨基酸密码子TTA替换为CTG;
(29)将第81、83、88、110、145、199、249、336、345、404、458和518位氨基酸密码子AAA替换为AAG;
(30)将第85、216、323和387位氨基酸密码子GCA替换为GCC;
(31)将第95、238和450位氨基酸密码子CAT替换为CAC;
(32)将第101和115位氨基酸密码子CTC替换为CTG;
(33)将第103和388位氨基酸密码子AGG替换为AGA;
(34)将第107、242、263和431位氨基酸密码子CAA替换为CAG;
(35)将第117、301、302和530位氨基酸密码子TCG替换为TCC;
(36)将第128、188和422位氨基酸密码子CCC替换为CCA;
(37)将第133位氨基酸密码子CGC替换为AGA;
(38)将第146和419位氨基酸密码子GGG替换为GGT;
(39)将第149位氨基酸密码子CTT替换为CTG;
(40)将第155和306位氨基酸密码子ACG替换为ACC;
(41)将第162、175、241、314、329、374、375、381、395、411、453、463、472、480、489和509位氨基酸密码子AAT替换为AAC;
(42)将第165位氨基酸密码子GCG替换为GCC;
(43)将第166、313、338、533和549位氨基酸密码子TTG替换为CTG;
(44)将第173、191、348、382、421、449和538位氨基酸密码子ATT替换为ATC;
(45)将第176、332、491、506和529位氨基酸密码子GGC替换为GGT;
(46)将第178位氨基酸密码子CGA替换为AGG;
(47)将第179、325和514位氨基酸密码子GTG替换为GTC;
(48)将第186、430和502位氨基酸密码子CAG替换为CAA;
(49)将第194和379位氨基酸密码子GTA替换为GTC;
(50)将第204、256和290位氨基酸密码子CGA替换为AGA;
(51)将第205、278和457位氨基酸密码子GCG替换为GCT;
(52)将第245、337和501位氨基酸密码子GTT替换为GTC;
(53)将第246、259和347位氨基酸密码子CGT替换为AGA;
(54)将第252和440位氨基酸密码子CCG替换为CCA;
(55)将第254、412和454位氨基酸密码子TCA替换为TCT;
(56)将第261、424和469位氨基酸密码子ACC替换为ACT;
(57)将第266、423和428位氨基酸密码子TCG替换为TCT;
(58)将第281、389和546位氨基酸密码子TGC替换为TGT;
(59)将第288位氨基酸密码子TCC替换为TCA;
(60)将第320和357位氨基酸密码子AGT替换为TCT;
(61)将第322和548位氨基酸密码子CGG替换为AGA;
(62)将第344和351位氨基酸密码子GTC替换为GTT;
(63)将第353位氨基酸密码子AGC替换为TCT;
(64)将第418位氨基酸密码子CCC替换为CCT;
(65)将第429位氨基酸密码子GGT替换为GGA;
(66)将第451和536位氨基酸密码子AGC替换为TCC;
(67)将第452位氨基酸密码子ATC替换为ATT;
(68)将第470位氨基酸密码子TCC替换为TCT;
(69)将第556位的终止密码子TGA替换为TAA。
本发明优化的玉足海参海藻糖酶基因序列所编码的氨基酸序列与原基因序列(SEQ ID NO.1)所编码的氨基酸序列一致,所述的氨基酸序列如SEQ ID NO.3所示。
优选地,本发明提供的一种密码子优化的玉足海参海藻糖酶基因,其核苷酸序列如SEQ ID NO.2所示。
本发明第二个目的是提供一种表达载体,含有上述的密码子优化的玉足海参海藻糖酶基因。优选地,所述的表达载体为pGAP9K。
本发明第三个目的是提供一种宿主细胞,含有上述的表达载体。
优选地,所述的宿主细胞为毕赤酵母GS115。
本发明第四个目的是提供上述密码子优化的玉足海参海藻糖酶基因、扩增引物、表达载体或宿主细胞在制备海藻糖酶或其制剂中的应用。
优选地,包括以下步骤:将密码子优化的玉足海参海藻糖酶基因连接至表达载体如pGAPZαA上,筛选得到重组表达载体;再将重组表达载体转化宿主细胞如毕赤酵母,筛选获得阳性重组子;将阳性重组子进行诱导培养,其培养物经分离纯化后得到海藻糖酶。
优选地,所述的诱导培养,其条件为:培养液含2.5-3.5%(质量分数)NaCl;pH 5-6;28-32℃培养60-84h。
更优选地,所述的诱导培养,其条件为:培养液含2.8%(质量分数)NaCl;pH 5.5;30℃培养72h。
与现有技术相比,本发明的优势在于:
(1)本发明优化的玉足海参海藻糖酶基因序列编码的氨基酸序列与原基因序列编码一致,但能在酵母表达系统(毕赤酵母GS115)中成功表达出具有海藻糖酶活性的蛋白;
(2)本发明提供了毕赤酵母表达阳性克隆的最佳产酶条件,在此条件下,玉足海参海藻糖酶组成型重组表达蛋白的活性达到A-THL=418.1U/ml;
(3)本发明为人工养殖海参提供了一种有助于摄食和消化的饲料调节剂,同时也可应用于大型海藻的精深加工中的海藻营养提取与释放。
附图说明
图1为玉足海参海藻糖酶基因的氨基酸、核苷酸序列及密码子优化位点信息,其中AA:玉足海参海藻糖酶氨基酸序列;S1:玉足海参海藻糖酶核苷酸序列;S2:密码子优化后的海藻糖酶核苷酸序列)。
图2为人工合成的EcoR I-Hl-Tre-Not I基因结构图。
图3为EcoR I-Hl-Tre-Not I基因扩增电泳图。
图4为pGAP9K-Hl-Tre菌落PCR鉴定电泳图。
图5为pGAP9K-Hl-TrePme I线性化琼脂糖凝胶电泳检测图。
图6为pGAP9K-Hl-Tre GS115电转化菌落PCR鉴定电泳图。
图7为玉足海参海藻糖酶组成型重组表达蛋白(P-Hl-Tre)的表达情况,其中M:分子量Marker;S1-S3:玉足海参海藻糖酶组成型重组表达菌株1-3用YPD培养基培养3天后的上清样品;C:毕赤酵母GS115用YPD培养基培养3天后的上清样品。
图8为玉足海参海藻糖酶组成型重组表达菌株的产酶条件优化。
图9为玉足海参海藻糖酶组成型重组表达菌株的酶活测定。
具体实施方式
以下结合实施例对本发明作进一步的说明,但并不局限于此。
下述实施例中的实验方法,如无特殊说明,均为常规方法或者按照试剂盒说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径获得。引物合成和测序由生工生物工程(上海)股份有限公司完成。
实施例1目的基因密码子优化及基因合成
为了构建更适宜的毕赤酵母表达载体pGAPZαA,对发明人前期克隆获得的Hl-Tre基因序列(GenBank:MG765267)利用密码子数据库(http://gcua.schoedl.de/)进行序列分析,发现Hl-Tre基因中有多处是毕赤酵母的稀有密码子。本发明对密码子进行优化,在不改变氨基酸序列的前提下,使其与毕赤酵母GS115的密码子使用频率相匹配(图1),密码子优化后的玉足海参海藻糖酶基因序列如SEQ ID NO.2所示(atggacggtccagttttggaggctgttcaattggctgagttgtacgaggactccaagaccttcgttgacatgaccttgaaagagtcctccgacactgttttggatgctttcgctgatatcgacgacccaactgacaaggctgctatcgaagagttcgttgctacttacttcgagggtccagacttggagttcgatgattacgaacctaccgactggaaagagaaccctttgttcctggctaagatcaaggacgccacttacaagaagtgggctgaagatctgcacagattgtggttcaccctgggtagaaagatcaagcaggatgttaaggacgataccgacctgtactccctgatctacgttgacgagtacttcgttgttccaggtggtagattcagagagttcttctactgggacacctactggatcatcaagggtttgttgctgtccgagatgtacgacaccgttagaggtatgttgagaaactgggctgccctgattgacagatacggtaagatcttgaacggtaacagggtctacttcgagaacagatcccaaccaccattcttcatcccaaccgtcaacgagttctacaaggctactgaggacagagctttcttggctgagatgatgccattcatggaagccgagtacaccttctggatgaacgagagaactgttgagatctacgacgagtccaagcagactaaccacaccttgaaccagtacaacgtcagaatgggtaagccaagaccagagtctttcagagaggatagagagactactcaggacttgtctgaagatgaggctgctgttgtttacgctgagattgcttctgcttgtgaatccggttgggacttctcatccagatggttgggtcctgaagaagatgctccattgtcctccatgagaaccaccaagatcgttccagctgacctgaactccatcatgtgtttgtctgagagagccatggtcaccttgcacaacgttatgggtaacttcgacaaggtcctggaatactccaacgctgttaaggctagaatctccgctgttgagtctgttttgtggtctgaggatgctggtacttggttggacttcgacttggaaactggtgagggtaacaacaagttctccgtcgctaacatcttcccattgtgggccagatgtttcgttgaggacgaaaacggtggtgtcaccaacttcgagactaaggttttggagtacctgctgaactctgaggttctggattaccctggtggtatcccatctactttggttgactctggacaacagtgggactacccaaatgtttggccaccattgatggaagttgctatccaggctatccactccattaactctgacgaggctaagactatcgcctacaacttggctgctaactggacttcttccaactgggaaaagtacgacgagactaacgtcatgtacgagaagtacaactgcaacgacggtactccaggttctggtggtgagtacattgtccaagatggtttcggttggaccaacggtgttatcttggtcttgttggacaagtacggtgacactttggaatccactggtgatggttccagattgctgactttgtccccaatcgttttgctgggtttctgcatgtgttccagactgcatcatcaccaccatcactaa)。在目的基因两端分别引入EcoR I和Not I酶切位点,将优化后的基因序列交生工生物工程(上海)股份有限公司,委托其人工合成基因序列;并将基因合成的EcoR I-Hl-Tre-Not I(图2)连接到pPICZαA载体,得到重组质粒pPICZαA-Hl-Tre。
实施例2玉足海参海藻糖酶组成型毕赤酵母表达载体pGAP9K-Hl-Tre的构建
以商业化的pGAPZαA载体为模板,以引物F1:5’-CGG ATC CGA TCT TTT TTG TAGAAA TGT CTT GGT GTC C-3’(下划线为BamH I酶切位点);R1:5’-GAA TTC AGC TTC AGCCTC TCT TTT CTC GAG AGATAC C-3’(下划线为EcoR I酶切位点)为引物进行GAP启动子序列扩增。PCR产物纯化后,与pMD19-T simple载体连接,转化至E.coli DH5α感受态细胞。转化子提质粒,经BamH I和EcoR I双酶切,胶回收后得到pGAP片段。
用BamH I和EcoR I双酶切商业化的pPIC9K质粒,再将酶切产物用T4 DNA连接酶与pGAP片段连接过夜。将连接产物转化到E.coli DH5α感受态细胞。转化子提质粒,获得组成型表达载体pGAP9K质粒。质粒送生工生物工程(上海)股份有限公司测序,并与NCBI的数据进行比对,以核验表达载体序列的正确性。
以基因合成的玉足海参海藻糖酶重组质粒pPICZαA-Hl-Tre为模板,F2:5’-GACTGGTTCCAATTGACAAGC-3’,R2:5’-GCAAATGGCATTCTGACATCC-3’为引物进行PCR扩增,获取含有目的基因片段EcoR I-Hl-Tre-Not I的扩增产物。扩增产物经1%琼脂糖凝胶电泳检测后(图3),送生工生物工程(上海)股份有限公司测序,并与NCBI的数据进行比对,以核验人工合成基因的正确性。测序正确的扩增产物经PCR产物纯化试剂盒纯化后,用内切酶EcoR I和Not I双酶切,酶切反应体系为:10×Fast digestbuffer 5μL,Fast digest EcoR I 2μL,Fast digestNot I 2μL,PCR回收产物41μL,总体积50μL;酶切条件为37℃3h。
同时,用内切酶EcoR I和Not I双酶切pGAP9K质粒,酶切反应体系为:10×Fastdigest buffer 5μL,Fast digestEcoR I 2μL,Fast digestNot I 2μL,pGAP9K质粒25μL,ddH2O 16μL,总体积50μL;酶切条件为37℃1h。
pPICZαA-Hl-Tre和pGAP9K质粒的双酶切产物经PCR产物纯化后,用T4 DNA连接酶进行连接,连接反应体系为:10×T4 DNA ligase buffer 1μL,T4 DNA ligase 1μL,pPICZαA-Hl-Tre酶切回收片段6μL,pGAP9K质粒酶切回收片段2μL,总体积10μL;连接条件为16℃连接过夜。连接产物转化E coli DH5α感受态细胞,并涂布于卡那霉素(10μg/ml)抗性平板。待菌落长出后,挑取单克隆至10μL无菌水中混匀做模板,用引物F3:5’-GTCCCTATTTCAATCAATTGAAC-3’和R2:5’-GCAAATGGCATTCTGACATCC-3’进行菌落PCR扩增检测。扩增产物经1%琼脂糖凝胶电泳检测后,将阳性克隆送生工生物工程(上海)股份有限公司测序(图4);测序结果与NCBI的数据进行比对,以确认玉足海参海藻糖酶组成型毕赤酵母表达载体pGAP9K-Hl-Tre构建的正确性。
实施例3 pGAP9K-Hl-Tre电转化及重组子筛选
测序正确的玉足海参海藻糖酶组成型毕赤酵母表达载体pGAP9K-Hl-Tre用Pme I限制性内切酶酶切,使其线性化(图5)。酶切完毕后的线性化产物用PCR产物纯化试剂盒回收后,溶于5-10μl TE中备用。取80μl毕赤酵母GS115感受态细胞与5-20μg线性化pGAP9K-Hl-Tre混合,转入预冷的0.2cm电转杯中;在冰上放置5min,1500V电转化,立即加入1ml预冷的1M山梨醇,复苏后涂布MD平板,30℃培养24-48h。用无菌牙签挑取阳性转化子点种到含有4mg/ml G418的MD平板上进行高拷贝克隆筛选。筛选出的阳性克隆用菌落PCR进行鉴定,鉴定引物为F3:5’-GTCCCTATTTCAATCAATTGAAC-3’和R2:5’-GCAAATGGCATTCTGACATCC-3’。扩增产物经1%琼脂糖凝胶电泳检测后,将阳性克隆送生工生物工程(上海)股份有限公司测序(图6),测序正确的克隆即为玉足海参海藻糖酶组成型毕赤酵母表达菌株。
实施例4玉足海参海藻糖酶组成型毕赤酵母表达阳性克隆摇瓶诱导培养
将玉足海参海藻糖酶组成型毕赤酵母表达阳性克隆用牙签挑取至0.5ml MD液体培养基,30℃,200rpm摇床培养至OD600为1;再以1%比例接种于含20mLYPD培养基的250ml三角瓶中,200rpm、30℃培养3天。将培养液转移至50ml离心管中,4℃,12000g离心5min,取上清进行SDS-PAGE蛋白电泳分析,检测玉足海参海藻糖酶组成型重组表达蛋白(P-Hl-Tre)的表达情况,结果见图7。
实施例5玉足海参海藻糖酶组成型重组表达蛋白的活性检测
利用生工生物工程(上海)股份有限公司生产的海藻糖酶活性检测试剂盒(D799409-0050)检测摇瓶培养的粗酶活性,具体检测方法参考该试剂盒说明书。通过检测不同培养温度、时间、培养基盐度及pH下的海藻糖酶活性,获得最佳产酶量的培养条件如下:按质量分数计,培养液含1%Yeast Extract(酵母膏),2%Peptone(蛋白胨),2%glucose(葡萄糖),2.8%NaCl(氯化钠),pH 5.5;30℃培养72h,具体见图8。
按照说明书的操作方法建立标准曲线,公式为y=0.9599x+0.2194(R2=0.9883)。一个单位酶活定义为:在最优产酶条件下,每ml上清在反应体系中每分钟催化产生1μg葡萄糖定义为一个酶活力单位。因此,海藻糖酶活力A-THL(U/ml)=1000×y。玉足海参海藻糖酶组成型重组表达蛋白在540nm(A540)处的吸光度为A540=0.207,代入标准曲线中得到y=0.4181mg/ml,经过计算确定玉足海参海藻糖酶组成型重组表达蛋白的活性为A-THL=418.1U/ml,结果见图9。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国科学院南海海洋研究所
南方海洋科学与工程广东省实验室(广州)
<120> 一种密码子优化的玉足海参海藻糖酶基因及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1668
<212> DNA
<213> 玉足海参(Holothuria leucospilota)
<400> 1
atggatggac ctgttcttga ggcagtacaa ctcgctgaac tgtatgaaga ttctaagaca 60
tttgtggata tgacactgaa ggagagttca gacacggttc tagacgcctt tgccgatatc 120
gacgatccta cagataaggc tgccatagaa gaatttgtgg caacttattt cgaaggtcct 180
gatttagaat ttgatgatta tgagccaact gattggaaag aaaacccttt gttcttagca 240
aaaataaaag atgcaacata caaaaagtgg gccgaggatc tgcatagact ctggtttact 300
ctcggaagga agatcaagca agacgtgaaa gatgacactg atctctactc gttaatatat 360
gtagatgaat actttgttgt tcccggtgga agatttcgcg aattcttcta ttgggacact 420
tactggataa tcaaagggct tttactttcc gagatgtacg atacggtgag aggaatgtta 480
agaaattggg cagcgttgat tgacagatac ggtaagattc tgaatggcaa ccgagtgtac 540
tttgagaaca gaagtcagcc accctttttc attccaactg taaacgaatt ttacaaagct 600
acggaagatc gagcgttcct tgcagaaatg atgccattta tggaagcaga gtacacattt 660
tggatgaacg agagaacagt agagatatat gacgaatcaa agcagacaaa ccatacttta 720
aatcaatata acgttcgtat gggtaaacca agaccggaat catttcgaga agaccgtgaa 780
accacacaag atctttcgga agatgaagcc gctgtggttt atgctgaaat tgcgtctgcc 840
tgcgagagtg gttgggactt ttccagtcga tggctaggtc ctgaggagga cgctcctctt 900
tcgtcgatga gaacaacgaa gatagttcct gctgatttga atagtataat gtgtctaagt 960
gaacgggcaa tggtgaccct ccacaatgta atgggcaact ttgacaaagt tttggaatat 1020
tcaaacgctg tcaaagcccg tattagtgct gtcgagagcg tattgtggag tgaagacgca 1080
ggaacatggc tcgatttcga cttggaaaca ggagagggaa ataataagtt ctcagtagcc 1140
aatatttttc ctctctgggc aaggtgcttt gtggaagacg aaaatggagg agtcacaaac 1200
tttgagacaa aagtattaga atacctgctg aattcagaag ttctggatta tcccggggga 1260
attccctcga cccttgtgga ttcgggtcag caatgggact acccaaatgt ttggcctccg 1320
ttgatggaag tagcaatcca ggctattcat agcatcaatt cagatgaagc gaaaactatc 1380
gcctacaatc ttgccgccaa ctggacctcc tcaaattggg aaaagtacga cgaaacgaat 1440
gtcatgtacg aaaagtacaa ctgcaatgat ggcacgcctg gatctggagg agaatacatt 1500
gttcaggatg gatttggctg gactaatgga gtaatactgg tgttgctgga taaatatggt 1560
gacacactag agagtacagg agatggctcg agacttttga cacttagccc aattgtgttg 1620
ctgggttttt gcatgtgcag tcggttgcac caccaccacc accactga 1668
<210> 2
<211> 1668
<212> DNA
<213> 玉足海参(Holothuria leucospilota)
<400> 2
atggacggtc cagttttgga ggctgttcaa ttggctgagt tgtacgagga ctccaagacc 60
ttcgttgaca tgaccttgaa agagtcctcc gacactgttt tggatgcttt cgctgatatc 120
gacgacccaa ctgacaaggc tgctatcgaa gagttcgttg ctacttactt cgagggtcca 180
gacttggagt tcgatgatta cgaacctacc gactggaaag agaacccttt gttcctggct 240
aagatcaagg acgccactta caagaagtgg gctgaagatc tgcacagatt gtggttcacc 300
ctgggtagaa agatcaagca ggatgttaag gacgataccg acctgtactc cctgatctac 360
gttgacgagt acttcgttgt tccaggtggt agattcagag agttcttcta ctgggacacc 420
tactggatca tcaagggttt gttgctgtcc gagatgtacg acaccgttag aggtatgttg 480
agaaactggg ctgccctgat tgacagatac ggtaagatct tgaacggtaa cagggtctac 540
ttcgagaaca gatcccaacc accattcttc atcccaaccg tcaacgagtt ctacaaggct 600
actgaggaca gagctttctt ggctgagatg atgccattca tggaagccga gtacaccttc 660
tggatgaacg agagaactgt tgagatctac gacgagtcca agcagactaa ccacaccttg 720
aaccagtaca acgtcagaat gggtaagcca agaccagagt ctttcagaga ggatagagag 780
actactcagg acttgtctga agatgaggct gctgttgttt acgctgagat tgcttctgct 840
tgtgaatccg gttgggactt ctcatccaga tggttgggtc ctgaagaaga tgctccattg 900
tcctccatga gaaccaccaa gatcgttcca gctgacctga actccatcat gtgtttgtct 960
gagagagcca tggtcacctt gcacaacgtt atgggtaact tcgacaaggt cctggaatac 1020
tccaacgctg ttaaggctag aatctccgct gttgagtctg ttttgtggtc tgaggatgct 1080
ggtacttggt tggacttcga cttggaaact ggtgagggta acaacaagtt ctccgtcgct 1140
aacatcttcc cattgtgggc cagatgtttc gttgaggacg aaaacggtgg tgtcaccaac 1200
ttcgagacta aggttttgga gtacctgctg aactctgagg ttctggatta ccctggtggt 1260
atcccatcta ctttggttga ctctggacaa cagtgggact acccaaatgt ttggccacca 1320
ttgatggaag ttgctatcca ggctatccac tccattaact ctgacgaggc taagactatc 1380
gcctacaact tggctgctaa ctggacttct tccaactggg aaaagtacga cgagactaac 1440
gtcatgtacg agaagtacaa ctgcaacgac ggtactccag gttctggtgg tgagtacatt 1500
gtccaagatg gtttcggttg gaccaacggt gttatcttgg tcttgttgga caagtacggt 1560
gacactttgg aatccactgg tgatggttcc agattgctga ctttgtcccc aatcgttttg 1620
ctgggtttct gcatgtgttc cagactgcat catcaccacc atcactaa 1668
<210> 3
<211> 555
<212> PRT
<213> 玉足海参(Holothuria leucospilota)
<400> 3
Met Asp Gly Pro Val Leu Glu Ala Val Gln Leu Ala Glu Leu Tyr Glu
1 5 10 15
Asp Ser Lys Thr Phe Val Asp Met Thr Leu Lys Glu Ser Ser Asp Thr
20 25 30
Val Leu Asp Ala Phe Ala Asp Ile Asp Asp Pro Thr Asp Lys Ala Ala
35 40 45
Ile Glu Glu Phe Val Ala Thr Tyr Phe Glu Gly Pro Asp Leu Glu Phe
50 55 60
Asp Asp Tyr Glu Pro Thr Asp Trp Lys Glu Asn Pro Leu Phe Leu Ala
65 70 75 80
Lys Ile Lys Asp Ala Thr Tyr Lys Lys Trp Ala Glu Asp Leu His Arg
85 90 95
Leu Trp Phe Thr Leu Gly Arg Lys Ile Lys Gln Asp Val Lys Asp Asp
100 105 110
Thr Asp Leu Tyr Ser Leu Ile Tyr Val Asp Glu Tyr Phe Val Val Pro
115 120 125
Gly Gly Arg Phe Arg Glu Phe Phe Tyr Trp Asp Thr Tyr Trp Ile Ile
130 135 140
Lys Gly Leu Leu Leu Ser Glu Met Tyr Asp Thr Val Arg Gly Met Leu
145 150 155 160
Arg Asn Trp Ala Ala Leu Ile Asp Arg Tyr Gly Lys Ile Leu Asn Gly
165 170 175
Asn Arg Val Tyr Phe Glu Asn Arg Ser Gln Pro Pro Phe Phe Ile Pro
180 185 190
Thr Val Asn Glu Phe Tyr Lys Ala Thr Glu Asp Arg Ala Phe Leu Ala
195 200 205
Glu Met Met Pro Phe Met Glu Ala Glu Tyr Thr Phe Trp Met Asn Glu
210 215 220
Arg Thr Val Glu Ile Tyr Asp Glu Ser Lys Gln Thr Asn His Thr Leu
225 230 235 240
Asn Gln Tyr Asn Val Arg Met Gly Lys Pro Arg Pro Glu Ser Phe Arg
245 250 255
Glu Asp Arg Glu Thr Thr Gln Asp Leu Ser Glu Asp Glu Ala Ala Val
260 265 270
Val Tyr Ala Glu Ile Ala Ser Ala Cys Glu Ser Gly Trp Asp Phe Ser
275 280 285
Ser Arg Trp Leu Gly Pro Glu Glu Asp Ala Pro Leu Ser Ser Met Arg
290 295 300
Thr Thr Lys Ile Val Pro Ala Asp Leu Asn Ser Ile Met Cys Leu Ser
305 310 315 320
Glu Arg Ala Met Val Thr Leu His Asn Val Met Gly Asn Phe Asp Lys
325 330 335
Val Leu Glu Tyr Ser Asn Ala Val Lys Ala Arg Ile Ser Ala Val Glu
340 345 350
Ser Val Leu Trp Ser Glu Asp Ala Gly Thr Trp Leu Asp Phe Asp Leu
355 360 365
Glu Thr Gly Glu Gly Asn Asn Lys Phe Ser Val Ala Asn Ile Phe Pro
370 375 380
Leu Trp Ala Arg Cys Phe Val Glu Asp Glu Asn Gly Gly Val Thr Asn
385 390 395 400
Phe Glu Thr Lys Val Leu Glu Tyr Leu Leu Asn Ser Glu Val Leu Asp
405 410 415
Tyr Pro Gly Gly Ile Pro Ser Thr Leu Val Asp Ser Gly Gln Gln Trp
420 425 430
Asp Tyr Pro Asn Val Trp Pro Pro Leu Met Glu Val Ala Ile Gln Ala
435 440 445
Ile His Ser Ile Asn Ser Asp Glu Ala Lys Thr Ile Ala Tyr Asn Leu
450 455 460
Ala Ala Asn Trp Thr Ser Ser Asn Trp Glu Lys Tyr Asp Glu Thr Asn
465 470 475 480
Val Met Tyr Glu Lys Tyr Asn Cys Asn Asp Gly Thr Pro Gly Ser Gly
485 490 495
Gly Glu Tyr Ile Val Gln Asp Gly Phe Gly Trp Thr Asn Gly Val Ile
500 505 510
Leu Val Leu Leu Asp Lys Tyr Gly Asp Thr Leu Glu Ser Thr Gly Asp
515 520 525
Gly Ser Arg Leu Leu Thr Leu Ser Pro Ile Val Leu Leu Gly Phe Cys
530 535 540
Met Cys Ser Arg Leu His His His His His His
545 550 555
Claims (10)
1.一种密码子优化的玉足海参海藻糖酶基因,其特征在于,其核苷酸序列是以SEQ IDNO.1所示的序列为基础,作出以下(1)~(69)任意一项或多项的改变:
(1)将第2、17、23、42、45、61、71、84、111、114、122、154、203、264、312、365、427、455、490和517位氨基酸密码子GAT替换为GAC;
(2)将第3、102、130、158、361、371、373、396、397、420、494、496、497、504、510和527位氨基酸密码子GGA替换为GGT;
(3)将第4、43、60、299、310、384、439和493位氨基酸密码子CCT替换为CCA;
(4)将第6、147、207、265、300、425、464、532和535位氨基酸密码子CTT替换为TTG;
(5)将第8、54、80、164、208、360和445位氨基酸密码子GCA替换为GCT;
(6)将第9、121、227、330、354、405、444和511位氨基酸密码子GTA替换为GTT;
(7)将第11、97、327、364和385位氨基酸密码子CTC替换为TTG;
(8)将第13、16、51、58、63、74、123、134、196、202、209、232、253、257、260、269、276、321、358、392、407、413、456、478、484和498位氨基酸密码子GAA替换为GAG;
(9)将第14、26、174、513和516位氨基酸密码子CTG替换为TTG;
(10)将第15、56、67、120、137、230、243、274、340、417和519位氨基酸密码子TAT替换为TAC;
(11)将第18位氨基酸密码子TCT替换为TCC;
(12)将第20、25、219、305和399位氨基酸密码子ACA替换为ACC;
(13)将第21、37、52、64、99、125、132、181、189、197、213、220、255、287、334、383、390、401、505和543位氨基酸密码子TTT替换为TTC;
(14)将第22、53、109、156、272、391、426和539位氨基酸密码子GTG替换为GTT;
(15)将第27位氨基酸密码子AAG替换为AAA;
(16)将第29、185、283、289、315、349、525和547位氨基酸密码子AGT替换为TCC;
(17)将第30、233、341、378和471位氨基酸密码子TCA替换为TCC;
(18)将第32、201、479和492位氨基酸密码子ACG替换为ACT;
(19)将第34、292、319和523位氨基酸密码子CTA替换为TTG;
(20)将第35、108、112、258、297和359位氨基酸密码子GAC替换为GAT;
(21)将第36、38、48、91、270、280、346、380、465和466位氨基酸密码子GCC替换为GCT;
(22)将第44、56、226、236、262、362、370、403、522、526和534位氨基酸密码子ACA替换为ACT;
(23)将第49、82、119、143、229、308、316和512位氨基酸密码子ATA替换为ATC;
(24)将第62、148、160、240和406位氨基酸密码子TTA替换为TTG;
(25)将第68、282、295、296和524位氨基酸密码子GAG替换为GAA;
(26)将第69位氨基酸密码子CCA替换为CCT;
(27)将第70、100、113、140、193、239和508位氨基酸密码子ACT替换为ACC;
(28)将第79和118位氨基酸密码子TTA替换为CTG;
(29)将第81、83、88、110、145、199、249、336、345、404、458和518位氨基酸密码子AAA替换为AAG;
(30)将第85、216、323和387位氨基酸密码子GCA替换为GCC;
(31)将第95、238和450位氨基酸密码子CAT替换为CAC;
(32)将第101和115位氨基酸密码子CTC替换为CTG;
(33)将第103和388位氨基酸密码子AGG替换为AGA;
(34)将第107、242、263和431位氨基酸密码子CAA替换为CAG;
(35)将第117、301、302和530位氨基酸密码子TCG替换为TCC;
(36)将第128、188和422位氨基酸密码子CCC替换为CCA;
(37)将第133位氨基酸密码子CGC替换为AGA;
(38)将第146和419位氨基酸密码子GGG替换为GGT;
(39)将第149位氨基酸密码子CTT替换为CTG;
(40)将第155和306位氨基酸密码子ACG替换为ACC;
(41)将第162、175、241、314、329、374、375、381、395、411、453、463、472、480、489和509位氨基酸密码子AAT替换为AAC;
(42)将第165位氨基酸密码子GCG替换为GCC;
(43)将第166、313、338、533和549位氨基酸密码子TTG替换为CTG;
(44)将第173、191、348、382、421、449和538位氨基酸密码子ATT替换为ATC;
(45)将第176、332、491、506和529位氨基酸密码子GGC替换为GGT;
(46)将第178位氨基酸密码子CGA替换为AGG;
(47)将第179、325和514位氨基酸密码子GTG替换为GTC;
(48)将第186、430和502位氨基酸密码子CAG替换为CAA;
(49)将第194和379位氨基酸密码子GTA替换为GTC;
(50)将第204、256和290位氨基酸密码子CGA替换为AGA;
(51)将第205、278和457位氨基酸密码子GCG替换为GCT;
(52)将第245、337和501位氨基酸密码子GTT替换为GTC;
(53)将第246、259和347位氨基酸密码子CGT替换为AGA;
(54)将第252和440位氨基酸密码子CCG替换为CCA;
(55)将第254、412和454位氨基酸密码子TCA替换为TCT;
(56)将第261、424和469位氨基酸密码子ACC替换为ACT;
(57)将第266、423和428位氨基酸密码子TCG替换为TCT;
(58)将第281、389和546位氨基酸密码子TGC替换为TGT;
(59)将第288位氨基酸密码子TCC替换为TCA;
(60)将第320和357位氨基酸密码子AGT替换为TCT;
(61)将第322和548位氨基酸密码子CGG替换为AGA;
(62)将第344和351位氨基酸密码子GTC替换为GTT;
(63)将第353位氨基酸密码子AGC替换为TCT;
(64)将第418位氨基酸密码子CCC替换为CCT;
(65)将第429位氨基酸密码子GGT替换为GGA;
(66)将第451和536位氨基酸密码子AGC替换为TCC;
(67)将第452位氨基酸密码子ATC替换为ATT;
(68)将第470位氨基酸密码子TCC替换为TCT;
(69)将第556位的终止密码子TGA替换为TAA。
2.根据权利要求1所述的密码子优化的玉足海参海藻糖酶基因,其特征在于,其核苷酸序列如SEQ ID NO.2所示。
3.一种表达载体,其特征在于,含有权利要求1或2所述的密码子优化的玉足海参海藻糖酶基因。
4.根据权利要求3所述的表达载体,其特征在于,所述的表达载体为pGAP9K。
5.一种宿主细胞,其特征在于,含有权利要求3或4所述的表达载体。
6.根据权利要求5所述的宿主细胞,其特征在于,所述的宿主细胞为毕赤酵母GS115。
7.权利要求1或2所述的密码子优化的玉足海参海藻糖酶基因、权利要求3或4所述的表达载体或权利要求5或6所述的宿主细胞在制备海藻糖酶或其制剂中的应用。
8.根据权利要求7所述的应用,其特征在于,包括以下步骤:将权利要求1或2所述的密码子优化的玉足海参海藻糖酶基因连接至权利要求3或4所述的表达载体上,筛选得到重组表达载体;再将重组表达载体转化至权利要求5或6所述的宿主细胞,筛选获得阳性重组子;将阳性重组子进行诱导培养,其培养物经分离纯化后得到海藻糖酶。
9.根据权利要求8所述的应用,其特征在于,所述的诱导培养,其条件为:培养液含2.5-3.5%(质量分数)NaCl;pH 5-6;28-32℃培养60-84h。
10.根据权利要求9所述的应用,其特征在于,所述的诱导培养,其条件为:培养液含2.8%(质量分数)NaCl;pH 5.5;30℃培养72h。
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