CN111690580B - 用于生产冰核蛋白的重组大肠杆菌及其构建方法与应用 - Google Patents

用于生产冰核蛋白的重组大肠杆菌及其构建方法与应用 Download PDF

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CN111690580B
CN111690580B CN201910199203.0A CN201910199203A CN111690580B CN 111690580 B CN111690580 B CN 111690580B CN 201910199203 A CN201910199203 A CN 201910199203A CN 111690580 B CN111690580 B CN 111690580B
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刘桂明
张子娟
赖小勤
晏礼明
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Abstract

本发明提供一种用于生产冰核蛋白的重组大肠杆菌及其构建方法与应用。所述重组大肠杆菌是利用基因工程手段将冰核蛋白基因或冰核蛋白基因表达盒整合到大肠杆菌的染色体上构建得到的。本发明通过将冰核蛋白基因整合到大肠杆菌的染色体中,并除去抗性,得到可用于生产冰核蛋白,且不含任何已知抗生素抗性的重组大肠杆菌。该重组大肠杆菌表达的冰核蛋白可以在稀释为万分之一的水中显著提高冰点,在过冷至‑2℃左右即可结冰,该重组大肠杆菌具有很好的成冰核活性,应用前景广阔。

Description

用于生产冰核蛋白的重组大肠杆菌及其构建方法与应用
技术领域
本发明属于基因工程领域,具体地说,涉及一种用于生产冰核蛋白的重组大肠杆菌及其构建方法与应用。
背景技术
冰核蛋白最早发现于丁香假单胞菌(Psudomonas syringae)中,冰核蛋白的存在使得该菌能够在过冷水中诱导形成冰晶。随后,在欧文氏菌属(Erwinia)和黄单胞菌属(Xanthomonas)中的一些菌种也发现存在冰核蛋白。表达有冰核蛋白的细菌可以在较高的温度(-2~-5℃)下形成规则、细腻、微小异质冰晶的能力。因此,冰核蛋白可以用于人工造雪,食品冷冻浓缩和冻干等。
丁香假单胞菌是一种植物病害菌,该菌引起的植物病害发生率位居十大细菌性植物病害之首。如果直接将该菌应用于人工造雪,可能会引起造雪地附近的植物病害。利用大肠杆菌重组表达冰核蛋白则可以很好地解决这个问题。
发明内容
本发明的目的是提供一种用于生产冰核蛋白的重组大肠杆菌及其构建方法与应用。
为了实现本发明目的,第一方面,本发明提供一种用于生产冰核蛋白的重组大肠杆菌,所述重组大肠杆菌是利用基因工程手段将冰核蛋白基因或冰核蛋白基因表达盒整合到大肠杆菌的染色体上构建得到的。
本发明中,所述冰核蛋白基因来自于丁香假单胞菌(Psudomonas syringae),或者泛菌属(Pantoea sp.)、黄单胞菌属(Xanthanmonas sp.)中的菌种。优选丁香假单胞菌。丁香假单胞菌冰核蛋白基因的核苷酸序列及其编码蛋白的氨基酸序列分别如SEQ ID NO:1和2所示。
本发明提供的用于生产冰核蛋白的重组大肠杆菌中不含有抗生素抗性基因。所述抗生素包括氨苄青霉素、羧苄青霉素、氨基糖苷类(链霉素、庆大和卡那霉素)、四环素族,以及所有喹诺酮类抗生素等已知抗革兰氏阴性菌的抗生素。
进一步地,所述重组大肠杆菌的出发菌株为大肠杆菌B系菌株或K12系菌株,优选BW25113。
优选地,所述冰核蛋白基因表达盒中,所述冰核蛋白基因由阿拉伯糖启动子驱动。
优选地,所述重组大肠杆菌是利用CRISPR/Cas9系统将所述冰核蛋白基因或冰核蛋白基因表达盒整合到大肠杆菌基因组中得到的。
更优选地,所述CRISPR/Cas9系统靶向大肠杆菌galR基因。
所述CRISPR/Cas9系统中sgRNA作用位点的核酸序列为5′-TTGCACTATGCACAGCCAGC-3′(SEQ ID NO:7)。
第二方面,本发明提供用于生产冰核蛋白的重组大肠杆菌的构建方法,包括以下步骤:
1)从丁香假单胞菌中克隆得到冰核蛋白基因(SEQ ID NO:1);
2)将步骤1)所得基因构建到质粒pBAD/His A上,得到重组表达质粒;
3)以步骤2)所得重组表达质粒为模板扩增带有阿拉伯糖诱导启动子的冰核蛋白基因片段;同时以大肠杆菌基因组为模板PCR扩增得到galR基因上、下游片段knock-in-up和knock-in-down;
4)将步骤3)所得三个基因片段通过重叠延伸PCR得到基因编辑片段;
5)构建靶向大肠杆菌galR基因的CRISPR/Cas9基因编辑载体;
6)将步骤4)所得基因编辑片段和步骤5)所得CRISPR/Cas9打靶载体共同导入大肠杆菌BW25113中,得到用于生产冰核蛋白的重组大肠杆菌。
其中,步骤3)中用于PCR扩增所述knock-in-up和knock-in-down的引物分别如SEQID NO:3-4和SEQ ID NO:5-6所示。
步骤5)中所述CRISPR/Cas9基因编辑载体是基于CRISPR-Cas9系统的sgRNA表达载体,其中,sgRNA作用位点的核酸序列为5′-TTGCACTATGCACAGCCAGC-3′。
在本发明的一个具体实施方式中,所述重组大肠杆菌的构建方法如下:
(1)从丁香假单胞菌中克隆冰核蛋白基因;
(2)将步骤(1)所得的基因连接到重组表达质粒pBAD/His A上;
(3)以步骤(2)所得的重组表达质粒为模板扩增带有阿拉伯糖诱导启动子的冰核蛋白基因片段。以大肠杆菌基因组为模板扩增出拟插入位点基因上下游片段knock-in-up和knock-in-down;
(4)将步骤(3)所得的三个基因片段进行重叠延伸PCR以得到基因编辑片段;
(5)利用CRISPR/Cas9技术及步骤(4)所得基因编辑片段将带有阿拉伯糖诱导启动子的冰核蛋白基因整合到大肠杆菌中的基因组中得到重组大肠杆菌。
所述步骤(1)所述冰核蛋白基因inp的GenBank登录序列号为MK347225。
其中,所述产冰核蛋白的重组大肠杆菌的具体步骤如下:
①从丁香假单胞菌中克隆冰核蛋白基因inp;
②将步骤①所得的基因连接到pBAD/His A质粒上得到重组表达质粒pBAD-inp;
③以步骤②所得的重组表达质粒为模板扩增带有阿拉伯糖诱导启动子的冰核蛋白基因片段ara-inp,以大肠杆菌BW25113基因组DNA为模板扩增galR基因上下游片段galR-up和galR-down。将galR的基因序列导入sgRNA在线设计网站,设计得到其对应的N20
④将步骤③所得的三个基因片段ara-inp、galR-up和galR-down进行重叠延伸PCR以得到基因编辑片段ΔgalR∷ara-inp。将步骤③所得的N20连接入pTargetF(Addgene62226)得到质粒pTargetF-galR;
⑤将步骤④所得基因编辑片段ΔgalR∷ara-inp和质粒pTargetF-galR电转入含有pCas(Addgene 62225)质粒的大肠杆菌BW25113中;
⑥筛选步骤⑤所得重组菌得到没有抗生素抗性的重组大肠杆菌BW25113ΔgalR∷ara-inp。
第三方面,本发明提供所述重组大肠杆菌或按照上述方法构建得到的重组大肠杆菌的以下任一应用:
i)用于人工造雪;
ii)用于冷冻干燥工艺。
第四方面,本发明提供一种人工造雪剂,有效成分为所述重组大肠杆菌或按照上述方法构建得到的重组大肠杆菌,以及它们的死菌。
第五方面,本发明提供一种制冷剂,有效成分为所述重组大肠杆菌或按照上述方法构建得到的重组大肠杆菌,以及它们的死菌。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明通过将冰核蛋白基因整合到大肠杆菌的染色体中,并除去抗性,得到可用于生产冰核蛋白,且不含任何已知抗生素抗性的重组大肠杆菌。该重组大肠杆菌表达的冰核蛋白可以在稀释为万分之一的水中显著提高冰点,在过冷至-2℃左右即可结冰,该重组大肠杆菌具有很好的成冰核活性,应用前景广阔。
附图说明
图1为本发明实施例2中重组INP菌株TDTC-INP01发酵的蛋白胶图(SDS-PAGE),显示INP大小为150KD左右。INP为可溶性蛋白。
图2为本发明实施例3中TDTC-INP01产冰核蛋白INP的活性。8OD的菌体浓度稀释100万倍后,-2.7℃已开始结冰,-3.5℃冻结率达到一半,-4℃达到60%。
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用于生产冰核蛋白的重组大肠杆菌的构建
1.1冰核蛋白基因inp的扩增
以丁香假单胞菌(Psudomonas syringae)TDTC-IN 13的基因组DNA为模板,用以下引物进行PCR扩增:
inp-F:ctaacaggaggaattaaccatgaatctcgacaaggcgttg
inp-R:gagctcggatccccatcgatctactcgacctctatccagtc
采用南京诺唯赞生物科技有限公司(Vazyme Biotech Co.,Ltd)的高效保真酶Phanta Max Super-Fidelity DNA Polymerase进行PCR扩增。PCR扩增程序为:95℃3min;95℃15s、58℃15s、72℃2min,30个循环;72℃5min。将所得的PCR产物跑电泳后切胶,使用Omega公司的凝胶回收试剂盒进行切胶回收(根据试剂盒说明书操作),得到基因片段inp。
1.2重组表达载体的构建
将步骤1.1所得的基因片段inp与经过限制性内切酶NcoI和ClaI双酶切处理的pBAD/His A质粒经Gibson连接(质粒与DNA片段的摩尔比约为1:3,50℃1h),然后将连接产物转化DH5α。转化后从平板上挑取5-10个的单克隆,取37℃摇床培养1h之后的菌液,利用相应的引物进行PCR验证并对正确的转化子进行测序,从验证正确的转化子提取得到重组表达质粒pBAD-inp。
1.3以步骤1.2所得的重组表达质粒pBAD-inp为模板,用以下引物进行PCR扩增:
ara-inp-F:cattcccacgatgaaaacacgccgtgcctgtcaaatggacgaag
ara-inp-R:ggcgctggaattgctttaactgcctactcgacctctatccagtc
PCR扩增程序为:95℃3min;95℃15s、58℃15s、72℃2.5min,30个循环;72℃5min。将所得的PCR产物经过切胶回收后得到ara-inp。
以大肠杆菌BW25113基因组DNA为模板,用以下引物进行PCR扩增:
galRuF:ccttcaatacgcacaggaataac
galRuR:cttcgtccatttgacaggcacggcgtgttttcatcgtgggaatg
PCR扩增程序为:95℃3min;95℃15s、58℃15s、72℃30s,30个循环;72℃5min。将所得的PCR产物经过切胶回收后得到galR基因上游片段galR-up。
用以下引物进行PCR扩增:
galRdF:gactggatagaggtcgagtaggcagttaaagcaattccagcgcc
galRdR:agatgttatcgatcaggcgacgc
PCR扩增程序为:95℃3min;95℃15s、58℃15s、72℃30s,30个循环;72℃5min。将所得的PCR产物经过切胶回收后得到galR基因下游片段galR-down。
将galR的基因序列导入sgRNA在线设计网站(http://crispr.dbcls.jp/),设计得到其对应的N20为:TTGCACTATGCACAGCCAGC,用以下引物进行退火延伸:
galRN20-F:gtcctaggtataatactagtttgcactatgcacagccagcgttttagagctagaaatagc
galRN20-R:gctatttctagctctaaaacgctggctgtgcatagtgcaaactagtattatacctaggac
20μL反应体系含有2μL galRN20-F,2μL galRN20-R和16μL ddH2O,反应条件为90℃10min,Touch down程序降温降至16℃得到galRN20
1.4打靶基因片段和pTargetF-galR的构建
将步骤1.3所得的三个基因片段ara-inp、galR-up和galR-down进行重叠延伸PCR,先在10μL体系中取galR-up、galR-down和ara-inp各100ng,其它如加酶等一切照常,但先不加两端引物。扩增条件为95℃30s;95℃15s,50℃15s,72℃1min,5个循环;72℃5min。然后,以上述PCR产物为模板,取1μL至50μL体系中(加入引物galRuF和galRdR),扩增条件为95℃30s;95℃15s,52℃30s,每循环降0.5℃,72℃3.5min,10个循环;95℃15s,48℃30s,72℃3.5min,25个循环;72℃5min。将所得的PCR产物经过切胶回收后得到打靶基因片段ΔgalR∷ara-inp。
以pTargetF(Addgene 62226)质粒为模板,用以下引物进行PCR扩增:
Target-F:gttttagagctagaaatagc
Target-R:actagtattatacctaggac
PCR扩增程序为:95℃30s;95℃15s,58℃15s,72℃1min,30个循环;72℃5min。将所得的PCR产物经过切胶回收后得到pTarget。将步骤1.3所得的galRN20与pTarget经Gibson连接(质粒与DNA片段的摩尔比约为1:3,50℃1h),然后将连接产物转化DH5α。转化后从平板上挑取5-10个单克隆,取37℃摇床培养1h之后的菌液,利用相应的引物进行PCR验证,从验证正确的菌株提取得到pTargetF-galR质粒。
1.5电转打靶基因片段至大肠杆菌BW25113
先将pCas(Addgene 62225)质粒转化菌株E.coli BW25113,涂布卡那霉素(50μg/mL)抗性平板。然后将含有pCas的BW25113菌株制备成电转感受态,电转感受态制备方法如下:挑取平板上单克隆,接入含卡那霉素(50μg/mL)的LB液体培养基(氯化钠10g/L,胰蛋白胨10g/L,酵母粉5g/L)30℃培养过夜。将过夜培养的菌液以1%的接种量接种至装有50mL液体LB培养基(含50μg/mL卡那霉素)中。30℃,220rpm条件下培养1-2h,待OD600生长至0.15-0.2左右,加入阿拉伯糖(终浓度为0.2%)。当OD600值达到0.5~0.6时,取出摇瓶冰浴10-15min。然后,4℃,4100rpm离心10min收集菌体。弃上清液,加入25mL预冷的10%甘油重悬菌体。然后重复离心并重悬菌体两次。弃上清液,用10%甘油重悬浮细胞至最终体积为500μL,即为电转感受态细胞。然后,按100μL每份装入1.5mL ep管,-80℃冻存备用。
添加步骤1.4得到的pTargetF-galR质粒约100ng和打靶片段ΔgalR∷ara-inp约500-1000ng至100μL电转感受态细胞中,冰上放置约5min。然后,转移至2mm电转杯中。调整电转仪(Bio-Rad MicroPluser)为细菌模式,加载EC2,对电转杯进行电击。电击后立即添加1mL的LB液体培养基至电转杯中。转移电转杯中菌液至新的ep管中,置于30℃,180rpm,复苏约2h。取全部菌液涂布到含有卡那霉素(50μg/mL)和壮观霉素(50μg/mL)的LB平板,对转化子进行PCR验证。
用以下引物进行PCR扩增:
galRu1239:ctccgaagcggtacattg
araCF113:accgctgggaatgaaagg
PCR扩增程序为:95℃30s;95℃15s,58℃15s,72℃1min,30个循环;72℃5min。能够扩增出大小为2.0kb条带的转化子是已经将冰核蛋白基因整合到大肠杆菌基因组的正确转化子。
1.6产冰核蛋白重组大肠杆菌的获得
将步骤1.5验证正确的转化子接入3mL的LB液体培养基(含有50μg/mL卡那霉素和1mM IPTG,isopropyl-β-D-thiogalactopyranoside),置于30℃摇床200rpm培养过夜。将菌液稀释后涂布含有卡那霉素(50μg/mL)的LB平板。将获得的单克隆,分别点种在含卡那霉素(50μg/mL)或壮观霉素(50μg/mL)的LB平板以得到对壮观霉素敏感的菌株。将对壮观霉素敏感的转化子接入3mL的LB液体培养基,置于42℃摇床200rpm培养过夜。将菌液稀释后涂布LB平板。将获得的单克隆分别点种在含有或不含卡那霉素(50μg/mL)LB平板以得到对卡那霉素敏感的菌株。对卡那霉素敏感的菌株就是不携带抗生素抗性基因的产冰核蛋白的重组大肠杆菌TDTC-inp001。
实施例2产冰核蛋白重组大肠杆菌的发酵
挑取实施例1构建好的TDTC-inp001单克隆至3mL的LB液体培养基,置于37℃摇床200rpm培养过夜。以1:100的比例接种菌液至10mL ZYM自诱导培养基(蛋白胨10g/L,酵母粉5g/L,Na2HPO4·12H2O 8.95g/L,KH2PO4 3.4g/L,NH4Cl 2.67g/L,Na2SO4 0.71g/L,甘油5ml/L,MgSO4 0.24g/L,50%葡萄糖1ml/L,终浓度0.2%的阿拉伯糖,微量元素(1000倍储液)1ml/L;其中微量元素(1000倍储液)为FeCl3 50mM,CaCl2 20mM,MnCl2 10mM,ZnSO4 10mM,CoCl2、NiCl2、NaMO4、NaSeO3和H3BO3各2mM)中,置于37℃摇床200rpm培养过夜以诱导冰核蛋白的表达。表达后的INP蛋白如图1所示。
实施例3成冰核活性检测
将实施例2发酵得到的TDTC-inp001菌液进行冷冻液滴实验以检测成冰核活性。具体方法如下:取菌培养好的该重组大肠杆菌用水重悬至1OD左右(精确测量OD),并以此为基础用1mL ddH2O稀释10倍,102倍,103倍,104倍和105倍。用Dry Cooling/Heating Reactor半导体精密控温冻滴仪检测冰点,将1ml上述稀释浓度的水以每滴10μl总计100滴均匀滴在冻滴仪的铝合金导热板上,设置所需温度,待降温到所需温度后开始计时,统计5分钟后结冰的水滴,50%的结冰率的温度为冰点。8OD发酵液稀释100万倍后冰点为-3.5℃(图2)。
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> 中国科学院微生物研究所
<120> 用于生产冰核蛋白的重组大肠杆菌及其构建方法与应用
<130> KHP191111281.5
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4023
<212> DNA
<213> 丁香假单胞菌(Pseudomonas syringae)
<400> 1
atgaatctcg acaaggcgtt ggtgctgcgt acctgtgcaa ataacatggc cgatcattgc 60
ggccttatat ggcccgcttc tggcacggtg gaatccaaat actggcagtc aaccaggcgg 120
catgagaatg gtctggtcgg tttactgtgg ggcgctggaa ccagcgcttt tctgagcgtg 180
catgccgatg cgcgatggat tgtctgtgaa gtcgccgttg ccgacatcat cagtctggaa 240
gaaccgggaa tggtcaagtt tccgcgggcc gaggtggttc atgtcggcga caggatcagc 300
gcgtcacact ttatttcggc acgtcaggcc gatcctgcat caacgccaac gccaatgacc 360
gcggccacgc ccccacccac gcccgcgaca gcaaatgtca cgttaccggt ggccgaacag 420
gccagtcatg aagtgttcga tgtggcgttg gtcagcgcgg ctgccccccc ggtaaatacc 480
ctgccggtga cgaccccgca gaatttgcag accgccactt acggcagcac gttgagcggc 540
gacaaccaca gtcgactcat tgccggttat ggcagtaacg agactgctgg caaccacagt 600
gatctgattg ccggttatgg aagtacgggc accgccggct ccgacagctc gctggtagca 660
ggctatggaa gcacccagac cgccggtggg gacagcgcgc tgacggcggg ttacggcagc 720
acccagaccg cccgcgaagg cagcaacctg acggccgggt atggcagtac gggaacagct 780
ggctcggata gttcgttgat cgccggttac ggcagtactc agacttccgg ggaagacagc 840
tcgctcacag cgggttacgg cagcacgcaa acggctcagg aaggcagcaa cctgacggcc 900
gggtacggca gcaccggcac ggcgggctcc gatagctcgc tgatcgccgg ttatggcagt 960
acacagacct ctggaggtga cagctcgctg acggcgggtt acggcagcac gcagacggcc 1020
caggaaggca gtaacctgac ggccgggtac ggcagcacag gcacggcggg ctcggatagt 1080
tcgttgatcg ccggttacgg cagtactcag acttccggag aagacagctc gctcacggcg 1140
ggttacggca gcacgcaaac ggctcaggaa ggcagcaatc tcaccgcagg gtatggcagt 1200
accggcactg cgggctcgga tagctcgttg atcgccggtt acggcagtac ccaaacttcg 1260
ggcggcgaca gttcgctgac cgcaggctac ggcagcacgc agacggctca ggaaggcagc 1320
aacctgacct ccgggtacgg cagcactggt accgcaggtg ccgacagctc gttgatcgcc 1380
ggctatggca gcacgcagac ttcgggaagc gacagcgccc tgaccgcagg ttacggcagc 1440
acgcaaactg ctcaggaagg cagcaatctc actgcagggt atggcagcac cggcacggca 1500
ggttccgaca gctcgctgat cgccggtcac ggcagcacgc aaacctcggg cagcgacagt 1560
tcgctcacgg cgggttacgg cagtacgcag acggcccagg agggcagcaa tctgacggcg 1620
gggtacggca gcacgagtac agcaggtgtc gacagctctc tgatcgcggg atacggcagc 1680
acgcagacct cgggaagtga cagtgcgctg acagcaggtt acggcagtac acagacggct 1740
caggaaggca gcaacctgac tgcgggctac ggcagcactg gcacagcagg tgccgacagt 1800
tcgctgatcg cgggatacgg cagcacgcag acctcgggaa gtgacagtgc gctgacggca 1860
ggttacggca gtacacagac cgcccaggag ggcagcaacc tgactgcggg ctacggcagt 1920
actggaacgg caggtgccga cagttcgctg atcgcaggat atggcagcac gcagacatca 1980
ggcagcgaaa gttcgctcac cgcaggctat ggcagtaccc agactgcccg tgaaggcagc 2040
accctgacgg ccgggtatgg cagtacggga acagctggcg ctgacagctc gttgatcgcc 2100
ggttatggca gcacacaaac ctcgggcagt gaaagctcgc tcacggcagg ttatggcagt 2160
acccagaccg cacagcaggg cagcgtactc acctcaggct atggcagtac gcaaacggcc 2220
ggggctgcca gtaacctcac caccggctac ggaagtacag gcaccgcagg gcatgaaagc 2280
ttcatcatcg cgggttacgg gagtacacag acagcgggcc acaaaagtat cctgaccgct 2340
ggttatggca gtacccagac ggccagggac ggtagcgacc tgattgcggg ctatggcagt 2400
accggaaccg caggctcagg cagttcgctg atcgcaggtt atggcagcac ccagaccgcg 2460
agctacagaa gcatgctgac cgccggctat ggcagtaccc agaccgccag agaacacagt 2520
gaccttgtca caggctatgg cagcacttcg acggcagggt caaacagttc gctgatcgcc 2580
ggctacggca gcactcagac ggcgggtttc aaaagcatac tgaccgcagg ttatggcagt 2640
acacagacag cacaggagcg cagcgacctg gtcgcaggct acggcagcac gtcgactgcg 2700
ggctattcca gttccttgat cgccggctat ggcagcacgc agacggcagg ctacgggagc 2760
accttgacga ccggttatgg cagtacgcaa accgctcagg aaaacagctc gctcaccaca 2820
ggttacggaa gtacctctac tgcgggctat tccagctcgc tgatcgcggg ttacggcagt 2880
acccagacgg caggctacga gagcacgttg accgccggtt acggcagtac gcaaaccgcg 2940
caggagcgca gtgacctggt gacaggttat ggcagtactt ccactgctgg ctacgcgagt 3000
tcgttgattg cgggttatgg cagtacgcag actgcggggt acgagagcac cttgaccgcc 3060
ggttacggca gtacgcaaac cgcacaggaa aacagctcgc tcaccaccgg ctacggaagt 3120
acttccactg ccggctttgc cagctcgctg atcgccggtt atggcagtac gcagacagcc 3180
ggctataaaa gtacccttac ggccggttac ggcagtactc agaccgcaga gtacggaagc 3240
tcgcttactg cgggctacgg cagcactgca acggccgggc aggacagttc attgatagcc 3300
ggttatggca gctccctgac cagcggaatc aggagttttc tgaccgcagg ctatggcagt 3360
acgctgatcg ccggacttcg cagcgttttg atcgccggct atggcagtag ccttacatcg 3420
ggcattcgca gcacattgac tgcgggttat ggcagtaacc agattgcaag ttatggcagc 3480
tcgttgattg caggccatga aagcattcag gtcgccggaa ataaaagcat gctgatcgcc 3540
ggcaagggca gctcgcagac agcaggtttt cgcagcacgc tgattgccgg tgcgggcagc 3600
gtacaactgg cgggtgatcg cagcaggttg attgccggtg cagacagtaa tcagaccgcg 3660
ggtgaccgca gcaaactact ggccggtaat aacagttatc tgactgccgg cgatagaagc 3720
aaactgaccg gcggacatga ctgcaccttg atggcgggag accaaagcag attgaccgct 3780
ggaaagaaca gtatcttgac ggcaggcgcg cgtagcaagc ttattggcag tgaaggctcg 3840
acgctctcgg cgggagaaga ctccacgctt attttcaggc tctgggacgg gaagaggtac 3900
aggcaattgg ttgccagaac gggtgagaac ggtgttgaag ccgacatacc gtattacgtg 3960
aacgaagagg acgatattgt cgataaaccc gacgaggatg atgactggat agaggtcgag 4020
tag 4023
<210> 2
<211> 1340
<212> PRT
<213> 丁香假单胞菌(Pseudomonas syringae)
<400> 2
Met Asn Leu Asp Lys Ala Leu Val Leu Arg Thr Cys Ala Asn Asn Met
1 5 10 15
Ala Asp His Cys Gly Leu Ile Trp Pro Ala Ser Gly Thr Val Glu Ser
20 25 30
Lys Tyr Trp Gln Ser Thr Arg Arg His Glu Asn Gly Leu Val Gly Leu
35 40 45
Leu Trp Gly Ala Gly Thr Ser Ala Phe Leu Ser Val His Ala Asp Ala
50 55 60
Arg Trp Ile Val Cys Glu Val Ala Val Ala Asp Ile Ile Ser Leu Glu
65 70 75 80
Glu Pro Gly Met Val Lys Phe Pro Arg Ala Glu Val Val His Val Gly
85 90 95
Asp Arg Ile Ser Ala Ser His Phe Ile Ser Ala Arg Gln Ala Asp Pro
100 105 110
Ala Ser Thr Pro Thr Pro Met Thr Ala Ala Thr Pro Pro Pro Thr Pro
115 120 125
Ala Thr Ala Asn Val Thr Leu Pro Val Ala Glu Gln Ala Ser His Glu
130 135 140
Val Phe Asp Val Ala Leu Val Ser Ala Ala Ala Pro Pro Val Asn Thr
145 150 155 160
Leu Pro Val Thr Thr Pro Gln Asn Leu Gln Thr Ala Thr Tyr Gly Ser
165 170 175
Thr Leu Ser Gly Asp Asn His Ser Arg Leu Ile Ala Gly Tyr Gly Ser
180 185 190
Asn Glu Thr Ala Gly Asn His Ser Asp Leu Ile Ala Gly Tyr Gly Ser
195 200 205
Thr Gly Thr Ala Gly Ser Asp Ser Ser Leu Val Ala Gly Tyr Gly Ser
210 215 220
Thr Gln Thr Ala Gly Gly Asp Ser Ala Leu Thr Ala Gly Tyr Gly Ser
225 230 235 240
Thr Gln Thr Ala Arg Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
245 250 255
Thr Gly Thr Ala Gly Ser Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
260 265 270
Thr Gln Thr Ser Gly Glu Asp Ser Ser Leu Thr Ala Gly Tyr Gly Ser
275 280 285
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
290 295 300
Thr Gly Thr Ala Gly Ser Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
305 310 315 320
Thr Gln Thr Ser Gly Gly Asp Ser Ser Leu Thr Ala Gly Tyr Gly Ser
325 330 335
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
340 345 350
Thr Gly Thr Ala Gly Ser Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
355 360 365
Thr Gln Thr Ser Gly Glu Asp Ser Ser Leu Thr Ala Gly Tyr Gly Ser
370 375 380
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
385 390 395 400
Thr Gly Thr Ala Gly Ser Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
405 410 415
Thr Gln Thr Ser Gly Gly Asp Ser Ser Leu Thr Ala Gly Tyr Gly Ser
420 425 430
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ser Gly Tyr Gly Ser
435 440 445
Thr Gly Thr Ala Gly Ala Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
450 455 460
Thr Gln Thr Ser Gly Ser Asp Ser Ala Leu Thr Ala Gly Tyr Gly Ser
465 470 475 480
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
485 490 495
Thr Gly Thr Ala Gly Ser Asp Ser Ser Leu Ile Ala Gly His Gly Ser
500 505 510
Thr Gln Thr Ser Gly Ser Asp Ser Ser Leu Thr Ala Gly Tyr Gly Ser
515 520 525
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
530 535 540
Thr Ser Thr Ala Gly Val Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
545 550 555 560
Thr Gln Thr Ser Gly Ser Asp Ser Ala Leu Thr Ala Gly Tyr Gly Ser
565 570 575
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
580 585 590
Thr Gly Thr Ala Gly Ala Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
595 600 605
Thr Gln Thr Ser Gly Ser Asp Ser Ala Leu Thr Ala Gly Tyr Gly Ser
610 615 620
Thr Gln Thr Ala Gln Glu Gly Ser Asn Leu Thr Ala Gly Tyr Gly Ser
625 630 635 640
Thr Gly Thr Ala Gly Ala Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
645 650 655
Thr Gln Thr Ser Gly Ser Glu Ser Ser Leu Thr Ala Gly Tyr Gly Ser
660 665 670
Thr Gln Thr Ala Arg Glu Gly Ser Thr Leu Thr Ala Gly Tyr Gly Ser
675 680 685
Thr Gly Thr Ala Gly Ala Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
690 695 700
Thr Gln Thr Ser Gly Ser Glu Ser Ser Leu Thr Ala Gly Tyr Gly Ser
705 710 715 720
Thr Gln Thr Ala Gln Gln Gly Ser Val Leu Thr Ser Gly Tyr Gly Ser
725 730 735
Thr Gln Thr Ala Gly Ala Ala Ser Asn Leu Thr Thr Gly Tyr Gly Ser
740 745 750
Thr Gly Thr Ala Gly His Glu Ser Phe Ile Ile Ala Gly Tyr Gly Ser
755 760 765
Thr Gln Thr Ala Gly His Lys Ser Ile Leu Thr Ala Gly Tyr Gly Ser
770 775 780
Thr Gln Thr Ala Arg Asp Gly Ser Asp Leu Ile Ala Gly Tyr Gly Ser
785 790 795 800
Thr Gly Thr Ala Gly Ser Gly Ser Ser Leu Ile Ala Gly Tyr Gly Ser
805 810 815
Thr Gln Thr Ala Ser Tyr Arg Ser Met Leu Thr Ala Gly Tyr Gly Ser
820 825 830
Thr Gln Thr Ala Arg Glu His Ser Asp Leu Val Thr Gly Tyr Gly Ser
835 840 845
Thr Ser Thr Ala Gly Ser Asn Ser Ser Leu Ile Ala Gly Tyr Gly Ser
850 855 860
Thr Gln Thr Ala Gly Phe Lys Ser Ile Leu Thr Ala Gly Tyr Gly Ser
865 870 875 880
Thr Gln Thr Ala Gln Glu Arg Ser Asp Leu Val Ala Gly Tyr Gly Ser
885 890 895
Thr Ser Thr Ala Gly Tyr Ser Ser Ser Leu Ile Ala Gly Tyr Gly Ser
900 905 910
Thr Gln Thr Ala Gly Tyr Gly Ser Thr Leu Thr Thr Gly Tyr Gly Ser
915 920 925
Thr Gln Thr Ala Gln Glu Asn Ser Ser Leu Thr Thr Gly Tyr Gly Ser
930 935 940
Thr Ser Thr Ala Gly Tyr Ser Ser Ser Leu Ile Ala Gly Tyr Gly Ser
945 950 955 960
Thr Gln Thr Ala Gly Tyr Glu Ser Thr Leu Thr Ala Gly Tyr Gly Ser
965 970 975
Thr Gln Thr Ala Gln Glu Arg Ser Asp Leu Val Thr Gly Tyr Gly Ser
980 985 990
Thr Ser Thr Ala Gly Tyr Ala Ser Ser Leu Ile Ala Gly Tyr Gly Ser
995 1000 1005
Thr Gln Thr Ala Gly Tyr Glu Ser Thr Leu Thr Ala Gly Tyr Gly Ser
1010 1015 1020
Thr Gln Thr Ala Gln Glu Asn Ser Ser Leu Thr Thr Gly Tyr Gly Ser
1025 1030 1035 1040
Thr Ser Thr Ala Gly Phe Ala Ser Ser Leu Ile Ala Gly Tyr Gly Ser
1045 1050 1055
Thr Gln Thr Ala Gly Tyr Lys Ser Thr Leu Thr Ala Gly Tyr Gly Ser
1060 1065 1070
Thr Gln Thr Ala Glu Tyr Gly Ser Ser Leu Thr Ala Gly Tyr Gly Ser
1075 1080 1085
Thr Ala Thr Ala Gly Gln Asp Ser Ser Leu Ile Ala Gly Tyr Gly Ser
1090 1095 1100
Ser Leu Thr Ser Gly Ile Arg Ser Phe Leu Thr Ala Gly Tyr Gly Ser
1105 1110 1115 1120
Thr Leu Ile Ala Gly Leu Arg Ser Val Leu Ile Ala Gly Tyr Gly Ser
1125 1130 1135
Ser Leu Thr Ser Gly Ile Arg Ser Thr Leu Thr Ala Gly Tyr Gly Ser
1140 1145 1150
Asn Gln Ile Ala Ser Tyr Gly Ser Ser Leu Ile Ala Gly His Glu Ser
1155 1160 1165
Ile Gln Val Ala Gly Asn Lys Ser Met Leu Ile Ala Gly Lys Gly Ser
1170 1175 1180
Ser Gln Thr Ala Gly Phe Arg Ser Thr Leu Ile Ala Gly Ala Gly Ser
1185 1190 1195 1200
Val Gln Leu Ala Gly Asp Arg Ser Arg Leu Ile Ala Gly Ala Asp Ser
1205 1210 1215
Asn Gln Thr Ala Gly Asp Arg Ser Lys Leu Leu Ala Gly Asn Asn Ser
1220 1225 1230
Tyr Leu Thr Ala Gly Asp Arg Ser Lys Leu Thr Gly Gly His Asp Cys
1235 1240 1245
Thr Leu Met Ala Gly Asp Gln Ser Arg Leu Thr Ala Gly Lys Asn Ser
1250 1255 1260
Ile Leu Thr Ala Gly Ala Arg Ser Lys Leu Ile Gly Ser Glu Gly Ser
1265 1270 1275 1280
Thr Leu Ser Ala Gly Glu Asp Ser Thr Leu Ile Phe Arg Leu Trp Asp
1285 1290 1295
Gly Lys Arg Tyr Arg Gln Leu Val Ala Arg Thr Gly Glu Asn Gly Val
1300 1305 1310
Glu Ala Asp Ile Pro Tyr Tyr Val Asn Glu Glu Asp Asp Ile Val Asp
1315 1320 1325
Lys Pro Asp Glu Asp Asp Asp Trp Ile Glu Val Glu
1330 1335 1340
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccttcaatac gcacaggaat aac 23
<210> 4
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cttcgtccat ttgacaggca cggcgtgttt tcatcgtggg aatg 44
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gactggatag aggtcgagta ggcagttaaa gcaattccag cgcc 44
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agatgttatc gatcaggcga cgc 23
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttgcactatg cacagccagc 20

Claims (3)

1.用于生产冰核蛋白的重组大肠杆菌,其特征在于,其构建方法包括以下步骤:
1)从丁香假单胞菌中克隆得到冰核蛋白基因;
2)将步骤1)所得基因构建到质粒pBAD/His A上,得到重组表达质粒;
3)以步骤2)所得重组表达质粒为模板扩增带有阿拉伯糖诱导启动子的冰核蛋白基因片段;同时以大肠杆菌基因组为模板PCR扩增得到galR基因上、下游片段knock-in-up和knock-in-down;
4)将步骤3)所得三个基因片段通过重叠延伸PCR得到基因编辑片段;
5)构建靶向大肠杆菌galR基因的CRISPR/Cas9基因编辑载体;
6)将步骤4)所得基因编辑片段和步骤5)所得CRISPR/Cas9基因编辑载体共同导入大肠杆菌BW25113中,得到用于生产冰核蛋白的重组大肠杆菌;
其中,步骤3)中用于PCR扩增所述knock-in-up和knock-in-down的引物分别如SEQ IDNO:3-4和SEQ ID NO:5-6所示;和/或
步骤5)中所述CRISPR/Cas9打靶载体是基于CRISPR-Cas9系统的sgRNA表达载体,其中,sgRNA作用位点的核酸序列为5′-TTGCACTATGCACAGCCAGC-3′;
丁香假单胞菌冰核蛋白基因编码的蛋白的氨基酸序列如SEQ ID NO:2所示。
2.权利要求1所述重组大肠杆菌的以下任一应用:
i)用于人工造雪;
ii)用于冷冻干燥工艺。
3.人工造雪剂,其特征在于,有效成分为权利要求1所述重组大肠杆菌及其死菌。
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