CN114717253B - 一种猪链球菌高效转座突变系统及其应用 - Google Patents
一种猪链球菌高效转座突变系统及其应用 Download PDFInfo
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Abstract
本发明公开了一种猪链球菌高效转座突变系统，所述转座突变系统，即pSET4s‑Tn质粒，包括：(一)猪链球菌温敏复制元件Ts ori；(二)大肠杆菌复制元件ColE1ori；(三)Mariner转座酶表达盒；(四)壮观霉素抗性表达盒(SpcR)；(五)上游和下游均包含一个转座酶识别序列(IR)的红霉素抗性表达盒(ErmR)。利用该转座突变系统可实现在猪链球菌中的高效转座突变，在单次实验中能够达到98％以上的猪链球菌为转座突变体，转座子插入在猪链球菌基因组中TA碱基位点，且转座插入位点无明显偏好性。
Description
技术领域
本发明属于基因突变技术领域，具体涉及一种猪链球菌高效转座突变系统及其应用。
背景技术
猪链球菌是一种重要的人兽共患病原细菌，可引起猪的脑膜炎、关节炎和败血症等，给养猪业造成巨大经济损失，也可感染人导致中毒休克样综合征，死亡率较高，危害公共卫生安全。开展猪链球菌的病原生物学和功能基因组学研究对鉴定新型抗菌药物靶点以及发现疫苗候选抗原具有重要意义。
对病原菌进行高效、快捷的遗传操作是研究病原菌生长、代谢和致病机制的关键，因此开发猪链球菌的遗传操纵工具对该病原菌的病原学和致病机制研究具有重要意义。目前，在猪链球菌中，常用的基因突变菌株的构建方法是采用传统的同源重组来实现，其可以定向缺失基因组的特定片段或将目的片段定向插入染色体特定位点。但是，该方法无法实现高通量突变体构建，也不能实现随机突变。
随机转座突变技术是通过转座子随机插入到染色体中，破坏插入位置原有基因的结构，导致基因突变的一种遗传操作。近年来，将随机转座突变技术与二代DNA测序技术相结合开发出的转座插入测序技术为全基因组范围内筛选功能基因提供了新的思路。在猪链球菌中，有研究使用Tn917转座子进行转座突变，但该转座子被认为具有一定的插入偏好性。因此，本研究构建了一种猪链球菌高效转座突变系统pSET4s-Tn，其携带Mariner转座酶以及包含红霉素抗性表达盒的插入元件，通过本研究构建的转座突变系统可以实现在猪链球菌中高效构建转座突变体，转座子插入在细菌染色体的TA位点，且插入位点无明显偏好性。目前并未检索到猪链球菌中有类似的高效随机转座突变系统。
发明内容
本发明的一个目的在于提供一种猪链球菌高效转座突变系统，利用该系统可高效构建猪链球菌转座突变体及突变体文库。
为了达到上述目的，本发明技术方案如下：
一种Mariner转座酶表达盒，包括一个gapdh基因启动子和Mariner转座酶的编码序列，其在猪链球菌中表达Mariner转座酶，所述gapdh基因启动子的核苷酸序列如SEQ IDNO.14所示，所述转座酶基因Mariner的核苷酸序列如SEQ ID NO.15所示。
一种猪链球菌高效转座突变系统，包含由上述的Mariner转座酶表达盒、抗性表达盒组成的基因片段Tn，以及质粒，所述抗性表达盒由抗性基因及两侧的转座酶识别序列组成，两侧的所述转座酶识别序列分别如SEQ ID NO.16和SEQ ID NO.17所示，抗性表达盒可被Mariner转座酶识别后将该抗性表达盒插入至猪链球菌染色体的TA碱基位点。
进一步地，所述抗性表达盒为红霉素抗性表达盒，其核苷酸序列如SEQ ID NO.18所示。
而且，所述红霉素抗性表达盒使得含有该质粒的猪链球菌具有红霉素抗性。
进一步地，所述基因片段Tn的核苷酸序列如SEQ ID NO.2所示。
进一步地，所述基因片段Tn插入pSET4s质粒中形成所述猪链球菌高效转座突变系统，即pSET4s-Tn质粒。
进一步地，所述pSET4s质粒的核苷酸序列如SEQ ID NO.1所示，含有如下基因片段：猪链球菌温敏复制元件Ts ori、大肠杆菌复制元件ColE1 ori、壮观霉素抗性表达盒。
而且，所述猪链球菌温敏复制元件Ts ori使得pSET4s-Tn质粒在低温(28℃以下)条件下能够在猪链球菌细胞内复制，当培养温度超过37℃时，质粒无法在猪链球菌细胞内复制，导致质粒丢失。
而且，所述大肠杆菌复制元件ColE1 ori，使所述质粒能够在大肠杆菌如DH5α菌株中复制，便于质粒的制备。
而且，所述壮观霉素抗性表达盒(SpcR)使得含有该质粒的猪链球菌具有壮观霉素抗性。
而且，所述猪链球菌高效转座突变系统在猪链球菌中表达转座酶后得到的含突变体的猪链球菌具有红霉素抗性，但不具有壮观霉素抗性，能区别于不含突变体的猪链球菌、含有pSET4s和pSET4s-Tn质粒被筛选出来。
本发明的另一个目的是提供一种猪链球菌高效转座突变系统的构建方法，包括以下步骤：将所述pSET4s质粒用限制性内切酶BglII+HindIII双酶切后用DNA回收试剂盒回收大片段，人工合成所述基因片段Tn，在37℃的条件下利用同源重组酶将酶切后的pSET4s质粒与Tn片段连接，转化大肠杆菌DH5α感受态，37℃过夜培养，挑选阳性克隆过夜培养。
本发明的另一个目的是提供一种猪链球菌高效转座突变系统的在猪链球菌转座子突变库构建中的应用，其构建步骤如下：
首先将所述pSET4s-Tn质粒转入猪链球菌感受态中，涂布于含终浓度为5％的牛血清，10μg/mL的红霉素和100μg/mL的壮观霉素的TSA培养平板上，28℃温箱中静置培养过夜。将转化子接种至含终浓度为5％的牛血清，10μg/mL的红霉素和100μg/mL的壮观霉素的TSB液体培养基中，28℃过夜培养，次日按1:20的比例转接至含终浓度为5％的牛血清和5μg/mL的红霉素的TSB液体培养基中，37℃培养12h；将生长的单菌落分别转点含终浓度为5％的牛血清和10μg/mL的红霉素的TSA以及含终浓度为5％的牛血清和100μg/mL的壮观霉素的TSA平板，37℃过夜培养，具有红霉素抗性而无壮观霉素抗性的菌落即为猪链球菌转座突变体。
相对于现有技术，本发明具有以下优势：通过单次实验，获得的细菌克隆98％以上均为转座子突变体。转座子插入在猪链球菌基因组的TA碱基处，插入位置不具有明显的偏好性。利用此转座突变体系统和方法，可快速获得大量转座突变体文库。
附图说明
图1为pSET4s-Tn质粒的构建过程图；
图2为10个转座子突变体的分析结果。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
本发明中所使用的原料，如无特殊说明，均为常规的市售产品；本发明中所使用的方法，如无特殊说明，均为本领域的常规方法。
实施例1
本发明的猪链球菌基因诱导表达系统，由gapdh基因启动子与转座酶基因Mariner组成的Mariner转座酶表达盒，以及转座酶识别序列IR为边界的红霉素抗性表达盒(ErmR)连接组成(如SEQ ID NO.2所示)，命名为Tn片段，gapdh基因启动子的核苷酸序列如SEQ IDNO.14所示，转座酶基因Mariner的核苷酸序列如SEQ ID NO.15所示，红霉素抗性表达盒(ErmR)的核苷酸序列如SEQ ID NO.18所示,红霉素抗性表达盒(ErmR)包括红霉素抗性基因、红霉素抗性基因与Mariner转座酶表达盒之间的转座酶识别序列(SEQ ID NO.17所示)、及另一侧的转座酶识别序列(如SEQ ID NO.16所示)。
本发明的猪链球菌高效转座突变系统(pSET4s-Tn质粒)，由Tn片段连接入温度敏感性质粒pSET4s(如SEQ ID NO.1所示)，构建成转座诱变质粒pSET4s-Tn。
pSET4s质粒包含以下元件：猪链球菌温敏复制元件Ts ori、大肠杆菌复制元件ColE1 ori和壮观霉素抗性表达盒(SpcR)。
Gapdh表示甘油醛-3-磷酸脱氢酶，是一种糖分解酶，负责将磷酸甘油醛GAP磷酸化产生1，3-二磷酸甘油酯DPG，甘油醛-3-磷酸脱氢酶是猪链球菌的一种表面蛋白，与细菌黏附宿主细胞有关，与细胞毒力密切相关，上述改造预期结果是猪链球菌表面的甘油醛-3-磷酸脱氢酶与gapdh基因启动子结合，起到启动基因高效转录的作用，在猪链球菌中表达Mariner高效转座酶，以转座酶识别序列IR为边界的红霉素抗性表达盒(ErmR)可被Mariner转座酶识别后将该红霉素抗性基因插入至猪链球菌染色体的TA碱基位点。猪链球菌温敏复制元件Ts ori使得pSET4s-Tn质粒在低温(28℃以下)条件下能够在猪链球菌细胞内复制，当培养温度超过37℃时，pSET4s-Tn质粒无法在猪链球菌细胞内复制，导致质粒丢失；大肠杆菌复制元件ColE1 ori，使pSET4s-Tn质粒能够在大肠杆菌如DH5α菌株中复制，便于pSET4s-Tn质粒的制备；壮观霉素抗性表达盒(SpcR)使得含有pSET4s-Tn质粒的猪链球菌具有壮观霉素抗性；
pSET4s-Tn质粒的具体构建过程如下：
(1)将SEQ ID NO.1所示的pSET4s质粒用限制性内切酶BglII和HindIII双酶切，酶切体系如下表1：
表1
将该体系放于PCR仪内，在37℃条件下反应15min，随后将酶切产物使用1％的琼脂糖凝胶电泳，用DNA回收试剂盒回收得到线性化载体片段(4060bp)，
(2)由武汉天一华煜基因科技有限公司合成如SEQ ID NO.2所示的Tn片段(2500bp)，该片段包含Mariner转座酶表达盒以及上游和下游均具有一个转座酶识别序列(IR)的红霉素抗性表达盒。利用如SEQ ID NO.4和SEQ ID NO.5所示的：引物Tn-F：5’-ATGTTGCGATTAATAGATCTGTCGACCTGCAGTAAG-3’引物Tn-R：5’-ACCATGATTACGCCAAGCTTTAACAGGTTGGATGATAAGTCC-3’扩增Tn片段，用于扩增上述Tn片段的PCR反应体系(50μL)如下表2：
表2
扩增反应条件如下：
1＝95.0℃FOR 5:00
2＝94.0℃FOR 0:30
3＝58.0℃FOR 0:30
4＝72.0℃FOR 2:30
5＝GOTO 2，30TIMES
6＝72.0℃FOR 7:00
7＝4.0℃FOR 10:00
8＝END
PCR扩增反应后，扩增产物于1％琼脂糖凝胶电泳，并凝胶回收目的片段。
(3)将线性化的pSET4s载体片段与Tn片段用同源重组酶连接，连接体系如下表3：
表3
在PCR仪中执行如下循环：
1＝37℃FOR 30:00
2＝4℃FOR 10:00
3＝END
(4)随后将连接产物转化大肠杆菌DH5α感受态中，37℃过夜培养。从含壮观霉素的LA平板上挑取单菌落若干，并用如SEQ ID NO.6和SEQ ID NO.7所示的引物Erm-F：5’-GAAACCGATACCGTTTACG-3’和Erm-R：5’-GACGATATTCTCGATTGACC-3’进行菌落PCR扩增以鉴定阳性克隆，使用质粒小提试剂盒提取pSET4s-Tn质粒，进行DNA测序以鉴定重组质粒pSET4s-Tn，序列如SEQ ID NO.3。
实施例2利用pSET4s-Tn质粒构建猪链球菌转座突变体
(1)将约100ng的pSET4s-Tn质粒加入猪链球菌感受态中，于冰上混合均匀，进行点击转化，之后加入400μl TSB培养基于28℃下培养2h，然后将培养物涂布含终浓度为5％的牛血清，10μg/mL的红霉素和100μg/mL的壮观霉素的TSA培养平板上，28℃温箱中静置过夜培养；
(2)利用如SEQ ID NO.6和SEQ ID NO.7所示的引物对及如SEQ ID NO.8和SEQ IDNO.9所示的引物对：Erm-F：5’-GAAACCGATACCGTTTACG-3’和Erm-R：5’-GACGATATTCTCGATTGACC-3’以及Spc-F：5’-GATCAGGAGTTGAGAGTG-3’和Spc-R：5’-CTCTTCTCACATCAGAAAATGG-3’进行菌落PCR，以确定质粒成功转化至猪链球菌；
(3)将经含pSET4s-Tn质粒的猪链球菌单菌落接种至含终浓度为5％的牛血清，10μg/mL的红霉素和100μg/mL的壮观霉素的TSB液体培养基中，28℃过夜培养。次日按1:20的比例转接至含终浓度为5％的牛血清和5μg/mL的红霉素的TSB液体培养基中，37℃培养12h；
(4)取适量菌液涂布于含终浓度为5％的牛血清和10μg/mL的红霉素的TSA培养平板上，37℃静置过夜培养至生长出单菌落；
(5)将单菌落分别转点含终浓度为5％的牛血清和10μg/mL的红霉素的TSA以及含终浓度为5％的牛血清和100μg/mL的壮观霉素的TSA平板，37℃过夜培养，具有红霉素抗性而无壮观霉素抗性的菌落即为猪链球菌转座突变体。
实施例3鉴定转座子插入位点
(1)随机挑选10个猪链球菌转座子突变体，接种至含终浓度为5％的牛血清和5μg/mL的红霉素的TSB液体培养基中，37℃培养12h，利用基因组DNA提取试剂盒提取细菌的基因组。
(2)以上述提取的基因组DNA为模板，使用如SEQ ID NO.10和SEQ ID NO.11所示的引物arb-2：5’-GGCCACGCGTCGACTAGTCANNNNNNNNNNGATCA-3’和erm5-4：5’-GGTTGAGTACTTTTTCACTCG-3’进行第一轮PCR。反应体系为：上下游引物各0.5nM，基因组DNA2μL，PCR Mix 10μL，ddH2O补充至20μL；PCR反应条件为94℃，2min；94℃，30s；42℃，30s；72℃，3min，以上反应重复6个循环；94℃，30s；65℃，30s；72℃，3min，以上反应重复25个循环；4℃，10min；
(3)以一轮PCR产物作为模板，使用如SEQ ID NO.12和SEQ ID NO.13所示的引物arb-3：5’-GGCCACGCGTCGACTAGTCA-3’和erm5-2：5’-GGAATACCAACATGACGAATC-3’进行第二轮PCR，PCR反应条件为94℃，30s；65℃，30s；72℃，1min，以上反应重复30个循环；72℃，5min，4℃，10min。
(4)对第二轮PCR产物使用引物erm5-2进行DNA测序，将测序结果与猪链球菌基因组进行BLAST比对，确定转座子插入位点，如图2和表4所示10个转座子突变体插入位点位于基因组不同的位置。
表4部分转座子突变体的插入位点
序列表
<110> 华中农业大学
<120> 一种猪链球菌高效转座突变系统及构建方法及其应用
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4506
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
actagttatc ggcataatcg ttaaaacagg cgttatcgta gcgtaaaagc ccttgagcgt 60
agcgtggctt tgcagcgaag atgttgtctg ttagattatg aaagccgatg actgaatgaa 120
ataataagcg cagcgccctt ctatttcggt tggaggaggc tcaagggagt atgagggaat 180
gaaattccct catgggtttg attttaaaaa ttgcttgcaa ttttgccgag cggtagcgct 240
ggaaaatttt tgaaaaaaat ttggaatttg gaaaaaaatg gggggaaagg aagcgaattt 300
tgcttccgta ctacgacccc ccattaagtg ccgagtgcca atttttgtgc caaaaacgct 360
ctatcccaac tggctcaagg gtttaagggg tttttcaatc gccaacgaat cgccaacgtt 420
ttcgccaacg ttttttataa atctatattt aagtagcttt attgttgttt ttatgattac 480
aaagtgatac actaacttta taaaattatt tgattggagt tttttaaatg gtgatttcag 540
aatcgaaaaa aagagttatg atttctctga caaaagagca agataaaaaa ttaacagata 600
tggcgaaaca aaaaggtttt tcaaaatctg cggttgcggc gttagctata gaagaatatg 660
caagaaagga atcagaacaa aaaaaataag cgaaagctcg cgtttttaga aggatacgag 720
ttttcgctac ttgtttttga taaggtaatt atatcatggc tattaaaaat actaaagcta 780
gaaattttgg atttttatta tatcctgact caattcctaa tgattggaaa gaaaaattag 840
agagtttggg cgtatctatg gctgtcagtc ctttacacga tatggacgaa aaaaaagata 900
aagatacatg gaataatagt aatattatac aaaatggaaa gcactataaa aaaccacact 960
atcacgttat atatattgca cgaaatcctg taacaataga aagcgttagg aacaagatta 1020
agcgaaaatt ggggaatagt tcagttgctc atgttgagat acttgattat atcaaaggtt 1080
catatgaata tttgactcat gaatcaaagg acgctattgc taagaataaa catatatacg 1140
acaaaaaaga tattttgaac attaatgatt ttgatattga ccgctatata acacttgatg 1200
aaagccaaaa aagagaattg aagaatttac ttttagatat agtggatgac tataatttgg 1260
taaatacaaa agatttaatg gcttttattc gccttagggg agcggagttt ggaattttaa 1320
atacgaatga tgtaaaagat attgtttcaa caaactctag cgcctttaga ttatggtttg 1380
agggcaatta tcagtgtgga tatagagcaa gttatgcaaa ggttcttgat gctgaaacgg 1440
gggaaataaa atgacaaaca aagaaaaaga gttatttgct gaaaatgagg aattaaaaaa 1500
agaaattaag gacttaaaag agcgtattga aagatacaga gaaatggaag ttgaattaag 1560
tacaacaata gatttattga gaggagggat tattgaataa ataaaagccc cctgacgaaa 1620
gtcgaagggg gtttttattt tggtttgatg ttgcgattaa tagatctcgg tgatgacggt 1680
gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 1740
gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 1800
aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 1860
cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac 1920
tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga 1980
tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa 2040
acgacggcca gtgaattcga gctcggtacc cggggatcct ctagagtcga cctgcaggca 2100
tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 2160
aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 2220
gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 2280
gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 2340
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 2400
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 2460
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 2520
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 2580
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 2640
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 2700
aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg 2760
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 2820
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 2880
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 2940
gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 3000
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 3060
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 3120
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 3180
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 3240
taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 3300
tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 3360
cgtgtagata actagtgttc gtgaatacat gttataataa ctataactaa taacgtaacg 3420
tgactggcaa gagatatttt taaaacaatg aataggttta cacttacttt agttttatgg 3480
aaatgaaaga tcatatcata tataatctag aataaaatta actaaaataa ttattatcta 3540
gataaaaaat ttagaagcca atgaaatcta taaataaact aaattaagtt tatttaatta 3600
acaactatgg atataaaata ggtactaatc aaaatagtga ggaggatata tttgaataca 3660
tacgaacaaa ttaataaagt gaaaaaaata cttcggaaac atttaaaaaa taaccttatt 3720
ggtacttaca tgtttggatc aggagttgag agtggactaa aaccaaatag tgatcttgac 3780
tttttagtcg tcgtatctga accattgaca gatcaaagta aagaaatact tatacaaaaa 3840
attagaccta tttcaaaaaa aataggagat aaaagcaact tacgatatat tgaattaaca 3900
attattattc agcaagaaat ggtaccgtgg aatcatcctc ccaaacaaga atttatttat 3960
ggagaatggt tacaagagct ttatgaacaa ggatacattc ctcagaagga attaaattca 4020
gatttaacca taatgcttta ccaagcaaaa cgaaaaaata aaagaatata cggaaattat 4080
gacttagagg aattactacc tgatattcca ttttctgatg tgagaagagc cattatggat 4140
tcgtcagagg aattaataga taattatcag gatgatgaaa ccaactctat attaacttta 4200
tgccgtatga ttttaactat ggacacgggt aaaatcatac caaaagatat tgcgggaaat 4260
gcagtggctg aatcttctcc attagaacat agggagagaa ttttgttagc agttcgtagt 4320
tatcttggag agaatattga atggactaat gaaaatgtaa atttaactat aaactattta 4380
aataacagat taaaaaaatt ataaaaaaat tgaaaaaatg gtggaaacac ttttttcaat 4440
ttttttgttt tattatttaa tatttgggaa atattcattc taattggtaa tcagatttta 4500
gaaaac 4506
<210> 2
<211> 2500
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gtcgacctgc agtaagatga accggtaagc agttgcaatt ttcggtaaat agttatataa 60
taaaaacgtt gaaaagatgc ttgtaggctt acaagttaat attttcacaa aaggattaaa 120
aggcttgcct tttaaaataa agaactcgat tttcataagg aggaaatcat tcatggaaaa 180
aaaggaattt cgtgttttga taaaatactg ttttctgaag ggaaaaaata cagtggaagc 240
aaaaacttgg cttgataatg agtttccgga ctctgcccca gggaaatcaa caataattga 300
ttggtatgca aaattcaagc gtggtgaaat gagcacggag gacggtgaac gcagtggacg 360
cccgaaagag gtggttaccg acgaaaacat caaaaaaatc cacaaaatga ttttgaatga 420
ccgtaaaatg aagttgatcg agatagcaga ggccttaaag atatcaaagg aacgtgttgg 480
tcatatcatt catcaatatt tggatatgcg gaagctctgt gcgaaatggg tgccgcgcga 540
gctcacattt gaccaaaaac aacgacgtgt tgatgattct aagcggtgtt tgcagctgtt 600
aactcgtaat acacccgagt ttttccgtcg atatgtgaca atggatgaaa catggctcca 660
tcactacact cctgagtcca atcgacagtc ggctgagtgg acagcgaccg gtgaaccgtc 720
tccgaagcgt ggaaagactc aaaagtccgc tggcaaagta atggcctctg ttttttggga 780
tgcgcatgga ataattttta tcgattatct tgagaaggga aaaaccatca acagtgacta 840
ttatatggcg ttattggagc gtttgaaggt cgaaatcgcg gcaaaacggc cccacatgaa 900
gaagaaaaaa gtgttgttcc accaagacaa cgcaccgtgc cacaagtcat tgagaacgat 960
ggcaaaaatt catgaattgg gcttcgaatt gcttccccac ccgccgtatt ctccagatct 1020
ggcccccagc gactttttct tgttctcaga cctcaaaagg atgctcgcag ggaaaaaatt 1080
tggctgcaat gaagaggtga tcgccgaaac tgaggcctat tttgaggcaa aaccgaagga 1140
gtactaccaa aatggtatca aaaaattgga aggtcgttat aatcgttgta tcgctcttga 1200
agggaactat gttgaataag aattcgagct cggtacccgg ggatcctaac aggttggatg 1260
ataagtcccc ggtctatcga tacaaattcc ccgtaggcgc tagggacctc tttagcttct 1320
tggaagctgt cagtagtata tctaataatt tatctccatt ccctttagta acgtgtaact 1380
ttccaaattt aaaaaagcga ctcatagaat tatttcctcc cgttaaataa tagataacta 1440
ttaaaaatag acaatacttg ctcataagta atggtactta aattgtttac tttggcgtgt 1500
ttcattgctt gatgaaactg atttttagta aacagttgac gatattctcg attgacccat 1560
tttgaaacaa agtacgtata tagcttccaa tatttatctg gaacatctgt ggtatggcgg 1620
gtaagtttta ttaagacact gtttactttt ggtttaggat gaaagcattc cgctggcagc 1680
ttaagcaatt gctgaatcga gacttgagtg tgcaagagca accctagtgt tcggtgaata 1740
tccaaggtac gcttgtagaa tccttcttca acaatcagat agatgtcaga cgcatggctt 1800
tcaaaaacca cttttttaat aatttgtgtg cttaaatggt aaggaatact cccaacaatt 1860
ttatacctct gtttgttagg gaattgaaac tgtagaatat cttggtgaat taaagtgaca 1920
cgaatgttca gttttaattt ttctgacgat aagttgaata gatgactgtc taattcaata 1980
gacgttacct gtttacttat tttagccagt ttcgtcgtta aatgcccttt acctgttcca 2040
atttcgtaaa cggtatcggt ttcttttaaa ttcaattgtt ttattatttg gttgagtact 2100
ttttcactcg ttaaaaagtt ttgagaatat tttatatttt tgttcatgta attactcctg 2160
aagtgattac atctgtaaat aaatacagaa gttaaacgat ttgtttgtaa ttttagttat 2220
ctgtttaaaa agtcataaga ttagtcactg gtaggaatta atctaacgta tttatttatc 2280
tgcgtaatca ctgtttttag tctgtttcaa aacagtagat gttttatcta cattacgcat 2340
ttggaatacc aacatgacga atccctcctt cttaattaca aatttttagc atctaattta 2400
acttcaattc ctattataca caaaatttta agatactgca ctatcaacac actcttaagt 2460
ttgcttctaa gagaccgggg acttatcatc caacctgtta 2500
<210> 3
<211> 6570
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
actagttatc ggcataatcg ttaaaacagg cgttatcgta gcgtaaaagc ccttgagcgt 60
agcgtggctt tgcagcgaag atgttgtctg ttagattatg aaagccgatg actgaatgaa 120
ataataagcg cagcgccctt ctatttcggt tggaggaggc tcaagggagt atgagggaat 180
gaaattccct catgggtttg attttaaaaa ttgcttgcaa ttttgccgag cggtagcgct 240
ggaaaatttt tgaaaaaaat ttggaatttg gaaaaaaatg gggggaaagg aagcgaattt 300
tgcttccgta ctacgacccc ccattaagtg ccgagtgcca atttttgtgc caaaaacgct 360
ctatcccaac tggctcaagg gtttaagggg tttttcaatc gccaacgaat cgccaacgtt 420
ttcgccaacg ttttttataa atctatattt aagtagcttt attgttgttt ttatgattac 480
aaagtgatac actaacttta taaaattatt tgattggagt tttttaaatg gtgatttcag 540
aatcgaaaaa aagagttatg atttctctga caaaagagca agataaaaaa ttaacagata 600
tggcgaaaca aaaaggtttt tcaaaatctg cggttgcggc gttagctata gaagaatatg 660
caagaaagga atcagaacaa aaaaaataag cgaaagctcg cgtttttaga aggatacgag 720
ttttcgctac ttgtttttga taaggtaatt atatcatggc tattaaaaat actaaagcta 780
gaaattttgg atttttatta tatcctgact caattcctaa tgattggaaa gaaaaattag 840
agagtttggg cgtatctatg gctgtcagtc ctttacacga tatggacgaa aaaaaagata 900
aagatacatg gaataatagt aatattatac aaaatggaaa gcactataaa aaaccacact 960
atcacgttat atatattgca cgaaatcctg taacaataga aagcgttagg aacaagatta 1020
agcgaaaatt ggggaatagt tcagttgctc atgttgagat acttgattat atcaaaggtt 1080
catatgaata tttgactcat gaatcaaagg acgctattgc taagaataaa catatatacg 1140
acaaaaaaga tattttgaac attaatgatt ttgatattga ccgctatata acacttgatg 1200
aaagccaaaa aagagaattg aagaatttac ttttagatat agtggatgac tataatttgg 1260
taaatacaaa agatttaatg gcttttattc gccttagggg agcggagttt ggaattttaa 1320
atacgaatga tgtaaaagat attgtttcaa caaactctag cgcctttaga ttatggtttg 1380
agggcaatta tcagtgtgga tatagagcaa gttatgcaaa ggttcttgat gctgaaacgg 1440
gggaaataaa atgacaaaca aagaaaaaga gttatttgct gaaaatgagg aattaaaaaa 1500
agaaattaag gacttaaaag agcgtattga aagatacaga gaaatggaag ttgaattaag 1560
tacaacaata gatttattga gaggagggat tattgaataa ataaaagccc cctgacgaaa 1620
gtcgaagggg gtttttattt tggtttgatg ttgcgattaa tagatctgtc gacctgcagt 1680
aagatgaacc ggtaagcagt tgcaattttc ggtaaatagt tatataataa aaacgttgaa 1740
aagatgcttg taggcttaca agttaatatt ttcacaaaag gattaaaagg cttgcctttt 1800
aaaataaaga actcgatttt cataaggagg aaatcattca tggaaaaaaa ggaatttcgt 1860
gttttgataa aatactgttt tctgaaggga aaaaatacag tggaagcaaa aacttggctt 1920
gataatgagt ttccggactc tgccccaggg aaatcaacaa taattgattg gtatgcaaaa 1980
ttcaagcgtg gtgaaatgag cacggaggac ggtgaacgca gtggacgccc gaaagaggtg 2040
gttaccgacg aaaacatcaa aaaaatccac aaaatgattt tgaatgaccg taaaatgaag 2100
ttgatcgaga tagcagaggc cttaaagata tcaaaggaac gtgttggtca tatcattcat 2160
caatatttgg atatgcggaa gctctgtgcg aaatgggtgc cgcgcgagct cacatttgac 2220
caaaaacaac gacgtgttga tgattctaag cggtgtttgc agctgttaac tcgtaataca 2280
cccgagtttt tccgtcgata tgtgacaatg gatgaaacat ggctccatca ctacactcct 2340
gagtccaatc gacagtcggc tgagtggaca gcgaccggtg aaccgtctcc gaagcgtgga 2400
aagactcaaa agtccgctgg caaagtaatg gcctctgttt tttgggatgc gcatggaata 2460
atttttatcg attatcttga gaagggaaaa accatcaaca gtgactatta tatggcgtta 2520
ttggagcgtt tgaaggtcga aatcgcggca aaacggcccc acatgaagaa gaaaaaagtg 2580
ttgttccacc aagacaacgc accgtgccac aagtcattga gaacgatggc aaaaattcat 2640
gaattgggct tcgaattgct tccccacccg ccgtattctc cagatctggc ccccagcgac 2700
tttttcttgt tctcagacct caaaaggatg ctcgcaggga aaaaatttgg ctgcaatgaa 2760
gaggtgatcg ccgaaactga ggcctatttt gaggcaaaac cgaaggagta ctaccaaaat 2820
ggtatcaaaa aattggaagg tcgttataat cgttgtatcg ctcttgaagg gaactatgtt 2880
gaataagaat tcgagctcgg tacccgggga tcctaacagg ttggatgata agtccccggt 2940
ctatcgatac aaattccccg taggcgctag ggacctcttt agcttcttgg aagctgtcag 3000
tagtatatct aataatttat ctccattccc tttagtaacg tgtaactttc caaatttaaa 3060
aaagcgactc atagaattat ttcctcccgt taaataatag ataactatta aaaatagaca 3120
atacttgctc ataagtaatg gtacttaaat tgtttacttt ggcgtgtttc attgcttgat 3180
gaaactgatt tttagtaaac agttgacgat attctcgatt gacccatttt gaaacaaagt 3240
acgtatatag cttccaatat ttatctggaa catctgtggt atggcgggta agttttatta 3300
agacactgtt tacttttggt ttaggatgaa agcattccgc tggcagctta agcaattgct 3360
gaatcgagac ttgagtgtgc aagagcaacc ctagtgttcg gtgaatatcc aaggtacgct 3420
tgtagaatcc ttcttcaaca atcagataga tgtcagacgc atggctttca aaaaccactt 3480
ttttaataat ttgtgtgctt aaatggtaag gaatactccc aacaatttta tacctctgtt 3540
tgttagggaa ttgaaactgt agaatatctt ggtgaattaa agtgacacga atgttcagtt 3600
ttaatttttc tgacgataag ttgaatagat gactgtctaa ttcaatagac gttacctgtt 3660
tacttatttt agccagtttc gtcgttaaat gccctttacc tgttccaatt tcgtaaacgg 3720
tatcggtttc ttttaaattc aattgtttta ttatttggtt gagtactttt tcactcgtta 3780
aaaagttttg agaatatttt atatttttgt tcatgtaatt actcctgaag tgattacatc 3840
tgtaaataaa tacagaagtt aaacgatttg tttgtaattt tagttatctg tttaaaaagt 3900
cataagatta gtcactggta ggaattaatc taacgtattt atttatctgc gtaatcactg 3960
tttttagtct gtttcaaaac agtagatgtt ttatctacat tacgcatttg gaataccaac 4020
atgacgaatc cctccttctt aattacaaat ttttagcatc taatttaact tcaattccta 4080
ttatacacaa aattttaaga tactgcacta tcaacacact cttaagtttg cttctaagag 4140
accggggact tatcatccaa cctgttaaag cttggcgtaa tcatggtcat agctgtttcc 4200
tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 4260
taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 4320
cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 4380
gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 4440
ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 4500
agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4560
ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4620
caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4680
gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 4740
cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 4800
tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 4860
gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 4920
cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 4980
tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 5040
tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 5100
caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 5160
aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 5220
cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 5280
ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 5340
tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 5400
atccatagtt gcctgactcc ccgtcgtgta gataactagt gttcgtgaat acatgttata 5460
ataactataa ctaataacgt aacgtgactg gcaagagata tttttaaaac aatgaatagg 5520
tttacactta ctttagtttt atggaaatga aagatcatat catatataat ctagaataaa 5580
attaactaaa ataattatta tctagataaa aaatttagaa gccaatgaaa tctataaata 5640
aactaaatta agtttattta attaacaact atggatataa aataggtact aatcaaaata 5700
gtgaggagga tatatttgaa tacatacgaa caaattaata aagtgaaaaa aatacttcgg 5760
aaacatttaa aaaataacct tattggtact tacatgtttg gatcaggagt tgagagtgga 5820
ctaaaaccaa atagtgatct tgacttttta gtcgtcgtat ctgaaccatt gacagatcaa 5880
agtaaagaaa tacttataca aaaaattaga cctatttcaa aaaaaatagg agataaaagc 5940
aacttacgat atattgaatt aacaattatt attcagcaag aaatggtacc gtggaatcat 6000
cctcccaaac aagaatttat ttatggagaa tggttacaag agctttatga acaaggatac 6060
attcctcaga aggaattaaa ttcagattta accataatgc tttaccaagc aaaacgaaaa 6120
aataaaagaa tatacggaaa ttatgactta gaggaattac tacctgatat tccattttct 6180
gatgtgagaa gagccattat ggattcgtca gaggaattaa tagataatta tcaggatgat 6240
gaaaccaact ctatattaac tttatgccgt atgattttaa ctatggacac gggtaaaatc 6300
ataccaaaag atattgcggg aaatgcagtg gctgaatctt ctccattaga acatagggag 6360
agaattttgt tagcagttcg tagttatctt ggagagaata ttgaatggac taatgaaaat 6420
gtaaatttaa ctataaacta tttaaataac agattaaaaa aattataaaa aaattgaaaa 6480
aatggtggaa acactttttt caattttttt gttttattat ttaatatttg ggaaatattc 6540
attctaattg gtaatcagat tttagaaaac 6570
<210> 4
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgttgcgat taatagatct gtcgacctgc agtaag 36
<210> 5
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
accatgatta cgccaagctt taacaggttg gatgataagt cc 42
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gaaaccgata ccgtttacg 19
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gacgatattc tcgattgacc 20
<210> 8
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gatcaggagt tgagagtg 18
<210> 9
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctcttctcac atcagaaaat gg 22
<210> 10
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggccacgcgt cgactagtca nnnnnnnnnn gatca 35
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggttgagtac tttttcactc g 21
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggccacgcgt cgactagtca 20
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggaataccaa catgacgaat c 21
<210> 14
<211> 160
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
taagatgaac cggtaagcag ttgcaatttt cggtaaatag ttatataata aaaacgttga 60
aaagatgctt gtaggcttac aagttaatat tttcacaaaa ggattaaaag gcttgccttt 120
taaaataaag aactcgattt tcataaggag gaaatcattc 160
<210> 15
<211> 1047
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggaaaaaa aggaatttcg tgttttgata aaatactgtt ttctgaaggg aaaaaataca 60
gtggaagcaa aaacttggct tgataatgag tttccggact ctgccccagg gaaatcaaca 120
ataattgatt ggtatgcaaa attcaagcgt ggtgaaatga gcacggagga cggtgaacgc 180
agtggacgcc cgaaagaggt ggttaccgac gaaaacatca aaaaaatcca caaaatgatt 240
ttgaatgacc gtaaaatgaa gttgatcgag atagcagagg ccttaaagat atcaaaggaa 300
cgtgttggtc atatcattca tcaatatttg gatatgcgga agctctgtgc gaaatgggtg 360
ccgcgcgagc tcacatttga ccaaaaacaa cgacgtgttg atgattctaa gcggtgtttg 420
cagctgttaa ctcgtaatac acccgagttt ttccgtcgat atgtgacaat ggatgaaaca 480
tggctccatc actacactcc tgagtccaat cgacagtcgg ctgagtggac agcgaccggt 540
gaaccgtctc cgaagcgtgg aaagactcaa aagtccgctg gcaaagtaat ggcctctgtt 600
ttttgggatg cgcatggaat aatttttatc gattatcttg agaagggaaa aaccatcaac 660
agtgactatt atatggcgtt attggagcgt ttgaaggtcg aaatcgcggc aaaacggccc 720
cacatgaaga agaaaaaagt gttgttccac caagacaacg caccgtgcca caagtcattg 780
agaacgatgg caaaaattca tgaattgggc ttcgaattgc ttccccaccc gccgtattct 840
ccagatctgg cccccagcga ctttttcttg ttctcagacc tcaaaaggat gctcgcaggg 900
aaaaaatttg gctgcaatga agaggtgatc gccgaaactg aggcctattt tgaggcaaaa 960
ccgaaggagt actaccaaaa tggtatcaaa aaattggaag gtcgttataa tcgttgtatc 1020
gctcttgaag ggaactatgt tgaataa 1047
<210> 16
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agaccgggga cttatcatcc aacctgtta 29
<210> 17
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
taacaggttg gatgataagt ccccggtct 29
<210> 18
<211> 735
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tttcctcccg ttaaataata gataactatt aaaaatagac aatacttgct cataagtaat 60
ggtacttaaa ttgtttactt tggcgtgttt cattgcttga tgaaactgat ttttagtaaa 120
cagttgacga tattctcgat tgacccattt tgaaacaaag tacgtatata gcttccaata 180
tttatctgga acatctgtgg tatggcgggt aagttttatt aagacactgt ttacttttgg 240
tttaggatga aagcattccg ctggcagctt aagcaattgc tgaatcgaga cttgagtgtg 300
caagagcaac cctagtgttc ggtgaatatc caaggtacgc ttgtagaatc cttcttcaac 360
aatcagatag atgtcagacg catggctttc aaaaaccact tttttaataa tttgtgtgct 420
taaatggtaa ggaatactcc caacaatttt atacctctgt ttgttaggga attgaaactg 480
tagaatatct tggtgaatta aagtgacacg aatgttcagt tttaattttt ctgacgataa 540
gttgaataga tgactgtcta attcaataga cgttacctgt ttacttattt tagccagttt 600
cgtcgttaaa tgccctttac ctgttccaat ttcgtaaacg gtatcggttt cttttaaatt 660
caattgtttt attatttggt tgagtacttt ttcactcgtt aaaaagtttt gagaatattt 720
tatatttttg ttcat 735
Claims (3)
1.一种猪链球菌高效转座突变系统，其特征在于，所述猪链球菌高效转座突变系统的核苷酸序列如SEQ ID NO.3所示，包含Mariner转座酶表达盒、抗性表达盒组成的基因片段Tn，以及pSET4s质粒，所述Mariner转座酶表达盒，包括一个gapdh基因启动子和Mariner转座酶的编码序列，其在猪链球菌中表达Mariner转座酶，所述gapdh基因启动子的核苷酸序列如SEQ ID NO.14所示，转座酶基因Mariner的核苷酸序列如SEQ ID NO.15所示，所述抗性表达盒为红霉素抗性表达盒，其核苷酸序列如SEQ ID NO.18所示，该抗性表达盒由抗性基因及两侧的转座酶识别序列组成，两侧的所述转座酶识别序列分别如SEQ ID NO.16和SEQID NO.17所示，抗性表达盒可被Mariner转座酶识别后将该抗性表达盒插入至猪链球菌染色体的TA碱基位点，所述基因片段Tn的核苷酸序列如SEQ ID NO.2所示，所述质粒为pSET4s质粒，其核苷酸序列如SEQ ID NO.1所示，含有如下基因片段：猪链球菌温敏复制元件Tsori、大肠杆菌复制元件ColE1 ori、壮观霉素抗性表达盒，所述基因片段Tn插入pSET4s质粒中形成所述猪链球菌高效转座突变系统，即pSET4s-Tn质粒。
2.如利用权利要求1所述的猪链球菌高效转座突变系统的构建方法，其特征在于，其步骤如下：将所述pSET4s质粒用限制性内切酶BglII+HindIII双酶切后用DNA回收试剂盒回收大片段，人工合成所述基因片段Tn，在37 ℃的条件下利用同源重组酶将酶切后的pSET4s质粒与Tn片段连接，转化大肠杆菌DH5α感受态，37 ℃过夜培养，挑选阳性克隆过夜培养。
3.如权利要求1所述的猪链球菌高效转座突变系统在猪链球菌转座子突变库构建中的应用。
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