CN112522266A - 家蚕微粒子虫诱导表达基因BmPUGT2的启动子及其应用 - Google Patents
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Abstract
本发明属于家蚕转基因技术领域，具体涉及一种家蚕微粒子虫诱导表达基因BmPUGT2的启动子及其应用，所述启动子的核苷酸基因如SEQ ID NO：6所示，该启动子可以驱动外源基因在家蚕中经家蚕微粒子虫诱导下表达，不但适用于基因功能分析等分子生物学理论研究，同时适用于利用基因工程进行家蚕品种改良，特别是家蚕抗微粒子病的品种培育，具有良好的应用前景。
Description
技术领域
本发明属于家蚕转基因技术领域，具体涉及一种家蚕微粒子虫诱导表达基因BmPUGT2的启动子及其应用。
背景技术
微孢子虫病是由细胞内专性寄生的微孢子虫引起的，危害人类健康、经济动物产业发展及生态环境稳定。蚕丝业作为我国的传统优势产业，亦长期饱受微孢子虫病害的侵扰。当前，我国每年因微粒子病而带来的直接经济损失即达上亿元，全国主要蚕种场用于防控该病的花费占蚕种生成总支出的一半。然而，目前生产上尚未发现对该病有抗性的家蚕材料。
随着家蚕转基因技术的突破和完善，转基因技术已成为家蚕抗性材料选育的强有力工具。但是，目前通过转基因家蚕育种方面利用的还是组成型表达的启动子。利用组成型启动子一方面外源基因的表达效率会降低，另一方面会对家蚕本身带来负担。目前鲜有关于家蚕微粒子虫诱导型启动子的报道。因此，开发家蚕微粒子虫诱导型启动子对在构建家蚕微粒子虫抗性育种素材中减少外源基因组成型表达导致的生理负荷，提高转基因家蚕的抗性以及开发新的微粒子虫抗性系统具有重大意义。
发明内容
有鉴于此，本发明的目的之一在于提供一种家蚕微粒子虫诱导表达基因BmPUGT2的启动子，目的之二在于提供一种重组载体，目的之三在于提供一种重组表达载体的构建方法，目的之四在于提供一种重组细胞，目的之五在于提供一种外源蛋白，目的之六在于提供一种家蚕微粒子虫诱导表达基因BmPUGT2的启动子在培育抗微粒子虫家蚕中的应用。
为达到上述目的，本发明提供如下技术方案：
1、一种家蚕微粒子虫诱导表达基因BmPUGT2的启动子，所述启动子的核苷酸序列如SEQ ID NO：6所示。
2、一种重组表达载体，所述重组表达载体包含所述家蚕微粒子虫诱导表达基因BmPUGT2的启动子。
3、一种重组表达载体的构建方法，所述方法为，根据所述家蚕微粒子虫诱导表达基因BmPUGT2的启动子的核苷酸序列，设计含有表达载体酶切位点的上、下游引物，以家蚕基因组DNA为模板，进行PCR扩增，获得含所述启动子的目的片段，将所述目的片段连接在表达载体上，构建重组表达载体。
作为优选的技术方案之一，所述表达载体为pSLfa1180fa载体。
作为优选的技术方案之一，所述上游引物的核苷酸序列如SEQ ID NO：7所示，所述下游引物的核苷酸序列如SEQ ID NO：8所示。
4、一种重组细胞，所述重组细胞是由所述重组表达载体转化受体细胞而得。
5、一种外源蛋白，所述外源蛋白是由所述重组细胞所表达。
6、所述家蚕微粒子虫诱导表达基因BmPUGT3的启动子在培育抗微粒子虫家蚕中的应用。本发明的有益效果在于：
本发明所构建的BmPUGT2启动子可以驱动外源基因在家蚕中经家蚕微粒子虫诱导下表达，不但适用于基因功能分析等分子生物学理论研究，同时适用于利用基因工程进行家蚕品种改良，特别是用于家蚕抗微粒子病的品种培育，比如诱导表达内外源致死基因以及诱导型的基因编辑系统，具有良好的应用前景。
附图说明
图1为BmPUGT2的基因序列图，斜体加粗的ATG表示翻译起始位点，TGA表示终止密码子，灰色框代表非翻译区；
图2为RT-PCR检测结果图，A中N3～N48分别代表家蚕微粒子虫感染后3h、6h、12h、24h及48h取材组，C3～C48为其清水对照组；B为未感染家蚕微粒子虫时家蚕各组织BmPUGT2的转录表达检测；
图3为启动子PPUGT2的序列图，小写斜体灰底的序列为BmPUGT2的5’UTR区域，大写斜体加粗的ATG代表翻译起始位点；
图4为BmN-SWU1细胞转染pSL[PPUGT2-mCherry-SV40]表达载体后的红色荧光的发光情况结果图，A为转染表达载体后添加家蚕微粒子虫诱导的荧光观察结果图，B为转染表达载体后添加家蚕微粒子虫诱导的白光观察结果图，C为转染表达载体添加PBS的荧光观察结果图，D为转染表达载体添加PBS作对照的白光观察结果图。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等编著)中所述的条件，或者按照制造商所建议的条件。
实施例1
家蚕微粒子虫诱导BmPUGT2基因表达
根据家蚕基因组数据库SilkDB(https://silkdb.bioinfotoolkits.net/main/species-info/-1)及NCBI(https://www.ncbi.nlm.nih.gov)，获得BmPUGT2(AK378453.1)基因的CDS序列(SEQ ID NO:1)，如图1所示，BmPUGT2的基因序列图，斜体加粗的ATG表示翻译起始位点，TGA表示终止密码子，灰色框代表非翻译区，根据BmPUGT2的基因序列设计该基因的检测引物BmPUGT2-F及BmPUGT2-R，家蚕Actin 3基因为内参，引物为BmA3-F及BmA3-R。
BmPUGT2-F：5’-ATCATTCACAGTACGTAT-3’(SEQ ID NO:2)
BmPUGT2-R：5’-GAAACATTATTTATTGGT-3(SEQ ID NO:3)
BmA3-F：5'-ATGGTGCGCTCCTCCAAGAACG-3'(SEQ ID NO:4)
BmA3-R：5'-CTACAGGAACAGGTGGTGGCGG-3'(SEQ ID NO:5)
将正常家蚕大造品种以人工饲料在标准环境中饲养(温度：25℃，湿度：80％)，5龄起蚕，一部分添食家蚕微粒子虫，另一部分添食清水作为对照。在添食3h，6h，12h，24h，48h后分别取其中肠，用RNA提取试剂盒(R6934，OMEGA)提取RNA，然后用反转录试剂盒(A5001，Promega)将RNA反转录为cDNA，再以cDNA为模板，BmPUGT2-F及BmPUGT2-R为引物，BmA3-F和BmA3-R引物对为对照，用rTaq酶(R004,Takara)进行PCR扩增，扩增条件为：94℃预变性3min，94℃变性30s，55℃退火30s，72℃延伸30s；共30个循环，最后终延伸10min。对PCR产物进行琼脂糖凝胶检测。结果如图2中A、B所示，BmPUGT2仅在感染后24及48小时检测到其转录，而未感染的情况下没有检测到该基因的转录；并且在正常家蚕的各个组织中均未检测到该基因的转录，由此证明BmPUGT2确实可以被微粒子虫诱导表达。
实施2
BmPUGT2启动子载体构建
根据家蚕基因组数据库(https://silkdb.bioinfotoolkits.net/main/species-info/-1)，获得BmPUGT2基因翻译起始位点ATG前1247bp的基因组序列(SEQ ID NO:6)，如图3所示，小写斜体灰底的为BmPUGT2的5’UTR区域，大写斜体加粗的ATG代表翻译起始位点，利用网站分析这段基因组序列(https://www.fruitfly.org/seq_tools/promoter.html)，发现在这段序列中含有典型的启动子结构区域，据此设计该区域特异性引物PPUGT2-F-EcoRI及PPUGT2-R-BamHI扩增该段序列，序列如下:
基因组提取试剂盒(D3396,OMEGA)提取家蚕大造品系的基因组DNA，并以家蚕基因组DNA为模板，用引物PPUGT2-F-EcoRI、PPUGT2-R-BamH及Q5高保真酶(M0493，NEB)进行PCR扩增，其扩增条件为：98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸2min；共30个循环，最后终延伸10min。用胶回收试剂盒(D2500,OMEGA)回收该PCR产物，获得PPUGT2启动子片段，用T4连接酶(M0202,NEB)将目的片段与pMD19-T载体连接，转化至DH5a感受态细胞，获得阳性克隆后测序，测序结果表明已成功将PPUGT2启动子连接到pMD19-T载体上，且与预期序列一致。
利用已经构建好的pSL[MCS-mCherry-SV40]载体(pSLfa1180fa载体上SV40终止信号序列上下游酶切位点分别是Not I和Hind III，mCherry上下游酶切位点分别是有BamH I和Not I)，通过EcoR I和BamH I酶对pMD19/PPUGT2进行酶切，回收PPUGT2启动子片段。同时用EcoR I和BamH I酶对pSL[MCS-mCherry-SV40]载体酶切，回收载体大片段。将BmPUGT2启动子片段和pSL[MCS-mCherry-SV40]骨架用T4 DNA连接酶在16℃下连接过夜，进而转化至DH5α感受态细胞，获得pSL[PPUGT2-mCherry-SV40]表达载体的阳性克隆。
实施3
BmPUGT2启动子的功能验证
将实施例2中构建的pSL[PPUGT2-mCherry-SV40]表达载体按质粒：脂质体＝1：2，ug:ul的比例转染至BmN-SWU1细胞系，12h后添加家蚕微粒子虫，家蚕微粒子虫与细胞的个数比例为10:1，以添加PBS作对照，72h后通过荧光显微镜检测红色荧光情况。结果如图4所示，在转染pSL[PPUGT2-mCherry-SV40]的细胞中，当添加家蚕微粒子虫时，启动子PPUGT2被激活并诱导其下游mCherry基因的表达而使细胞呈红色荧光，而对照组PBS则没有红色荧光，由此说明启动子PPUGT2是可以被家蚕微粒子虫所诱导激活的，实现调控的目的。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 西南大学
<120> 家蚕微粒子虫诱导表达基因BmPUGT2的启动子及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2300
<212> DNA
<213> 天然序列(Natural sequence)
<400> 1
atcattcaca gtacgtatta agaacgcgag cagcgtaaca caaaaataca atgaattttc 60
aaacgattca tttgttggtg ttaagtgcgc tcgcttgcga tgcatataag atattgctcg 120
tgtttccgtt cccttcgaaa agccacgcta ttcttggaga gggttacgtc aggaatctcc 180
tcaaagcagg acatgaagtg acctacataa cgccatatcc gagagatccg gctcccaatt 240
tacggatcat ccaagtttca cagcatgact tcgaggaaaa aattaactcc acgttaacca 300
tagaaaagct aatagactac agttttacag taatggaaat gtttaacatt acgaagtctt 360
ttatttatac agctaatgat acagtagcta atacggaagt acaacagctg atgttagatc 420
cacagacaca ttttgatgtt gttgttgcag aatggatgat aactgaaatt tttagtggct 480
tcagtgtaat tttcaactgt ccactcatat ggtcttcttc aatggaacca catagctgga 540
tattgccttt gatagacgag attccccatc ctgcctattc atcaaatata ttagggcttt 600
ttgaaccgcc atacaatttc gtccaaagag caataaacac ttcgttggag attgcgttaa 660
aagtaattaa atggcttgtc actttaatag aagaacagat ttataaagaa ggatttgcag 720
ctgcattcaa agcaaaaggt cttattcagc caagcttaga ggaattgaga tactctgctg 780
ctttggtttt gggaaattcc cacatttctt ctggagctcc gctgaaattg ccacaaaatt 840
acaaggctat tggcggttat catatagatg aacaatctaa gccattgccc aaggatttta 900
agaacattct agacaactcg aagcacggcg ttatttattt cagtctagga tcgatggctc 960
caagtaaatc gatgcctgca gcaataaaaa acggattatt tgaaatgttt aggagtttaa 1020
aatatactgt tatatggaaa ttcgaagatg aatttcaaaa tgttcctgac aacgttcaca 1080
tcgtaaaatg ggctccacag caaagcatac tagcacaccc taactgcatt ctcttcatca 1140
cccacggtgg cttattgtct acaacggaaa cattacatta cggtgttcct attattggaa 1200
tgcctatgtt tggagatcag gtcatgaata tcaagaaggc tgtccataaa ggctttggac 1260
tagaagtgaa acttaacttc gatactccaa agaacttgaa agcagctata aatgaggttt 1320
tgtccaatca aaagtatcgc gatcgagtta aggagttgtc gttgatatat cacgatcgtc 1380
cggtgtctcc gggtgctgag ctggtccact gggtggagca tgtcgtcaag actaaaggag 1440
ctctccatct gcgctcacag gcgctgcacg tgcctttgta ccagaagctg ttattagata 1500
taatcttcgt gtcgttattg ttgttccttg gttttgtatt cttcatcaaa tatatggtga 1560
cccggtgttt gaagaagaaa atagatatta gaaaaaaaac tttgtagctt taaaccttat 1620
actaattata tttcaaaacg aactgcaaat tcctaagtac aactcgacca agctaagttt 1680
gcacctcgct ggtatgcggc gcccgcccac ttccccgctc accttcccgc gatcgccctg 1740
tcgcgtcagt acagttagca tctttgttgc tgacacgcgt catacgagtt ttcgtctccc 1800
tatctgttga attttaaatt ttttaactgt tcttgttagg ttttttgttt atgtttagct 1860
atattattga actatttttg ttaagttttt gttgaactgt taactgttgt tactgaacgc 1920
ttaacgttca ttttttttta cttcgagagg attgttgtgg gcaacaaaaa tgaaaggaaa 1980
ggtgatacaa agtgaaggga gaaacataat catgaaaaag aaacataaag gaaaattatt 2040
actgagcaat tgtgacgtaa tcgatgcgaa gtgcaaactt agcttggtcg agttgtaatg 2100
tatacttatc tataattttt taatattata aagccgaaga gtttgttggt ttgaacgcgc 2160
taatctcaag aactacttac tggtccgatt tgaaaaaata tttcagtatt agataactca 2220
tttatcgagg aaggctatat atacgttata caacatcacg ctaagaccaa cgcaagcgga 2280
gcaccaataa ataatgtttc 2300
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atcattcaca gtacgtat 18
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gaaacattat ttattggt 18
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggtgcgct cctccaagaa cg 22
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctacaggaac aggtggtggc gg 22
<210> 6
<211> 1250
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtacaatttc acgggaccca caatgattgt gcgtttttcg ttgatgcacg ttgttttaat 60
ttgcaagtcc acgcctctcc tccgcttccg ccgcggtgag gaaaatgggt tgctatgttt 120
cgaacagaaa tttaagttac aaaaaaaaca aaagcaacac aatacacaca ttattttgta 180
catcaactta cactataaga ctatatataa tactcatcgg actaaatcac cttataaata 240
ttttaaaata gcttttctat atgctataag taccaagcaa aagatgtcag cattagaaaa 300
gttagcatca tacaacctac aagatatacg gacgaaagag ttagaagtat atttcgttcg 360
agccgtggac atgtggaaat aatgtggaag gtctgtttag aatgcgcgga aatacgacta 420
catttaaatg aaaatttgaa aagaaggtag ctagtatcaa tctcattatt tattatttta 480
tataaaaaca tttgatccct cagtagccta cgatcgacta gagagataaa ttatgtcgat 540
tctggtgagt caaagcgacg acaatggtat aagatatgtt taaaaaactt tttttggact 600
ctttccagtc gctcaatata aatattatat tggggattcc aggcgacgga gccatattcc 660
aatatagatc gtacaaaact gttgtacaac agaatcagag tagatggtct tttaaaatct 720
ttaccaactc taagtataaa gcctaacatt tgaaaatcct tatttattat ttcattaact 780
tgaacataaa atcaaactgg aatccaaata aatacccaaa tcacggataa ctgtggtgcg 840
taccaaagtt tgaatattga tagaatagtc atacaagata ttcgatctac cacgtgagaa 900
tgtgataaca gtacttttta taaaaatatt tttttcttat actattttta atttaaattt 960
attaagattt attttctatt ttttagtagg attttctata aaagcgtatt tttttagttt 1020
ttttaaacta ttattttatt tttttgtagt tcattcaaga tatgtgatgt ttttaaacta 1080
tcttacttac gtcaacttaa ttattataat agattactaa gtctgactta aagaaagata 1140
agagtcgcat agcgattatg aaagcgatac aaatacaaat tgttttagtt atcgtgtatc 1200
attcacagta cgtattaaga acgcgagcag cgtaacacaa aaatacaatg 1250
<210> 7
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccggaattcg tacaatttca cgggacccac 30
<210> 8
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgcggatccc attgtatttt tgtgttacgc tg 32
Claims (8)
1.一种家蚕微粒子虫诱导表达基因BmPUGT2的启动子，其特征在于，所述启动子的核苷酸序列如SEQ ID NO：6所示。
2.一种重组表达载体，其特征在于，所述重组表达载体包含权利要求1中所述的启动子。
3.权利要求2所述重组表达载体的构建方法，其特征在于，所述方法为，根据权利要求1所述的启动子的核苷酸序列，设计含有表达载体酶切位点的上、下游引物，以家蚕基因组DNA为模板，进行PCR扩增，获得含所述启动子的目的片段，将所述目的片段连接在表达载体上，构建重组表达载体。
4.如权利要求3所述的构建方法，其特征在于，所述表达载体为pSLfa1180fa载体。
5.如权利要求3或4任一项所述的构建方法，其特征在于，所述上游引物的核苷酸序列如SEQ ID NO：7所示，所述下游引物的核苷酸序列如SEQ ID NO：8所示。
6.一种重组细胞，其特征在于，所述重组细胞是由权利要求2所述重组表达载体转化受体细胞而得。
7.一种外源蛋白，其特征在于，所述外源蛋白是由权利要求6所述的重组细胞所表达。
8.权利要求1所述的启动子在培育抗微粒子虫家蚕中的应用。
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