CN114606255A - 一种多重基因调控下游基因检测方法 - Google Patents
一种多重基因调控下游基因检测方法 Download PDFInfo
- Publication number
- CN114606255A CN114606255A CN202210282034.9A CN202210282034A CN114606255A CN 114606255 A CN114606255 A CN 114606255A CN 202210282034 A CN202210282034 A CN 202210282034A CN 114606255 A CN114606255 A CN 114606255A
- Authority
- CN
- China
- Prior art keywords
- gene
- vector
- trp
- pro
- met
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims description 10
- 239000013598 vector Substances 0.000 claims abstract description 36
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 26
- 230000003993 interaction Effects 0.000 claims abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 230000001105 regulatory effect Effects 0.000 claims abstract description 5
- 230000007547 defect Effects 0.000 claims abstract description 3
- KLSJWNVTNUYHDU-UHFFFAOYSA-N Amitrole Chemical compound NC1=NC=NN1 KLSJWNVTNUYHDU-UHFFFAOYSA-N 0.000 claims description 20
- 238000013518 transcription Methods 0.000 claims description 10
- 230000035897 transcription Effects 0.000 claims description 10
- 102100039556 Galectin-4 Human genes 0.000 claims description 9
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 claims description 8
- 101150105382 MET gene Proteins 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- 230000006801 homologous recombination Effects 0.000 claims description 6
- 238000002744 homologous recombination Methods 0.000 claims description 6
- 108700008625 Reporter Genes Proteins 0.000 claims description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 5
- 101150077230 GAL4 gene Proteins 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 102000003960 Ligases Human genes 0.000 claims description 2
- 108090000364 Ligases Proteins 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 108010001515 Galectin 4 Proteins 0.000 claims 1
- 239000013604 expression vector Substances 0.000 claims 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 abstract description 22
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 229930182817 methionine Natural products 0.000 abstract description 2
- 102000040945 Transcription factor Human genes 0.000 description 13
- 108091023040 Transcription factor Proteins 0.000 description 13
- 239000000243 solution Substances 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 9
- 238000001976 enzyme digestion Methods 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000000499 gel Substances 0.000 description 5
- 230000004568 DNA-binding Effects 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001712 DNA sequencing Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000006916 protein interaction Effects 0.000 description 2
- 238000010079 rubber tapping Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 101150009006 HIS3 gene Proteins 0.000 description 1
- 101100285000 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) his-3 gene Proteins 0.000 description 1
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 1
- 102000006290 Transcription Factor TFIID Human genes 0.000 description 1
- 108010083268 Transcription Factor TFIID Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种多重基因调控下游基因检测方法,在多重基因调控下游基因检测实验中,本发明以酿酒酵母为宿主,使用载体为双表达框,其中第二表达框以pMET25为启动子,通过甲硫氨酸缺陷与否来调控第二表达框基因表达与否,再通过第二表达框基因表达与否来检测第一表达框表达基因(TF)和第二表达框表达基因(基因)的相互作用来检测对下游基因Pro的调控。本发明技术方案成本低、工作量小、可行性高,可广泛应用于多重基因调控下游基因检测。
Description
技术领域
本发明属分子生物学技术领域,具体涉及一种多重基因调控下游基因检测方法。
背景技术
酵母单杂交是研究蛋白质相互作用的重要方法之一。原理是:真核生物基因的转录起始需转录因子参与,转录因子通常由一个DNA特异性结合功能域和一个或多个其他调控蛋白相互作用的激活功能域组成,即DNA结合结构域(DNA—binding domain,BD)和转录激活结构域(activation domain,AD)。用于酵母单杂交系统的酵母GAL4蛋白是一种典型的转录因子,GAL4的DNA结合结构域靠近羧基端,含有几个锌指结构,可激活酵母半乳糖苷酶的上游激活位点(UAS),而转录激活结构域可与RNA聚合酶或转录因子TFIID相互作用,提高RNA聚合酶的活性。在这一过程中,DNA结合结构域和转录激活结构域可完全独立地发挥作用。据此可将GAL4的DNA结合结构域置换为文库蛋白编码基因,只要其表达的蛋白能与目的基因相互作用,同样可通过转录激活结构域激活RNA聚合酶,启动下游报告基因的转录,但目前还未见多重基因调控下游基因检测方法的报道。
发明内容
本发明的目的在于提供了一种多重基因调控下游基因检测方法,该方法操作简单成本较低、工作量小。为了实现上述的目的,本发明采用以下技术措施,包括如下步骤:
(1)以pBridge载体扩增Met25启动子表达框,并通过T4连接酶连接至经NotI和NheI线性化后的pGADT7载体,重组载体命名为pGADT7-Met;
(2)将TF(转录因子)以同源重组的方式构建到pGADT7-Met,并与AD融合;
(3)将另一个未知基因以同源重组的方式克隆至步骤(2)载体中的Met25启动子启动的表达框,构建好的基因和载体命名为:pGADT7-TF&Met-基因;
(4)全基因合成GAL4基因,用SacI和PteI线性化pHis载体,以同源重组方式构建至pHis2载体上,将报告基因组氨酸缺陷变为GAL4,载体命名为pGAL4;
(5)将下游基因启动子pro构建到所构建的pGAL4载体多克隆位点处,构建好的载体命名为pGAL4-pro;
(6)pGAL4-pro转化AH109或Y2Hgold宿主,涂布SD/-Trp平板,并检测启动子在酵母中内源基因的转录抑制浓度;
(7)将pGADT7-TF&Met-基因载体转化AH109(pGAL4-pro)或Y2Hgold(pGAL4-pro)酵母菌株,涂布SD-Trp/-Leu平板,并进行阳性克隆的筛选;
(8)将生长出来菌斑加入NaCl溶液中,并稀释OD600值至0.01涂布到SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板和SD-Met/-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板,观察酵母生长状态,若TF和pro相互作用,那么在SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上正常生长且变蓝,若TF通过基因相互作用修饰后与pro相互作用,那么在SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上不生长,在SD-Met/-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上生长且变蓝。
采用本发明的产生的有益效果为:
本方法具有成本低、工作量较小、可行性高等优点,可广泛应用于多重基因调控下游基因检测。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下对本发明进行进一步的详细说明。应当理解,此处所描述的具体实施例仅用于解释本发明,并不用于限定本发明。
在多重基因调控下游基因检测实验中,本发明以酿酒酵母为宿主,使用载体为双表达框,其中第二表达框以pMET25为启动子,通过甲硫氨酸缺陷与否来调控第二表达框基因表达与否,再通过第二表达框基因表达与否来检测第一表达框表达基因和第二表达框表达基因的相互作用来检测对下游基因的调控。
实施例1重组pGADT7-TF&Met-基因载体的构建
(1)PCR扩增Met25启动子表达框:
根据DNAman软件合成两条特异性引物(上海生工进行合成):Met25-F:gttatattaagggttccggatcTTATTTTTTGCTTTTTCTCTTGAGGTCAC
Met25-R:TTACGTCCAGCCAAGCTAGCAACGCAGAATTTTCGAGT,合成后的引物溶解成10μM浓度的引物溶液,备用。
PCR仪预热到95℃,取2μL模板DNA(pBridge),70μL ddH2O,10μL PCR缓冲液,2μL10×GC-Melt溶液,2μL 50×dNTP混合物,2μL 5’PCR引物,2μL 3’PCR引物和2μL 50×Advantage 2聚合酶,混匀后,稍稍离心放入预热到95℃的PCR仪中,运行PCR程序;反应扩增反应结束后,等量合并扩增产物进行后续割胶纯化步骤,配制质量比1%的琼脂糖凝胶,将扩增好的样品进行电泳,140V电压,30-40分钟,电泳结束后,使用QIAGEN公司的胶回收试剂盒QIAquick Gel Extraction kit(Cat.No.28706)进行胶回收;
(2)胶回收纯化的PCR产物采用NotI和NheI双酶切,再经过电泳回收纯化后与经过同样处理的pGADT7连接,连接产物转化E.coli感受态DH5α细胞,挑选单菌落,小提质粒后经PCR及双酶切分析验证后,进行DNA测序鉴定。
(3)将与启动子结合的TF(转录因子)以相同的PCR扩增、双酶切、连接至pGADT7-Met载体上,并与AD融合;将另一个基因克隆至Met25启动子启动的表达框,构建好的基因和载体命名为:pGADT7-TF&Met-基因,质粒大提以后将PCR及双酶切鉴定均为阳性的重组质粒送测序。并且分析测序结果与NCBI中有关该基因的数据库序列的比对效果。
实施例2重组pGAL4-pro载体的构建
(1)PCR扩增GAL4基因:
根据DNAman软件合成两条特异性引物(上海生工进行合成):A-F:AGGGC-GAATTCCCGGGGAGCTCACGCGTTCGCGAATCGAT
A-R:AAAGAAAAAAGGAAAGCGCGCCTCGTTCAGAATGACACGTATAG,合成后的引物溶解成10μM浓度的引物溶液,备用。
PCR仪预热到95℃,取2μL模板DNA(pBridge),70μL ddH2O,10μL PCR缓冲液,2μL10×GC-Melt溶液,2μL 50×dNTP混合物,2μL 5’PCR引物,2μL 3’PCR引物和2μL 50×Advantage 2聚合酶,混匀后,稍稍离心放入预热到95℃的PCR仪中,运行PCR程序;反应扩增反应结束后,等量合并扩增产物进行后续割胶纯化步骤,配制质量比1%的琼脂糖凝胶,将扩增好的样品进行电泳,140V电压,30-40分钟,电泳结束后,使用QIAGEN公司的胶回收试剂盒QIAquick Gel Extraction kit(Cat.No.28706)进行胶回收;
(2)胶回收纯化的PCR产物采用SacI和PteI双酶切,再经过电泳回收纯化后与经过同样处理的pHis载体连接,连接产物转化E.coli感受态DH5α细胞,挑选单菌落,小提质粒后经PCR及双酶切分析验证后,进行DNA测序鉴定。
(3)将下游基因启动子pro通过PCR扩增、双酶切、连接至pGAl4载体上,构建好的基因和载体命名为:pGAl4-pro,质粒大提以后将PCR及双酶切鉴定均为阳性的重组质粒送测序。并且分析测序结果与NCBI中有关该基因的数据库序列的比对效果。
实施例3 pGAL4-pro质粒转化酵母宿主Y2HGold,生成pGAL4-pro酵母菌株
采用电转化技术将pGAL4-pro质粒转化至酵母宿主细胞,并检测检测启动子在酵母中内源基因的转录抑制浓度。
3-氨基-1,2,4-三唑是一种组氨酸的竞争性抑制剂,可以抑制His3的泄露表达和轻微自激活现象。诱饵酵母中含有GAL4转录因子启动的组氨酸合成报告基因(His),当基因通过与TF相互作用后使TF能与下游基因基因启动子结合进而促使GAL4表达时,GAL4就会激活HIS3编码组氨酸合成途径的一个蛋白。
将1000ng的pBait-Pro-GAL4转入诱饵酵母菌株中,转化方法参见Yeast makerYeast Transformation System 2;快速离心转化液,用600μL 0.9%的NaCl溶液分别悬浮转化菌体Y2HGold(pBait-Pro-GAL4),调节菌液的OD600值为0.002,取8μL悬浮液分别滴在SD/-Trp/-His/3-AT*和SD/-Trp培养基上,30℃培养3d;设计3-AT的浓度梯度,取100μL菌液分别涂布在含不同3-AT浓度的SD/-Trp/-His培养基上;观察菌落生长状况,确定抑制诱饵酵母菌株自激活的最小3-AT浓度。
经检测,启动子在酵母中内源基因的转录抑制浓度为20mM/mL。
实施例3 pGADT7-TF&Met-基因载体转化Y2HGold(pBait-Pro-GAL4)酵母菌株,验证互作蛋白
采用电转化技术将pGADT7-TF&Met-基因质粒转化至酵母宿主细胞Y2HGold(pBait-Pro-GAL4),30℃培养2-3天,菌斑长出以后,挑取菌斑加入NaCl溶液中,并稀释OD600值至0.01涂布到SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板和SD-Met/-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板,观察酵母生长状态,若TF和pro相互作用,那么在SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上正常生长且变蓝,若TF通过基因相互作用修饰后与pro相互作用,那么在SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上不生长,在SD-Met/-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上生长且变蓝。
以上内容是结合具体实施例对本发明所作的进一步详细说明,不能认定本发明的具体实施只限于此。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下做出若干等同替代或明显变型,且性能或用途相同,都应当视为属于本发明由所提交的权利要求书确定的专利保护范围。
Met25启动子表达框
TTATTTTTTGCTTTTTCTCTTGAGGTCACATGATCGCAAAATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCCTTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAATACAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCCATACAATGGGCCATATGGCTTCTAGCTATCCTTATGACGTGCCTGACTATGCCAGCCTGGGAGGACCTTCTAGTCCTAAGAAGAAGAGAAAGGTGGCGGCCGCATTAGCCCGAAGATCTTCGGGCTGATCTCCCATGTCTCTACTGGTGGTGGTGCTTCTTTGGAATTATTGGAAGGTAAGGAATTGCCAGGTGTTGCTTTCTTATCCGAAAAGAAATAAATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCTGCGTT
全基因合成GAL4基因及部分载体序列B:
ACGCGTTCGCGAATCGATCCGCGGTCTAGAAATTCCTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGATTGACTCGGCAGCTCATCATGATAACTCCACAATTCCGTTGGATTTTATGCCCAGGGATGCTCTTCATGGATTTGATTGGTCTGAAGAGGATGACATGTCGGATGGCTTGCCCTTCCTGAAAACGGACCCCAACAATAATGGGTTCTTTGGCGACGGTTCTCTCTTATGTATTCTTCGATCTATTGGCTTTAAACCGGAAAATTACACGAACTCTAACGTTAACAGGCTCCCGACCATGATTACGGATAGATACACGTTGGCTTCTAGATCCACAACATCCCGTTTACTTCAAAGTTATCTCAATAATTTTCACCCCTACTGCCCTATCGTGCACTCACCGACGCTAATGATGTTGTATAATAACCAGATTGAAATCGCGTCGAAGGATCAATGGCAAATCCTTTTTAACTGCATATTAGCCATTGGAGCCTGGTGTATAGAGGGGGAATCTACTGATATAGATGTTTTTTACTATCAAAATGCTAAATCTCATTTGACGAGCAAGGTCTTCGAGTCAGGTTCCATAATTTTGGTGACAGCCCTACATCTTCTGTCGCGATATACACAGTGGAGGCAGAAAACAAATACTAGCTATAATTTTCACAGCTTTTCCATAAGAATGGCCATATCATTGGGCTTGAATAGGGACCTCCCCTCGTCCTTCAGTGATAGCAGCATTCTGGAACAAAGACGCCGAATTTGGTGGTCTGTCTACTCTTGGGAGATCCAATTGTCCCTGCTTTATGGTCGATCCATCCAGCTTTCTCAGAATACAATCTCCTTCCCTTCTTCTGTCGACGATGTGCAGCGTACCACAACAGGTCCCACCATATATCATGGCATCATTGAAACAGCAAGGCTCTTACAAGTTTTCACAAAAATCTATGAACTAGACAAAACAGTAACTGCAGAAAAAAGTCCTATATGTGCAAAAAAATGCTTGATGATTTGTAATGAGATTGAGGAGGTTTCGAGACAGGCACCAAAGTTTTTACAAATGGATATTTCCACCACCGCTCTAACCAATTTGTTGAAGGAACACCCTTGGCTATCCTTTACAAGATTCGAACTGAAGTGGAAACAGTTGTCTCTTATCATTTATGTATTAAGAGATTTTTTCACTAATTTTACCCAGAAAAAGTCACAACTAGAACAGGATCAAAATGATCATCAAAGTTATGAAGTTAAACGATGCTCCATCATGTTAAGCGATGCAGCACAAAGAACTGTTATGTCTGTAAGTAGCTATATGGACAATCATAATGTCACCCCATATTTTGCCTGGAATTGTTCTTATTACTTGTTCAATGCAGTCCTAGTACCCATAAAGACTCTACTCTCAAACTCAAAATCGAATGCTGAGAATAACGAGACCGCACAATTATTACAACAAATTAACACTGTTCTGATGCTATTAAAAAAACTGGCCACTTTTAAAATCCAGACTTGTGAAAAATACATTCAAGTACTGGAAGAGGTATGTGCGCCGTTTCTGTTATCACAGTGTGCAATCCCATTACCGCATATCAGTTATAACAATAGTAATGGTAGCGCCATTAAAAATATTGTCGGTTCTGCAACTATCGCCCAATACCCTACTCTTCCGGAGGAAAATGTCAACAATATCAGTGTTAAATATGTTTCTCCTGGCTCAGTAGGGCCTTCACCTGTGCCATTGAAATCAGGAGCAAGTTTCAGTGATCTAGTCAAGCTGTTATCTAACCGTCCACCCTCTCGTAACTCTCCAGTGACAATACCAAGAAGCACACCTTCGCATCGCTCAGTCACGCCTTTTCTAGGGCAACAGCAACAGCTGCAATCATTAGTGCCACTGACCCCGTCTGCTTTGTTTGGTGGCGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGTAACACCGATTATTTAAAGCTGCAGCATACGATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTATGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTCATTCTGAACGAG
Claims (3)
1.一种多重基因调控下游基因检测方法,其特征在于,包括以下步骤:
(1)以pBridge载体扩增Met25启动子表达框,并通过T4连接酶连接至经NotI和NheI线性化后的pGADT7载体,重组载体命名为pGADT7-Met;
(2)将TF以同源重组的方式构建到pGADT7-Met,并与AD融合;
(3)将另一个基因以同源重组的方式克隆至步骤(2)载体中的Met25启动子启动的表达框,构建好的基因和载体命名为pGADT7-TF&Met-基因;
(4)全基因合成GAL4基因,用SacI和PteI线性化pHis载体,以同源重组方式构建至pHis2载体上,将报告基因组氨酸缺陷变为GAL4,载体命名为pGAL4;
(5)将下游基因启动子pro构建到步骤(4)所构建的pGAL4载体多克隆位点处,构建好的载体命名为pGAL4-pro;
(6)pGAL4-pro转化酵母宿主,涂布SD/-Trp平板,并检测启动子在酵母中内源基因的转录抑制浓度;
(7)将pGADT7-TF&Met-基因载体转化步骤(6)酵母菌株,涂布SD-Trp/-Leu平板,并进行阳性克隆的筛选;
(8)将步骤(7)生长出来菌斑加入NaCl溶液中,并稀释OD600值至0.01涂布到SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板和SD-Met/-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板,观察酵母生长状态,若TF和pro相互作用,那么在SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上正常生长且变蓝,若TF通过基因相互作用修饰后与pro相互作用,那么在SD-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上不生长,在SD-Met/-Trp/-Leu/-His/-Ade/X-a-gal/3-AT平板上生长且变蓝。
2.如权利要求1所述的检测方法,其特征在于:所述的双框表达载体第二表达框为Met25启动子表达框。
3.如权利要求1所述的检测方法,其特征在于:所述TF通过基因相互作用修饰后与pro相互作用的报告基因为GAL4。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210282034.9A CN114606255A (zh) | 2022-03-21 | 2022-03-21 | 一种多重基因调控下游基因检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210282034.9A CN114606255A (zh) | 2022-03-21 | 2022-03-21 | 一种多重基因调控下游基因检测方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114606255A true CN114606255A (zh) | 2022-06-10 |
Family
ID=81864629
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210282034.9A Pending CN114606255A (zh) | 2022-03-21 | 2022-03-21 | 一种多重基因调控下游基因检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114606255A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6406863B1 (en) * | 2000-06-23 | 2002-06-18 | Genetastix Corporation | High throughput generation and screening of fully human antibody repertoire in yeast |
CN101037707A (zh) * | 2006-03-13 | 2007-09-19 | 中国医学科学院基础医学研究所 | 一种验证性筛选目的结构域结合配体的方法 |
CN108559715A (zh) * | 2018-04-19 | 2018-09-21 | 西南大学 | 筛选与G蛋白βγ二聚体相互作用蛋白质的酵母转化子及其筛选方法 |
-
2022
- 2022-03-21 CN CN202210282034.9A patent/CN114606255A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6406863B1 (en) * | 2000-06-23 | 2002-06-18 | Genetastix Corporation | High throughput generation and screening of fully human antibody repertoire in yeast |
CN101037707A (zh) * | 2006-03-13 | 2007-09-19 | 中国医学科学院基础医学研究所 | 一种验证性筛选目的结构域结合配体的方法 |
CN108559715A (zh) * | 2018-04-19 | 2018-09-21 | 西南大学 | 筛选与G蛋白βγ二聚体相互作用蛋白质的酵母转化子及其筛选方法 |
Non-Patent Citations (2)
Title |
---|
FRANZISKA GLASS ET AL: "The Yeast Three-Hybrid System for Protein Interactions", 《METHODS IN MOLECULAR BIOLOGY》, pages 3 * |
JEROEN WAGEMANS ET AL: "Identification of Protein-Protein Interactions by Standard Gal4p-Based Yeast Two-Hybrid Screening", 《METHODS IN MOLECULAR BIOLOGY》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3556860B1 (en) | Type i-b crispr-cas system gene cas3-based gene editing method | |
KR102229968B1 (ko) | 메틸영양성 효모를 유전적으로 조작하는 발현 구축물 및 방법 | |
JP6833812B2 (ja) | プロモーター変異体 | |
US5776689A (en) | Protein recruitment system | |
US20170088845A1 (en) | Vectors and methods for fungal genome engineering by crispr-cas9 | |
CN113462582B (zh) | 一株过表达Spt7基因的黑曲霉工程菌株与应用 | |
WO2022193849A1 (zh) | 液态酵母单双杂高通量筛库方法及其应用 | |
US20110129874A1 (en) | Pichia Pastoris Das Promoter Variants | |
Zhang et al. | CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans | |
CN110004171B (zh) | 一种酵母双杂交方法 | |
CN114606255A (zh) | 一种多重基因调控下游基因检测方法 | |
CN116218846B (zh) | 一种调控猪Ptrf基因表达的增强子序列的鉴定及其应用 | |
CN109321618B (zh) | 一种通过糖多孢红霉菌sace_5717基因提高红霉素产量的方法 | |
JPWO2006046694A1 (ja) | 誘導可能に発現するエルゴステロール合成酵素を有する酵母を利用した遺伝子のスクリーニング法 | |
CN115838713A (zh) | 一种蛋白酶及其在l-肌肽合成中的应用 | |
WO2021103528A1 (zh) | 一种pdgfb启动子活性报告质粒的构建方法 | |
CN110669117B (zh) | 一个草菇乙烯受体蛋白 | |
CN112981002B (zh) | 高通量筛选构巢曲霉启动子与启动子库的构建方法 | |
WO2012177499A1 (en) | Systems for induction of gene expression and protein depletion in yeast | |
Janus et al. | Identification of a minimal cre1 promoter sequence promoting glucose-dependent gene expression in the β-lactam producer Acremonium chrysogenum | |
JP2001513989A (ja) | 酵母における調節した遺伝子発現 | |
Spasskaya et al. | Escherichia coli Dam-methylase as a molecular tool for mapping binding sites of the yeast transcription factor Rpn4 | |
Maria et al. | Conservation of a DNA replication motif among phylogenetically distant budding yeast species | |
CN111088278B (zh) | 一个具有自主转座活性的逆转录转座子Ra-RARE-1及其应用 | |
CN107828790B (zh) | 一种在酸条件下诱导表达的启动子 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |