陈相好1,2, 张峥嵘1,3, 刘芳1, 陈峥宏1,3, 洪伟4, 綦廷娜1,3, 谷俊莹2,5
, 崔古贞1,3 1. 贵州医科大学基础医学院, 贵州 贵阳 550025;
2. 贵州医科大学医学检验学院, 贵州 贵阳 550004;
3. 贵州省普通高等学校病原生物学特色重点实验室, 贵州 贵阳 550025;
4. 贵州医科大学分子生物学重点实验室, 贵州 贵阳 550004;
5. 贵州医科大学附属医院, 贵州 贵阳 550004
收稿日期：2019-01-28；修回日期：2019-04-04；网络出版日期：2019-04-22
基金项目：国家自然科学基金（31760318，31500078，31560318，31601012）；贵州省科技计划项目（黔科合基础[2018]1132，[2019]1441）；贵州省教育厅自然科学研究项目（黔教合KY字[2014]216）；贵州省研究生科研基金立项项目（11348）
*通信作者：Tel:+86-851-88174015, E-mail:谷俊莹, 921469044@qq.com;
崔古贞, E-mail:cuiguzhen@hotmail.com.
摘要：[目的] 筛选影响Ll.LtrB内含子编码蛋白（Intron encoded protein，IEP）反转录功能的关键催化位点，并获得无反转录活性的IEP突变体。[方法] 首先，利用NCBI数据库，通过序列比对及同源建模方法筛选影响IEP反转录功能的关键氨基酸催化位点；然后，对筛选获得的关键催化位点进行定点突变，同时以Targetron载体为模板，构建无反转录功能的突变型Targetron打靶系统；最后，以大肠杆菌lacZ基因为例，体内验证IEP突变体的功能及其对Ⅱ型内含子"归巢"效率的影响。[结果] 筛选到C164和G214两个位点是影响内含子编码蛋白反转录功能的关键氨基酸残基，并获得C164K和G214W两个突变体。体内功能分析表明，此两个位点突变完全失活了Ⅱ型内含子的"归巢"功能。[结论] 筛选并获得了失活反转录功能的Ll.LtrB内含子编码蛋白突变体，为深入研究Ⅱ型内含子的结构和"归巢"机理奠定了基础。
关键词：Ll.LtrBⅡ型内含子内含子编码蛋白反转录Targetron
Key catalytic sites in the reverse transcription domain of Ll.LtrB intron encoded protein
Xianghao Chen1,2, Zhengrong Zhang1,3, Fang Liu1, Zhenghong Chen1,3, Wei Hong4, Tingna Qi1,3, Junying Gu2,5
, Guzhen Cui1,3 1. School of Basic Medical Science, Guizhou Medical University, Guiyang 550025, Guizhou Province, China;
2. School of Clinical Laboratory Science, Guizhou Medical University, Guiyang 550004, Guizhou Province, China;
3. Key Laboratory of Medical Microbiology and Parasitology of Education Department of Guizhou, Guiyang 550025, Guizhou Province, China;
4. Key Laboratory of Molecular Biology, Guizhou Medical University, Guiyang 550004, Guizhou Province, China;
5. Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
Received: 28 January 2019; Revised: 4 April 2019; Published online: 22 April 2019
*Corresponding author: Tel:+86-851-88174015, E-mail: Junying Gu, 921469044@qq.com;
Guzhen Cui, E-mail:cuiguzhen@hotmail.com.
Foundation item: Supported by the National Natural Science Foundation of China (31760318, 31500078, 31560318, 31601012), by the Science and Technology Plan Program of Guizhou Province ([2018]1132, [2019]1441), by the Natural Science Research Project of Guizhou Education Department ([2014]216) and by the Project of Guizhou Graduate Research Fund (11348)
Abstract: [Objective] To screen the key catalytic sites that affect the reverse transcription function of intron-encoded protein (IEP) from Ll.LtrB, and to obtain the IEP mutant without reverse transcription activity. [Methods] The key catalytic sites affecting the reverse transcription of IEP were screened by sequence alignment and homology modeling methods. Then, the screened key catalytic sites were subjected to site-directed mutagenesis. The mutated IEP was combined with the Targetron vector to construct the mutated Targetron targeting system without reverse transcription function. Finally, the function of mutant IEP was verified by using the lacZ gene in Escherichia coli. [Results] The sites C164 and G214 of IEP were screened and mutated, completely inactivated the "retrohoming" function in vivo. [Conclusion] The IEP mutants of Ll.LtrB obtained in our study laid a solid foundation for further research on the structure and mechanism of group Ⅱ intron.
Keywords: Ll.LtrBgroup Ⅱ intronintron encode proteinreverse transcriptionTargetron
Ⅱ型内含子(group Ⅱ intron)是一类反转录转座子，由具有催化功能的内含子RNA和具有反转录酶活性的内含子编码蛋白(intron encoded protein，IEP)两部分组成[1-4]。其中，内含子RNA具有核酶活性，能够通过碱基互补配对原则识别双链DNA，并切割DNA靶位点；IEP蛋白具有反转录酶功能，与内含子RNA结合成为核糖核蛋白复合体，辅助Ⅱ型内含子的识别和切割；而且，IEP蛋白能以内含子RNA为模板合成互补cDNA，从而实现内含子RNA在染色体上的“归巢”(retrohoming)[5-7]。
Ⅱ型内含子主要存在于细菌、真核生物细胞器(线粒体、叶绿体)及部分古细菌中[8]。目前，研究较多的是来源于乳酸乳球菌(Lactococcus lactis)的Ll.LtrB的Ⅱ型内含子，其“归巢”过程如图 1所示。首先，内含子RNA与内含子编码蛋白组装为核糖核蛋白复合体，通过碱基互补配对原则识别双链DNA靶位点；然后，内含子RNA的核酶活性反向切割并整合于DNA靶位点；随后，IEP蛋白的核酸内切酶活性切开双链DNA的反义链，并以切口处下游的3'末端为引物，以插入的内含子RNA为模板合成cDNA；最后，受损伤的DNA借助细胞自身修复机制，实现内含子RNA在靶位点的“归巢”[6, 9-10]。由于Ⅱ型内含子的特异性主要是由内含子RNA的碱基配对决定的，因此，通过设计内含子RNA的识别序列，即可实现内含子RNA在染色体不同位点“归巢”[9]。基于此原理，目前已开发出高效基因打靶技术——Targetron技术，该技术已在革兰阴性菌(如大肠杆菌、鼠伤寒沙门氏菌、弗氏志贺菌)[11-13]和革兰阳性菌(如艰难梭菌、肉毒梭菌、产气夹膜梭菌、金黄色葡萄糖菌、乳酸乳球菌)[14-18]以及人类细胞[19]等多个物种中获得广泛应用。
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| 图 1 Ll.LtrB Ⅱ型内含子“归巢”原理示意图 Figure 1 Retrohoming of group Ⅱ intron. A: target recognition, the complex composed of intron RNA and IEP recognizes target sites through base complementary pairing; B: reverse splicing, with the assistance of IEP, intron RNA is inserted into a single DNA strand; C: double-strand break, the endonuclease of IEP cleaves the other single strand of DNA; D: reverse transcription, group Ⅱ introns synthesize cDNA using intron RNA as template with the reverse transcriptase activity of IEP; E: DNA repair, cells synthesize complementary DNA using cDNA as template through DNA repair mechanism. |
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然而，上述Ⅱ型内含子的“归巢”过程是根据现有实验数据进行的推测，仍缺乏详细的实验证据。近年来，由于冷冻电镜技术的发展，Ⅱ型内含子复合体的结构得以解析，为其机理研究提供了更深入的依据[20]。由于Ⅱ型内含子“归巢”过程中靶位点的识别、切割和反转录等过程是一个连续发生的过程，在时间上无法分离，在空间上彼此接近，因此，很难深入了解其每一步的详细过程。如果能够将Ⅱ型内含子识别、切割、反转录等过程加以分离，就有可能深入了解其“归巢”机制。
Ⅱ型内含子“归巢”过程中最关键的一步是反转录，如果能够失活IEP蛋白的反转录功能，而又不影响IEP蛋白的其他功能，就有可能将Ⅱ型内含子的“归巢”过程加以分离。因此，本研究首先利用序列分析方法，分析并筛选了影响IEP蛋白反转录活性的关键氨基酸残基，然后通过定点突变获得失活反转录功能的IEP蛋白突变体，最后，通过构建突变型Targetron系统，在大肠杆菌中验证突变型IEP蛋白的功能，为深入了解Ⅱ型内含子的“归巢”机制奠定了基础。
1 材料和方法 1.1 材料
1.1.1 主要试剂与仪器: TRYPTONE、YEAST EXTRACT、Agar Powder购自OXOID公司；IPTG、X-gal、抗生素购自北京索莱宝公司；Trans Start FastPfu DNA Polymerase、DNA Marker和T4 DNA连接酶购自北京全式金生物技术有限公司；限制性内切酶购自Thermo Fisher Scientific；PCR产物纯化试剂盒、琼脂糖凝胶DNA回收试剂盒、定点突变试剂盒、质粒提取试剂盒购自北京天根公司。
PCR仪和高速冷冻离心机，Thermo Fisher Scientific公司；电泳仪和凝胶成像仪，北京六一公司；恒温仪，杭州米欧公司。
1.1.2 培养基及培养方法: LB液体培养基(10 g/L TRYPTONE、5 g/L YEAST EXTRACT、10 g/L NaCl)；LB固体培养基(10 g/L TRYPTONE、5 g/L YEAST EXTRACT、10 g/L NaCl，15 g/L Agar Powder)。液体培养基置于37 ℃、200 r/min摇床中培养，固体培养基置于37 ℃恒温箱培养。需要时加入氨苄青霉素(100 μg/mL)进行筛选，IPTG (100 μmol/L)和X-gal (40 μg/mL)加入LB固体培养基中用于蓝白斑筛选和菌落计数。
1.1.3 引物: 本研究用到的引物由生工生物工程(上海)股份有限公司合成，引物及序列详见表 1。
表 1. 本研究所用引物 Table 1. The primers used in this study
| Primers name | Sequence(5′→3′) | Description |
| lacZ-F | GGCCCGCACCGATCGCCCTTC | Detection primers of lacZ |
| lacZ-R | GCCATTTTTTGATGGACCATTTC | |
| IEP-F | ATCGAGGCTAGCGCTATATGCGTTGATG | Amplification primers of IEP, the underline indicates enzyme loci of Nhe Ⅰ |
| IEP-R | CGTTCCAGATCTCCTTACTCGTA | Amplification primers of IEP, the underline indicates enzyme loci of Bgl Ⅱ |
| C164K-F | GGAGATATAAAAGGCAAATTCGATAATATAGAC | Mutation primers of C164K |
| C164K-R | TTTGCCTTTTATATCTCCCTCCACAAACCATCT | |
| G214W-F | AGCGGAACACCTCAATGGGGAATTCTATCTCCT | Mutation primers of G214W |
| G214W-R | CCATTGAGGTGTTCCGCTGTAAGTTTTGTGATA |
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1.1.4 菌株及质粒: 本研究中用的菌株及质粒详见表 2。
表 2. 本研究所用到的菌株及质粒 Table 2. The strains and plasmids used in this study
| Strains and plasmids | Relevant features | Source or reference |
| Strains | ||
| E. coli DH5α | Clone strain, FˉlacZΔM15Δ (lacZYA-argF) relA1 | TakaRa |
| E. coli BL21(DE3) | F-, ompT, hsdS (rBB-mB), gal, dcm (DE3) | TakaRa |
| BL21-pSY7-lacZ-635s | Derived from BL21(DE3), carrying plasmid pSY7-lacZ-635s | This lab[21] |
| BL21-pSY7-lacZ-1063a | Derived from BL21(DE3), carrying plasmid pSY7-lacZ-1063a | This lab[21] |
| BL21-pSY7-M1-lacZ-635s | Derived from BL21(DE3), carrying plasmid pSY7-M1-lacZ-635s | This study |
| BL21-pSY7-M2-lacZ-635s | Derived from BL21(DE3), carrying plasmid pSY7-M2-lacZ-635s | This study |
| BL21-pSY7-M1-lacZ-1063a | Derived from BL21(DE3), carrying plasmid pSY7-M1-lacZ-1063a | This study |
| BL21-pSY7-M2-lacZ-1063a | Derived from BL21(DE3), carrying plasmid pSY7-M2-lacZ-1063a | This study |
| Plasmids | ||
| pMD19T | TA clone vector, Ampr | TakaRa |
| pMD19T-IEP | pMD19T ligated with part IEP sequence | This study |
| pMD19T-M1-IEP | Derived from pMD19T-IEP, IEP C164K mutation | This study |
| pMD19T-M2-IEP | Derived from pMD19T-IEP, IEP G214W mutation | This study |
| pSY7 | Derived from pSY6, lacI, T7 promotor, Ampr | This lab[21] |
| pSY7-lacZ-635s | Derived from pSY7, targeting the sense strand 635 site of lacZ in BL21(DE3) | This lab[21] |
| pSY7-lacZ-1063a | Derived from pSY7, targeting the antisense strand 1063 site of lacZ in BL21(DE3) | This lab[21] |
| pSY7-M1-lacZ-635s | Derived from pSY7-lacZ-635s, IEP C164K site mutation | This study |
| pSY7-M2-lacZ-635s | Derived from pSY7-lacZ-635s, IEP G214W site mutation | This study |
| pSY7-M1-lacZ-1063a | Derived from pSY7-lacZ-1063a, IEP C164K site mutation | This study |
| pSY7-M2-lacZ-1063a | Derived from pSY7-lacZ-1063a, IEP G214W site mutation | This study |
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1.2 内含子编码蛋白生物信息学分析 以来源于乳酸乳球菌Ll.LtrB的Ⅱ型内含子编码蛋白序列为模板，利用NCBI数据库进行同源序列比对和分析，筛选可能影响Ⅱ型内含子反转录功能的关键氨基酸残基，同时在https://swissmodel.expasy.org/检索获得IEP蛋白晶体结构，利用PyMOL和Swiss-PdbViewer软件分析IEP蛋白三维结构图。
1.3 定点突变及突变型Targetron载体构建 以质粒pSY7为模板，IEP-F/IEP-R为引物，利用TaqDNA聚合酶扩增获得1.2 kb IEP蛋白的基因序列(包含拟突变核苷酸位点)，将所获PCR产物与pMD19T载体连接获得pMD19T-IEP载体。
以pMD19T-IEP为模板，用定点突变引物扩增整个质粒。扩增条件为：95 ℃ 2 min；95 ℃ 30s，55 ℃ 30 s，72 ℃ 4 min，循环15次；72 ℃ 5 min。反应结束后，PCR产物用Dpn Ⅰ消化除去甲基化的质粒模板；然后，转化E. coli DH5α，测序获得突变型pMD19T-M-IEP质粒。
用Nhe Ⅰ和Bgl Ⅱ双酶切pMD19T-M-IEP质粒，回收目的片段，与经同样双酶切的pSY7-lacZ- 635s、pSY7-lacZ-1063a载体连接，获得内含子编码蛋白反转录催化位点突变的Targetron基因打靶载体pSY7-M-lacZ-635s和pSY7-M-lacZ-1063a，详细构建流程如图 2所示。
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| 图 2 突变型Tgrgetron载体构建流程 Figure 2 Flow chart of mutant Targetron vectors construction. The red asterisk indicates the mutation sites. |
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1.4 遗传筛选及打靶效率计算 将质粒pSY7-lacZ-635s、pSY7-lacZ-1063a及IEP蛋白突变的质粒pSY7-M-lacZ-635s、pSY7-M-lacZ-1063a分别转化E. coli BL21(DE3)，在含100 μg/mL氨苄青霉素的LB液体培养基中37 ℃、200 r/min过夜培养。40 μL的过夜培养物转接到4 mL含氨苄青霉素的LB培养基中37 ℃、200 r/min培养1 h，加入IPTG使其终浓度为0.5 mmol/L，于37 ℃、200 r/min诱导培养45 min，离心收集菌体，以不同的稀释浓度涂布LB固体培养基(含氨苄青霉素100 μg/mL、X-gal 40 μg/mL、IPTG 0.1 mmol/L)，置于37 ℃过夜培养，通过蓝白斑计数计算其“归巢”效率，“归巢”效率=白斑/(白斑+蓝斑)×100%。
1.5 菌落PCR鉴定 分别挑取蓝色菌落和白色菌落，以lacZ-F/ lacZ-R为引物进行菌落PCR扩增，检测内含子RNA是否特异性插入到DNA靶位点，进一步验证lacZ基因突变情况。
2 结果和分析 2.1 内含子编码蛋白同源序列分析及反转录核心催化位点筛选 以乳酸乳球菌Ll.LtrB的Ⅱ型内含子编码蛋白氨基酸序列为模板，利用同源序列比对分析，结合其蛋白结构解析，筛选到13个可能影响内含子编码蛋白反转录功能的核心催化位点，分别是：D160、I161、K162、G163、C164、F165、Q213、G214、Y306、D308、D309、L357、G358(图 3亮黄色背景显示序列)，其中，预测D160、I161、K162、G163、C164、F165、Q213、D308可能与底物NTP结合，G214可能与染色体上的核酸结合。
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| 图 3 内含子编码蛋白同源序列分析 Figure 3 Analysed of homology sequence with intron encoded protein. The yellow background are key amino acids which related to the activity of reverse transcriptase, the grey number indicates the number of amino acids omitted. |
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2.2 C164和G214在IEP蛋白中的相互作用分析 IEP蛋白的空间结构已被解析，将IEP蛋白氨基酸序列提交至https://swissmodel.expasy.org/检索获得IEP蛋白晶体结构，利用PyMOL软件分析得到IEP三维结构图(图 4)。本研究分别选择与底物NTP结合的C164残基和与DNA核酸结合的G214残基为靶点，分别将其定点突变为C164K和G214W，研究此两个位点突变对Ⅱ型内含子反转录功能的影响。
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| 图 4 IEP候选位点野生型与突变型的空间结构图 Figure 4 Spatial structure of wild and mutant IEP candidate sites. A: Cartoon and spherical diagram of the three-dimensional structure of IEP; B: The three-dimensional structure of C164 wild type and mutant type, the dotted line in purple represents hydrogen bonds, the red number indicates the hydrogen bond distance; C: The three-dimensional structure of G214 wild type and mutant type, the dotted line in purple represents hydrogen bonds, the red number indicates the hydrogen bond distance. |
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然后，我们分别将C164K和G214W两个位点突变的IEP蛋白氨基酸序列提交至https://swissmodel.expasy.org/，以野生型IEP蛋白为模板进行同源建模，分别获得C164K和G214W突变的IEP蛋白三维结构，利用Swiss-PdbViewer软件分析C164和G214位点突变前后空间结构的变化(图 4)。从空间结构上看，C164与Y208通过氢键相互作用，当C164突变后，C164K仍与Y208保持氢键的连接，但其氢键的距离在空间结构上较突变前缩短0.11 Å；G214与T211、L217、S218通过氢键相互作用，当G214突变后失去与L217和S218的氢键相互作用。因此从结构上推测C164和G214两个位点可能影响IEP蛋白反转结构域的空间结构。
2.3 IEP蛋白反转录结构域关键氨基酸位点突变对内含子“归巢”效率的影响 以筛选到的IEP蛋白C164和G214为靶位点，利用定点突变方法构建IEP蛋白突变体，构建流程如图 2所示。将野生型Targetron载体及突变型Targetron载体分别转化E. coli BL21(DE3)，通过菌落PCR检测(图 5)及蓝白斑筛选(图 6)，分析内含子编码蛋白反转录结构域关键氨基酸位点突变对Ⅱ型内含子“归巢”的影响。菌落PCR结果显示，经过诱导表达后，野生型Ⅱ型内含子可通过“归巢”插入到DNA靶位点，突变型Ⅱ型内含子不能插入DNA靶位点(图 5)。前期的统计结果表明，野生型Ⅱ型内含子在lacZ-635s及lacZ-1063a位点的归巢效率分别为90.845%±6.792%、92.582%±2.898%[21]；然而，当IEP蛋白反转录结构域中两个关键催化位点(C164、G214)突变后，其归巢效率均为0%，归巢功能完全丧失(图 6)。上述结果表明C164和G214两个位点是IEP蛋白反转录结构域关键催化位点，该位点突变完全失活了IEP蛋白的反转录功能，Ⅱ型内含子不能通过反转录合成cDNA而插入到靶位点。
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| 图 5 突变型Ⅱ型内含子功能验证电泳图 Figure 5 Functional verification of mutant group Ⅱ introns. A: Diagram of primer binding sites. B: Functional verification electrophoresis of mutant group Ⅱ intron; M: 8000 bp DNA marker; 1: White colony of BL21-pSY7-lacZ-635s; 2: Blue colony of BL21-pSY7-lacZ-635s; 3: Blue colony of BL21-pSY7-M1-lacZ-635s; 4: Blue colony of BL21-pSY7-M2-lacZ-635s; 5: BL21(DE3); 6: White colony of BL21-pSY7-lacZ-1063a; 7: Blue colony of BL21-pSY7-lacZ-1063a; 8: Blue colony of BL21-pSY7-M1-lacZ-1063a; 9: Blue colony of BL21-pSY7-M2-lacZ-1063a; 10: BL21(DE3). |
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| 图 6 突变型Ⅱ型内含子对归巢效率的影响 Figure 6 Effect of mutant group Ⅱ introns on retrohoming efficiency. A: Blue and white spot screening; B: Comparison of retrohoming efficiency between wild and mutant group Ⅱ introns. |
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3 讨论 本研究中，我们筛选到影响Ⅱ型内含子“归巢”的两个关键氨基酸位点(C164和G214)，并通过定点突变和体内功能验证，确定此两个位点的突变完全失活了Ⅱ型内含子“归巢”的功能。此外，由于IEP蛋白是由反转录结构域、成熟酶结构域、DNA结合域、核酸内切酶结构域四个不同的部分组成的多功能蛋白[1, 6, 22]，且不同结构域在空间上和功能上相对独立[1]，因此，IEP蛋白反转录结构域关键催化位点的定点突变，理论上不会影响其他结构域的功能，即：反转录结构域突变，只可能影响IEP蛋白的反转录功能，不会影响其与DNA结合及核酸内切酶的功能。因此，本研究获得的突变体，理论上能够将Ⅱ型内含子识别、切割和反转录的连续过程加以分离。
在后续研究中，如果能够获得上述IEP蛋白突变体与内含子RNA、底物NTP和DNA复合体的结晶体，我们将有可能从其空间结构上进一步解析Ⅱ型内含子不同过程的作用机理，为深入研究Ⅱ型内含子的“归巢”机制和广泛应用提供理论模型。
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