CN113943750A - 一种秀丽隐杆线虫转基因质粒、转基因秀丽隐杆线虫的构建方法及应用 - Google Patents
一种秀丽隐杆线虫转基因质粒、转基因秀丽隐杆线虫的构建方法及应用 Download PDFInfo
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Abstract
本发明属于生物技术领域和环境科学领域,具体涉及一种秀丽隐杆线虫转基因质粒、转基因秀丽隐杆线虫的构建方法及应用。本发明制备了秀丽隐杆线虫rps‑30;rfp::rps‑30 Ubl 转基因品系;该转基因品系可用于环境污染物毒性评估。rps‑30;rfp::rps‑30 Ubl 转基因品系既具备已有荧光蛋白转基因品系快速、操作简便、灵敏度高、可连续监测的优点,又因棒状结构的量化而弥补了已有荧光蛋白转基因品系荧光强度的量化受到曝光强度、曝光时间长短影响的不足。
Description
技术领域
本发明属于生物技术领域和环境科学领域,具体涉及一种秀丽隐杆线虫转基因质粒、转基因秀丽隐杆线虫的构建方法及应用。
背景技术
随着我国社会经济的高速发展,环境污染问题越来越受到人们的关注。在工业生产和日常生活中,人们都可能接触各种环境污染物,其中以化学污染物(如苯、铅、汞、砷)最常见。环境污染物可通过饮食、饮水、呼吸等途径进入人体内,对人体健康产生严重影响,如铅中毒、哮喘、癌症、神经行为障碍等,尤其对儿童健康影响最为严重。
目前对环境污染物的检测方法主要包括高效液相色谱、气相色谱、化学分析等方法,这些方法具有可精确分析环境污染物成分及浓度的优点,但其缺点是实验周期长、实验代价高,因而不能广泛、快速的用于环境污染物的检测[1,2],且由于生命有机体具有自我调节功能,物理、化学检测方法难以明确环境污染物对生命有机体的毒性作用。近年来生物检测方法逐渐被开发应用于环境污染物毒性评估中,其具有操作简单、成本低廉、测试周期短以及可直接衡量环境污染物对生物体的毒性作用等优点,如模式生物秀丽隐杆线虫、斑马鱼;微生物细菌、真菌、藻类等[1,3]。
秀丽隐杆线虫因其具有明确的生活史、繁殖周期短、通体透明、饲养简单、遗传操作方便、基因组高度保守性以及对人员无害等优点,作为经典模式生物在很多科研领域被广泛应用。目前,秀丽隐杆线虫已成功被应用于环境污染物毒性评估中,建立了秀丽隐杆线虫环境污染物毒性评估体系[4,5]。在该评估体系中,环境污染物毒性评估指标包括:致死率、最长寿命、半数致死天数、细胞凋亡、个体发育、生殖、运动行为、乙酰胆碱酯酶活性、基因表达模式、β半乳糖苷酶(LacZ)转基因品系、hsp16::gfp转基因品系、mtl-2::gfp转基因品系等[6,7]。该评估体系在环境污染物对衰老、致死、发育毒理、生殖毒理、神经毒性等方面发挥重要评估作用,尤其在重金属检测方面,如银、镉、镍、铅、钡、铬、锌、铜等。
但在秀丽隐杆线虫环境污染物评估体系中,现有评估指标也存在一些缺点,如表型检测具有耗时长、计算繁琐等缺点;分子生物学检测方法具有虫体致死、检测不连续等缺点;酶活性检测具有步骤繁琐、操作要求高等缺点;hsp16::gfp转基因品系和mtl-2::gfp转基因品系是将绿色荧光蛋白(GFP)分别构建于hsp16基因启动子和mtl-2基因启动子下游;虫体受到环境污染物毒性作用后,增强hsp16和mtl-2启动子转录活动,导致GFP表达水平升高;因此,可根据虫体内GFP荧光强度的变化指示环境污染物毒性的强弱,为秀丽隐杆线虫环境污染物评估体系提供了一种快速、操作简便、灵敏度高、可连续监测的检测方法[1,6]。但该转基因品系中受污染物种类的限制,且荧光强度的量化受到曝光强度、曝光时间长短的影响,导致评价结果的科学性受到限制。
RPS-30Ubl蛋白为秀丽隐杆线虫核糖体蛋白小亚基30(RPS-30)在细胞质中的裂解产物。RPS-30是嵌合蛋白,在细胞质中被裂解产生泛素样蛋白(RPS-30Ubl)和核糖体蛋白S30(RPS-30S30),RPS-30Ubl蛋白C-末端的两个甘氨酸(Gly-Gly)是RPS-30的裂解位点[8]。裂解产生的RPS-30S30进入细胞核中形成核糖体小亚基[9];RPS-30Ubl蛋白具有类泛素化修饰功能[8]。我们研究发现,在秀丽隐杆线虫野生型标准虫株N2中过表达RPS-30Ubl,构建的N2; rfp::rps-30 Ubl 转基因品系,虫体寿命显著缩短,且产卵量显著减少[10],不适于对环境污染物毒性长时间连续监测及虫体大量繁殖应用。
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发明内容
本发明的目的是为了克服现有技术存在的缺点和不足,而提供一种秀丽隐杆线虫转基因质粒、转基因秀丽隐杆线虫的构建方法及应用。
本发明所采取的技术方案如下:一种秀丽隐杆线虫转基因质粒,其为rps-30启动子序列、RFP cDNA序列、RPS-30Ubl核苷酸序列通过PCR、酶切、连接步骤克隆到秀丽隐杆线虫转基因质粒pPD95.77中构建得到的;rps-30启动子序列如SEQ ID NO.1所示;RFP cDNA序列如SEQ ID NO.2所示;RPS-30Ubl核苷酸序列如SEQ ID NO.3所示。
克隆所述RPS-30Ubl的引物序列如下所示:
UblF,5' GAAGATCTCAGATTTTCCTCCTTGGC 3';
UblR,5' CTAGCTAGCTCCTCCGAGAAGTCTTCC 3'。
克隆所述RFP cDNA序列的引物序列如下所示:
RFPF,5' GAGGGTACCGGTAGAAAAAATGGTGAGCAAGGGCGAGGAG 3';
RFPR,5' GGAATTCTACGAATGCTAGCTAGCTAGGAAGATCTCTTGTACAGCTCGTCCAT 3'。
克隆所述rps-30启动子序列的引物序列如下所示:
prps30F,5' CCCAAGCTTGTGTCACCAACATCCATCCCTC 3';
prps30R,5' TCCCCCGGGTCCTGTAAAAAAAATTTTAAG 3'。
一种转基因秀丽隐杆线虫的构建方法,其特征在于:其通过在秀丽隐杆线虫体内注射转染如上所述的秀丽隐杆线虫转基因质粒培养后经紫外辐照构建稳转转基因品系得到的。
如上所述的转基因秀丽隐杆线虫的构建方法所构建的转基因秀丽隐杆线虫用于环境污染物毒性评估的应用。
一种环境污染物毒性评估的方法,包括以下步骤:
(1)将如上所述的转基因秀丽隐杆线虫的构建方法所构建的转基因秀丽隐杆线虫与被测环境污染物相作用;
(2)观测RFP棒状结构数量,RFP棒状结构数量与染毒液浓度和作用时间呈正相关。
本发明的有益效果如下:本发明研究发现,秀丽隐杆线虫在氧化应激、重金属毒性等生存环境中,其体内泛素样蛋白RPS-30Ubl聚集形成棒状结构,且棒状结构数量与氧化应激程度及重金属毒性强度呈正相关。因此,通过RPS-30Ubl与红色荧光蛋(RFP)融合表达,制备了rfp::rps-30 Ubl 转基因品系。通过量化秀丽隐杆线虫体内棒状结构数量,指示环境污染物毒性的强弱,弥补了已有荧光蛋白转基因品系荧光强度的量化受到曝光强度、曝光时间长短影响的不足;且使用了持家基因rps-30的启动子,其转录活性不受污染物种类的限制。且与N2; rfp::rps-30 Ubl 转基因品系相比rps-30基因敲除虫体rps-30(tm6034/nt1) 中拯救表达RPS-30Ubl,构建的rps-30;rfp::rps-30 Ubl 转基因品系,虫体寿命显著延长,产卵量显著提高。因此,rps-30;rfp::rps-30 Ubl 转基因品系更适于对环境污染物毒性长时间连续监测及虫体大量繁殖应用。
综上所述,本发明制备了秀丽隐杆线虫rps-30;rfp::rps-30 Ubl 转基因品系;该转基因品系可用于环境污染物毒性评估。rps-30;rfp::rps-30 Ubl 转基因品系既具备已有荧光蛋白转基因品系快速、操作简便、灵敏度高、可连续监测的优点,又因棒状结构的量化而弥补了已有荧光蛋白转基因品系荧光强度的量化受到曝光强度、曝光时间长短影响的不足。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,根据这些附图获得其他的附图仍属于本发明的范畴。
图1为转基因质粒构建示意图;A:pPD95.77质粒示意图;B:pPD95.77::promoter(rps-30)::RFP::RPS-30Ubl质粒示意图;
图2为 转基因虫体中RFP/GFP表达情况;A-C:promoter(rps-30)::GFP在rps-30虫体中表达;D-F:promoter(rps-30)::RFP::RPS-30Ubl在rps-30虫体中表达;G-I:promoter(rps-30)::RFP::RPS-30Ubl在N2虫体中表达;
图3为转基因虫体寿命、产卵量检测;A:各虫体寿命检测;B:各虫体产卵量检测;
图4为不同浓度敌敌畏染毒液作用4h后rps-30;rfp::rps-30 Ubl 转基因虫体RFP聚集成棒状结构,(箭头所示为RFP棒状结构);A、B:0.55×10-6 μg/mL敌敌畏染毒液作用4h后对照组rps-30;gfp转基因虫体中GFP无聚集;C、D:0μg/mL敌敌畏染毒液作用4h后rps-30; rfp::rps-30 Ubl 转基因虫体内RFP无聚集;E、F:0.55×10-8μg/mL敌敌畏染毒液作用4h后rps- 30;rfp::rps-30 Ubl 转基因虫体内RFP聚集;G、H:0.55×10-6μg/mL敌敌畏染毒液作用4h后rps-30;rfp::rps-30 Ubl 转基因虫体内RFP聚集;I、J:0.55×10-4μg/mL敌敌畏染毒液作用4h后rps-30;rfp::rps-30 Ubl 转基因虫体内RFP聚集;K、L:0.55×10-2μg/mL敌敌畏染毒液作用4h后rps-30;rfp::rps-30 Ubl 转基因虫体内RFP聚集;
图5 为6种污染物、不同浓度、作用不同时间后rps-30;rfp::rps-30 Ubl 转基因虫体内RPF棒状结构定量检测;A:不同浓度敌敌畏作用不同时间后rps-30;rfp::rps-30 Ubl 虫体内RPF棒状结构数量;B:不同浓度二氯百草枯作用不同时间后rps-30;rfp::rps-30 Ubl 虫体内RPF棒状结构数量;C:不同浓度亚砷酸钠作用不同时间后rps-30;rfp::rps-30 Ubl 虫体内RPF棒状结构数量;D:不同浓度氯化镉作用不同时间后rps-30;rfp::rps-30 Ubl 虫体内RPF棒状结构数量;E:不同浓度甲基氯化汞作用不同时间后rps-30;rfp::rps-30 Ubl 虫体内RPF棒状结构数量;F:不同浓度氯化铅作用不同时间后rps-30;rfp::rps-30 Ubl 虫体内RPF棒状结构数量。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
以下实施例所采用的材料如下所示:
1材料
1.1实验所用秀丽隐杆线虫
①rps-30基因敲除秀丽隐杆线虫杂合体rps-30(tm6034/nt1)购自日本东京女子医科大学,由本实验室保存培养。
②秀丽隐杆线虫野生型标准株N2由本实验室保存培养。
主要试剂
Escherichia coli尿嘧啶突变菌株(OP50)、E. coli TOP10感受态菌株、pPD95.77秀丽隐杆线虫表达载体由本实验室保存,克隆载体T-Vector pMD 19 (Simple);限制性内切酶HindIII、SmaI、KpnI、BglII、NheI、EcoRI;DNA连接酶;LA Taq DNA聚合酶;DNA Marker(3427Q)均购自宝生物工程(大连)有限公司(TaKaRa)。琼脂糖、Tris购自Promega公司;蛋白胨、酵母提取物购自OXOID公司;鼠源反转录酶(M-MLV)、TRIzol购自上海英骏生物技术有限公司(Invitrogen)。显微注射用油Halocarbon oil 700、敌敌畏、二氯百草枯、亚砷酸钠、氯化镉、甲基氯化汞、氯化铅购自西格玛奥德里奇(上海)贸易有限公司(Sigma)。
主要仪器和设备
PCR仪、凝胶成像系统仪、台式离心机(日本)、倒置荧光显微镜(Olympus IX71)、恒温水浴锅、微操作注射器 (IM300,Narishige)、MOTIC体式解剖镜(中国)、P-2000水平程控激光微电极拉制仪(美国Sutter公司)、生化培养箱。
主要溶液
(1)M9 缓冲液的配制:使用分析天平准确称量3g KH2PO4、6g Na2HPO4、5g NaCl,添加1ml 1M MgSO4后用ddH2O定容至1L,混合均匀并进行高温灭菌。
(2)溶菌肉汤(Lysogeny Broth,LB)培养基的配制:使用分析天平准确称量10g胰蛋白胨,5g酵母浸出粉和5g NaCl,配制LB 固体培养基需添加15g琼脂,用ddH2O定容至1L并混合均匀,使用1M NaOH 将pH值调至7.0(液体培养)或7.5(固体培养基)。
(3)线虫生长培养基(Nematode Growth Medium,NGM)的配制:使用分析天平准确称量3g NaCl,2.5g胰蛋白胨、17g琼脂,添加975ml ddH2O并混合均匀,高温灭菌。待灭菌后的NGM降温至55℃时,在无菌操作台中依次添加1ml 1M CaCl2,1ml 5mg/ml胆固醇溶液(溶剂为乙醇),1ml 1M MgSO4和25ml 1M K2HPO4-KH2PO4缓冲液,混合均匀后倒平板(10 ml NGM/35mm培养皿)。
(4)冷冻保存培养基的配制:使用分析天平准确称量1.17g NaCl和1.36g KH2PO4,添加60ml 100%甘油和140ml ddH2O混合均匀并高温灭菌。灭菌后的冻存培养基降温至55℃后添加60μl已灭菌的1M MgSO4溶液,混合均匀在室温下保存备用。
(5)K培养液的配制:51mM NaCl、32 mM KCl溶于ddH2O中,高温灭菌后室温保存备用。
(6)毒物检测液的配制:100ml LB培养基中接种1ml OP50大肠杆菌,250 rpm振荡培养3h至OD600约为0.4时,将菌液移至4个50ml离心管中,4000g离心4min,弃上清;用K培养液吹打润洗3次,最后加入100ml K培养液将沉淀悬起,吹打均匀;在该溶液(K培养液+OP50)中分别添加至不同浓度的敌敌畏、二氯百草枯、亚砷酸钠、氯化镉、甲基氯化汞、氯化铅,并调节pH=4.5-6.5,用于毒物毒性检测试验。
实施例1:构建转基因质粒
(1)引物设计
参照NCBI数据库中rps-30基因序列(NM_072606.7),设计rps-30 Ubl上下游引物,两端分别加入酶切位点BglII和NheI,用于克隆rps-30 Ubl基因序列;参照秀丽隐杆线虫基因组序列(BX284605.5),设计引物,两端分别加入酶切位点HindIII和SmaI,用于克隆rps-30启动子区2064bp序列;参照ptdTomato-N1质粒中红色荧光蛋白(RFP)核酸序列,设计RFP 上下游引物,上游引物加入酶切位点KpnI,下游引物加入酶切位点BglII、NheI、EcoRI,用于克隆rfp基因序列。引物见表1。
(2)质粒构建
将rps-30启动子序列(SEQ ID NO.1)、RFP cDNA序列(SEQ ID NO.2)、RPS-30Ubl核苷酸序列(SEQ ID NO.3)通过PCR、酶切、连接步骤克隆到秀丽隐杆线虫转基因质粒pPD95.77中,构建pPD95.77::promoter(rps-30)::RFP::RPS-30Ubl质粒,如图1;同时将rps- 30启动子序列单独克隆到pPD95.77中,构建pPD95.77::promoter(rps-30)::GFP质粒,通过rps-30启动子启始下游绿色荧光蛋白(GFP),作为后续实验对照。
1)PCR:
①PCR体系:
②PCR程序:
2)酶切:
①酶切体系:
②酶切条件:37℃,3小时。
3)胶回收(使用AxyPrep DNA Gel Extraction Kit)
将PCR产物或酶切产物目的DNA条带切割下来,胶块约100mg,置于1.5ml EP管中,加入300μl DE-A,将EP管置于75℃水浴中10min(每个2-3min迅速震荡混匀一次)。EP管中加入150μl DE-B,震荡1min;将液体移入置于2ml离心管的回收柱中,离心12000g 1min,弃掉液体,用500μl缓冲液W1洗一次,12000g离心1min,弃掉液体;用700μl缓冲液W2洗二次,分别12000g离心1min,弃掉液体;12000g空离2min,弃掉离心管,将回收柱置于新的1.5ml EP管中,加入25μl ddH2O,孵育2min,12000g离心1min,1.5ml EP管中获得回收产物。
4)连接(使用DNA Ligation Kit <Mighty Mix>)
①连接体系:
②连接条件:16℃,过夜
5)转化涂板
-80℃中取出E. coli TOP10感受态细菌,置于冰上融化,将25μl连接产物加入其中,轻轻吹打混匀,冰浴30min;42℃热应激90s,然后迅速冰浴3min,加入1ml LB培养,37℃,250rpm摇床培养1h;4000g离心4min,弃上清,留下约30μl残液,轻轻捶打沉淀,并涂板(含AMP的固体LB培养板),37℃过夜培养。
6)测序
挑取单克隆于3-5ml LB液体培养基(含AMP),通过菌液PCR进行初步鉴定(方法同1));PCR阳性菌液送上海生工生物工程有限公司测序。
7)质粒提取(使用AxyPrep Plasmid Miniprep Kit)
取1ml菌液于1.5ml EP管中,12000g离心1min,弃上清,加入250μl S1溶液,吹打混匀,加入250μl S2溶液, 温和颠倒混匀6-8次,加入350μl S3溶液,温和颠倒混匀6-8次,12000g离心10min,取上清至吸附柱,12000g离心1min,弃滤液,加入500μl W1缓冲液,12000g离心1min,弃滤液,加入700μl W2缓冲液洗2次,12000g离心1min,弃滤液;将吸附柱放置在新的1.5mlEP管上,开盖干燥3min,加入50μl ddH2O,室温静置5min,12000g离心1min,获得质粒。测质粒浓度,用ddH2O调制成50ng/μl,备用于显微注射。
构建得到的质粒测序序列如SEQ ID NO.10所示测序结果显示pPD95.77质粒中HindIII和SmaI酶切位点间插入了rps-30基因的启动子序列,其下游为rfp基因序列和rps- 30 Ubl基因序列,表明质粒构建成功。
实施例2:构建转基因虫体
(1)显微注射准备工作
Pad的制作:将2%琼脂糖滴于盖玻片上,迅速用另一个盖玻压成极薄的琼脂糖薄片,放置室温干燥过夜,4℃保存备用。
针的制备:使用P-200水平程控光微电极拉制仪(美国Sutter公司)将毛细玻璃管(FHC, Borosil 1.0mm OD×0.75 mm ID/Fiber)制成显微注射用针。
显微注射用油:Halocarbon oil 700。
(2)C. elegans虫体准备
C. elegans野生型标准株N2和 rps-30基因敲除秀丽隐杆线虫rps-30(tm6034/ nt1) 均用NGM培养基,于20 ℃培养;挑取四期幼虫(L4)于新的NGM培养基中,培养12-14 h后进行显微注射。
(3)显微注射质粒的制备
将实验组重组质粒pPD95.77::promoter(rps-30)::RFP::RPS-30Ubl、对照组为重组质粒pPD95.77::promoter(rps-30)::GFP 8000 g离心10 min以去除可能的微小杂质。
(4)显微注射程序(使用显微注射仪:Narishige,IM300)
a. 打开显微注射仪,按下mode键,调节至action界面。
b. 打开N2钢瓶总阀,然后打开压力阀使指针指向0.4-0.5。
c. 按下显微注射仪上的balance键。
d. 脚踏踏板检查载针器出气是否流畅。
e. 装针。将载针器的端部轻轻拧松,然后把已装有注射质粒的玻璃针的尾部轻轻插入载针器中,拧紧端部使针固定。
f. 找针。先将10×物镜调至最上端,调节针尖使其成30°~45°位于物镜中心垂直上方较近的距离,打开光源,在视野下调节物镜使其缓慢下降,直至视野中出现针尖为止,然后调节针尖在40×视野下的最佳位置。
g. 撞针。将预先制备好的撞针载玻片放到载物台上,调节40×物镜至能够清晰看到玻璃管,并调节载玻片使玻璃管垂直与注射针。调节载针器,使针缓慢下移至视野里出现针尖,并与玻璃管处于同一平面。轻轻用针撞击玻璃管(最好撞击同时踩踏板),然后踩踏板检查是否液体迅速流出成液滴,以示成功。
h. 挑虫。在制备好的pad上滴一滴卤化碳油(Halocarbon oil 700),观察NGM培养基中的虫体,挑取含有5个以上虫卵的虫体,将其固定在pad上的卤化碳油中(头部不要固定)。
i. 注射。视野下尾部上缘和咽部下缘有两处较明显的透明处为虫体的性腺部位,将针尖斜插进性腺合胞体区域,踩踏踏板进行注射(最好当针尖进入合胞体同时踩踏,以免堵塞针尖)。
j. 注射后,调节载针器使针抬起,取下pad在显微镜下迅速将虫体挑到NGM培养基中。
(5)转基因虫体的筛选
经显微注射转染后的虫体(F0)放于NGM培养基中,4条/皿。3天后于荧光显微镜下挑取发红/绿荧光L4虫体为F1代,置于新的NGM培养基中,4条/皿;4天后于荧光显微镜下挑取发红/绿荧光L4虫体为F2代。用F2代虫体进行下一步紫外辐照构建稳转转基因品系。
(6)紫外辐照构建稳转转基因品系(使用Spectrolinker紫外交联仪XLE-1000)
挑取60条发红/绿荧光的L4虫体,放入新NGM培养板中(无OP50),20条/板。15℃培养2h后,打开皿盖,在紫外交联仪中以120J/m2紫外照射量对虫体进行辐照。辐照后的虫体为F0代,于20℃培养。于荧光显微镜下挑取100条发红/绿荧光的F1代L4虫体,1条/板,20℃培养。观察F2代虫体,选择产生大量(约70%)具有红/绿荧光F2代的F1代平板,每板挑取4条F2代L4虫体,1条/板,20℃培养。观察F3代虫体,找到100%具有红/绿荧光F3代的F2代平板,每板挑取8条具有红/绿荧光的F3代L4虫体,1条/板,20℃培养。持续扩繁虫体,至虫体所发荧光强度不变,则建立了稳定遗传的rps-30;gfp和rps-30;rfp::rps-30 Ubl 转基因品系。
(7)转基因虫体的保存、复苏及饲养
采用线虫冷冻培养液保存法可以将rps-30;rfp::rps-30 Ubl 转基因虫体在-80℃条件下保存数年,步骤如下:
①选取3-5个长满大量L1-L2期幼虫并已无大肠杆菌存在的 NGM平板,使用M9缓冲液将上面的全部线虫冲洗下来并放入15ml离心管中;
②加入等体积的冷冻保存培养液并且混合均匀。
③将混合液分装于已标记好线虫名称的1.5ml离心管并放入冻存盒中(含异丙醇),以保证在冷冻过程中以 1℃/min 的速度缓慢降温,将冻存盒直接放入-80℃超低温冰箱内。
冻存线虫的复苏是将冰箱中取出的线虫直接放在室温下进行解冻,并将解冻后的线虫液倒入长有E. coli OP50的NGM平板上轻轻晃动,待液体被完全吸干后将线虫放入20℃恒温培养箱中培养。2-3d后挑取已经复活且健康度较好的线虫于新的NGM培养基中继续培养。
(8)rps-30;rfp::rps-30 Ubl 转基因品系虫体同期化处理
在NGM培养基中,将秀丽隐杆线虫培养至成虫阶段,用3ml M9缓冲液将虫体洗下,转移到5ml离心管中,调整溶液总体积达到2.8ml,然后依次加入0.8ml NaOCl溶液,0.4ml5M NaOH溶液,充分混匀,待约有2/3的虫体裂解时,5500rpm离心1min,弃上清液,用M9缓冲液洗两遍,获得成虫体内虫卵;将制备好的虫卵置于4ml M9缓冲液中孵化过夜,即可获得L1期幼虫;将L1期幼虫接种于NGM培养基中,20℃培养,约48h即可获得L4期虫体,约60h即可获得成虫。
通过显微注射将pPD95.77::promoter(rps-30)::RFP::RPS-30Ubl质粒分别转染到N2虫体和rps-30虫体中;将pPD95.77::promoter(rps-30)::GFP质粒转染到rps-30虫体中;将转染成功的虫体经紫外辐照及多代次筛选,成功建立了稳定遗传的N2;rfp::rps-30 Ubl 、rps-30;rfp::rps-30 Ubl 、rps-30;gfp转基因品系。通过荧光显微镜观察:各转基因虫体中,RFP/GFP均呈全身性表达,如图2所示。
实施例三:转基因品系寿命、产卵量分析
(1)寿命检测:rps-30;rfp::rps-30 Ubl 转基因品系和N2;rfp::rps-30 Ubl 转基因品系各10条L4期虫体分别放置在NGM培养基中20°C培养15h。虫体产卵后,挑出成虫。虫卵孵化后,幼虫放置于新的NGM培养基中,1条/皿20°C培养。每天观察一次,直至虫体死亡。产卵期,则要将虫体每两天更换新的培养基。判定线虫死亡的标准为用铂金丝触碰虫体无反应即为死亡,记录每条虫体存活时间,本试验重复3次,每次检测虫体量30-40条。
(2)产卵量检测:rps-30;rfp::rps-30 Ubl 转基因品系和N2;rfp::rps-30 Ubl 转基因品系各40条L4期虫体分别放置在NGM培养基中,1条/皿20°C培养。待虫体产卵期,每天更换一次新培养基;旧的培养基3天后待虫卵孵化、发育至成虫后,数出虫体数量,即为虫体每天产卵量,直至产卵期停止,最后计算出每条虫体总产卵量,本试验重复3次。
寿命检测结果显示:与N2;rfp::rps-30 Ubl 转基因虫体相比,rps-30;rfp::rps- 30 Ubl 转基因虫体寿命显著延长,如图3A所示;产卵量检测结果显示:rps-30;rfp::rps-30 Ubl 转基因虫体产卵量显著高于N2;rfp::rps-30 Ubl 转基因虫体,如图3B所示。表明rps-30; rfp::rps-30 Ubl 转基因品系更适于对环境污染物毒性长时间连续监测及虫体大量繁殖应用。
实施例4:环境污染物毒性评估
选取了环境中常见的污染物敌敌畏、百草枯、砷、镉、汞、铅。
污染物24h rps-30;rfp::rps-30 Ubl 转基因品系L4期虫体半数致死浓度(LC50)测定
在K培养液中添加敌敌畏、二氯百草枯、亚砷酸钠、氯化镉、甲基氯化汞、氯化铅配制不同浓度的染毒液,染毒液终浓度见表2。
在24孔培养板中,每孔添加300μl染毒液,并放入10条rps-30;rfp::rps-30 Ubl 转基因品系L4期虫体,每组设置4个平行孔,20℃培养24h后,在体式解剖镜下观察,判定线虫死亡的标准为用铂金丝触碰虫体无反应即为死亡,记录死亡虫体数量,本试验重复3次。根据染毒液浓度及虫体死亡率,通过SPSS软件计算每种污染物24h rps-30;rfp::rps-30 Ubl 转基因品系L4期虫体LC50值。
检测结果如表3所示。
污染物染毒后,rps-30;rfp::rps-30 Ubl 转基因品系虫体RFP表型变化
根据已测定的各污染物24h rps-30;rfp::rps-30 Ubl 转基因品系L4期虫体LC50值,每种污染物分别配置终浓度为0、LC50×10-8、LC50×10-6、LC50×10-4、LC50×10-2的染毒液;在24孔培养板中,每孔添加300μl染毒液,并放入10条rps-30;rfp::rps-30 Ubl 转基因品系L4期虫体,每个浓度4个平行孔;设5个检测时间点:0h、2h、4h、6h、8h。每个时间点分别取虫体,于倒置荧光显微镜下观察RFP形态变化。
荧光显微镜观察结果显示,rps-30;rfp::rps-30 Ubl 转基因虫体在不同浓度(0.55×10-8μg/mL、0.55×10-6μg/mL、0.55×10-4μg/mL、0.55×10-2μg/mL)的敌敌畏染毒液中作用4h后,虫体内RFP聚集,形成棒状结构,且棒状结构数量随着敌敌畏染毒液浓度增高而增多;在0μg/mL敌敌畏染毒液中作用4h后,虫体内RFP无聚集;而对照组rps-30;gfp转基因虫体在0.55×10-6 μg/mL敌敌畏染毒液中作用4h后,虫体内GFP无聚集。如图4所示。
RPF棒状结构定量结果显示,rps-30;rfp::rps-30 Ubl 转基因虫体在6种污染物、不同浓度、作用不同时间后,RPF聚集形成的棒状结构数量呈现不同;RPF棒状结构数量与染毒液浓度呈正相关,与染毒液作用时间呈正相关;rps-30;rfp::rps-30 Ubl 转基因虫体在染毒液中作用2h后,即可观测到RPF棒状结构;随着作用时间的延长,RPF棒状结构数量可能会出现平台期;而不同种类污染物导致的RPF棒状结构数量平台期时,棒状结构数量可能不同。如图5所示。
综上所述,本发明成功制备了rps-30;rfp::rps-30 Ubl 秀丽隐杆线虫转基因品系。研究发现该转基因虫体在敌敌畏、百草枯、砷、镉、汞、铅等常见的环境污染物染毒液作用后,RFP聚集成棒状结构,且RFP棒状结构数量与染毒液浓度和作用时间呈正相关。因此,rps-30;rfp::rps-30 Ubl 秀丽隐杆线虫转基因品系可以用于环境污染物毒性评估。
已有用于环境污染物毒性评估秀丽隐杆线虫转基因品系,如hsp16::gfp和mtl- 2::gfp,均是将GFP构建于hsp16基因启动子和mtl-2基因启动子下游,通过环境因素调控hsp16或mtl-2基因启动子的转录活性,进而通过荧光显微镜检测GFP荧光强度变化,指示环境污染物毒性的强弱。该转基因品系评估结果不仅受曝光强度、曝光时间长短的影响,还受检测物种类的限制[6,7]。本研究中的rps-30;rfp::rps-30 Ubl 秀丽隐杆线虫转基因品系,启动子为持家基因rps-30的启动子,其转录活性不受污染物种类的影响;而且通过对RFP棒状结构进行定量计数,弥补了曝光强度、曝光时间长短对结果的影响。
RPS-30Ubl为泛素样蛋白,可以修饰凋亡蛋白Bcl-G,调控细胞凋亡[8]。我们通过在线粒体标记虫株CB5600中进行定位研究,显示RPS-30Ubl可以定位在线粒体上,表明RFP棒状结构的形成可能与线粒体凋亡途径有关(该研究结果未展示)。RPS-30Ubl C-末端的两个甘氨酸(Gly-Gly)为类泛素化修饰位点,因此,本研究将RFP构建于RPS-30Ubl的N-末端,不影响RPS-30Ubl的类泛素化修饰功能。
rps-30;rfp::rps-30 Ubl 秀丽隐杆线虫转基因品系在环境污染物染毒液作用2h后,即可观察到RFP棒状结构;且可以检测到LC50×10-8浓度的环境污染物毒性,表明该转基因品系用于环境污染物毒性评估还具有快速、灵敏度高的优势。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
序列表
<110> 温州医科大学
<120> 一种秀丽隐杆线虫转基因质粒、转基因秀丽隐杆线虫的构建方法及应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2064
<212> DNA
<213>人工序列
<400> 1
gtgtcaccaa catccatccc tcattccgcg ctgaagaata gtttcttgag gagagaaaca 60
cgacccactt cacctaaaaa atgagacact cattgacact tgttgtggtc attctttccc 120
tttcattata ttctgtaaat ggttttttct ttggtgctgc aggaggtgga ggtgcatgtg 180
gatgtgctcc gagacctgca tgtccacctc caccaccttc aggatgttcc ggttcgggag 240
tgagagcggt ggcaagaggt gctaaaacta tggtaatgta ttcttcaaat ttgttttttt 300
ttctaaatcc acatttttga tattcagaca tttgatcagc caatttattc acctactcct 360
caatcttatg cacaaccacc acgggtatat tctgttccag atgaacttga cttggtatgt 420
gaataaaaac tagtctagat gcttacttca ccctatcaga acggatacaa tcttttttat 480
ctctcccttc aacgttatta tgtaattcgc tagttaattg aacttgtttt actcgcaaca 540
ggccgtcgtt ttatagagag ctgctgcatt tggagtgccg attcagccac aaccacagta 600
cacgattttc gacattctgc ataacttggg agaaagtaac gacaatccag cacagcagtt 660
ggtcgtcgca gacggaagta cgaacaccaa ctacttcccg aaagagaatg acgaggatct 720
cactgtatcc gacatgacag gcgatggagg cttgtatcgg tcgaaaaata ttcgaagagc 780
acctgtagag ccaacggtcc atgttcatcg agacagttcg gttgaaacca ctactatttc 840
gtctgaaata tctggaatgg aaacggatga aactctagac aaaaataaat gcagcagctc 900
agttttacga aaactcatga tcgaggtaac ttctaatggg catttcataa ttaaagtctt 960
gtgaacttca gaacatcagc gacagctctt cggaatctaa aagaaatatc aacctagcag 1020
ctgagggtaa attcggaggc aatgttgacg ttatttgctc tcgggggcac ttttcctata 1080
tcttcacctc aaatctatat tgcgaggcaa caagagggct gacaacgtgc atcgctttcc 1140
gacaatccga caaatctcga cgaagaagat gaattaatcg cttcttgctt gtacatttta 1200
ataattttaa tcaattcgcc gtcttttcaa taaaaatatt tattgaaaat tgaacaacag 1260
ataaggcgaa aggtgacggg atgatccggt gaccgatcag cagcttagag cttcagcttg 1320
gtgcgtctcc agtggcgtct cttggcattg tacttcatct gaaaatggaa aagttaccaa 1380
tagaatttga caattaatga aattttaaaa ataaaaaaat tacaaataat tttctgagaa 1440
aagttaatac aaaaacttac ggtgtttcca gtcttcatgc ggacccattg tggcattgga 1500
cgattttgct tttgcttctt agcaagcttg cgcttgatga aggatttctt caaggcagac 1560
attacctgaa aaattgaata tttaacgaaa aattgtggaa gcggtagaag gttaaaaatt 1620
actattggaa aatcacaaga agcaagtaaa cagtaaattt aataacaaga ttttcacatt 1680
tttatgctga aaaatactca aaaacgcgtt cgaaaaaaga aaaaatcggc aaaatatttt 1740
aggattgaaa agaaaatcga ggcagtagcg gcgaagacgg agaaaaaacg gctcttcgct 1800
gcgagacccg cgctttcgct gcgtacttgc ctgagtattc aacgttaatg cgtgtgtctg 1860
ccgccctcct cggttttggt cgcttttccg atttttcttt tcgggttttt cacgattttt 1920
ttgaaaattt gtagatattg cgcgatattg gctgataaaa atgaaattat ttgttgattc 1980
tgtacatttt cttcgatatt cgcatattta aaacccttaa atttgtcgaa attattgatt 2040
ttttcttaaa atttttttta cagg 2064
<210> 2
<211> 705
<212> DNA
<213>人工序列
<400> 2
atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60
ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120
ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180
atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240
cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300
gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360
tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag 420
accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480
gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540
atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600
gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660
cgccaccacc tgttcctgta cggcatggac gagctgtaca agtaa 705
<210> 3
<211> 213
<212> DNA
<213>人工序列
<400> 3
atgcagattt tcctccttgg cctcgataac actacgcaca cgctcgacgt ggatgcctcc 60
accaccctct ccgctattaa gggagtgatc ggagccggag aagaattctc gatctcctac 120
ggatccaagg ttcttagcga ggagctcact cttggagaat gccaaattga gagcctctcc 180
accctttccg tcaacggaag acttctcgga gga 213
<210> 4
<211> 26
<212> DNA
<213>人工序列
<400> 4
gaagatctca gattttcctc cttggc 26
<210> 5
<211> 27
<212> DNA
<213>人工序列
<400> 5
ctagctagct cctccgagaa gtcttcc 27
<210> 6
<211> 31
<212> DNA
<213>人工序列
<400> 6
cccaagcttg tgtcaccaac atccatccct c 31
<210> 7
<211> 30
<212> DNA
<213>人工序列
<400> 7
tcccccgggt cctgtaaaaa aaattttaag 30
<210> 8
<211> 40
<212> DNA
<213>人工序列
<400> 8
gagggtaccg gtagaaaaaa tggtgagcaa gggcgaggag 40
<210> 9
<211> 53
<212> DNA
<213>人工序列
<400> 9
ggaattctac gaatgctagc tagctaggaa gatctcttgt acagctcgtc cat 53
<210> 10
<211> 3109
<212> DNA
<213>人工序列
<400> 10
aagcttgtgt caccaacatc catccctcat tccgcgctga agaatagttt cttgaggaga 60
gaaacacgac ccacttcacc taaaaaatga gacactcatt gacacttgtt gtggtcattc 120
tttccctttc attatattct gtaaatggtt ttttctttgg tgctgcagga ggtggaggtg 180
catgtggatg tgctccgaga cctgcatgtc cacctccacc accttcagga tgttccggtt 240
cgggagtgag agcggtggca agaggtgcta aaactatggt aatgtattct tcaaatttgt 300
ttttttttct aaatccacat ttttgatatt cagacatttg atcagccaat ttattcacct 360
actcctcaat cttatgcaca accaccacgg gtatattctg ttccagatga acttgacttg 420
gtatgtgaat aaaaactagt ctagatgctt acttcaccct atcagaacgg atacaatctt 480
ttttatctct cccttcaacg ttattatgta attcgctagt taattgaact tgttttactc 540
gcaacaggcc gtcgttttat agagagctgc tgcatttgga gtgccgattc agccacaacc 600
acagtacacg attttcgaca ttctgcataa cttgggagaa agtaacgaca atccagcaca 660
gcagttggtc gtcgcagacg gaagtacgaa caccaactac ttcccgaaag agaatgacga 720
ggatctcact gtatccgaca tgacaggcga tggaggcttg tatcggtcga aaaatattcg 780
aagagcacct gtagagccaa cggtccatgt tcatcgagac agttcggttg aaaccactac 840
tatttcgtct gaaatatctg gaatggaaac ggatgaaact ctagacaaaa ataaatgcag 900
cagctcagtt ttacgaaaac tcatgatcga ggtaacttct aatgggcatt tcataattaa 960
agtcttgtga acttcagaac atcagcgaca gctcttcgga atctaaaaga aatatcaacc 1020
tagcagctga gggtaaattc ggaggcaatg ttgacgttat ttgctctcgg gggcactttt 1080
cctatatctt cacctcaaat ctatattgcg aggcaacaag agggctgaca acgtgcatcg 1140
ctttccgaca atccgacaaa tctcgacgaa gaagatgaat taatcgcttc ttgcttgtac 1200
attttaataa ttttaatcaa ttcgccgtct tttcaataaa aatatttatt gaaaattgaa 1260
caacagataa ggcgaaaggt gacgggatga tccggtgacc gatcagcagc ttagagcttc 1320
agcttggtgc gtctccagtg gcgtctcttg gcattgtact tcatctgaaa atggaaaagt 1380
taccaataga atttgacaat taatgaaatt ttaaaaataa aaaaattaca aataattttc 1440
tgagaaaagt taatacaaaa acttacggtg tttccagtct tcatgcggac ccattgtggc 1500
attggacgat tttgcttttg cttcttagca aggttgcgct tgatgaagga tttcttcaag 1560
gcagacatta cctgaaaaat tgaatattta acgaaaaatt gtggaagcgg tagaaggtta 1620
aaaattacta ttggaaaatc acaagaagca agtaaacagt aaatttaata acaagatttt 1680
cacattttta tgctgaaaaa tactcaaaaa cgcgttcgaa aaaagaaaaa atcggcaaaa 1740
tattttagga ttgaaaagaa aatcgaggca gtagcggcga agacggagaa aaaacggctc 1800
ttcgctgcga gacccgcgct ttcgctgcgt acttgcctga gtattcaacg ttaatgcgtg 1860
tgtctgccgc cctcctcggt tttggtcgct tttccgattt ttcttttcgg gtttttcacg 1920
atttttttga aaatttgtag atattgcgcg atattggctg ataaaaatga aattatttgt 1980
tgattctgta cattttcttc gatattcgca tatttaaaac ccttaaattt gtcgaaatta 2040
ttgatttttt cttaaaattt tttttacagg acccgggatt ggccaaagga cccaaaggta 2100
tgtttcgaat gatactaaca taacatagaa cattttcagg aggacccttg agggtaccgg 2160
tagaaaaaat ggtgagcaag ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc 2220
gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc 2280
cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg 2340
cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg 2400
ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga 2460
tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca 2520
cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc 2580
agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc 2640
tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt 2700
tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca 2760
ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct 2820
ccgagggccg ccaccacctg ttcctgtacg gcatggacga gctgtacaag agatctcaga 2880
ttttcctcct tggcctcgat aacactacgc acacgctcga cgtggatgcc tccaccaccc 2940
tctccgctat taagggagtg atcggagccg gagaagaatc ctcgatctcc tacggatcca 3000
aggttcttag cgaggagctc actcttggag aatgccaaat tgagagcctc tccacccttt 3060
ccgtcaacgg aagacttctc ggaggagcta gctaggattc gtagaattc 3109
Claims (7)
1. 一种秀丽隐杆线虫转基因质粒,其特征在于:其为rps-30启动子序列、RFP cDNA序列、RPS-30Ubl核苷酸序列通过PCR、酶切、连接步骤克隆到秀丽隐杆线虫转基因质粒pPD95.77中构建得到的;rps-30启动子序列如SEQ ID NO.1所示;RFP cDNA序列如SEQ IDNO.2所示;RPS-30Ubl核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的秀丽隐杆线虫转基因质粒,其特征在于:克隆所述RPS-30Ubl的引物序列如下所示:
UblF,5' GAAGATCTCAGATTTTCCTCCTTGGC 3';
UblR,5' CTAGCTAGCTCCTCCGAGAAGTCTTCC 3'。
3. 根据权利要求1所述的秀丽隐杆线虫转基因质粒,其特征在于:克隆所述RFP cDNA序列的引物序列如下所示:
RFPF,5' GAGGGTACCGGTAGAAAAAATGGTGAGCAAGGGCGAGGAG 3';
RFPR,5' GGAATTCTACGAATGCTAGCTAGCTAGGAAGATCTCTTGTACAGCTCGTCCAT 3'。
4.根据权利要求1所述的秀丽隐杆线虫转基因质粒,其特征在于:克隆所述rps-30启动子序列的引物序列如下所示:
prps30F,5' CCCAAGCTTGTGTCACCAACATCCATCCCTC 3';
prps30R,5' TCCCCCGGGTCCTGTAAAAAAAATTTTAAG 3'。
5.一种转基因秀丽隐杆线虫的构建方法,其特征在于:其通过在秀丽隐杆线虫体内注射转染如权利要求1-4任一项所述的秀丽隐杆线虫转基因质粒培养后经紫外辐照构建稳转转基因品系得到的。
6.如权利要求5所述的转基因秀丽隐杆线虫的构建方法所构建的转基因秀丽隐杆线虫用于环境污染物毒性评估的应用。
7.一种环境污染物毒性评估的方法,其特征在于包括以下步骤:
(1)将如权利要求5所述的转基因秀丽隐杆线虫的构建方法所构建的转基因秀丽隐杆线虫与被测环境污染物相作用;
(2)观测RFP棒状结构数量,RFP棒状结构数量与染毒液浓度和作用时间呈正相关。
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