CN116676329A - 一种油茶sqs基因的克隆及表达载体构建的方法 - Google Patents
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Abstract
本发明属于生物工程技术领域，具体涉及一种油茶SQS基因的克隆及表达载体构建的方法：(1)提取油茶RNA，合成cDNA并进行PCR扩增；(2)构建油茶SQS基因的过表达载体；(3)利用油茶SQS基因的过表达载体将重组产物转化至大肠杆菌DH5α细胞；(4)将重组表达载体转化烟草；(5)测定成熟烟草种子中SQS基因的相对表达量。本发明首次将油茶种子中的SQS基因通过克隆与构建表达载体的方法成功转化到烟草。并测定烟草种子中角鲨烯的含量和SQS基因的相对表达量。可以实现从转化的烟草植株中筛选得到高表达SQS基因及高含量的角鲨烯单株植株，为进一步探究油茶种子中角鲨烯合成与高积累的机制方面提供了技术依据。
Description
技术领域
本发明涉及生物工程技术领域，具体涉及一种油茶SQS基因的克隆及表达载体构建的方法。
技术背景
油茶(Camelliaoleifera)是我国南方重要的木本油料，2019年我国油茶籽产量267.9万吨，茶油年产量65万吨，综合产值达千亿元。油茶种子中不仅富含油脂(约45％)和油酸(约75％)，而且特别含有100～700μg/g的角鲨烯，角鲨烯是一种开链三萜类化合物，是所有类固醇类物质的生物合成前体，研究表明，其具有抗氧化、清除人体自由基和增强免疫力等作用，长期食用茶油可有效预防心血管和肝脏疾病。角鲨烯应用广泛，近年来已有多篇文献报道了关于角鲨烯合成通路中的相关酶和关键基因的研究。
角鲨烯合成酶(squalene synthase,SQS)可催化异戊二烯途径中的碳流向三萜生物合成，SQS活性与角鲨烯等三萜化合物的产量相关，是角鲨烯等三萜类化合物合成过程中的关键酶。近年来已有研究对不同植物如人参、水稻、重楼等植物的SQS基因进行了克隆与载体构建，实现了在分子水平上探讨植物中三萜类化合物合成的机理和通过基因转化来探究基因表达的最佳条件。而目前对于油茶中SQS基因的克隆与载体构建的相关研究极少。
发明内容
为解决角鲨烯来源少、供不应求的情况，本发明从基因克隆和基因载体构建的分子角度探究角鲨烯在油茶种子中产生以及高积累的机制。目前本发明已完成油茶SQS基因的测序工作，通过生物信息学方法分析SQS基因编码蛋白的理化性质，并以不同品种油茶的种子为试验材料，研究发现多个不同品种油茶SQS基因表达量与油茶种子中积累的角鲨烯含量之间呈正相关关系。本发明在前期研究成果的基础上，进一步对油茶SQS基因进行克隆，构建植物转化载体，将载体转化至烟草，以实现从转化的植株中筛选得到高表达SQS基因及高含量的角鲨烯单株植株。
本发明的技术方案如下：
一种油茶SQS基因的克隆及表达载体构建的方法，包括以下步骤：
(1)提取油茶RNA，合成cDNA并进行PCR扩增；
(2)构建油茶SQS基因的过表达载体；
(3)利用油茶SQS基因的过表达载体将重组产物转化至大肠杆菌DH5α细胞；
(4)将重组表达载体转化烟草；
(5)测定烟草种子中SQS基因的相对表达量和角鲨烯含量。
进一步的，步骤(2)具体为：表达载体构建的过程中，采用的酶切反应体系为2μL10×K Buffer、5μL载体质粒、2μL BamHI、不多于40μL的ddH2O，重组反应试剂盒采用的是Vazyme公司ClonExpress-II One Step Cloning Kit，重组连接反应体系(总体积10μL)为4μL线性化载体、1μL插入片段、2μL 5×CE II Buffer、15μL Exnase II以及补足10μLddH2O。
进一步的，步骤(3)具体为：重组产物转化至大肠杆菌DH5α细胞过程中，在100μL大肠杆菌感受态细胞中加入10μL连接产物，反应条件为，冰浴30min；42℃热激60-90s；冰浴2min；加入800μL LB液体培养基，反应条件为，37℃摇床培养30min；6000rpm离心3min。
进一步的，步骤(4)转化烟草过程中使用的MS预培养固体培养基组成为：4.74g/LMS培养基粉末、30g/L蔗糖、8g/L琼脂粉、0.5mg/L6-苄氨基腺嘌呤(6-BA)、0.1mg/Lα.萘乙酸(NAA)，pH6.0。
进一步的，步骤(4)：SQS基因表达载体转化至烟草过程中，使用的分化培养基组成为：4.74g/LMS培养基粉末、30g/L蔗糖、8g/L琼脂粉、0.5mg/L 6-苄氨基腺嘌呤(6-BA)、0.1mg/Lα/萘乙酸(NAA)、50mg/L卡那霉素(Kan)、500mg/L羧苄青霉素(Car)，pH6.0。
油茶SQS基因的序列如下：
ATGGGAAGTTTGGGGGCAATTTTGAAGCATCCGGATGATTTCTATCCAT
TGATGAAGTTGAAGATGGCGGCGAGGCGAGCGGAGAAGAATATTCCTCCG
GAGCCTCACTGGGGCTTCTGTTACTCCATGCTTCACAAGGTTTCTCGCAGT
TTCGCCCTCGTAATTCAACAGCTCGACACCGAGCTTCGCAACGCTGTATGC
ATTTTTTATTTGGTTCTTCGAGCCCTCGACACCGTTGAGGATGATACAAGCA
TAGCTACAGAGGTTAAAGTACCTATTCTGATGGCTTTTCATCGTCATATATAT
GACCGTGACTGGCATTTTTCATGTGGTACAAAGGAGTACAAGGTTCTCATG
GATGAGTTCCATCATGTTTCAACTGCTTTTTCGGAGCTTGGGAGAGGTTATC
AGGAGGCAATCGAGGACATTACTATGAGAATGGGTGCAGGAATGGCAAAG
TTTATATGCAAGGAGGTAGAAACAATTGATGACTATGATGAATATTGTCACT
ACGTAGCAGGACTGGTTGGGTTAGGGTTATCAAAGCTTTTCCATGCCTCTG
GGTCTGAAGATTTGGCATCAGATTCTCTCTCCAATTCAATGGGTTTATTTCTT
CAGAAAACAAACATTATTCGGGACTATTTGGAGGATATAAACGAGATACCA
AAGTCACGCATGTTTTGGCCTCGCCAGATTTGGAGTAAATATGTTAACAAA
CTTGAGGACTTAAAAGACAAGGAAAACTCAGTCAAAGCTGTGGAATGCCT
TAATGACATGGTGACAAATGCTTTGATACATGTGGAGGATTGCCTGACATAC
ATGTCAGCTTTGCGAGATCCTTCAATTTTCCGATTTTGTGCAATTCCACAGA
TCATGGCAATTGGAACCTTGGCTTTATGCTACAACAATATTGAAGTCTTCAG
AGGTGTTGTAAAAATGAGGCGTGGTCTTACTGCTAAAGTTATTGACCGGAC
AAAAACAATGTCGGATGTCTATGGTGCTTTCTTCGATTTTTCTTGTATGCTG
AAGTCAAAGGTCAACAAGAGTGACCCAAATGCTATGAAAGCATTAAGCAG
GCTAGAGGCAATTCAGAAAATTTGCAGGGAATCTGGAACCCTAAACAAAA
GGAAATCTTACATAATCAAGAGCGAACCAAGATACAACTCAACTCTGGTTT
TTGTATTATTCATTATACTCGCAATCCTGTTTGCATATCTATCTGCAAACAGA
CCAAGTAATATGTGA。
与现有技术相比，本发明的有益效果如下：
本发明首次将油茶种子中的SQS基因克隆与构建表达载体的方法成功转化到烟草。并测定烟草种子中角鲨烯的含量和SQS基因的相对表达量。该方法首次从生物工程的角度去探究油茶种子中角鲨烯合成与积累的机制。
附图说明
图1为基因克隆电泳图；
图2为转化菌落PCR电泳图；
图3为SQS表达载体3个克隆测序比对；
图4为pCambia1301-ky质粒图谱。
具体实施方式
下面结合实施例对本发明做进一步的说明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的的实验方法均为常规方法，所用实验器材、材料、试剂等均可从商业途径获得。
实施例1提取油茶种子RNA，并获得SQS基因cDNA
首先将油茶种子用液氮研磨成粉，根据上海生物工程公司柱式植物总RNA抽提纯化试剂盒推荐方法改进后进行油茶总RNA的提取，得到总的RNA提取液之后，采用NanoDrop对其进行浓度和纯度检测，测定OD260/280以及OD260/230比值；再用1.5％琼脂糖凝胶电泳检测总RNA完整性，根据其亮度与Marker相比，初步估计提取的油茶RNA的浓度。参照Vazyme公司的HiScript 1st Strand cDNA Synthesis Kit反转录试剂盒的说明书将已提取的油茶RNA反转录为cDNA。其反应体系如下：
反应程序为50℃反应45min；85℃反应5min。
再以cDNA为模板，以上述引物进行PCR扩增，采用的是Vazyme公司的phanta maxsuper-fidelity DNAPolymerase试剂盒提供的说明进行PCR扩增。扩增反应体系为如下：
反应程序为：95℃30s；39个循环：95℃15s，退火温度45-55℃15s，延伸72℃1min；循环完毕后最终进行延伸72℃，5min。将PCR产物进行凝胶电泳鉴定其大小。结果显示扩增到单一条带，大小为750左右，与期望的大小基本相当，说明克隆得到的DNA片段大小正确，基因克隆电泳结果如图1所示。
实施例2构建油茶种子SQS基因过表达载体
用KpnI酶切载体pCambia1301-KY线性化，采用的是Thermo公司或Takara公司相应限制性内切酶产品。酶切反应体系如下：
酶切产物纯化后与上述PCR产物进行重组反应，重组反应试剂盒采用的是Vazyme公司ClonExpress-II One Step Cloning Kit。重组连接反应体系(总体积10μL)如下：
上述反应液使用移液器轻轻吸打混匀，短暂离心将反应液收集至管底。放置于37℃反应30min，随后立即置于冰上冷却。转化菌落PCR电泳图如图2所示，记为1301-SQS 1-8号克隆质粒。从1至8号中选取2、3、7号这3个SQS表达载体克隆质粒进行测序对比，结果如图4所示，2号与7号克隆一致，结果说明目的基因已插入载体，表达载体构建准确。
实施例3利用油茶SQS基因过表达载体转化大肠杆菌DH5α细胞
将重组产物转化至大肠杆菌DH5α细胞，具体转化步骤为：
(1)在100μL大肠杆菌感受态细胞中加入10μL连接产物；
(2)冰浴30min；
(3)42℃热激60-90s；
(4)冰浴2min；
(5)加入800μL LB液体培养基；
(6)37℃摇床培养30min；
(7)6000rpm离心3min，弃上清，涂布Kana(50mg/L)抗性培养基平板；
(8)37℃倒置培养12-16h后，挑取抗性菌落；
(9)在96孔板中，每孔加入100μL LB(含Kana)液体培养基；
(10)各平板取4-8个菌落，37℃，2h，180rpm扩大培养；
(11)取1μL菌液进行PCR阳性检测。
挑取PCR阳性的转化子摇菌培养提取质粒，同时扩增产物送测序。扩增和测序引物为插入目的基因两侧的载体序列，分别为
35S-F：5’-GACGCACAATCCCACTATCC-3’
F：5’-GCTTCCGGCTCGTATGTTG-3’
实施例4重组表达载体转化烟草
采用叶盘法将构建的SQS基因表达载体转化到烟草中，具体方法如下：
将烟草种子消毒，消毒后将烟草种子播种于MS固体培养基上，条件为25±3℃温度下光照16h或黑暗处理8h，培养5周。5周后，剪取烟草叶片，近轴面朝上置于MS预培养固体培养基上(4.74g/L MS培养基粉末+30g/L蔗糖+8g/L琼脂粉+0.5mg/L 6-BA+0.1mg/LNAA，pH6.0)，25±3℃条件下黑暗培养2天。将烟草叶片置于含有pBI121-T-SQS重组表达载体的大肠杆菌菌液中浸泡5min，然后吸干叶片表面菌液，再将其近轴面朝上重新放回培养基，25±3℃条件下黑暗共培养2天。将共培养2天的烟草叶片用含500mg/L Car的无菌水清洗两遍，无菌滤纸吸干表面液体后置于含有抗生素的MS固体筛选分化培养基上(4.74g/L MS培养基粉末+30g/L蔗糖+8g/L琼脂粉+0.5mg/L 6-BA+0.1mg/L NAA+50mg/LKan+500mg/L Car，pH6.0)，25±3℃条件下16h光照/8h黑暗进行筛选和分化培养，直到外植体切口处形成愈伤并分化出小苗。选取带有生长点且发育状态良好的抗性苗，将其切下后转移到1/2MS培养基中(2.47g/L1/2MS培养基粉末+20g/L蔗糖+8g/L琼脂条+50mg/LKan+250mg/L Car，pH5.8)进行继代增殖和生根培养。最后通过提取基因组DNA和PCR扩增验证是否成功转化至烟草。
以上所述实施方式仅为本发明的优选实施例，而并非本发明可行实施的全部实施例。对于本领域一般技术人员而言，在不背离本发明原理和精神的前提下对其所作出的任何显而易见的改动，都应当被认为包含在本发明的权利要求保护范围之内。
Claims (7)
1.一种油茶SQS基因的克隆及表达载体构建的方法，其特征在于，包括如下步骤：
(1)提取油茶RNA，合成cDNA并进行PCR扩增；
(2)构建油茶SQS基因的过表达载体；
(3)利用油茶SQS基因的过表达载体将重组产物转化至大肠杆菌DH5α细胞；
(4)将重组表达载体转化烟草；
(5)测定烟草种子中SQS基因的相对表达量。
2.如权利要求1所述的油茶SQS基因的克隆及表达载体构建的方法，其特征在于，步骤(2)表达载体构建的过程中，采用的酶切反应体系为2μL10×KBuffer、5μL载体质粒、2μLBamHI、不多于40μL的ddH2O；重组连接反应体系的总体积10μL，包括4μL线性化载体、1μL插入片段、2μL5×CEIIBuffer、15μLExnaseII，其余为10μLddH2O。
3.如权利要求2所述的油茶SQS基因的克隆及表达载体构建的方法，其特征在于，步骤(2)表达载体构建的过程中，重组反应试剂盒采用的是Vazyme公司ClonExpress-IIOneStepCloningKit。
4.如权利要求1所述的油茶SQS基因的克隆及表达载体构建的方法，其特征在于，步骤(3)重组产物转化至大肠杆菌DH5α细胞过程中，在100μL大肠杆菌感受态细胞中加入10μL连接产物，冰浴30min；42℃热激60-90s；冰浴2min；加入800μLLB液体培养基，37℃摇床培养30min；6000rpm离心3min。
5.如权利要求1所述的油茶SQS基因的克隆及表达载体构建的方法，其特征在于，步骤(4)转化烟草过程中使用的MS预培养固体培养基组成为：4.74g/LMS培养基粉末、30g/L蔗糖、8g/L琼脂粉、0.5mg/L6-苄氨基腺嘌呤、0.1mg/Lα-萘乙酸，pH6.0。
6.如权利要求1所述的油茶SQS基因的克隆及表达载体构建的方法，其特征在于，步骤(4)表达载体转化至烟草过程中使用的分化培养基组成为：4.74g/LMS培养基粉末、30g/L蔗糖、8g/L琼脂粉、0.5mg/L6-苄氨基腺嘌呤、0.1mg/Lα-萘乙酸、50mg/L卡那霉素、500mg/L羧苄青霉素，pH6.0。
7.如权利要求1所述的油茶SQS基因的克隆及表达载体构建的方法得到的油茶SQS基因的序列，其特征在于，基因序列如下：
ATGGGAAGTTTGGGGGCAATTTTGAAGCATCCGGATGATTTCTATCCATTGATGAAGTTGAAGATGGCGGCGAGGCGAGCGGAGAAGAATATTCCTCCGGAGCCTCACTGGGGCTTCTGTTACTCCATGCTTCACAAGGTTTCTCGCAGTTTCGCCCTCGTAATTCAACAGCTCGACACCGAGCTTCGCAACGCTGTATGCATTTTTTATTTGGTTCTTCGAGCCCTCGACACCGTTGAGGATGATACAAGCATAGCTACAGAGGTTAAAGTACCTATTCTGATGGCTTTTCATCGTCATATATATGACCGTGACTGGCATTTTTCATGTGGTACAAAGGAGTACAAGGTTCTCATGGATGAGTTCCATCATGTTTCAACTGCTTTTTCGGAGCTTGGGAGAGGTTATCAGGAGGCAATCGAGGACATTACTATGAGAATGGGTGCAGGAATGGCAAAGTTTATATGCAAGGAGGTAGAAACAATTGATGACTATGATGAATATTGTCACTACGTAGCAGGACTGGTTGGGTTAGGGTTATCAAAGCTTTTCCATGCCTCTGGGTCTGAAGATTTGGCATCAGATTCTCTCTCCAATTCAATGGGTTTATTTCTTCAGAAAACAAACATTATTCGGGACTATTTGGAGGATATAAACGAGATACCAAAGTCACGCATGTTTTGGCCTCGCCAGATTTGGAGTAAATATGTTAACAAACTTGAGGACTTAAAAGACAAGGAAAACTCAGTCAAAGCTGTGGAATGCCTTAATGACATGGTGACAAATGCTTTGATACATGTGGAGGATTGCCTGACATACATGTCAGCTTTGCGAGATCCTTCAATTTTCCGATTTTGTGCAATTCCACAGATCATGGCAATTGGAACCTTGGCTTTATGCTACAACAATATTGAAGTCTTCAGAGGTGTTGTAAAAATGAGGCGTGGTCTTACTGCTAAAGTTATTGACCGGACAAAAACAATGTCGGATGTCTATGGTGCTTTCTTCGATTTTTCTTGTATGCTGAAGTCAAAGGTCAACAAGAGTGACCCAAATGCTATGAAAGCATTAAGCAGGCTAGAGGCAATTCAGAAAATTTGCAGGGAATCTGGAACCCTAAACAAAAGGAAATCTTACATAATCAAGAGCGAACCAAGATACAACTCAACTCTGGTTTTTGTATTATTCATTATACTCGCAATCCTGTTTGCATATCTATCTGCAAACAGACCAAGTAATATGTGA。
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