CN117587045B - 一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因及应用 - Google Patents
一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因及应用 Download PDFInfo
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Abstract
本发明公开了一种藜芦胆固醇22(R)‑羟化酶VnCYP90B27基因及在制备22(R)‑羟基胆固醇的应用。该藜芦胆固醇22(R)‑羟化酶VnCYP90B27基因具有如SEQ ID No.1所示的核苷酸序列,能够表达该藜芦胆固醇22(R)‑羟化酶VnCYP90B27的转基因工程菌。以胆固醇为底物,在由所述的藜芦胆固醇22(R)‑羟化酶VnCYP90B27基因编码得到的22(R)羟化酶的催化下,在胆固醇的C‑22位上进行羟基化反应,生成22(R)‑羟基胆固醇。该基因在构建22(R)‑羟基胆固醇的生物合成途径中具有重要作用。
Description
技术领域
本发明属于生物技术领域,特别涉及一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因及应用。
背景技术
藜芦(Veratrum nigrum)是百合科(Liliaceae)藜芦属(Veratrum)多年生草本植物,分布于我国东北、河北、山东、陕西、云南和贵州等地,也分布于亚洲北部和欧洲中部。生于海拔1200-3300米的山坡林下或草丛中。作为我国传统中药,藜芦用于喉痹不通、中风痰壅,癫痫,及疥癣和恶疮等疾病的治疗。主要化学成分主要为藜芦甾体生物碱,如藜芦胺、环巴胺、介芬胺等。甾体生物碱为植物的次生代谢产物,参与植物通讯和防御的调节,同时也是一些传统中药的主要生物活性成分,如从藜芦中分离得到的环巴胺是一种抑制人肿瘤细胞的增殖的新型的抗癌药物质。然而,环巴胺等藜芦中的甾体类生物碱的获取主要依赖于植物提取和化学合成,但由于含量较低,提取困难,且化学结构复杂,化学合成效率也不高。这导致环巴胺供不应求,野生资源消耗量极大。
由于未进入规模化人工种植,完全依赖自然生长缓慢的野生资源,很难到达市场需求量,近年来野生藜芦资源趋向渐危。利用合成生物学生产药效单体是缓解野生藜芦资源紧张,是目前呼声较高的保护藜芦种源多样性的关键途径之一。胆固醇是大多数类固醇生物碱的常见前体,环巴胺等甾体生物碱的生物合成也被认为始于胆固醇。近年来,随着代谢工程和合成生物学的迅速发展,许多药用活性成分利用微生物细胞工厂实现了从头合成,为药效单体的工业化生产提供了可能,如紫杉醇前体、秋水仙碱、人参皂苷及药用托品烷生物碱等,这为甾体生物碱的生产提供了新的方向。22(R)-羟基胆固醇是环巴胺合成的一步重要前体,通过生物合成甾体生物碱的关键前体22(R)-羟基胆固醇,或许是环巴胺的生物大量合成的关键一步。
发明内容
针对上述问题,本发明的目的是提供一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因。
为实现上述目的,本发明所采用的技术方案是:
一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因,所述藜芦胆固醇22(R)-羟化酶VnCYP90B27基因的核酸序列如SEQ ID NO:1所示。
本发明还提供了一种所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因所编码的蛋白,所述蛋白的氨基酸序列如SEQ ID NO:2所示。
本发明还提供了一种重组质粒,通过将上述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因与Y33载体同源重组获得,命名为Y33-VnCYP90B27。
本发明还提供了一种转基因工程菌,含有上所述的重组质粒或所述基因工程菌的基因组中整合有上述外源的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因。
作为优选,所述转基因工程菌为大肠杆菌DH5α菌株和酿酒酵母菌BY4742菌株。
本发明还提供了一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因在制备22(R)-羟基胆固醇中的应用。
作为优选,所述制备22(R)-羟基胆固醇的方法包括以下步骤:以胆固醇为底物,在由所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因编码得到的胆固醇22(R)-羟化酶的催化下,在胆固醇的C-22位上进行羟基化反应,生成22(R)-羟基胆固醇。
本发明具有以下有益效果:
本发明所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因,是从藜芦中,通过转录组测序及生物信息学技术,经大量实验筛选后得以鉴定出来的;采用RNA试剂后提取藜芦的RNA,并反转成cDNA后进行PCR扩增获得。所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因的扩增引物如下所示:
5'F:ATGGCGATGGAGCTCCT
3'R:TCAGTCCCCGAGTTTTTCGAG
此外,与载体Y33进行同源重组时,VnCYP90B27基因则需要使用带同源臂的引物进行扩增及回收,带同源臂的引物如下:
5'F:cagtcgacctcgaatctagaATGGCGATGGAGCTCCTC
3'R:atgatgcggccctctagaTCAGTCCCCGAGTTTTTCGA
从藜芦中分离并鉴定的基因VnCYP90B27可作为藜芦分子辅助育种的重要标记基因,同时也可作为酵母底盘细胞构建中生产22(R)-羟基胆固醇的重要候选基因。
附图说明
图1为基于VnCYP90B27功能构建22(R)-羟基胆固醇的生物合成途径;
图2为重组表达质粒Y33-VnCYP90B27的构建示意图(用于表达编码藜芦胆固醇22(R)-羟化酶VnCYP90B27基因);
图3中A为基因克隆,B为重组完成电泳图;
图4中A为标品胆固醇与22(R)-羟基胆固醇的质谱出峰时间;B为胆固醇与22(R)-羟基胆固醇的质谱特征;C转化菌株(Vn1-14)摇瓶发酵产物与胆固醇底盘菌(Vg13)空白对照的LC-MS出峰时间图。
实施方式
下面结合对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
首先对藜芦进行了转录组测序,并进行组装分析,然后对转录组数据进行了深度挖掘,通过本地BLAST对转录组数据及其KEGG蛋白数据库注释结果的搜索,获得与22(R)-羟基胆固醇合成相关的候选基因,并从转录组数据中找出RPKM值,利用RPKM值对这些基因进行差异表达分析,并将差异基因表达量利用TBtool制作热图,分别比较相关基因在藜芦根、叶中的表达量,对候选CYP90B27进行功能鉴定使用在线工具ORF Finder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)识别CYP90B27的开放阅读框(ORF)和氨基酸序列。来自其他物种的CYP基因的氨基酸序列可从美国国家生物技术信息中心(NCBI)数据库下载,并由ClustalW跟候选基因进行比对,通过MEGA-X在默认参数条件下用邻接法(neighborjoining method)构建系统进化树。之后进行cDNA的制备、候选基因的扩增及回收、同源重组、酵母转化、菌水检测、摇瓶发酵及产物提取,以及HPLC及LC/MS检测等一系列工作后,最终鉴定出可以催化胆固醇的C-22位上进行羟基化反应,生成22(R)-羟基胆固醇(图1)的VnCYP90B27。各阶段操作步骤如下:
(1)cDNA模板的制备
取藜芦的新鲜根、叶,切片后液氮速冻,进行RNA提取。总RNA提取采用广州美基生物科技有限公司的HiPure HP Plant RNA Mini Kit试剂盒。按试剂盒说明书的操作步骤提取RNA,经检测合格后,使用TAKARA反转录试剂盒将RNA反转录成cDNA,-20ºC保存备用。
(2)基因扩增及回收
利用NCBI在线软件(https://www.ncbi.nlm.nih.gov/orffinder)找出藜芦的候选CYP基因的ORF(开放阅读框)。然后用SnapGene软件设计基因编码区的全长序列的特异性引物,设计的引物带有酵母表达载体Y33-VnCYP90B27的同源臂,载体通用引物:
5'F:cagtcgacctcgaatctagaATGGCGATGGAGCTCCTC;
3'R:atgatgcggccctctagaTCAGTCCCCGAGTTTTTCGA。
以之前反转录得到的cDNA为模板,采用DNA聚合酶(phanta酶)进行基因扩增。扩增体系为:cDNA 2μl,2 x phantaMax Master mix 25μl,上、下游引物各2μl,ddH2O补足至50μl,扩增体系为:95ºC、3min,95ºC、15s,58ºC、15s,72ºC、45s,35个循环;72ºC、5min。PCR扩增程序结束后,用1.2%的琼脂糖凝胶电泳检测扩增的基因条带与目的基因条带长度是否一致。PCR产物经琼脂糖凝胶电泳检测后在1 500 bp左右有一条清晰明亮的带,与目的基因条带(1464bp)长度相近,说明扩增效果好,利用GenStar公司的胶回收试剂盒回收纯化目的基因(图3)。利用仪器NanoDrop2000测定回收浓度,于在-20℃冰箱中保存。
(3)基因重组载体的构建与鉴定
A载体线性化:利用核酸内切酶XbaI对Y33载体质粒进行单酶切得到线性化载体Y33。使用omega公司的E.Z.N.A.®Cycle Pure Kit试剂盒进行纯化回收,测定其浓度后存-20℃冰箱中备用。
B基因重组:将使用生工生物工程(上海)股份有限公司的即用型无缝克隆酶试剂盒扩增得到的目的基因和与线性化的Y33载体连接重组,连接方法与转化方式见图2,转化菌株为E.coli(DH5α)感受态。
C菌水PCR检测:在超净工作台中从转化培养基上随机挑选出8个单菌落于20μlddH2O中,取3μl作为模板进行PCR扩增,PCR反应体系采用的是南京诺唯赞生物科技有限公司的2x Taq Master Mix酶反应体系,PCR程序为95℃,3min,95℃,30s,55℃,15s,72℃,30/kb,35个循环;72℃、5min反应程序结束后,用1%琼脂糖检测PCR产物的长度大小。经琼脂糖电泳检测后PCR产物在1 500 bp~2 000 bp有明显的条带,与目的片段(1 700bp)相近,与期望的结果一致(图3B)。之后将菌水送往擎科公司测序,结果显示基因连接成功,无突变,挑选单菌落为阳性菌,说明组装成功。用50%甘油与菌液按体积比1∶1的方法进行保种。
(4)质粒载体提取与酵母底盘醋酸锂转化
载体质粒提取: 将使用武汉艾瑞科生物科技有限公司的即用型Steadure质粒提取试剂盒提取得到带有目的基因的Y33-VnCYP90B27载体质粒。
酵母醋酸锂转化:利用醋酸锂转化法将带有目的基因Y33-VnCYP90B27的载体质粒导入到产胆固醇酵母底盘工程菌中,涂布于SC-His-Leu-Ura酵母缺陷固体培养基上,筛选阳性转化子。
(5)摇瓶发酵
将醋酸锂转化筛选到的阳性单菌落的种子液接种到50ml酵母SC-H-L-U培养基摇床震荡培养5d,收集发酵液。分别用10ml甲醇,10ml氯仿30min超声提取,氮气吹干后用1ml甲醇复溶。将复溶提取液经滤头(0.22μm)过滤后放入液相瓶,进行产物检测。
(6)产物检测
LC-MS检测条件如下:
为进一步确认HPLC的检测结果,采用Agilent1290 UPLC/6540 Q-TOF液相色谱质谱联用仪(LC/MS)进行检测:质谱条件:离子源采用的是正离子模式,电压:3500V;碎裂电压:135V;锥孔电压:60V;射频电压:750V,扫描范围:100-1000m/z,扫描方式:SRM。色谱条件:色谱柱为Agilent Extend-C18(250mm x 4.6mm,5μm),柱温:30℃,测定产物流动相为水溶液(A)-甲醇(B),梯度洗脱:0-40min,20%-20%A;40-45min,20%-5%A;45-70 min,5%-5% A。洗脱时间:70min;进样量:10μL;流速:1mL/min;检测波长203nm。检测结果见图4,从结果中可以看出,反应产物特征峰出峰时间(图4C(Vn1-14))和特征峰(图4C(Vn1-14))与标准品22(R)-羟基胆固醇特征峰出峰时间(图4A(PPD-UP))和特征峰(图4B)的结果相吻合,进一步确认生成的产物为22(R)-羟基胆固醇。
以上结合对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (10)
1.一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因,其特征在于,所述藜芦胆固醇22(R)-羟化酶VnCYP90B27基因的核酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因与Y33载体同源重组获得的重组质粒。
3.根据权利要求1所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因所编码的蛋白,其特征在于,所述蛋白的氨基酸序列如SEQ ID NO:2所示。
4.表达如权利要求3所述的蛋白的转基因工程菌。
5.如权利要求4所述的转基因工程菌,其特征在于,包含有如权利要求2所述的含有藜芦胆固醇22(R)-羟化酶VnCYP90B27基因的重组质粒。
6.如权利要求4所述的转基因工程菌,其特征在于,基因组中整合有如权利要求1所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因。
7.如权利要求4-6中任意一项所述的转基因工程菌,其特征在于,其原始菌为大肠杆菌DH5α菌株或酿酒酵母菌BY4742菌株。
8.如权利要求1所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因在制备22(R)-羟基胆固醇中的应用。
9.如权利要求4-6中任意一项所述的转基因工程菌在制备22(R)-羟基胆固醇中的应用。
10.根据权利要求9所述的转基因工程菌在制备22(R)-羟基胆固醇中的应用,其特征在于,所述制备22(R)-羟基胆固醇的方法包括以下步骤:以胆固醇为底物,在由所述的藜芦胆固醇22(R)-羟化酶VnCYP90B27基因编码得到的22(R)羟化酶的催化下,在胆固醇的C-22位上进行羟基化反应,生成22(R)-羟基胆固醇。
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