CN117965576B - 一种王百合苷生物合成基因LlHCT1及其应用 - Google Patents
一种王百合苷生物合成基因LlHCT1及其应用 Download PDFInfo
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Abstract
本发明公开了一种王百合苷生物合成基因LlHCT1及其应用，LlHCT1基因的核苷酸序列和编码的蛋白质的氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。本发明以卷丹百合为对象，克隆获得王百合苷生物合成关键基因LlHCT1，构建过表达载体，利用农杆菌介导法，转入卷丹百合叶片和鳞片中，使目标基因超量表达，对获得的瞬时过表达百合材料进行王百合苷定性定量分析，结果表明，多种王百合苷含量显著提高，说明LlHCT1参与多种王百合苷生物合成。本发明为王百合苷的合成调控提供了新的方法，对王百合苷的异源生物合成提供了首个基因，在王百合苷类医药成分的工业化生产领域具有重要意义。
Description
技术领域
本发明涉及生物基因工程技术领域，具体涉及一种王百合苷生物合成基因LlHCT1及其应用。
背景技术
百合是重要的观赏植物，同时也是我国传统中药和药食同源植物。王百合苷（regaloside）是百合中特有的一类重要活性成分，为苯丙烯酰基甘油葡萄糖苷类物质的统称（Zhou et al., 2021），目前研究者先后从百合属植物中检测到13种王百合苷。研究表明，王百合苷具抗氧化、抑菌、抗炎，以及延缓II型糖尿病、保护心肌细胞等功能（Luo etal., 2012; Murray et al., 2019; Kim et al., 2020）。但目前市场上王百合苷均从卷丹、龙牙百合等百合属植物中提取，产量低、价格昂贵，也不利于百合资源保护。
通过遗传改良的方法已成为提高生物活性成分含量及工业化生产的趋势。利用基因工程方法提高植物中关键酶基因的表达，或将关键酶基因导入其他植物/微生物中构建重要功能性代谢物生物合成通路是提高目标产物含量或目标产物的异源合成的常用方法。
研究者从不同百合中检测到多种王百合苷前体物质（苯丙烯酰基甘油酯类成分），表明王百合苷是先形成酯化产物，再经过糖基化产生（Tang et al., 2021；Liu et al.,2023）。因此，催化苯丙烯酰基-CoA与甘油成酯是王百合苷类成分生物合成的关键步骤（Tang et al., 2021; Luo et al., 2022）。鉴定生物活性成分生物合成途径中关键基因是进行遗传改良的前体和基础，而目前关于催化王百合苷生物合成关键成酯步骤反应的酶基因还未见报道。
发明内容
针对现有技术中的上述不足，本发明的目的在于提供一种王百合苷生物合成基因LlHCT1及其应用。
为了达到上述发明目的，本发明采用的技术方案为：
第一方面，提供一种王百合苷生物合成基因LlHCT1，其核苷酸序列如SEQ ID NO.1所示。
第二方面，提供一种王百合苷生物合成基因LlHCT1编码的蛋白质，其氨基酸序列如SEQ ID NO.2所示。
第三方面，提供一种含有王百合苷生物合成基因LlHCT1的载体或重组菌。
第四方面，提供王百合苷生物合成基因LlHCT1在王百合苷生物合成中的应用。
本发明的有益效果为：
结合转录组数据，克隆并鉴定到一个参与王百合苷生物合成成酯步骤的基因LlHCT1，并进行生物信息学分析。基于qRT-PCR测定LlHCT1在百合不同组织、空载及瞬时过表达百合材料中表达情况；并结合王百合苷定性定量分析验证LlHCT1功能。结果表明，LlHCT1促进多种王百合苷生物合成，为王百合苷生物合成通路解析奠定基础，同时为王百合苷的异源生物合成提供了可用基因。
附图说明
图1为LlHCT1全长克隆胶图；
图2为LlHCT1在卷丹百合不同组织表达情况示意图；
图3为LlHCT1在NCBI数据库中Blast比对情况示意图；
图4为LlHCT1的同源建模示意图；
图5为LlHCT1多序列比对示意图；
图6为侵染72 h后35S驱动下eGFP、LlHCT1-eGFP在烟草和百合叶片中表达情况示意图；
图7为过表达LlHCT1后卷丹叶片LlHCT1表达情况示意图；其中，CK为空载侵染后LlHCT1表达情况，OE为过表达载体侵染后LlHCT1表达情况；*表示P<0 .05时的统计学显著差异，**表示P<0 .01时的统计学显著差异；
图8为过表达LlHCT11后卷丹叶片不同王百合苷含量示意图；其中，CK为空载侵染后不同王百合苷含量，OE为过表达载体侵染后不同王百合苷含量；*表示P<0.05时统计学显著差异，**表示P<0.01时统计学显著差异；PGG11表示王百合苷(2S)-1-O-Z-p-coumaroyl-2-O-acetyl-3-O-β-D-glucopyranosylglycerol，PGG12表示王百合苷(2S)-1-O-E-p-coumaroyl-3-[O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl]glycerol，PGG13表示王百合苷(2S)-1-O-E-p-coumaroyl-2-O-acetyl-3-[O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl]glycerol；
图9 为过表达LlHCT1后卷丹鳞片LlHCT1表达情况示意图；其中，CK为空载侵染后LlHCT1表达情况，OE为过表达载体侵染后LlHCT1表达情况；*表示P<0 .05时的统计学显著差异，**表示P<0 .01时的统计学显著差异；
图10为过表达LlHCT1后卷丹鳞片不同王百合苷含量示意图；其中，CK为空载侵染后LlHCT1表达情况，OE为过表达载体侵染后LlHCT1表达情况；*表示P<0.05时的统计学显著差异，**表示P<0.01时的统计学显著差异；PGG11表示王百合苷(2S)-1-O-Z-p-coumaroyl-2-O-acetyl-3-O-β-D-glucopyranosylglycerol，PGG12表示王百合苷(2S)-1-O-E-p-coumaroyl-3-[O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl]glycerol，PGG13表示王百合苷(2S)-1-O-E-p-coumaroyl-2-O-acetyl-3-[O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl]glycerol。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例
材料与方法
菌株、载体及试剂
大肠杆菌DH5α和根癌农杆菌GV3101、EHA105感受态，克隆载体pEASY®-BluntCloning Vector购于北京全式金生物有限公司； pCAMBIA2300为实验室保存。
植物RNA提取试剂盒（天根生化生物有限公司），反转录试剂盒和荧光定量试剂盒（上海翊圣生物科技有限公司）、胶回收试剂盒、质粒回收试剂盒（北京全式金生物有限公司）、同源重组试剂盒、利福平、氨苄卡那霉素、内切酶、高保真酶等。
基因全长克隆
以卷丹珠芽为样本，提取RNA后反转录成cDNA保存于-20℃备用。根据全长转录本序列，利用Primer3Plus设计包含LlHCT1完整ORF的全长引物，于上海生工生物科技有限公司引物合成。以cDNA为模板用高保真酶进行PCR扩增，扩增产物进行胶回收后构建到pEASY®-Blunt Cloning Vector，转入大肠杆菌DH5α涂平板，挑单克隆小摇12 h后送北京生工生物有限公司测序鉴定。
载体构建
以鉴定正确的重组质粒为模板，用添加对应载体接头的引物扩增LlHCT1，胶回收后分别构建到酶切后的pCAMBIA2300载体上，转化大肠杆菌DH5α挑单克隆测序确定后提取pCAMBIA2300-LlHCT1-eGFP质粒，转入到根癌农杆菌EHA105感受态，涂平板、挑单克隆并小摇后PCR菌检，合格的转化单克隆保菌备用。
表1引物序列和用途
烟草瞬时过表达
将pCAMBIA2300-eGFP、pCAMBIA2300-LlHCT1-eGFP质粒转入EHA105菌株感受态，以培养4周龄左右的本氏烟草为材料，进行瞬时过表达。具体步骤如下：
a. 取导入pCAMBIA2300、pCAMBIA2300-LlHCT1的EHA105菌株100 μL涂布于添加Rif和Kan抗生素的LB培养基上，28℃导致培养至形成明显可见单菌落；
b. 挑取单菌落吹打于1 mL LB 液体培养基中，于28℃、200 rpm培养过夜；
c. 取0.5 mL菌液吹打于50 mL液体LB培养基中，于28℃、200 rpm震荡培养至OD600= 0.8 ~ 1.0；
d. 所得菌液于5000 rpm离心10 min收集菌体，用重悬液（MS + 10 mM MES + 10mM MgCl2+ 200 μM乙酰丁香酮）重悬菌体、调整OD600= 0.6，黑暗孵育2 ~3 h，得侵染液；
e. 用1 mL一次性注射器将侵染液注入烟草叶片背面，至形成明显水渍状；
f. 将注射过得烟草至于25℃黑暗培养；
g. 注射24 h后，每隔12 h进行拍照观察。
百合瞬时过表达
将pCAMBIA2300、pCAMBIA2300-LlHCT1质粒转入EHA105菌株感受态，分别以卷丹叶片（剪去两端）或直径为1 cm卷丹鳞片圆盘（打孔器制备，现打现用）为材料进行瞬时过表达，具体步骤如下：
a. 将导入pCAMBIA2300、pCAMBIA2300-LlHCT1载体的菌株涂布于添加Rif和Kan抗生素的LB培养基平板，28℃导致培养至形成明显可见单菌落；
b. 挑取单菌落吹打于1 mL LB 液体培养基中，于28℃、200 rpm培养过夜；
c. 取0.5 mL菌液吹打于50 mL液体LB培养基中，于28℃、200 rpm震荡培养至OD600= 0.8 ~ 1.0；
d. 所得菌液于5000 rpm离心10 min收集菌体，用重悬液（MS + 10 mM MES + 10mM MgCl2+ 200 μM乙酰丁香酮）重悬菌体、调整OD600= 0.6，黑暗孵育2 ~3 h，得侵染液；
e. 取卷丹叶片（剪去两端）或直径为1 cm卷丹鳞片圆盘（打孔器制备，现打现用）装入广口瓶，倒入对应侵染液，于- 50 kpa真空渗透5 min（重复2次），用蒸馏水洗去表面侵染液后纱布保湿，于培养箱25℃黑暗培养；
f. 注射24 h后，每隔12 h进行拍照观察。
qRT-PCR验证
根据前期观察，侵染后5 d后取材，用于总RNA提取和qRT-PCR。以lilyActin（GeneBank Accession Number: JX826390）基因作为内参进行标准化。相对表达量以2-ΔΔCT计算。
王百合苷定性定量分析
根据观察结果，分别取侵染5 d后的百合瞬时过表达鳞片盘和叶片液氮研磨后，精确称取0.1 g于2 mL离心管，加入1 mL70%甲醇，400W、30℃超声助提30 mim（中间上下颠倒混匀2次），冷却至室温后8000 rmp离心5 min，取上清液，用70%甲醇稀释10倍后，过0.22 μm有机滤膜，收集于2 ml于色谱试剂瓶得提取液。
主要仪器与参数设置
优化后使用的仪器及相关参数如下：
仪器：Agilent 1290 Infinity Ⅱ 超高效液相色谱配备6495 LC/TQ三重四极杆质谱检测仪（UPLC-MS/MS）；色谱柱：Thermo Acclaim 120 C18 (3 μm, 2.1 × 150 mm)；流动相：A：0.1 FA水，B：0.1%FA乙腈；流速：0.2 mL/min；进样量：1 μL；柱温：35℃；电离源：ESI。
流动相设置：起始，90% A，10% B；0 ~ 10 min，线性变化为50% A，50% B；10 ~ 11min，线性变化为20% A，80% B；11 ~ 12 min 线性变化为90% A，10% B，并保持1 min。
进样时每10个检测样本插入一个QC。结果以mg/100 g 鲜重（FW）表示，每个处理3次生物学重复。
结果与分析
LlHCT1基因克隆与分析
基于全长转录组数据，克隆了LlHCT1全长序列（图1）。组织表达分析显示LlHCT1在卷丹根、鳞片和珠芽中表达量较高，花瓣中表达量次之，叶中表达量最低，这一趋势与卷丹不同组织中王百合苷含量类似（图2）。序列分析结果表明LlHCT1全长1290 bp，编码429个氨基酸残基。Blast比对结果表明其属于Transferase superfamily；具有PLN02481保守结构域（Omega-hydroxypalmitate O-feruloyl transferase），暗示其可能具有O-feruloyltransferase（O-阿魏酰基转移酶）活性（图3），进一步证明其可能参与王百合苷生物合成前体物质苯丙烯酰基甘油酯的形成。
基于LlHCT1氨基酸序列，多序列比对结果表明，LlHCT1具有2个HXXXD motifs和1个DFGWG motif（图4）；基于LlHCT1氨基酸序列，利用SWISS-MODEL进行蛋白建模。结果表明，LlHCT1与拟南芥羟基肉桂酰辅酶A：莽草酸羟基肉桂酰转移酶（AtHCT）具有相似的构象，暗示LlHCT1蛋白可能具有羟基肉桂酰辅酶A结合位点（图5），进一步证明LlHCT1可能具有催化王百合苷生物合成反应中成酯反应步骤。
LlHCT1在烟草和百合中表达情况
用导入pCAMBIA2300-eGFP或pCAMBIA2300-LlHCT1-eGFP质粒的农杆菌EHA105注射烟草叶片，并用真空渗透法将导入pCAMBIA2300-eGFP、pCAMBIA2300-LlHCT1-eGFP质粒的农杆菌EHA105分别渗入卷丹百合叶片，侵染72 h后pCAMBIA2300-eGFP和pCAMBIA2300-LlHCT1-eGFP在烟草和卷丹百合叶片中大量表达，表明目标基因成功导入烟草和卷丹百合中（图6）。
LlHCT1促进多种王百合苷的生物合成
用真空渗透法将导入pCAMBIA2300-eGFP、pCAMBIA2300-LlHCT1-eGFP质粒的农杆菌EHA105分别渗入卷丹百合叶片和鳞片，选择eGFP、LlHCT1-eGFP过表达5 d后的卷丹叶片和鳞片盘取材，进行qRT-PCR分析和进行王百合苷定性定量分析（以空载处理为对照）。qRT-PCR结果表明，过表达LlHCT1显著提高百合叶片中LlHCT1表达量，达到对照的近100倍（图7）；而鳞片中提高为对照的1.5倍（图8）。王百合苷定性定量分析结果表明，卷丹叶片中过表达LlHCT1提高了多种王百合苷含量，包括王百合苷A、顺式王百合苷A、王百合苷C、王百合苷F、王百合苷K和顺式王百合苷A等（图9）；在卷丹鳞片中，尽管LlHCT1表达量也显著促进了以上多种王百合苷的积累，包括王百合苷A、顺式王百合苷A、王百合苷C、王百合s苷F、王百合苷K、PGG13和顺式王百合苷A等（图10）。以上结果表明LlHCT1参与了多种王百合苷的生物合成。
综上，本发明结合转录组数据，克隆并鉴定到一个百合LlHCT1基因，并进行生物信息学分析。基于qRT-PCR测定LlHCT1在百合不同组织、空载及瞬时过表达百合材料中表达情况；并结合王百合苷定性定量分析验证LlHCT1功能。结果表明，LlHCT1促进多种王百合苷生物合成，为王百合苷生物合成通路解析奠定基础，同时为王百合苷的异源生物合成提供了可用基因。
Claims (4)
1.一种王百合苷生物合成基因LlHCT1，其特征在于，所述基因的核苷酸序列如SEQ IDNO.1所示。
2.一种权利要求1所述的王百合苷生物合成基因LlHCT1编码的蛋白质，其特征在于，所述蛋白质的氨基酸序列如SEQ ID NO.2所示。
3.一种含有权利要求1所述的王百合苷生物合成基因LlHCT1的载体或重组菌。
4.权利要求1所述的王百合苷生物合成基因LlHCT1在王百合苷生物合成中的应用。
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