药学学报

Purpose	Primer sequence (5′→3′)
5′/3′-RACE	UPM-S: 5′-CTAATACGACTCACTATAGGGC-3′
	UPM-L: 5′-ctaatacgactcactatagggcAAGCAGTGG TATCAACGCAGAGT-3′
	5′-f1: GACTTGTTCAGCGATTTCAGGGGACG
	5′-f2: GGTGGAGGTTACCTATCAGGGGCAGC
	3′-f1: TCCCAGCCAAGACCCAAGTCATAGTG
	3′-f2: GGAGGAGGATTTGCCCTGGGATTG
LD-PCR	QCf: ATGGAGCAGCAATTCTTTTCATCTC
	QCr: TCAGGTAATGTTAGGAACCAAAAGC
qRT-PCR	RT-f: GAAGAAAAGCCGCCGTTGATAAG
	RT-r: TAGATGATGTTTCAGCCCCACC
Internal control	GAPDH-f: CTGGTATGGCATTCCGTGTA
	GAPDH-r: AACCACATCCTCTTCGGTGTA

Purpose	Primer sequence (5′→3′)
5′/3′-RACE	UPM-S: 5′-CTAATACGACTCACTATAGGGC-3′
	UPM-L: 5′-ctaatacgactcactatagggcAAGCAGTGG TATCAACGCAGAGT-3′
	5′-f1: GACTTGTTCAGCGATTTCAGGGGACG
	5′-f2: GGTGGAGGTTACCTATCAGGGGCAGC
	3′-f1: TCCCAGCCAAGACCCAAGTCATAGTG
	3′-f2: GGAGGAGGATTTGCCCTGGGATTG
LD-PCR	QCf: ATGGAGCAGCAATTCTTTTCATCTC
	QCr: TCAGGTAATGTTAGGAACCAAAAGC
qRT-PCR	RT-f: GAAGAAAAGCCGCCGTTGATAAG
	RT-r: TAGATGATGTTTCAGCCCCACC
Internal control	GAPDH-f: CTGGTATGGCATTCCGTGTA
	GAPDH-r: AACCACATCCTCTTCGGTGTA

白木香细胞色素P450基因AsCYP71D1的克隆及表达分析

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高世玺 ¹ , 戎梅 ¹ , 彭俊祥 ¹ , 徐艳红 ¹^,^* , 魏建和 ¹^,²^,^*

药学学报 | 研究论文 2023,58(10): 3123-3129

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药学学报 |研究论文 2023 , 58 (10) : 3123 -3129

白木香细胞色素P450基因AsCYP71D1的克隆及表达分析

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2. Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine & Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1198960245105066492, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656214818190154, authorId=1198960244710801881, language=CN, stringName=魏建和, firstName=建和, middleName=null, lastName=魏, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=¹^,²^,^*, address=1.中国医学科学院、北京协和医学院, 药用植物研究所, 中草药物质基础与资源利用教育部重点实验室, 濒危药材繁育国家工程实验室, 北京 100193
2.中国医学科学院、北京协和医学院, 药用植物研究所海南分所, 海南省南药资源保护与开发重点实验室, 国家中医药管理局沉香可持续利用重点研究室, 海南海口 570311, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1198960242777227500, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656214818190154, xref=null, ext=[AuthorCompanyExt(id=1198960242785616111, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656214818190154, companyId=1198960242777227500, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education & National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China), AuthorCompanyExt(id=1198960242794004720, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656214818190154, companyId=1198960242777227500, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.中国医学科学院、北京协和医学院, 药用植物研究所, 中草药物质基础与资源利用教育部重点实验室, 濒危药材繁育国家工程实验室, 北京 100193)]), AuthorCompany(id=1198960242911445246, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656214818190154, xref=null, ext=[AuthorCompanyExt(id=1198960242932416769, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656214818190154, companyId=1198960242911445246, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2. Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine & Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China), AuthorCompanyExt(id=1198960242944999684, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656214818190154, companyId=1198960242911445246, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.中国医学科学院、北京协和医学院, 药用植物研究所海南分所, 海南省南药资源保护与开发重点实验室, 国家中医药管理局沉香可持续利用重点研究室, 海南海口 570311)])])]

高世玺¹, 戎梅¹, 彭俊祥¹, 徐艳红¹^,^*, 魏建和¹^,²^,^*

作者信息

1.中国医学科学院、北京协和医学院, 药用植物研究所, 中草药物质基础与资源利用教育部重点实验室, 濒危药材繁育国家工程实验室, 北京 100193

2.中国医学科学院、北京协和医学院, 药用植物研究所海南分所, 海南省南药资源保护与开发重点实验室, 国家中医药管理局沉香可持续利用重点研究室, 海南海口 570311

通讯作者:

*徐艳红, Tel/Fax: 86-10-57833359, 86-10-57833358, E-mail: xuyanhong99@163.com;

魏建和, E-mail: wjianh@263.net

Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis (Lour.) Gilg

Shi-xi GAO¹, Mei RONG¹, Jun-xiang PENG¹, Yan-hong XU¹^,^*, Jian-he WEI¹^,²^,^*

Affiliations

1. Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education & National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China

2. Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine & Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China

出版时间: 2023-10-12 doi: 10.16438/j.0513-4870.2023-0404

文章导航

摘要

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细胞色素P450 (cytochrome P450 monooxygenases, CYP450) 是一类超基因家族编码的含有血红素的氧化酶类, 分布于各种需氧生物体内, 广泛参与萜类、生物碱、黄酮、脂肪酸等的生物合成。本研究依据课题组前期测得白木香转录组数据库中一个CYP450单加氧酶基因的部分转录本序列设计引物, 利用逆转录-聚合酶链反应(reverse transcription-PCR, RT-PCR) 和cDNA末端快速扩增(rapid-amplification of cDNA ends, RACE) 技术克隆该基因的全长cDNA序列; 并对其组织表达及亚细胞定位进行了研究。结果发现, 该基因全长cDNA序列为1 920 bp, 其中5′-非翻译区(untranslated region, UTR) 为88 bp, 3′-UTR为344 bp并具有21 bp的polyA尾, 开放阅读框为1 488 bp, 编码495个氨基酸。序列比对发现该蛋白属于CYP450家族的CYP71D家族成员, 命名为AsCYP71D1。组织表达分析的结果显示, AsCYP71D1基因主要在茎中表达。进一步洋葱表皮中亚细胞定位显示, AsCYP71D1编码的蛋白在细胞质、细胞核及细胞膜上都有表达。本研究为后续深入研究其在沉香倍半萜合成途径的功能奠定了基础。

关键词

白木香 / 倍半萜 / 细胞色素P450 / 基因克隆 / 表达分析

Abstract

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Cytochrome P450 (CYP450) is a kind of superfamily oxidase containing heme, which is distributed in various aerobic organisms. They are widely involved in the biosynthesis of terpenoids, alkaloids, flavonoids, fatty acids, etc. In this study, the full-length cDNA sequence of a P450 was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, with the specific primers that designed according to the sequence of a transcript annotated as P450 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The tissue expression and subcellular localization were also studied. The full-length cDNA of the cloned P450 gene is 1 920 bp, with 88 bp 5′-untranslated region (UTR), 344 bp 3′-UTR and a 21 bp polyA tail, and 1 488 bp open reading frame (ORF), encoding 495 amino acids. Sequence alignment revealed that the protein belonged to CYP71D family of cytochrome P450 family, and named AsCYP71D1. Tissue expression analysis indicated that AsCYP71D1 was mainly expressed in stem. Further subcellular localization of onion epidermis showed that AsCYP71D1 was expressed in cytoplasm, nucleus and cell membrane. This study will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Key words

Aquilaria sinensis / sesquiterpene / cytochrome P450 / gene clone / expression analysis

引用本文

高世玺, 戎梅, 彭俊祥, 徐艳红, 魏建和. 白木香细胞色素P450基因AsCYP71D1的克隆及表达分析. 药学学报, 2023 , 58 (10) : 3123 -3129 . DOI: 10.16438/j.0513-4870.2023-0404

Shi-xi GAO, Mei RONG, Jun-xiang PENG, Yan-hong XU, Jian-he WEI. Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis (Lour.) Gilg[J]. Acta Pharmaceutica Sinica, 2023 , 58 (10) : 3123 -3129 . DOI: 10.16438/j.0513-4870.2023-0404

正文

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细胞色素P450 (cytochrome P450 monooxygenases, CYP450) 是一类超基因家族编码的含有血红素的氧化酶类, 分布于各种需氧生物体内。在植物体内催化多种初生和次生代谢反应, 参与萜类、生物碱类、脂肪酸、甾醇类、植物激素、信号分子等的合成与代谢反应^{[1, 2]}。CYP450具有多种催化机制, 对多种底物表现出催化活性, 参与的反应包括羟基化、环氧化和脱烷基化反应等^{[3, 4]}。自1969年, Frear等^[5]在棉花中首次发现CYP450以来, 大量P450家族基因被鉴定出来。然而, 由于植物P450通常丰度很低, 不稳定且难以提纯, 使基因克隆及功能研究受到阻碍, 要从P450s超基因家族中, 鉴定到参与次生代谢途径的特异P450仍然具有极大的难度和挑战性。

植物中P450参与许多萜类化合物的生物合成。在单萜类衍生物中, CYP450催化烯丙基的羟基化^[6]; 二萜类衍生物如植物生长调节剂赤霉素的生物合成途径中, 由内-贝壳杉烯生成醛基-GA12的过程有5个连续的步骤都由P450催化^[7]; 在三萜皂苷的合成中, 更是有多个P450基因的参与^[8]。P450还参与多种植物中倍半萜的氧化生成, 棉花中的CYP706B1具有(+)δ-杜松烯-8-羟化酶活性, 催化棉酚生物合成的第二步反应, 是迄今为止第一个被分离的植物倍半萜羟化酶^[9]。另外, α-barbatene羟化酶CYP706A3^[10]、檀香烯氧化酶CYP76F39v1和香柠檬烯氧化酶CYP76F37v1^[11]也是催化倍半萜氧化的酶类。

沉香是沉香属白木香等物种由伤害诱导产生的含树脂木材, 是珍稀濒危南药和高价值的世界性资源。倍半萜和色酮类物质是沉香主要成分^[12-15]。在合成途径中, 倍半萜合酶催化FPP形成萜烯类中间产物, 然而, 沉香倍半萜终产物多为醇、酮类化合物, 说明萜烯类中间产物形成后下游还要经过一步或多步氧化修饰过程, 也就是需要加氧酶的参与才能形成沉香倍半萜终产物。CYP450家族蛋白可能参与沉香倍半萜合成的下游氧化步骤, 但迄今沉香属植物中CYP450的研究还鲜有报道。

为揭示伤害诱导沉香形成的分子机制, 课题组前期利用454测序技术对白木香转录组进行了分析^[16]。后续重点关注了沉香倍半萜的生物合成与调控相关基因, 分析了CYP450基因响应MeJA处理的表达特性, 发现序列Cluster57402.seq. Singlet1受MeJA诱导表达, 推测其可能参与沉香倍半萜的合成。本研究对该基因的部分cDNA片段进行了全长序列克隆, 并对其组织表达特性和编码蛋白的亚细胞定位进行研究, 为进一步揭示其功能及解析沉香倍半萜的生物合成途径奠定基础。

材料与方法

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试验材料及处理 中国医学科学院药用植物研究所温室栽培的健康三年生白木香由中国医学科学院药用植物研究所魏建和研究员鉴定, 用酒精消毒后的砍刀取生长良好的根、茎、叶, 取样后迅速放入液氮中冷冻, 置于-80 ℃冰箱中保存备用。

菌株、载体和试剂 瞬时表达载体pAN580为本实验室保存; 测序载体pEASY-Blunt Simple和大肠杆菌感受态Trans-T1均购自北京全式金生物技术有限公司; RNA提取纯化试剂盒购于Aidlab公司; Super SMART^TM PCR cDNA合成试剂盒、Advantage^® 2 PCR试剂盒均购自Clontech公司; MMLV逆转录酶、Oligod(T)₁₈、2×SYBR Premix Ex Taq^TM SpeI/BamHI和T4连接酶均购自TaKaRa公司。

RNA提取 利用RNA提取纯化试剂盒提取总RNA。1%琼脂糖凝胶电泳检测RNA完整性, 分光光度计NanoDrop 2000C (Thermo Scientific, USA) 测定RNA浓度。

cDNA第一链的合成 cDNA末端快速扩增法(rapid-amplification of cDNA ends, RACE) cDNA的合成: 取约2 μg RNA利用Super SMART^TM PCR cDNA合成试剂盒进行反转录, 利用Advantage^® 2 PCR试剂盒扩增。实时荧光定量PCR分析样品的cDNA的合成: 取约1 μg RNA, 以MMLV为逆转录酶, 以oligod (T)₁₈为引物, 反转录合成cDNA第一链。

5′-RACE和3′-RACE 454测序得到的unigene (Cluster57402.seq. Singlet1) 预测为含部分开放阅读框(open reading frame, ORF) 序列的CYP450基因, 属于CYP71D亚族, 序列比对发现ORF 5′和3′端都不完整。根据已知的ORF序列片段, 利用primer5.0软件设计特异性引物(表 1), 进行5′-RACE和3′-RACE。RACE方法参照文献^{[17, 18]}。将RACE测序结果与454测序结果拼接, 得到能够完整翻译的基因全长。RACE用到的引物见表 1。

长距离PCR (long-distance PCR, LD-PCR) 扩增 根据拼接的结果设计LD-PCR引物(表 1), 以反转录的单链cDNA为模板, PCR验证拼接序列的正确性。扩增程序为94 ℃预变性5 min; 然后94 ℃ 30 s, 60 ℃ 30 s, 72 ℃ 2 min, 30个循环; 最后72 ℃延伸10 min。扩增产物电泳检测、回收、克隆和测序验证。

实时荧光定量PCR (quantitative real-time PCR, qRT-PCR) 为了明确AsCYP71D1基因在不同组织及不同伤害处理下的表达特性, 利用qRT-PCR进行表达量的检测。仪器采用伯乐的CFX96TM C1000 qRT-PCR检测系统。反应体系为: 2×SYBR Premix Ex Taq^TM 5 μL; 正反向引物浓度均为0.25 μL; cDNA模板0.3 μL; 加水补足至10 μL, 每个反应体系至少重复3次。扩增程序如下: 95 ℃ 30 s, 95 ℃ 5 s, 60 ℃ 20 s, 40个循环。qRT-PCR反应均以三磷酸甘油醛脱氢酶基因(GAPDH) 为内参^[19]。

AsCYP71D1-GFP瞬时表达载体的构建及转化在AsCYP71D1 ORF两端分别设计含有SpeI和BamHI位点的PCR引物, 扩增AsCYP71D1全长编码区。PCR产物回收后连接测序载体pEASY-Blunt Simple。对测序正确的克隆提取质粒, 用SpeI/BamHI双酶切回收目的片段, 用T4连接酶与同时双酶切回收的表达载体pAN580片段连接。将连接产物转化大肠杆菌感受态Trans-T1, 得到的单克隆分别进行菌液PCR和酶切2种方法鉴定。阳性克隆为构建正确的pAN580-AsCYP71D1表达载体。构建正确的质粒基因枪法转化洋葱表皮, 黑暗培养16 h后激光显微镜观察。

结果与分析

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1 AsCYP71D1基因的5′/3′-RACE及全长cDNA的克隆

根据已知序列, 设计了基因特异引物5′-f1和巢式引物5′-f2, 进行5′-RACE实验。扩增结果见图 1, 第一轮PCR得到了弥散条带(图 1A条带1), 随后以第一轮PCR扩增产物为模板, 进行第二轮巢式PCR, 得到一条清晰的条带(图 1A条带2)。3′-RACE步骤同上, 结果见图 1B条带3和条带4。将第二次的条带回收, 连接到pMD19-T载体, 并克隆测序。结果表明, 测序序列和已有的序列有重叠片段, 确认该克隆片段为目的基因的5′/3′延伸序列。将两端RACE片段与已知中间片段拼接后得到含有完整ORF的全长cDNA序列, 其5′-非翻译区(untranslated region, UTR) 为88 bp, 3′-UTR为344 bp并具有21 bp的polyA尾, ORF1 488 bp, 编码495个氨基酸。为进一步验证拼接序列的可靠性, 根据拼接的ORF序列, 设计LD-PCR引物, 结果扩增出了1 488 bp的特异条带(图 1C条带5), 测序结果与拼接序列完全一致。将该基因命名为AsCYP71D1, GenBank登录号为OP581932。

2 AsCYP71D1的生物信息学分析

利用ExPASy Proteomics Server的在线软件Protparam (http://www.cn.expasy.org/tools/protparam.html) 对AsCYP71D1蛋白的理化性质进行预测分析。推测其分子式为C₂₅₀₄H₃₉₅₇N₆₇₉O₇₂₀S₁₇, 分子质量为55.6 kD, 带正电残基(Asp+Glu) 为59, 负电残基(Arg+Lys) 为59, 脂肪系数为92.81, 等电点pI 7.17, 该蛋白的不稳定系数为39.88, 为稳定性蛋白, 平均亲水性系数为-0.157, 预测该蛋白为亲水蛋白。

NCBI BLAST在线比对显示, AsCYP71D1氨基酸序列与来自茶树(Camellia sinensis)、木薯(Manihot esculenta) 以及夏栎(Quercus robur) 中的蛋白(GenBank登录号分别为XP_028065313.1、XP_021603267.1、XP_050246566.1) 序列一致性最高, 达到73%以上, 推测具有相似功能。为了预测白木香AsCYP71D1的生物学功能, 利用DNAMAN软件将其氨基酸序列和检索到的与其序列一致性最高的其他氨基酸序列进行多重序列比对(图 2), 发现蛋白序列具有高度的保守性, 均具有血红素结合域FxxGxRxCxG、电子传递通道残基PERF和K螺旋中的ExxR残序, 序列一致性大于55%, 说明AsCYP71D1属于CYP71D亚家族。然而, 结果中序列一致性较高的蛋白均未有功能验证的报道, 其具体催化功能仍需进一步实验验证。图 3所示为由不同物种的16个P450蛋白构建的系统发育树, AsCYP71D1编码的蛋白与木薯来源的CYP71D10 (GenBank number: XP021603267.2) 聚为一支, 说明其亲缘关系较近, 进一步证明AsCYP71D1属于CYP71D亚家族。但与其他CYP71D家族成员相距较远, 暗示其可能还有区别于其他CYP71D家族成员的特殊功能。

3 AsCYP71D1基因的组织表达特异性分析

利用实时荧光定量PCR检测AsCYP71D1基因的组织表达特异性, 结果表明该基因在茎中的表达量最高, 其次是叶片、花和根中(图 4), 说明AsCYP71D1可能主要在茎中发挥生物学功能。白木香受到伤害后主要在茎中合成倍半萜和色酮类化合物, 推测AsCYP71D1除了可能参与倍半萜的生物合成外, 还可能与植物的生长发育密切相关。

4 AsCYP71D1-GFP瞬时表达载体的构建

菌液PCR鉴定(图 5A) 及双酶切鉴定(图 5B) 结果表明, AsCYP71D1基因已连接到表达载体pAN580上, 进一步经过测序验证该瞬时表达载体构建是正确的。

5 AsCYP71D1-GFP质粒轰击洋葱表皮及荧光观察

将构建好的重组质粒pAN580-AsCYP71D1-GFP与金粉微载体混合制成金粉-DNA的复合体。1.1 kPa压力下, 在基因枪的轰击作用下, 将质粒快速转入MS培养的洋葱表皮细胞, 进行16~24 h的黑暗培养。在激光共聚焦显微镜蓝色光激发下观察, 发现洋葱表皮细胞核和细胞膜都有很强的绿色荧光发出(图 6), 证明AsCYP71D定位在细胞膜、细胞核和细胞质中。

讨论

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沉香具有很高的药用价值和经济价值, 倍半萜类化合物是其主要活性成分之一。倍半萜类化合物上游合成途径已经清晰, 然而其下游途径的关键酶还研究甚少。P450是一类含有血红素配基的多功能酶, 具有广泛的催化活性, 主要涉及萜类、生物碱类、甾醇类、黄酮/异黄酮类、植物激素等的合成和代谢, 在植物体内起着重要作用。对参与次生代谢的CYP450基因的分离、筛选和功能鉴定一直是研究的热点和难点问题。

基于白木香转录组数据, 本研究利用RT-PCR和RACE技术获得CYP450 AsCYP71D1的全长cDNA序列。多序列比对发现AsCYP71D1编码的蛋白中含有三个保守基序, 分别为: 血红素结合域FxxGxRxCxG、电子传递通道残基PERF和K螺旋中的ExxR残序。已有的研究表明, 在所有已知的P450蛋白中, 都含有一个保守的血红素结合域FxxGxRxCxG及螺旋K区的ExxR序列, 这也是鉴定P450的主要特征序列。说明本研究分离的AsCYP71D1编码的蛋白属于P450家族酶。系统发育树分析显示AsCYP71D1编码的蛋白属于CYP71D亚家族, 与木薯来源的CYP71D10聚为一支, 推测可能具有相似的功能, 然而木薯来源的CYP71D10蛋白暂未有功能验证的报道, AsCYP71D1的具体催化功能仍需进一步实验验证。根据Pateraki等^[20]的报道, 倍半萜类物质的合成通常由CYP71和/或CYP76家族的P450s参与, 推测AsCYP71D1有可能参与沉香倍半萜的下游合成反应。P450基因的表达调控主要在转录水平, 并具有组织器官的特异性。本研究中AsCYP71D1主要在茎中表达, 而且响应MeJA诱导。在健康组织中几乎不表达, 受MeJA诱导后表达量迅速升高, 这与之前鉴定的倍半萜合酶基因的表达具有相似的表达特性^[21], 表明其可能参与沉香倍半萜生物合成。CYP450多定位于细胞质^{[22, 23]}, 也有研究表明, 部分CYP450定位在细胞膜和细胞核, 如苦蘵中CYP450基因comp152181编码的蛋白在细胞质、细胞膜和细胞核中均有表达^[24], 这和AsCYP71D1编码蛋白相一致。

这些发现有助于进一步对参与沉香倍半萜生物合成的CYP450基因进行功能鉴定。虽然随着测序技术的进步, 很多物种中的P450基因家族被预测和注释出来, 但只有极少数的基因功能被鉴定, 它仍然是该领域研究的难点和焦点。这些酶基因功能鉴定的方法主要有两种, 一种是利用原核或真核表达系统异源表达酶蛋白, 进行体外催化反应, 以确定基因功能。另一种方法就是利用转基因技术, 分析转基因植株的化学成分变化; 或者是合成生物学的手段, 利用酵母体系进行底盘改造, 检测化学成分的变化。然而, 由于很多次生代谢合成途径并不完全清晰, 很难获得相应的底物和产物, 以及很多非模式植物缺乏转基因体系等, 诸多因素限制了P450基因功能的发掘。因此, 从数量众多的P450家族中鉴定一个基因的功能仍然存在极大的难度和挑战性。

作者贡献: 高世玺执行本项工作的实验、数据分析和论文初稿的撰写; 戎梅参与实验和数据分析; 彭俊祥参与实验材料取样和RNA提取; 徐艳红和魏建和负责实验设计、文章修改和课题资助。

利益冲突: 所有作者声明不存在任何利益冲突。

基金

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北京市自然基金项目(7222286)
国家自然科学基金项目(81573525)
中国医学科学院医学与健康科技创新工程--重大协同创新项目(2021-1-12M-032)

参考文献

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2023年第58卷第10期

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doi: 10.16438/j.0513-4870.2023-0404

接收时间：2023-04-03
首发时间：2025-11-21
出版时间：2023-10-12

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收稿日期：2023-04-03
修回日期：2023-07-07

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北京市自然基金项目(7222286)

国家自然科学基金项目(81573525)

中国医学科学院医学与健康科技创新工程--重大协同创新项目(2021-1-12M-032)

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1.中国医学科学院、北京协和医学院, 药用植物研究所, 中草药物质基础与资源利用教育部重点实验室, 濒危药材繁育国家工程实验室, 北京 100193

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*徐艳红, Tel/Fax: 86-10-57833359, 86-10-57833358, E-mail: xuyanhong99@163.com;

魏建和, E-mail: wjianh@263.net

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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