药学学报

Gene name	Forward primer sequence (5′-3′)	Reverse primer sequence (5′-3′)	Annealing temperature/℃
ACTIN	TTGTGTTGGACTCTGGTGATGG	CCGTTCAGCAGTGGTGGTG	60
PGK	TCGCTCTTGGAGAAGGTTGAC	CTTGTCCGCAATCACTACATCAG	59.5
rolB	GTCTGCTATCATCCTCCTA	GATATGGTTGAATCGTTGTC	58.8
PMT	CCTACTTACCCTACTGGTGTTATC	GCGAAAGATGGCAAAATAAAAGC	56.4
TRI	CCTGCTGCTTCTTATATTACGG	GCCCAAGGTACAAATCACAA	57
H6H	TTCCACTTGAGCAGAAAGCAAAGC	CCTCATGGTCAACTTCCTCACTTCC	56.4
ArAT4	CCTCTGCTTTCTCTATTTTGCATTG	TTTGTTTCATGATCATTCCCTTTCT	57
PYKS	TCACCTCGCATTACACCTAC	GCATTACCTCCTGGATGAAGAA	60
UGT1	CCATCTAATCCATATCCTGCCTTA	TCAGCACGAACGACTTGT	57
TRⅡ	AGCACTGGATCAACTCACAAGATG	TTAGGCTCTCCCATTCGGTGTAG	61.4

Gene name	Forward primer sequence (5′-3′)	Reverse primer sequence (5′-3′)	Annealing temperature/℃
ACTIN	TTGTGTTGGACTCTGGTGATGG	CCGTTCAGCAGTGGTGGTG	60
PGK	TCGCTCTTGGAGAAGGTTGAC	CTTGTCCGCAATCACTACATCAG	59.5
rolB	GTCTGCTATCATCCTCCTA	GATATGGTTGAATCGTTGTC	58.8
PMT	CCTACTTACCCTACTGGTGTTATC	GCGAAAGATGGCAAAATAAAAGC	56.4
TRI	CCTGCTGCTTCTTATATTACGG	GCCCAAGGTACAAATCACAA	57
H6H	TTCCACTTGAGCAGAAAGCAAAGC	CCTCATGGTCAACTTCCTCACTTCC	56.4
ArAT4	CCTCTGCTTTCTCTATTTTGCATTG	TTTGTTTCATGATCATTCCCTTTCT	57
PYKS	TCACCTCGCATTACACCTAC	GCATTACCTCCTGGATGAAGAA	60
UGT1	CCATCTAATCCATATCCTGCCTTA	TCAGCACGAACGACTTGT	57
TRⅡ	AGCACTGGATCAACTCACAAGATG	TTAGGCTCTCCCATTCGGTGTAG	61.4

Name	Leaf length/cm	Leaf width/cm	Leaf fresh weight/g	Flower diameter/cm
WT	14.5 ± 0.38	6.1 ± 0.29	0.9 ± 0.1	2.0 ± 0.12
B-1	20.0 ± 0.87^**	11.3 ± 0.74^**	4.1 ± 0.19^**	2.6 ± 0.09^**
B-2	19.5 ± 0.75^**	10.2 ± 0.21^**	3.9 ± 0.18^**	3.0 ± 0.09^**
B-3	20.0 ± 1.78^*	10.8 ± 0.54^**	3.7 ± 0.67^*	2.7 ± 0.23^**

Name	Leaf length/cm	Leaf width/cm	Leaf fresh weight/g	Flower diameter/cm
WT	14.5 ± 0.38	6.1 ± 0.29	0.9 ± 0.1	2.0 ± 0.12
B-1	20.0 ± 0.87^**	11.3 ± 0.74^**	4.1 ± 0.19^**	2.6 ± 0.09^**
B-2	19.5 ± 0.75^**	10.2 ± 0.21^**	3.9 ± 0.18^**	3.0 ± 0.09^**
B-3	20.0 ± 1.78^*	10.8 ± 0.54^**	3.7 ± 0.67^*	2.7 ± 0.23^**

rolB基因对颠茄表型发育和托品烷生物碱合成的影响

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敖雯雯 , 谭艾娟 , 马炳南 , 穆德会 , 路星星 , 潘航 , 张明生 , 强玮 ^*

药学学报 | 研究论文 2023,58(6): 1705-1712

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药学学报 |研究论文 2023 , 58 (6) : 1705 -1712

rolB基因对颠茄表型发育和托品烷生物碱合成的影响

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敖雯雯, 谭艾娟, 马炳南, 穆德会, 路星星, 潘航, 张明生, 强玮^*

作者信息

贵州大学生命科学学院/农业生物工程研究院, 山地植物资源保护与种质创新教育部重点实验室, 贵州贵阳 550025

通讯作者:

*强玮, Tel: 13678505784, E-mail: wayneqiang@126.com

Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna

Wen-wen AO, Ai-juan TAN, Bing-nan MA, De-hui MU, Xing-xing LU, Hang PAN, Ming-sheng ZHANG, Wei QIANG^*

Affiliations

Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), College of Life Sciences/Institute of Agro-bioengineering, Guizhou University, Guiyang 550025, China

出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1190

文章导航

摘要

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发根农杆菌pRiA4质粒上的rol基因是促进次生代谢的强效基因, 可利用rol基因对颠茄进行分子育种以提升颠茄中托品烷生物碱(tropane alkaloids, TAs) 含量。本研究将rolB基因在颠茄植株中进行超表达, 为了研究rolB基因对颠茄TAs生物合成的影响, 分析了转基因颠茄的表型、TAs含量及TAs合成途径中关键酶基因的表达水平。结果表明, 转基因颠茄根系发达, 叶片增大、叶片鲜重增加、叶片颜色加深, 花朵增大、花形改变, 雌蕊高度降低, 花粉活力下降。转基因颠茄茎中TAs的含量显著高于对照, 东莨菪碱、山莨菪碱、莨菪碱的含量分别为对照的2.11~2.91、1.23~2.37、4.88~5.20倍。与对照组相比, TAs合成途径中的关键酶基因N-甲基腐胺转移酶(PMT)、吡咯烷聚酮合酶(PYKS)、托品酮还原酶I (TRI)、芳香族氨基酸氨基转移酶4 (ArAT4)、UDP-糖基转移酶1 (UGT1) 和莨菪碱6-β-羟化酶(H6H) 的表达上调, 且托品酮还原酶Ⅱ (TRⅡ) 作为代谢分流基因表达下调。研究表明rolB基因通过增强TAs合成途径代谢流和减弱竞争支路代谢分流, 增强了颠茄根中TAs的合成能力和茎中的积累。本研究为利用rolB基因进行高产TAs颠茄的分子育种奠定了基础。

关键词

rolB基因 / 颠茄 / 东莨菪碱 / 山莨菪碱 / 莨菪碱

Abstract

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The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type Ⅲ polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase Ⅱ (TRⅡ) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

Key words

rolB gene / Atropa belladonna L. / scopolamine / anisodamine / hyoscyamine

引用本文

敖雯雯, 谭艾娟, 马炳南, 穆德会, 路星星, 潘航, 张明生, 强玮. rolB基因对颠茄表型发育和托品烷生物碱合成的影响. 药学学报, 2023 , 58 (6) : 1705 -1712 . DOI: 10.16438/j.0513-4870.2022-1190

Wen-wen AO, Ai-juan TAN, Bing-nan MA, De-hui MU, Xing-xing LU, Hang PAN, Ming-sheng ZHANG, Wei QIANG. Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1705 -1712 . DOI: 10.16438/j.0513-4870.2022-1190

正文

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颠茄(Atropa belladonna L.) 是茄科颠茄属多年生草本植物, 现代药学科学已揭示, 颠茄能产生具有重要医药价值的托品烷生物碱(tropane alkaloids, TAs), 包括莨菪碱(hyoscyamine)、东莨菪碱(scopolamine) 和药效稍低的山莨菪碱(anisodamine)。三种TAs作为抗胆碱药物在临床上被广泛使用, 市场需求巨大^[1]。莨菪碱可用于治疗心律失常、有机磷中毒、各种神经痛等; 山莨菪碱可用于治疗感染中毒性休克、血管痉挛、胃肠道绞痛等; 东莨菪碱药效最强, 毒副作用最弱, 可用于治疗帕金森病、抗晕动、止痛解痉、治疗农药中毒等^[2-4]。野生颠茄中TAs含量极低, 莨菪碱大约仅为干重的0.02%~0.17%, 东莨菪碱更低, 约为0.01%~0.08%。TAs的低产量严重限制了其工业化生产及临床应用, 研究者们尝试通过遗传、辐射、多倍体育种、化学合成、悬浮细胞培养等多种方法以期获得高产TAs却都未取得成功^{[5, 6]}。2020年, Srinivasan等^[7]利用合成生物学手段实现了通过工程酵母发酵生产莨菪碱和东莨菪碱, 但其合成能力远未达到工业化生产水平。TAs的供应仍将长期依赖于从颠茄中提取。利用分子育种手段培育高产优质TAs颠茄是TAs产业的共同目标。

rol基因是发根农杆菌pRi质粒上的基因群, 已鉴定出4种类型的rol基因, rolA、rolB、rolC、rolD。研究发现, rol基因是茄科、五加科、茜草科、葡萄科和蔷薇科中次生代谢的潜在激活剂, 能促进次生代谢物显著积累^[8]。rolA和rolD研究较少, rolB和rolC均能诱导次生代谢, 且rolB是rol基因中次级代谢最强的诱导剂^[9]。rolC使得黄花蒿中总类黄酮含量增加25.5%、青蒿素含量提高110.2%、茜草中蒽醌含量提高4.3倍^{[10, 11]}。rolB可提高山葡萄愈伤中白藜芦醇的产量达100倍^[12], 黄花蒿中青蒿素最大提高9倍, 青蒿琥酯最大提高12倍^[13], 番茄中番茄红素含量增加62%、抗坏血酸含量增加225%^[8]。可见, 利用rol基因进行代谢工程研究是目前的研究热点。

颠茄作为《中华人民共和国药典》收录的唯一TAs商业药源, 被广泛用于TAs生物合成相关研究^{[14, 15]}。然而目前针对颠茄开展的代谢工程的靶标全部是TAs合成途径基因, 随着莨菪醛脱氢酶(hyoscyamine dehydrogenase, HDH) 的发现^[5], TAs合成途径得以完全阐明。寻找新的TAs遗传改造靶标以培育高产TAs颠茄对推进TAs工业化生产具有重要意义。本研究以rolB基因为靶标对颠茄进行遗传改造, 获得了pBI121-rolB转基因颠茄植株, 极大地提升了TAs的含量, 同时对转基因颠茄植株进行表型分析、TAs合成途径基因表达量检测, 为利用rol基因进行高产TAs颠茄分子育种的进一步研究奠定基础。

材料与方法

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植物材料 颠茄种子(Atropa belladonna L.) 繁育并保存于贵州大学植物生理生化与分子生物学实验室。

仪器高效液相色谱仪(Waters e2695, 沃特世公司); 实时荧光定量PCR仪(Bio-Rad CFX 96, 伯乐公司); 凝胶成像仪(上海培清科技有限公司); 核酸蛋白分析仪(Thermo NanoDrop 2000, 赛默飞公司)。

试剂总RNA提取试剂盒RNAsimple Total RNA Kit、质粒提取试剂盒TIAprep Mini Plasmid Kit、胶回收试剂盒TIANgel Midi Purification Kit (北京天根公司); 逆转录cDNA试剂盒RNA PCR^TM Kit AMV Ver 3.0、荧光定量反转录试剂盒PrimeScript^TM RT reagent Kit Perfect Real Time、荧光定量染料TB Green^Ⓡ premix Ex Taq^TM Ⅱ、各限制性内切酶(TaKaRa公司); 高保真HiFi聚合酶TransTaq DNA Polymerase High Fidelity (北京全式金公司); 重组试剂盒ClonExpress Ⅱ One Step Cloning Kit (南京诺唯赞公司); Phenomenex Gemini C18 110A色谱柱(上海岛津GL公司); 遗传转化所用激素及抗生素: 赤霉素(gibberellic acid, GA3)、6-苄氨基腺嘌呤(6-benzylaminopurine, 6-BA)、萘乙酸(1-naphthaleneaceticacid, NAA)、乙酰丁香酮(acetosyringone, AS)、玉米素(zeatin, ZT)、吲哚-3-乙酸(indole-3-acetic acid, IAA) 和3-吲哚丁酸(3-indolebutyric acid, IBA)、头孢霉素(cefradine, Cef)、利福平(rifampicin, Rif)、卡那霉素(kanamycin, Kan)、链霉素(streptomycin, Str) (索莱宝公司)。本研究所用引物及测序服务由重庆擎科生物科技有限公司提供, 所有引物序列见表 1。其他试剂均为分析纯国产试剂。

rolB基因的克隆及超表达载体的构建从NCBI上的GenBank中获取已发表的发根农杆菌中pRiA4质粒的序列(其中包含rolB基因编码序列, 序列号: X03433), 设计rolB基因的特异性重组引物, 分别在上下游引物5′端引入BamH I和Sac I酶切位点, 引物序列如下: F-rolB: ACGGGGGACTCTAGAGGATCCGC TCTTGCAGTGCTAGATTT, R-rolB: CGATCGGGGA AATTCGAGCTCGAAGGTGCAAGCTACCTCTC。

利用高保真HiFi酶从C58C1农杆菌中的pRiA4质粒上克隆获取rolB基因, 并用BamH I和Sac I酶切质粒pBI121从而获得载体骨架, 扩增和酶切产物分别经1%琼脂糖凝胶电泳检测并回收后, 重组并转化至大肠杆菌DH5α感受态细胞, 菌落PCR筛选阳性克隆, 送往重庆擎科公司测序, 将测序正确的菌液扩大培养, 提取质粒, 命名为pBI121-rolB, 将其转化根癌农杆菌EHA105, 将转化成功的工程菌命名为EHA105-pBI121-rolB, 保存于-80 ℃超低温冰箱备用。

遗传转化获得pBI121-rolB颠茄 颠茄遗传转化参照Song等^[16]的方法。选取适量饱满的颠茄种子, 用1 g·L^-1 GA3溶液浸泡24~48 h, 清水冲洗数次除净GA3溶液及种子表面杂质后, 转移至超净台中进行消毒处理。无菌水冲洗种子数次后, 75%酒精消毒种子1 min, 无菌水冲洗数次除净残余酒精, 再用50%次氯酸钠溶液消毒种子8~10 min, 无菌水反复冲洗除净残余次氯酸钠, 并用无菌滤纸吸干种子表面水分后将其接种至MS (Murashige & Skoog medium) + Cef (200 mg·L^-1) 固体培养基上, 25 ℃、16 h/8 h (light/dark) 条件下培养萌发, 待颠茄种子长出两片子叶后剪取子叶及下胚轴用于遗传转化外植体材料。

将预存于-80 ℃的转化工程菌在YEP (yeast extract peptone medium) + Rif (40 mg·L^-1) + Kan (50 mg·L^-1) + Str (25 mg·L^-1) 固体培养基上进行划线培养, 挑取单克隆接种于15 mL相应抗性YEP液体培养基中, 28 ℃、200 r·min^-1活化48 h, 按1∶50比例吸取1 mL活化好的菌液加入到含50 mL相应抗性的YEP液体培养基中进行二次活化24~48 h, 常规台式离心机5 000 r·min^-1离心6 min收集菌体, 并用转化共培培养基MS + AS (100 μmol·L^-1) 悬浮菌体, 调A₆₀₀到0.4~0.6, 继续适应培养30 min后即可用于转化。剪取颠茄的子叶及下胚轴作为外植体, 在含有EHA105-pBI121-rolB工程菌的MS + AS (100 μmol·L^-1) 培养基中浸泡大约8 min, 无菌滤纸吸干外植体材料的残余菌液, 并将其转移至铺有无菌滤纸的共培培养基MS + 6-BA (1 mg·L^-1) + NAA (1 mg·L^-1) + AS (100 μmol·L^-1) 上, 25 ℃共培养4天后将外植体材料转移至除菌和再生培养基MS + ZT (1 mg·L^-1) + IAA (0.5 mg·L^-1) + Cef (400 mg·L^-1) + Kan (100 mg·L^-1), 25 ℃、16 h/8 h (light/dark) 光照条件下进行培养, 5天左右转接1次培养基, 筛选抗性芽。待外植体材料长出带抗性芽和芽点的抗性愈伤即转移到生根培养基MS + IBA (0.2 mg·L^-1) + Cef (400 mg·L^-1) 中继续培养直至长成完整植株。在组培瓶中继代扩培获得的pBI121-rolB转基因颠茄, 待其长势一致后移栽至室内育苗钵中生长2月后进行后续分析。

pBI121-rolB颠茄的表型记录及分析 观察pBI121-rolB颠茄生长各时期表型特征并拍照记录, 待将其移栽到温室进行生长至2月左右, 测量其叶长、叶宽、节间长及叶鲜重, 并利用IBM SPSS Statistics 26软件对记录的上述表型数据进行分析。

生物碱提取及HPLC检测 分别采取同时期生长2个月的颠茄茎、叶, 40 ℃烘干, 打磨成粉末, 过50目筛后称取0.2 g样品粉末进行生物碱提取, 具体方法参照Fattahi等^[17]进行, 稍有改动。利用HPLC进行生物碱含量检测。每个株系选取3个生物学重复, 混合取样处理, 分别检测东莨菪碱、莨菪碱及山莨菪碱的含量。色谱柱为Phenomenex Gemini 5u C18 110A液相色谱柱(250 mm × 4.6 mm); 流动相为甲醇乙腈等比例混合液∶乙酸铵缓冲液(0.1%甲酸, pH 4.0) = 17∶83 (v/v); 检测波长为215 nm; 柱温40 ℃; 流速为1.0 mL·min^-1; 检测时间20 min, 进样量为20 μL。

总RNA的提取及cDNA的合成取适量颠茄的须根洗净立即液氮速冻, 冰上快速研磨成粉末, 按照RNAsimple Total RNA Kit说明书提取总RNA, 1%琼脂糖凝胶电泳检测RNA完整性, NanoDrop 2000核酸蛋白分析仪检测其A₂₆₀/A₂₈₀及A₂₆₀/A₂₃₀以分析RNA的浓度及纯度, 将质量合格的RNA按照TaKaRa PrimeScript^TM RT reagent Kit (Perfect Real Time) 试剂盒说明书进行反转录, 每个样品反转录500 ng RNA以合成cDNA第一链, 反转录得到的cDNA保存于-20 ℃备用。

基因表达量检测 将按照荧光定量反转录试剂盒反转录得到的cDNA进行稀释5倍, 作为荧光定量PCR的模板, 使用Bio-Rad CFX 96 Real Time PCR仪, 参照TB Green^Ⓡ premix Ex Taq^TM Ⅱ说明书进行qPCR反应。反应体系为: TB Green Premix EX Taq (2×) 10 μL、Forward和Reverse Primer各0.8 μL、ddH₂O 6.4 μL、模板cDNA 2 μL, 每个样品设置3个重复, 反应程序为95 ℃预变性30 s, 95 ℃变性5 s, 适当温度退火30 s (在此收集荧光信号), 循环数40; PCR反应结束后, 95 ℃ 15 s, 60 ℃ 60 s, 95 ℃ 30 s, 60 ℃ 15 s。以ACTIN (序列号: JX154681) 和PGK (序列号: JX154676) 为内参基因, 检测各TAs合成途径基因的表达量。采用比较Ct值法2^-ΔΔCt法计算各基因的相对表达量。

数据分析及制图 利用Excel 2019对各数据进行处理, IBM SPSS Statistics 26软件对所有实验数据进行统计学分析, Dunnett's multiple comparisons test法对各数据进行多重比较, GraphPad Prism 6.0软件进行制图。

结果与分析

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1 pBI121-rolB超表达载体构建

利用rolB基因特异性重组引物, 以C58C1菌液作为模板, 用高保真酶HiFi酶扩增得到rolB基因, 片段长度为780 bp (图 1A), 用BamH I和Sac I酶切pBI121载体获得大骨架, 利用胶回收试剂盒进行纯化后重组并转化至大肠杆菌感受态细胞DH5α, 经菌落PCR鉴定并测序验证, 从而成功获得超表达载体pBI121-rolB (图 1B)。

2 rolB转基因颠茄的获得

以萌发15天左右长出两片子叶的颠茄幼苗为材料进行遗传转化, 将外植体在EHA105-pBI121-rolB工程菌液中侵染8 min, 无菌滤纸吸干外植体表面残余菌液后转移至共培培养基上共培4天(图 2A), 然后转移至含有100 mg·L^-1 Kan的再生和筛选培养基上, 待其长出抗性芽(图 2B), 将带有抗性芽或芽点的愈伤转移到含有0.2 mg·L^-1 IBA的MS固体培养基上诱导其生根(图 2C), 待其长成完整植株后进行继代扩培。将长势一致的转基因颠茄苗移栽至温室(图 2D), 剪取颠茄幼叶提取DNA, PCR鉴定转基因阳性颠茄植株。选取3个转基因阳性株系B-1、B-2、B-3进行后续分析。

3 rolB转基因颠茄的表型分析

对温室培养2个月的rolB转基因颠茄进行表型分析, 发现pBI121-rolB颠茄3个株系和对照WT颠茄相比, 株高、分支数、节间长、主茎粗等表型均无显著差异(图 3A~C), 但叶片、根系及花朵则出现了显著差异。pBI121-rolB颠茄根系更为茂密发达(图 3D), 叶片颜色加深呈深绿色, 叶长、叶宽、叶鲜重显著增加(表 2), 且出现了皱缩的性状(图 3E)。此外, pBI121-rolB颠茄花朵体形增大、花径变大(表 2)、花冠凹陷, 雌蕊高度降低, 且花朵形状也发生了改变, 野生型颠茄花冠呈钟形, 而转rolB颠茄花冠呈坛形(图 3F)。

4 托品烷生物碱含量测定

利用HPLC对生长2个月的转基因和对照颠茄茎、叶中的生物碱含量进行检测, 结果如图 4。pBI121-rolB颠茄和对照WT颠茄相比, 茎中生物碱含量均大量提高, 叶中生物碱含量对比无显著差异。pBI121-rolB颠茄叶片中, 除个别株系和个别种类生物碱含量有提高外, 总体上无提高。东莨菪碱仅B-3株系的含量[166.87 μg·g^-1 DW (dry weight)] 是对照(76.06 μg·g^-1 DW) 的2.19倍; 莨菪碱仅B-2株系的含量(425.32 μg·g^-1 DW) 是对照(337.88 μg·g^-1 DW) 的1.26倍; 山莨菪碱均没有提高。而pBI121-rolB颠茄茎中除B-3株系的山莨菪碱含量没有提高外, 各株系东莨菪碱、山莨菪碱、莨菪碱含量和对照相比均显著提高。东莨菪碱含量相比对照(29.02 μg·g^-1 DW) 分别提高2.11~2.91倍, B-2株系含量(84.5 μg·g^-1 DW) 提高最大; 山莨菪碱含量相比对照(23.27 μg·g^-1 DW) 提高1.23~2.37倍, B-1株系含量(55.23 μg·g^-1 DW) 提高最大; 莨菪碱含量相比对照(101.75 μg·g^-1 DW) 分别提高4.88~5.20倍, B-3株系含量(528.86 μg·g^-1 DW) 提高最大。

5 TAs合成途径基因表达量检测

TAs在颠茄须根中合成, 相关合成途径基因均特异性在须根中表达。以各株系颠茄须根为材料, 利用荧光定量PCR分析pBI121-rolB颠茄中rolB基因及TAs合成途径基因的表达情况。pBI121-rolB颠茄3个株系中均检测到rolB基因高水平表达, 而对照WT颠茄中未检测到rolB基因表达, 表明B-1、B-2、B-3三个株系均为rolB基因超表达颠茄(图 5)。

检测了转基因株系及WT颠茄须根中6个TAs合成途径基因和1个竞争支路基因的表达水平, 结果见图 6。rolB基因显著上调了3个转基因株系中PMT、PYKS、TRI、ArAT4、UGT1和H6H共6个TAs合成途径酶基因的表达量, 其中PMT除B-2株系外, B-1和B-3株系分别提高3.54和3.70倍; PYKS除B-2株系外, B-1和B-3株系分别提高3.95和3.0倍; TRI基因上调1.55~2.38倍; ArAT4除B-3株系外, B-1和B-2株系分别提高2.36和2.13倍; UGT1基因上调5.82~14.30倍; H6H基因上调4.34~11.8倍。TRⅡ基因作为TAs合成途径中游的分流基因, 将底物托品酮还原为假托品, 进一步生成TAs代谢副产物。TRⅡ基因表达量在转基因颠茄中显著下调, 为对照的18%~22%。

讨论

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TAs来源于少数茄科植物, 其中的莨菪碱(其外消旋体为阿托品) 和东莨菪碱作为抗胆碱药物具有重要的临床价值。发根农杆菌中rol基因由于能诱导植物产生发根, 而且合成高于母体植物数倍的次生代谢产物而广受关注^[18]。在研究较为充分的4个rol基因(rolA、rolB、rolC和rolD) 中, 已证实rolB和rolC基因是促进次生代谢的强效基因, 通常rolB的作用最强^[19]。在茜草、山葡萄、青蒿、拟南芥和番茄中, rolB分别促进了蒽醌、白藜芦醇、青蒿素、芥子油甙、番茄红素和酚类化合物等化合物的大量合成和积累^{[20, 21]}。利用rol基因作为颠茄分子育种的靶基因, 提升颠茄中TAs含量在代谢工程研究和TAs产业的发展中具有重要意义。

rolB基因作为古老的Plast基因家族成员, 在植物中具有多方面的作用, 已报道的有改变植物生长发育、调节激素和活性氧含量、影响植物免疫和胁迫响应、增强光合能力和促进次生代谢^[22]。Arshad等^[8]和Schmülling等^[23]报道了rolB转基因番茄植株高度降低、节间长度变短、果实变小, 同时引起转基因植株叶片及花朵增大、叶片皱缩、柱头增大、生根能力增强、顶端优势下降、花粉活力降低等表型, 本研究中rolB超表达颠茄也发生了部分类似的表型改变但不完全相同。rolB颠茄表现出根系发达, 叶片变大、颜色加深, 花朵形状改变及体积增大等优于野生型植株的园艺性状, 同时不影响植物的株高、节间长、分支数、主茎粗和果实大小。在繁育子一代rolB颠茄时发现, rolB颠茄虽可育, 但花粉活力下降, 种子体积增大, 表现为可育性降低。rolB可刺激多种器官(包括芽、根和花) 的分生组织形成, 在植物形态发生中发挥作用^[24]。rolB蛋白在体外被报道具有β-葡萄糖苷酶和酪氨酸磷酸酶活性, 能诱导转化细胞对生长素的敏感性, 可干扰激素的信号转导途径, 从而影响转化细胞的生理模式^[25]。这可能是导致转基因植株产生表型变异的原因。

有趣的是, 近期Lu等^[26]的研究中, 另一个rol基因rolC导致颠茄出现植株矮小、节间缩短、腋芽丛生、叶片狭长、开花提前且花朵变小、雄性不育等特征。可见不同的rol基因对同一物种引起的表型变化也有差异。但rolB和rolC均使得颠茄地下部分顶端优势下降, 须根生物量增大, 而须根作为TAs生物合成的场所^[27], 其生物量的增大有助于提高TAs的合成与积累。rolB还导致颠茄叶片的叶长、叶宽、叶鲜重增加, 而叶片作为颠茄中TAs的储藏器官之一, 其大小的增加也更有利于TAs的积累。综合来看, rolB基因的导入引起颠茄地上部分表型改变相较rolC基因导致的变异小, 且稍优于野生型, 有利于提高TAs在地上部分的积累量。

颠茄地上部分茎和叶是生产上的主要采收部位, rolB超表达没有影响叶中TAs的含量却极大提高了茎中3种TAs的积累, 尤其是莨菪碱含量提高了4.88~5.20倍。TAs主要在颠茄须根中合成, 经过长距离运输和跨膜转运在茎叶果中积累, 嫩茎、嫩叶和果萼等器官中含量最高^{[27, 28]}。rolB颠茄茎中生物碱含量积累显著提高而叶片中无显著变化应与TAs在地上部分的转运有关, 可能存在一个茎特异的跨膜转运蛋白被rolB特异性地激活。尼古丁的合成和积累过程与TAs类似, 在烟草根细胞中合成, 通过木质部向上转运至叶片中积累。目前已发现若干个尼古丁转运蛋白, 大多也能非特异性转运莨菪碱、小檗碱等生物碱^{[29, 30]}。而关于TAs在颠茄中的运输及转运蛋白研究目前还较少, 是否存在茎特异性的TAs转运和储存还有待深入研究。

rolB促进植物次生代谢的机制目前还未充分阐明。较多的研究集中在rolB降低了细胞中活性氧的积累^[22], 最新的报道显示拟南芥中rolB超表达激活了水杨酸和活性氧信号系统, 两者协同诱导了黄酮醇特异性转录因子MYB11的表达, 进而转录激活CHS和F3H基因的表达, 促进黄酮类化合物的合成^[31]。本研究发现rolB超表达促进了颠茄PMT、PYKS、TRI、ArAT4、UGT1和H6H这些TAs合成途径基因的表达, 降低了竞争途径TRⅡ基因的表达, UGT1和H6H分别最大提高11.8和14.3倍, 提高最为显著。PMT作为第一个限速酶, 其表达量的提高可为下游生物碱的合成提供更多的前体物质, 而H6H作为TAs合成途径的最下游基因, 可有效促进东莨菪碱的合成, 这两个基因对TAs合成的影响十分重要。Wang等^[4]和Xia等^[28]均利用PMT和H6H共同转化颠茄, 极大促进了颠茄中东莨菪碱的合成, 同时证实PMT和H6H的表达量与东莨菪碱的积累呈正相关。TRⅡ是合成途径中游和TRI竞争底物的支路途径基因, 诱导生成副产物, 其表达量降低有利于TAs的积累。总之, rolB基因主要通过上调合成途径基因表达和抑制竞争途径基因表达来提高颠茄TAs的合成。

综上, 本研究利用rolB基因为靶标对颠茄进行遗传改造得到rolB超表达颠茄。rolB基因的引入造成颠茄根和叶发生有益于生产的园艺形状改变, 同时通过上调PMT、PYKS、TRI、UGT1、ArAT4和H6H这6个TAs合成途径基因, 下调竞争支路TRⅡ基因表达从而提高颠茄根中东莨菪碱、山莨菪碱和莨菪碱的合成和在茎中的积累。本研究为基于rol基因进行颠茄分子育种研究奠定基础, 对深入探究rol基因作用机制和培育TAs高产的颠茄新品种具有重要意义。

作者贡献: 强玮、谭艾娟和张明生设计了本实验; 马炳南、穆德会和潘航构建了植物表达载体; 敖雯雯和路星星完成了颠茄的遗传转化、种植及相关指标的记录及检测; 敖雯雯完成了数据分析和论文初稿的写作; 强玮、谭艾娟、张明生共同指导论文的写作与修改。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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国家自然科学基金资助项目(32200225)
贵州省省级科技计划项目(黔科合基础-ZK [2022] 一般096)
贵州大学培育项目(贵大培育[2019] 51号)
贵州大学引进人才科研项目(贵大人基合字[2017] 58)
贵州省中药材现代产业技术体系建设项目(GZCYTX-02)
国家喀斯特石漠化防治工程技术研究中心建设项目(2012FU125X13)
贵州省生物学一流学科建设项目(GNYL [2017] 009)

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2023年第58卷第6期

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doi: 10.16438/j.0513-4870.2022-1190

接收时间：2022-11-07
首发时间：2025-11-21
出版时间：2023-06-12

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收稿日期：2022-11-07
修回日期：2022-12-29

基金

国家自然科学基金资助项目(32200225)

贵州省省级科技计划项目(黔科合基础-ZK [2022] 一般096)

贵州大学培育项目(贵大培育[2019] 51号)

贵州大学引进人才科研项目(贵大人基合字[2017] 58)

贵州省中药材现代产业技术体系建设项目(GZCYTX-02)

国家喀斯特石漠化防治工程技术研究中心建设项目(2012FU125X13)

贵州省生物学一流学科建设项目(GNYL [2017] 009)

作者信息

贵州大学生命科学学院/农业生物工程研究院, 山地植物资源保护与种质创新教育部重点实验室, 贵州贵阳 550025

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*强玮, Tel: 13678505784, E-mail: wayneqiang@126.com

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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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TxT

Gene name	Forward primer sequence (5′-3′)	Reverse primer sequence (5′-3′)	Annealing temperature/℃
ACTIN	TTGTGTTGGACTCTGGTGATGG	CCGTTCAGCAGTGGTGGTG	60
PGK	TCGCTCTTGGAGAAGGTTGAC	CTTGTCCGCAATCACTACATCAG	59.5
rolB	GTCTGCTATCATCCTCCTA	GATATGGTTGAATCGTTGTC	58.8
PMT	CCTACTTACCCTACTGGTGTTATC	GCGAAAGATGGCAAAATAAAAGC	56.4
TRI	CCTGCTGCTTCTTATATTACGG	GCCCAAGGTACAAATCACAA	57
H6H	TTCCACTTGAGCAGAAAGCAAAGC	CCTCATGGTCAACTTCCTCACTTCC	56.4
ArAT4	CCTCTGCTTTCTCTATTTTGCATTG	TTTGTTTCATGATCATTCCCTTTCT	57
PYKS	TCACCTCGCATTACACCTAC	GCATTACCTCCTGGATGAAGAA	60
UGT1	CCATCTAATCCATATCCTGCCTTA	TCAGCACGAACGACTTGT	57
TRⅡ	AGCACTGGATCAACTCACAAGATG	TTAGGCTCTCCCATTCGGTGTAG	61.4

Gene name

Forward primer sequence (5′-3′)

Reverse primer sequence (5′-3′)

Annealing temperature/℃

ACTIN

TTGTGTTGGACTCTGGTGATGG

CCGTTCAGCAGTGGTGGTG

PGK

TCGCTCTTGGAGAAGGTTGAC

CTTGTCCGCAATCACTACATCAG

59.5

rolB

GTCTGCTATCATCCTCCTA

GATATGGTTGAATCGTTGTC

58.8

PMT

CCTACTTACCCTACTGGTGTTATC

GCGAAAGATGGCAAAATAAAAGC

56.4

TRI

CCTGCTGCTTCTTATATTACGG

GCCCAAGGTACAAATCACAA

H6H

TTCCACTTGAGCAGAAAGCAAAGC

CCTCATGGTCAACTTCCTCACTTCC

56.4

ArAT4

CCTCTGCTTTCTCTATTTTGCATTG

TTTGTTTCATGATCATTCCCTTTCT

PYKS

TCACCTCGCATTACACCTAC

GCATTACCTCCTGGATGAAGAA

UGT1

CCATCTAATCCATATCCTGCCTTA

TCAGCACGAACGACTTGT

TRⅡ

AGCACTGGATCAACTCACAAGATG

TTAGGCTCTCCCATTCGGTGTAG

61.4

Name	Leaf length/cm	Leaf width/cm	Leaf fresh weight/g	Flower diameter/cm
WT	14.5 ± 0.38	6.1 ± 0.29	0.9 ± 0.1	2.0 ± 0.12
B-1	20.0 ± 0.87^**	11.3 ± 0.74^**	4.1 ± 0.19^**	2.6 ± 0.09^**
B-2	19.5 ± 0.75^**	10.2 ± 0.21^**	3.9 ± 0.18^**	3.0 ± 0.09^**
B-3	20.0 ± 1.78^*	10.8 ± 0.54^**	3.7 ± 0.67^*	2.7 ± 0.23^**

Name

Leaf length/cm

Leaf width/cm

Leaf fresh weight/g

Flower diameter/cm

14.5 ± 0.38

6.1 ± 0.29

0.9 ± 0.1

2.0 ± 0.12

B-1

20.0 ± 0.87^**

11.3 ± 0.74^**

4.1 ± 0.19^**

2.6 ± 0.09^**

B-2

19.5 ± 0.75^**

10.2 ± 0.21^**

3.9 ± 0.18^**

3.0 ± 0.09^**

B-3

20.0 ± 1.78^*

10.8 ± 0.54^**

3.7 ± 0.67^*

2.7 ± 0.23^**