CN114990081B - 闽楠PbPLR2基因，其编码的蛋白和应用 - Google Patents

闽楠PbPLR2基因，其编码的蛋白和应用 Download PDF

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CN114990081B
CN114990081B CN202210692646.5A CN202210692646A CN114990081B CN 114990081 B CN114990081 B CN 114990081B CN 202210692646 A CN202210692646 A CN 202210692646A CN 114990081 B CN114990081 B CN 114990081B
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张俊红
张毓婷
韩潇
童再康
王倩清
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Abstract

本发明属于植物分子生物学领域，具体涉及闽楠PbPLR2基因，其编码的蛋白和应用。该基因的cDNA序列全长如SEQ ID No.1所示，其编码蛋白质的氨基酸序列如SEQ ID No.2所示。通过根癌农杆菌介导法转化拟南芥，筛选拟南芥后代植株，并对其进行分子检测，对拟南芥过表达植株进行木脂素的提取和PbPLR2基因的表达分析，结果表明野生型拟南芥中只检测到松脂素，而PbPLR2过表达植株中检测到含量更高的松脂素和落叶松脂素。

Description

闽楠PbPLR2基因，其编码的蛋白和应用
技术领域
本发明属于植物生物技术领域，涉及闽楠PbPLR2基因，其编码的蛋白和应用。
背景技术
闽楠(Phoebe bournei)属樟科(Lauraceae)楠属植物，为中国特有珍稀阔叶树种，为“金丝楠木”原种植物之一，其木材具有极高耐腐性。有研究表明从木材分离的木脂素类化合物，具有抗菌活性，参与植物防御系统，可增强木材耐久性和抗真菌能力。如北美乔柏(Thuja plicata)心材中提取的木脂素通过清除自由基和螯合亚铁来干扰真菌氧化还原循环提高木材耐腐性，南洋杉(Araucaria araucana)中提取的松脂素和开环落叶松脂素对木材腐朽菌具有较好抗性。从3种智利柏科心材提取物中分离到开环落叶松脂素、落叶松脂素和罗汉松脂素等木脂素，均表现出抗DPPH和铁螯合能力的抗氧化活性，有利于保护木材抵抗腐生真菌的侵染。此外，木脂素的药理活性具有抗病毒、保肝、抗肿瘤和抗氧化等功效，已在药用植物中进行了长期研究。例如，从菘蓝(Isatis indigotica)根中分离的落叶松脂素和落叶松脂素苷可有效抑制流感病毒诱导的炎症反应。这些结果表明木脂素类化合物可作为杀菌剂和氧化抑制剂防止木材腐烂，成为新型木材耐腐剂加以利用，同时具有重要的医药开发价值。
木脂素的生物合成是一系列苯丙烷类物质的代谢过程。该类化合物起始于苯丙氨酸(Phe)，经由一系列羟化、甲基化和氧化还原反应，生成第一个前体化合物松柏醇，两分子松柏醇在Dirigent蛋白作用下通过氧化耦合反应生成一分子的松脂素。PLR基因作为木脂素生物合成途径中的重要限速酶，在大部分植物中松脂素还原酶(Pinoresinol-lariciresinol reductase，PLR)连续催化松脂素到落叶松脂素再到异落叶松脂素的两个还原过程。PLR对8,8'-C原子的任一对映体构型都表现出对映特异性，即用于(+)-松脂素、(+)-落叶松树脂素和(-)-异落叶松脂素的R,R和S,S用于(-)-松脂素、(-)-落叶松树脂素和(+)-异落叶松脂素，表明PLR作为还原酶催化两个还原步骤的复杂性。然而，拟南芥还原酶(AtPrR)例外，它们仅催化还原反应的第一步，因此被命名为松脂醇还原酶(PrR)，其中AtPrR2对松脂素的亲和力比落叶松脂素高35倍，而AtPrR1不亲和落叶松脂素。虽然已利用基因组对来自不同物种的PLR进行了一定研究，但诸多同工酶仍未被识别。例如，亚麻基因组中存在5个PLR基因，仅2个PLR基因被证实有生物学功能。
发明内容
为了解决上述问题，本发明提供了闽楠PbPLR2基因，其编码的蛋白和应用。
首先，本发明提供闽楠PbPLR2蛋白，其为：
1)由SEQ ID No.2所示的氨基酸组成的蛋白质；或
2)在SEQ ID No.2所示的氨基酸序列中经取代、缺失或添加一个或几个氨基酸且具有同等活性的由1)衍生的蛋白质。
本发明还提供编码所述的闽楠PbPLR2蛋白的基因。
优选的，所述基因的序列如SEQ ID No.1所示。
本发明还提供含有所述基因的过表达载体，宿主细胞和工程菌。
本发明还提供所述基因在提高植物积累松脂素和/或落叶松脂素中的用途。
在本发明一个具体实施方案中，将所述基因转入植物基因组中，并在转基因植物中超量表达，提高植物积累松脂素和/或落叶松脂素。
本发明还提供一种转基因植株的构建方法，采用农杆菌介导的方法，将含有所述基因的载体转入植物基因组中，筛选获得转基因植株。其中，所述的转基因植株与野生型相比，积累更高含量的松脂素和/或落叶松脂素。
本发明克隆了闽楠PbPLR2基因，并通过根癌农杆菌介导法转化拟南芥，筛选拟南芥阳性植株，并对其进行分子检测，对拟南芥过表达植株根系进行PbPLR2的表达分析，结果表明PbPLR2在转基因植株根中显著高表达。进一步对拟南芥野生型和过表达植株的根提取总木脂素，应用高效液相色谱技术检测松脂素和落叶松脂素含量，并对照标准曲线，计算两种木脂素的绝对含量表明，野生型拟南芥只检测到松脂素(4号峰)，浓度为1.601μg/g，而过表达植株松脂素含量为3.034μg/g；野生型拟南芥中没检测到落叶松脂素(2号峰)，过表达植株中落叶松脂素含量为5.108μg/g。总之，我们首次在拟南芥中过表达闽楠PbPLR2基因，获得了较高浓度的落叶松脂素，同时提高极显著提高了松脂素含量，即人为调控植物体内落叶松脂素和松脂素合成是可行的，具有较大的运用前景和经济效益。
附图说明
图1所示为农杆菌菌液PCR检测。1-4：PbPLR2过表达载体转化根癌农杆菌；M：DL2000Marker。
图2所示为转基因拟南芥株系的GFP基因检测。注：1-3：PbPLR2过表达拟南芥阳性株系；4，野生型拟南芥；M：DL 2000Marker。
图3所示为拟南芥过表达植株根中PbPLR2基因表达。
图4所示为拟南芥根的木脂素测定光谱图。A：野生型WT；B：过表达植株；C：加了标准品2和4的过表达植株。2号峰为落叶松脂素；4号峰为松脂素。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1闽楠PbPLR2基因的克隆和过表达载体的构建
提取闽楠根、茎和叶总RNA，等量混合后反转录成cDNA(参照Takara公司的反转录试剂盒)。根据闽楠基因组PbPLR2序列，应用primer5软件设计基因克隆引物，根据过表达载体构建的原则，设计相应引物(表1)
表1闽楠PbPLR基因引物序列表
Figure BDA0003700769550000041
以混合cDNA为模板，以表1中的基因克隆引物进行PCR扩增，通过琼脂糖凝胶电泳检测，将目的片段进行凝胶回收。根据pEASY-Blunt Zero Cloning Vector说明书，在PCR管中将DNA PCR回收产物片段与载体进行连接构建克隆载体。转化大肠杆菌感受态细胞，菌液PCR后验证正确后提取大肠杆菌质粒并测序。测得的闽楠PbPLR的cDNA序列如SEQ ID No.1所示，其编码的蛋白的氨基酸序列如SEQ ID No.2所示。
以质粒为模板，以表1中的过表达引物进行PCR扩增。通过琼脂糖凝胶电泳检测，将目的片段进行凝胶回收。将目的片段连接到入门载体(参见PENTR/
Figure BDA0003700769550000042
Cloning Kit说明书)，转化大肠杆菌感受态细胞，验证正确后提取大肠杆菌质粒并测序。选择测序结果正确的质粒连接过表达终载体PK2GW7。反应体系如下：
Figure BDA0003700769550000043
Figure BDA0003700769550000051
将上述体系溶液离心混匀后，25℃连接过夜，然后转化DH5α感受态，在含有50mg/L的状观霉素(Spec)培养基上培养8-14h，挑取单克隆菌检送测。提取测序正确的菌液的质粒，转化农杆菌GV3101感受态细胞并进行阳性克隆鉴定(图1)。选择一个验证正确的菌液进行菌体扩繁，进行拟南芥的浸染实验。
实施例2闽楠PbPLR2过表达载体转化与验证
通过花序侵染法将实施例1制备的含闽楠PbPLR2过表达载体的农杆菌GV3101转化哥伦比亚野生型拟南芥。收集T0代拟南芥种子，用带有卡那霉素抗性的培养基筛选T1代拟南芥。将获得的T1代拟南芥提取DNA进行PCR检测，使用PK2GW7载体通用引物验证GFP基因(图2)，多个植株均检测到GFP基因，说明带GFP基因的PK2GW7载体已成功转入拟南芥中，得到3个过表达PbPLR2的T1代株系。
实施例3转基因拟南芥中闽楠PbPLR2基因表达定量分析
按株系培养获得T3代拟南芥，进行基因表达分析及木脂素提取。以野生型拟南芥为对照，3个转基因拟南芥株系为3个生物学重复。因后期木脂素测定材料为根系(已木质化)，需检验PbPLR2在根中表达水平，故提取根系RNA，反转录后进行定量实验(图3)。内参基因选用表达稳定的Actin基因，引物如表2。结果表明PbPLR2基因在拟南芥中过量表达，其根中表达水平显著高于野生型。
表2闽楠PbPLR2表达分析的定量PCR引物
Figure BDA0003700769550000052
实施例4转基因拟南芥木脂素含量测定
(1)木脂素含量计算方法
木脂素含量计算方法：外标一点发。
即根据标准品浓度与峰面积的比值来计算供试品浓度：C(供试品)＝C(标准品)×A(供试品)/A(标准品)，(C为浓度，A为峰面积)。
(2)液相条件
液相条件如表3，流动相为甲醇-水溶液，流速0.8ml/min，进样量5μL，检测时间1h，柱温30℃。
表3 HPLC测定木脂素的液相条件
Figure BDA0003700769550000061
对野生型和拟南芥过表达植株OE2的根进行木脂素提取，结果发现野生型拟南芥中只检测到松脂素，浓度为1.601μg/g，而PbPLR2过表达植株OE2中检测到含量更高的松脂素(3.034μg/g)；野生型拟南芥中没检测到落叶松脂素，过表达植株中落叶松脂素含量为5.108μg/g(图4)。总之，闽楠PbPLR2基因参与了植物体内松脂素和落叶松脂素的生物合成。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 浙江农林大学
<120> 闽楠PbPLR2基因，其编码的蛋白和应用
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Ile Lys Arg Phe Leu Pro Ser Glu Phe Gly Met Asp Val Asp Arg Met
115 120 125
Glu His Ala Ile Pro Pro Gly Ala Tyr Val Phe Thr Asp Lys Arg Val
130 135 140
Val Arg Arg Ala Ile Glu Lys Ala Asn Ile Pro Tyr Thr Tyr Ile Ser
145 150 155 160
Ala Asn Cys Cys Ala Gly Tyr Phe Leu Ala Ala Leu Ala Gln Leu Gly
165 170 175
His Phe Met Pro Pro Thr Asp His Ala Leu Ile Tyr Gly Asp Gly Asp
180 185 190
Lys Lys Cys Ile Trp Val Asp Glu Asp Asp Met Ala Met Tyr Ala Met
195 200 205
Met Ala Val Asp Asp Pro Arg Ala Leu Asn Lys Thr Leu Tyr Leu Arg
210 215 220
Pro Arg Gly Asn Ile Leu Thr Gln Met Glu Val Val Arg Thr Trp Glu
225 230 235 240
Lys Ile Ile Gly Lys Glu Leu Lys Lys Thr Phe Val Ser Gln Asp Glu
245 250 255
Trp Leu Ser Ser Ile Asp Lys Val Ala Pro Pro Leu Gln Ile Gly Val
260 265 270
Ala His Leu Tyr Gln Ile Phe Tyr His Gly Asp Leu Glu Phe Glu Val
275 280 285
Glu Gly Pro His Gly Val Asp Ser Asn Asp Leu Tyr Pro Asn His Lys
290 295 300
Tyr Val Thr Ala Glu Glu Tyr Leu Lys Arg Phe Ala
305 310 315
<210> 3
<211> 23
<212> DNA
<213> 闽南(Phoebe bournei)
<400> 3
atggctcttg ttttgggctc tgg 23
<210> 4
<211> 25
<212> DNA
<213> 闽南(Phoebe bournei)
<400> 4
ctagcaagcc tcttcatatg tgctc 25
<210> 5
<211> 27
<212> DNA
<213> 闽南(Phoebe bournei)
<400> 5
caccatggct cttgttttgg gctctgg 27
<210> 6
<211> 43
<212> DNA
<213> 闽南(Phoebe bournei)
<400> 6
ctaatggtga tggtgatggt ggcaagcctc ttcatatgtg ctc 43
<210> 7
<211> 20
<212> DNA
<213> 闽南(Phoebe bournei)
<400> 7
tctggattac gtgcaacagg 20
<210> 8
<211> 21
<212> DNA
<213> 闽南(Phoebe bournei)
<400> 8
agcgtttgag atagtccttc g 21
<210> 9
<211> 20
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 9
gaaatcacag cacttgcacc 20
<210> 10
<211> 20
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 10
aagcctttga tcttgagagc 20

Claims (10)

1.闽楠PbPLR2蛋白，其为由SEQ ID No.2所示的氨基酸组成的蛋白质。
2.编码权利要求1所述的闽楠PbPLR2蛋白的基因。
3.如权利要求2所述的基因，其特征在于，序列如SEQ ID No.1所示。
4.含有权利要求2或3所述基因的载体。
5.含有权利要求4所述载体的宿主细胞。
6.含有权利要求2或3所述基因的工程菌。
7.权利要求2或3所述基因在提高拟南芥积累松脂素和/或落叶松脂素中的用途。
8.如权利要求7所述的用途，其特征在于，将所述基因转入拟南芥基因组中，并在转基因拟南芥中超量表达，提高拟南芥积累松脂素和/或落叶松脂素。
9.一种转基因植株的构建方法，采用农杆菌介导的方法，将含有权利要求2或3所述基因的载体转入拟南芥基因组中，筛选获得转基因植株。
10.如权利要求9所述的构建方法，其特征在于，所述的转基因植株与野生型相比，积累更高含量的松脂素和/或落叶松脂素。
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