CN116083450A - 一种甘露醇合成代谢组合基因及组合酶 - Google Patents
一种甘露醇合成代谢组合基因及组合酶 Download PDFInfo
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Abstract
本发明提供了一种甘露醇合成代谢组合基因及组合酶，属于基因工程领域。本发明通过对坛紫菜中甘露醇合成代谢机制的研究，发现了1种甘露醇‑1‑磷酸脱氢酶PhMT1D和3种甘露醇‑1‑磷酸酶PhM1Pase，提供了一种新的甘露醇合成代谢过程，为研究甘露醇的合成代谢过程及红藻的生长和发育提供了理论基础。
Description
技术领域
本发明属于基因工程领域，具体涉及一种甘露醇合成代谢组合基因及组合酶。
背景技术
坛紫菜(Pyropia haitanensis)，俗名紫菜、乌菜，是一种可人工栽培的大型海藻。坛紫菜属红藻纲，紫球藻科，紫菜属，藻体暗紫绿略带褐色，披针形、亚卵形或长卵形，长12-30cm以上，基部心脏形、圆形或楔形，边缘稍有褶皱或无，具有稀疏的锯齿，藻体单层，局部双层，色素体单一或少数具双，基部细胞呈圆头形，雌雄异株，少数同株，暖温带性种类，为中国浙江、福建和广东沿岸的主要栽培藻类。富含蛋白质、多糖和维生素，可供食用或药用。
甘露醇(mannitol)是一种六元糖醇(C6H14O6)，与山梨醇(sorbitol)、艾杜糖醇(iditol)和塔里糖醇(talitd)等互为同分异构体。甘露醇为白色或无色针状晶体或结晶性粉末，无臭、味甜，其甜度相当于蔗糖的40-50％。对稀酸稀碱性质稳定，是唯一一种不吸湿的多元醇。
甘露醇是我国基本药物，已收载于《中国药典》。甘露醇注射液因其高渗特性，可作为脱水药用于治疗青光眼、脑水肿并可预防和治疗糖尿病、急性肾功能衰竭、腹水等。甘露醇化学性质稳定，无吸湿性，可作为片剂的赋形剂、填充剂，以及胶囊的增塑剂等。甘露醇在食品保健行业具有以下应用：作为无糖口香糖的甜味剂或防粘剂；作为糖果和冰淇淋等甜味食品的糖衣；作为食品的定型剂、防结块剂和品质改良剂；用于储藏水果和果脯，提升香味。另外，作为重要的化工原料，甘露醇可在塑料行业用作增塑剂，在化妆品中作保湿剂，并可作为植物生长调节剂，用于果品贮藏保鲜。
Bidwell等(1958)用14C同位素示踪实验，发现墨角藻中甘露醇被标记强度占总放射强度的90％以上。纪明侯等(1980)将14CO2通入暂养海带的海水中，检测到最初出现的放射性产物为甘露醇，推测其为光合作用的积累产物之一。Quillet等(1985)提出了以果糖-6-磷酸(F6P)为代谢中间物的甘露醇合成途径。其后，甘露醇代谢相关酶的分离和酶活解析成为研究重点。Ikawa等(1972)从网地藻(Dictyota dichotoma)和褐舌藻(Spatoglossumpacificum)中分离了甘露醇-1-磷酸脱氢酶(M1PDH)和甘露醇-1-磷酸酶(M1Pase)；Richter等(1987)发现扁藻中D-甘露醇脱氢酶和M1PDH在应对渗透胁迫中发挥作用；Karsten等(1997)分离并测定了鹧鸪菜中参与甘露醇代谢的四种酶的动力学参数。Iwamoto等(2005)提出藻类中甘露醇的合成与分解途径，其中参与合成的两步酶为M1PDH和M1Pase，参与分解的两步酶为甘露醇-2-脱氢酶(M2DH)和果糖激酶(FK)。目前，甘露醇代谢途径的研究数据很少，对甘露醇代谢相关酶的研究也较少。
甘露醇作为藻类主要的碳积累产物，研究红藻中甘露醇的合成代谢过程及其相关酶蛋白，对于红藻的生长、发育及品种选育具有重要的意义。
发明内容
针对上述不足，本发明提供了一种甘露醇合成代谢组合基因及组合酶。本发明通过对坛紫菜中甘露醇合成代谢机制的研究，发现了1种甘露醇-1-磷酸脱氢酶PhMT1D和3种甘露醇-1-磷酸酶PhM1Pase，为研究甘露醇的合成代谢过程及红藻的生长和发育提供了理论基础。
为了实现上述发明目的，本发明的技术方案如下：
一方面，本发明提供了一种甘露醇合成代谢组合基因，所述的组合基因包括甘露醇-1-磷酸脱氢酶PhMT1D基因和/或甘露醇-1-磷酸酶PhM1Pase基因。
具体地，所述的甘露醇-1-磷酸脱氢酶PhMT1D基因序列为SEQ ID No:1所示的核苷酸序列。
具体地，所述的甘露醇-1-磷酸酶PhM1Pase基因序列为SEQ ID No:2、SEQ ID No:3和/或SEQ ID No:4所示的核苷酸序列。
又一方面，本发明提供了一种甘露醇合成代谢组合酶，所述的组合酶由上述组合基因编码得到。
具体地，所述的组合酶包括甘露醇-1-磷酸脱氢酶PhMT1D和/或甘露醇-1-磷酸酶PhM1Pase。
进一步具体地，所述的甘露醇-1-磷酸脱氢酶PhMT1D的氨基酸序列为SEQ ID No:5所示的序列。
进一步具体地，所述的甘露醇-1-磷酸酶PhM1Pase的氨基酸序列为SEQ ID No:6、SEQ ID No:7和/或SEQ ID No:8所示的序列。
又一方面，本发明提供了一种载体，所述的载体包含上述组合基因。
又一方面，本发明提供了一种宿主细胞，所述的宿主细胞包含上述组合基因或载体。
又一方面，本发明提供了一种基因工程细胞，所述的基因工程细胞包含上述组合基因或载体或生产上述组合酶。
又一方面，本发明提供了上述组合基因、组合酶、载体、宿主细胞或基因工程细胞在甘露醇合成代谢中的应用。
又一方面，本发明提供了一种甘露醇合成代谢的方法，所述的方法包括以下步骤：果糖-6-磷酸Fru-6-P在甘露醇-1-磷酸脱氢酶PhMT1D催化下转化成甘露醇-1-磷酸Mannitol-1-P，接着在甘露醇-1-磷酸酶PhM1Pase的作用下去磷酸生成甘露醇Mannitol。
又一方面，本发明还提供了上述组合基因、组合酶、载体、宿主细胞或基因工程细胞在红藻和植物育种中的应用。
与现有技术相比，本发明的积极和有益效果在于：
本发明通过对坛紫菜中甘露醇合成代谢机制的研究，发现了一种甘露醇合成代谢组合基因及组合酶，所述的组合酶包括1种甘露醇-1-磷酸脱氢酶PhMT1D和3种甘露醇-1-磷酸酶PhM1Pase，提供了一种新的甘露醇合成代谢过程，为研究甘露醇的合成代谢过程及红藻的生长和发育提供了理论基础。
附图说明
图1为甘露醇合成代谢通路图。
图2为坛紫菜中果糖-6-磷酸和甘露醇含量检测图。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
样品为2019年12月11日在福建东甲岛采集的野生坛紫菜叶状体。将坛紫菜转移到室内充气培养系统中，用PES培养基(Provasoli,L.,1968)在黑暗、21℃条件下恢复培养48h。恢复培养后，将坛紫菜置于21℃，50mol·m-2s-1光照条件下(12h光照，12h黑暗)培养，培养基每2天更换一次。在培养第5天光照6h时候，选取几乎相同大小的坛紫菜进行基因分析和含量测定。
实施例2甘露醇合成代谢通路研究
坛紫菜中甘露醇合成代谢通路如图1所示，果糖-6-磷酸Fru-6-P在甘露醇-1-磷酸脱氢酶PhMT1D催化下转化成甘露醇-1-磷酸Mannitol-1-P，接着在甘露醇-1-磷酸酶PhM1Pase的作用下去磷酸生成甘露醇Mannitol。
实施例3基因分析
(1)从NCBI(https://www.ncbi.nlm.nih.gov/)，MGU(https://marinegenomics.oist.jp/algae/gallery)和Ensembl(http://plants.ensembl.org/index.html)数据库中下载甘露醇代谢通路中涉及的同源序列，保存为fasta格式；
(2)利用Secure FX和Putty软件连接主服务器上的坛紫菜基因组数据库，在Putty中运行命令“formatdb-iOneKP.faspF”对数据库进行格式化；
(3)利用Secure FX将下载到本地电脑上蛋白质序列文件上传到格式化好的数据库文件夹下；
(4)在Putty中利用“blastall-p tblastn-d(sequence name)-i(searchsequence)-oout-(gene name)-F F-e 1e-5”命令，进行以氨基酸为query的序列调取，结果文件以.txt格式打开并保存到本地电脑；
(5)利用Editplus(https://www.editplus.com/)、perl和Tbtools软件将结果文件中同源性较高的预选序列从数据库中分离出。
(6)将上一步筛选得到的序列提交到NCBI Conserved-Domains(https://www.ncbi.nlm.nih.gov/cdd/？term＝)进行保守结构域预测，剔除不含目标基因家族保守结构域的序列，确定目标基因。
利用ProtParam工具(http://web.expasy.org/protparam)分析基因的序列长度、分子量、等电点和不稳定性指数(Gasteiger et al.,2003)。经检测，甘露醇-1-磷酸脱氢酶PhMT1D基因的序列为SEQ ID No:1所示的核苷酸序列；甘露醇-1-磷酸酶PhM1Pase基因包括PhM1Pase1、PhM1Pase2和PhM1Pase，PhM1Pase1的序列为SEQ ID No:2所示的核苷酸序列，PhM1Pase2的序列为SEQ ID No:3所示的核苷酸序列，PhM1Pase3的序列为SEQ ID No:4所示的核苷酸序列。
基因特征结果如下表1所示。
表1坛紫菜红藻淀粉合成通路基因特征
| Gene name | SEQ ID No | Amino acids | ORF(bp) | MV(kDa) | pI | Introns | Instability index |
| PhMT1D | SEQ ID No:1 | 583 | 1752 | 58.40154 | 5.96 | 1 | 36.52 |
| PhM1Pase1 | SEQ ID No:2 | 253 | 762 | 26.28638 | 5.39 | 0 | 39.80 |
| PhM1Pase2 | SEQ ID No:3 | 313 | 942 | 31844.27 | 5.52 | 0 | 37.60 |
| PhM1Pase3 | SEQ ID No:4 | 317 | 954 | 32.66114 | 4.82 | 0 | 48.01 |
实施例4基因的克隆表达和蛋白纯化
采用大肠杆菌、pET载体系统、His-tag镍柱纯化系统对下述蛋白进行表达纯化，具体操作步骤参见相关菌株、试剂、试剂盒的操作手册：
1.甘露醇-1-磷酸脱氢酶PhMT1D：氨基酸序列为SEQ ID No:5所示的序列。
2.甘露醇-1-磷酸酶PhM1Pase：包括PhM1Pase1、PhM1Pase2和PhM1Pase3，PhM1Pase1的氨基酸序列为SEQ ID No:6所示的序列，PhM1Pase2的氨基酸序列为SEQ IDNo:7所示的序列，PhM1Pase3的氨基酸序列为SEQ ID No:8所示的序列。
基因克隆表达与蛋白纯化为本领域常规步骤，故在此不做赘述。
通过对纯化蛋白进行SDS-PAGE发现条带位置均与通过氨基酸预测的蛋白大小一致。
实施例5果糖-6-磷酸和甘露醇检测
1.甘露醇
称取0.5g样品加水至50mL，超声20min，过膜上机。
色谱参数：检测器：蒸发光检测器，流动相：乙腈:水＝70:30，流速1.5mL/min，色谱柱:APS-2HYPERSIL。。
2.果糖-6-磷酸
(1)取100mg鲜样破碎粉末于2.0mL旋口管中，加入1.2mL 50％乙醇，40℃震荡1h。
(2)12000rpm离心10min。
(3)吸取上清转移到新离心管中，加入700μL CHCl3，10000rpm离心3min。
(4)取上清上机检测。
色谱参数：
色谱系统采用的是Thermo ICS5000+离子色谱系统(ICS5000+，Thermo FisherScientific，USA)，采用DionexTM CarboPacTM PA10(250*4.0mm，10μm)液相色谱柱，进样量为20μL。流动相A(10mM NaOH)，流动相B(10mM NaOH，50mM NaAC)，柱温为30℃，利用电化学检测器对单糖组分进行分析检测。流动相梯度如下表2所示。
表2流动相梯度
经检测，坛紫菜中果糖-6-磷酸含量为7.44μg/g，甘露醇含量为88.04mg/g，检测结果如图2所示。
实施例6体外培养实验
采用体外培养检测本发明所述甘露醇合成代谢机制。在体系中加入实施例4制备得到的100μL甘露醇-1-磷酸脱氢酶PhMT1D和100μL甘露醇-1-磷酸酶PhM1Pase混合物(30μLPhM1Pase1+35μL PhM1Pase2+35μL PhM1Pase3)，以及底物果糖-6-磷酸(终浓度为3mM)，反应条件为30℃3h。反应完成后，经检测发现，果糖-6-磷酸含量下降，为0.1mM，甘露醇含量上升，为1.3mM。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种甘露醇合成代谢组合基因，其特征在于：所述的组合基因包括甘露醇-1-磷酸脱氢酶PhMT1D基因和/或甘露醇-1-磷酸酶PhM1Pase基因。
2.根据权利要求1所述的组合基因，其特征在于：
所述的甘露醇-1-磷酸脱氢酶PhMT1D基因序列为SEQ ID No:1所示的核苷酸序列；
所述的甘露醇-1-磷酸酶PhM1Pase基因序列为SEQ ID No:2、SEQ ID No:3和/或SEQ IDNo:4所示的核苷酸序列。
3.一种甘露醇合成代谢组合酶，其特征在于：所述的组合酶由权利要求1-2任一项所述的组合基因编码得到；所述的组合酶包括甘露醇-1-磷酸脱氢酶PhMT1D和/或甘露醇-1-磷酸酶PhM1Pase。
4.根据权利要求3所述的组合酶，其特征在于：
所述的甘露醇-1-磷酸脱氢酶PhMT1D的氨基酸序列为SEQ ID No:5所示的序列；
所述的甘露醇-1-磷酸酶PhM1Pase的氨基酸序列为SEQ ID No:6、SEQ ID No:7和/或SEQ ID No:8所示的序列。
5.一种载体，其特征在于：所述的载体包含权利要求1-2任一项所述的组合基因。
6.一种宿主细胞，其特征在于：所述的宿主细胞包含权利要求1-2任一项所述的组合基因或权利要求5所述的载体。
7.一种基因工程细胞，其特征在于：所述的基因工程细胞包含权利要求1-2任一项所述的组合基因或权利要求5所述的载体，或生产权利要求3-4任一项所述的组合酶。
8.权利要求1-2任一项所述的组合基因、权利要求3-4任一项所述的组合酶、权利要求5所述的载体、权利要求6所述的宿主细胞或权利要求7所述的基因工程细胞在甘露醇合成代谢中的应用。
9.权利要求1-2任一项所述的组合基因、权利要求3-4任一项所述的组合酶、权利要求5所述的载体、权利要求6所述的宿主细胞或权利要求7所述的基因工程细胞在红藻和植物育种中的应用。
10.一种甘露醇合成代谢的方法，其特征在于：所述的方法包括以下步骤：果糖-6-磷酸Fru-6-P在甘露醇-1-磷酸脱氢酶PhMT1D催化下转化成甘露醇-1-磷酸Mannitol-1-P，接着在甘露醇-1-磷酸酶PhM1Pase的作用下去磷酸生成甘露醇Mannitol。
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