CN109609523A - 两种催化桃udp-鼠李糖生物合成的基因及编码蛋白和应用 - Google Patents

两种催化桃udp-鼠李糖生物合成的基因及编码蛋白和应用 Download PDF

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CN109609523A
CN109609523A CN201910060742.6A CN201910060742A CN109609523A CN 109609523 A CN109609523 A CN 109609523A CN 201910060742 A CN201910060742 A CN 201910060742A CN 109609523 A CN109609523 A CN 109609523A
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李鲜
赵志康
解林峰
任传宏
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Abstract

本发明公开了两种催化桃UDP‑鼠李糖生物合成的基因及编码蛋白和应用，所述两种基因为PpRHM1和PpRHM2，是从桃中分离并克隆获得的分别具有如SEQ:NO.1和SEQ:NO.2所示的核苷酸序列，和如SEQ:NO.3和SEQ:NO.4所示的氨基酸序列。本发明首次克隆并验证了桃中UDP‑鼠李糖合成相关的PpRHM1和PpRHM2两个基因的功能。通过构建重组质粒，实现两个基因在大肠杆菌中的重组表达，并得到纯化的重组蛋白。在体外，两个重组蛋白可以高效地将UDP‑葡萄糖转化成UDP‑鼠李糖。本发明可用于植物UDP‑鼠李糖的生物合成调控，并为实现UDP‑鼠李糖商品化生产提供了代谢工程基础。

Description

两种催化桃UDP-鼠李糖生物合成的基因及编码蛋白和应用
技术领域
本发明属于植物分子生物技术和基因工程领域，涉及两种催化桃UDP-鼠李糖生物合成的基因及其编码蛋白和应用。
背景技术
核苷糖，是细胞壁生物合成和蛋白质和脂质糖基化等过程所必需的活化糖供体。UDP- 鼠李糖是众多核苷糖中的一种，在结构上是由一分子的尿苷二磷酸和一分子的鼠李糖所组成，是构建鼠李糖基衍生物的重要中间体。
由于植物体内UDP-鼠李糖含量低、纯化难度大且效率低，限制了UDP-鼠李糖的获取，而利用现代化学合成手段合成成本较高且过程繁琐，迄今为止，市场上还没有商品化的UDP-鼠李糖。而采用生物合成UDP-鼠李糖的方法，为UDP-鼠李糖的合成提供了新途径。因此鉴别出高效催化UDP-鼠李糖生物合成的基因，对于植物UDP-鼠李糖的生物合成调控和实现UDP-鼠李糖的商品化生产具有重要意义。
发明内容
本发明的目的是提供两种催化UDP-鼠李糖生物合成的基因及其编码蛋白，所述两种基因为PpRHM1和PpRHM2，所述PpRHM1基因的CDS序列如SEQ:NO.1所示，编码序列全长为2028个核苷酸，其编码蛋白的氨基酸序列如SEQ:NO.3所示，可编码一个含675个氨基酸的蛋白；所述PpRHM2基因的CDS序列如SEQ:NO.2所示，编码序列全长为2016个核苷酸，其编码蛋白的氨基酸序列如SEQ:NO.4所示，可编码一个含671个氨基酸的蛋白。
本发明的另一个目的是提供所述两个基因PpRHM1和PpRHM2及其编码蛋白在植物UDP-鼠李糖的生物合成中的应用，是在植物UDP-鼠李糖生物合成调控和商业化合成中的应用。通过将上述基因PpRHM1和PpRHM2分别连接到pET-28a载体的多克隆位点中构建获得重组质粒，命名为pET-28a-PpRHM1和pET-28a-PpRHM2。在大肠杆菌中表达pET-28a-PpRHM1 和pET-28a-PpRHM2，得到重组蛋白，可将UDP-葡萄糖转化为UDP-鼠李糖。
本发明提供了两种催化UDP-鼠李糖生物合成的基因PpRHM1和PpRHM2及其编码蛋白在植物UDP-鼠李糖生物合成调控和商业化合成中的应用。本发明首次克隆并验证了桃中UDP-鼠李糖合成相关PpRHM1和PpRHM2基因的功能。本发明还提供了分别含有PpRHM1 和PpRHM2基因的重组质粒pET-28a-PpRHM1和pET-28a-PpRHM2，为通过生物工程方法大量合成UDP-鼠李糖，进一步开展鼠李糖基衍生物生物合成调控研究奠定基础。
附图说明
图1：桃PpRHM1重组蛋白的SDS-PAGE凝胶电泳分析图；1为蛋白Marker，2为纯化所得PpRHM1重组蛋白。
图2：桃PpRHM2重组蛋白的SDS-PAGE凝胶电泳分析图；1为蛋白Marker，2为纯化所得PpRHM2重组蛋白。
图3：两种重组蛋白催化酶活产物结果图；其中，A：底物UDP-葡萄糖标准品；B：未加蛋白空白对照；C：PpRHM1重组蛋白催化反应；D：PpRHM2重组蛋白催化反应。
具体实施方式
下面结合具体实施例和附图对本发明做进一步的阐述，但实施例不限制本发明的保护范围。
实施例1：桃PpRHM1和PpRHM2基因克隆
以‘湖景蜜露’桃品种为材料，取根、茎、叶、花、果实，迅速用液氮冻透，然后放至-80℃冰箱中保存。利用CTAB法提取桃各组织的RNA，按照PrimeScriptTM RT reagent Kit withgDNA Eraser(Takara)试剂说明书操作合成cDNA。
以反转录产物cDNA各组织混样为模板，用SEQ:NO.5和SEQ:NO.6所示引物进行 PCR扩增PpRHM1，用SEQ:NO.7和SEQ:NO.8所示引物进行PCR扩增PpRHM2，PCR反应体系为50μL，组分分别为：2×Phanta Max Buffer 25μL，dNTP Mix(10mM each)1μL，DNA polymerse(1U/μL)1μL，上下游引物(10μM)各2μL，cDNA 1μL，H2O 18μL。PCR程序为：95℃预变性3min，95℃变性15s，58℃退火15s和72℃延伸1min 40s，72℃继续延伸5min，得到扩增产物。
将两种PCR扩增产物分别连接到T-easy载体，转化DH5α大肠杆菌感受态细胞，进行菌落PCR验证，获得阳性菌落进行测序。获得与基因组数据库相匹配的基因序列SEQ:NO.1和SEQ:NO.2。
实施例2：PpRHM1和PpRHM2的原核表达
设计带有表达载体pET-28a载体的多克隆酶切位点的特异引物，PpRHM1引物序列如SEQ:NO.9和SEQ:NO.10所示，PpRHM2引物序列如SEQ:NO.11和SEQ:NO.12所示。
以测序正确返还质粒为模板，用SEQ:NO.9和SEQ:NO.10所示引物进行PCR扩增PpRHM1，用SEQ:NO.11和SEQ:NO.12所示引物进行PCR扩增PpRHM2，PCR反应体系为 50μL，组分分别为：2×Phanta Max Buffer 25μL，dNTP Mix(10mM each)1μL，DNA polymerse (1U/μL)1μL，上下游引物(10μM)各2μL，cDNA 1μL，H2O 18μL。PCR程序为：95℃预变性3min，95℃变性15s，58℃退火15s和72℃延伸1min 40s，72℃继续延伸5min，得到扩增产物。
将两种PCR扩增产物分别连接到用SalI和HindIII双酶切过的pET-28a载体，获得pET-28a-PpRHM1和pET-28a-PpRHM2重组质粒。
将pET-28a-PpRHM1和pET-28-PpRHM2重组质粒分别转化到大肠杆菌BL21(DE3)PlysS(购于上海普洛麦格生物产品有限公司)表达宿主菌中，经菌落PCR验证，挑取阳性菌落接种到200mL LB液体培养基，37℃摇菌，直至OD600约为0.6-0.8，获得转基因工程菌。
在上述转基因的工程菌中加入IPTG至终浓度为0.5mM，16℃诱导24h，收集菌体，200mL收集1管，加入15mL1×PBS缓冲液，充分悬浮菌体，-80℃放置24h以上，将菌体置于30℃水浴锅解冻后，超声破碎10min，10000rpm离心30min，收集上清液。用Clontech HisTALON试剂盒进一步纯化目的蛋白。利用SDS-PAGE方法检测蛋白表达和纯化效果，结果如图1和图2所示。
图1和图2中可看出，pET-28a-PpRHM1和pET-28a-PpRHM2重组质粒转化到表达宿主大肠杆菌BL21(DE3)PlysS，经IPTG诱导后，有重组蛋白的表达，上清蛋白经ClontechHisTALON试剂盒纯化后得到较纯的重组蛋白，且重组蛋白条带大小与预测的一致，加上重组标签后在75kDa左右有明显的重组蛋白条带。纯化的蛋白可用于进一步的酶学分析。
实施例3：PpRHM1和PpRHM2重组蛋白的酶学活性检测分析
对于UDP-鼠李糖底物的酶活检测，反应体系为200μL，含有100mM的Na2HPO4/Na H2PO4(pH＝9)缓冲液包含1mM UDP-葡萄糖作为反应底物，2mM的NAD辅酶和2mM的 NADPH辅酶，20μL纯化后的重组蛋白。
所有酶反应体系在37℃反应1h后停止反应，获得酶反应产物。酶反应产物经产物标准品结合HPLC进行检测鉴定，所述HPLC检测条件如下：Waters 2695-2996DAD检测器，ODS C18柱(4.6×250mm)色谱柱。1.5％三乙胺水溶液(甲酸调pH＝7.5)为流动相，等度洗脱25min，检测波长为260nm，柱温为25℃，流速为1mL/min，进样体积为10μL。
结果如图3所示，可以看出PpRHM1和PpRHM2重组蛋白以UDP-葡萄糖为反应底物，可催化UDP-葡萄糖生成UDP-鼠李糖，催化流程如下所示：
序列表
<110> 浙江大学
<120> 两种催化桃UDP-鼠李糖生物合成的基因及编码蛋白和应用
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gtggttaata tccctaacag cttgaccatc ttggatgaat tactacccat ctctattgag 1740
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aatgagattc tggagatgta caagcaatac attgacccaa agtttacatg ggcaaatttc 1860
acaattgaag agcaagccaa ggttatagtt gcagctcgaa gcaacaatga aatggatgca 1920
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<213> 桃(Prunus persica)
<400> 3
Met Ala Thr Ala Tyr Lys Pro Lys Asn Ile Leu Ile Thr Gly Ala Ala
1 5 10 15
Gly Phe Ile Ala Ser His Val Cys Asn Arg Leu Ile Arg Asn Tyr Pro
20 25 30
Glu Tyr Lys Ile Val Val Leu Asp Lys Leu Asp Tyr Cys Ser Asn Leu
35 40 45
Lys Asn Leu His Pro Ser Arg Ser Ser Ser Asn Phe Lys Phe Ile Lys
50 55 60
Gly Asp Ile Gly Ser Ala Asp Leu Val Asn Phe Ile Leu Leu Thr Glu
65 70 75 80
Ser Ile Asp Thr Ile Met His Phe Ala Ala Gln Thr His Val Asp Asn
85 90 95
Ser Phe Gly Asn Ser Phe Glu Phe Thr Lys Asn Asn Ile Tyr Gly Thr
100 105 110
His Val Leu Leu Glu Ala Cys Lys Val Thr Gly Gln Ile Lys Arg Phe
115 120 125
Ile His Val Ser Thr Asp Glu Val Tyr Gly Glu Thr Asp Glu Asp Ala
130 135 140
Val Val Gly Asn His Glu Ala Ser Gln Leu Leu Pro Thr Asn Pro Tyr
145 150 155 160
Ser Ala Thr Lys Ala Gly Ala Glu Met Leu Val Met Ala Tyr Gly Arg
165 170 175
Ser Tyr Gly Leu Pro Val Ile Thr Thr Arg Gly Asn Asn Val Tyr Gly
180 185 190
Pro Asn Gln Phe Pro Glu Lys Met Ile Pro Lys Phe Ile Leu Leu Ala
195 200 205
Met Lys Gly Lys Pro Leu Pro Ile His Gly Asp Gly Ser Asn Val Arg
210 215 220
Ser Tyr Leu Tyr Cys Glu Asp Val Ala Glu Ala Phe Glu Val Ile Leu
225 230 235 240
His Lys Gly Glu Val Gly His Val Tyr Asn Ile Gly Thr Lys Lys Glu
245 250 255
Arg Arg Val Val Asp Val Ala Lys Glu Ile Cys Gln Leu Phe Ser Leu
260 265 270
Asn Pro Asp Thr Gln Ile Lys Phe Val Glu Asn Arg Pro Phe Asn Asp
275 280 285
Gln Arg Tyr Phe Leu Asp Asp Gln Lys Leu Lys Asn Leu Gly Trp Ser
290 295 300
Glu Arg Thr Ser Trp Glu Glu Gly Leu Arg Lys Thr Met Asp Trp Tyr
305 310 315 320
Val Lys Asn Pro Glu Trp Trp Gly Asp Val Ser Gly Ala Leu Leu Pro
325 330 335
His Pro Lys Met Leu Met Val Pro Gly Ile Glu Arg Lys Phe Asp Gly
340 345 350
Thr Asp Thr Gly Ala Ser Ala Phe Ser Leu Ser Ala Ser Asp Ser Arg
355 360 365
Glu Ser His Met Val Val Pro Pro Pro Lys Asn Asn Pro Ser Thr Gln
370 375 380
Lys Pro Ser Leu Lys Phe Leu Ile Tyr Gly Lys Thr Gly Trp Ile Gly
385 390 395 400
Gly Leu Leu Gly Lys Ile Cys Glu Lys Gln Gly Ile Pro Tyr Glu Tyr
405 410 415
Gly Gln Gly Arg Leu Gln Glu Arg Ser His Leu Leu Ala Asp Ile Gln
420 425 430
Ser Val Lys Pro Thr His Val Phe Asn Ala Ala Gly Val Thr Gly Arg
435 440 445
Pro Asn Val Asp Trp Cys Glu Ser His Lys Pro Glu Thr Ile Arg Thr
450 455 460
Asn Val Val Gly Thr Leu Thr Leu Ala Asp Val Cys Arg Asp His Asn
465 470 475 480
Leu Leu Met Ile Asn Tyr Ala Thr Gly Cys Ile Phe Glu Tyr Asp Ala
485 490 495
Ala His Pro Ser Arg Ser Gly Ile Gly Phe Lys Glu Glu Asp Thr Pro
500 505 510
Asn Phe Thr Gly Ser Phe Tyr Ser Lys Thr Lys Ala Met Val Glu Glu
515 520 525
Leu Leu Lys Glu Tyr Asp Asn Val Cys Thr Leu Arg Val Arg Met Pro
530 535 540
Ile Ser Ser Asp Leu Ser Asn Pro Arg Asn Phe Ile Thr Lys Ile Ser
545 550 555 560
Arg Tyr Asp Lys Val Val Asp Ile Pro Asn Ser Met Thr Ile Leu Asp
565 570 575
Glu Leu Leu Pro Ile Ser Val Glu Met Ala Lys Arg Asn Leu Arg Gly
580 585 590
Leu Trp Asn Phe Thr Asn Pro Gly Val Val Ser His Asn Glu Ile Leu
595 600 605
Glu Met Tyr Lys Lys Tyr Ile Asp Pro Ser Phe Lys Trp Thr Asn Phe
610 615 620
Thr Leu Glu Glu Gln Ala Lys Val Ile Val Ala Pro Arg Ser Asn Asn
625 630 635 640
Glu Met Asp Ala Ser Lys Leu Lys Lys Glu Phe Pro Glu Leu Leu Pro
645 650 655
Ile Lys Glu Ser Leu Ile Lys Tyr Val Phe Glu Pro Asn Lys Lys Ala
660 665 670
Phe Ser Gly
675
<210> 4
<211> 671
<212> PRT
<213> 桃(Prunus persica)
<400> 4
Met Gly Ser Tyr Thr Pro Lys Asn Ile Leu Ile Thr Gly Ala Ala Gly
1 5 10 15
Phe Ile Ala Ser His Val Ala Asn Arg Leu Ile Arg Ser Tyr Pro Asp
20 25 30
Tyr Asn Ile Val Val Leu Asp Lys Leu Asp Tyr Cys Ser Asn Leu Lys
35 40 45
Asn Leu Leu Pro Ser Lys Ser Ser Pro Asn Phe Lys Phe Val Lys Gly
50 55 60
Asp Ile Gly Ser Ala Asp Leu Val Asn Tyr Leu Leu Ile Thr Glu Ser
65 70 75 80
Ile Asp Thr Ile Met His Phe Ala Ala Gln Thr His Val Asp Asn Ser
85 90 95
Phe Gly Asn Ser Phe Glu Phe Thr Lys Asn Asn Ile Tyr Gly Thr His
100 105 110
Val Leu Leu Glu Ala Cys Lys Val Thr Gly Gln Ile Arg Arg Phe Ile
115 120 125
His Val Ser Thr Asp Glu Val Tyr Gly Glu Thr Asp Glu Asp Ala Val
130 135 140
Val Gly Asn His Glu Ala Ser Gln Leu Leu Pro Thr Asn Pro Tyr Ser
145 150 155 160
Ala Thr Lys Ala Gly Ala Glu Met Leu Val Met Ala Tyr Gly Arg Ser
165 170 175
Tyr Gly Leu Pro Val Ile Thr Thr Arg Gly Asn Asn Val Tyr Gly Pro
180 185 190
Asn Gln Phe Pro Glu Lys Leu Ile Pro Lys Phe Ile Leu Leu Ala Met
195 200 205
Gln Gly Lys Pro Leu Pro Ile His Gly Asp Gly Ser Asn Val Arg Ser
210 215 220
Tyr Leu Tyr Cys Glu Asp Val Ala Glu Ala Phe Glu Leu Ile Leu His
225 230 235 240
Lys Gly Glu Val Gly His Val Tyr Asn Ile Gly Thr Lys Lys Glu Arg
245 250 255
Arg Val Ile Asp Val Ala Lys Asp Ile Cys Arg Leu Phe Ser Val Asp
260 265 270
Pro Glu Thr Asn Ile Lys Phe Val Glu Asn Arg Pro Phe Asn Asp Gln
275 280 285
Arg Tyr Phe Leu Asp Asp Gln Lys Leu Lys Ile Leu Gly Trp Ser Glu
290 295 300
Arg Thr Ile Trp Gln Glu Gly Leu Lys Lys Thr Ile Glu Trp Tyr Thr
305 310 315 320
Lys Asn Pro Asn Trp Trp Gly Asp Val Ser Gly Ala Leu Leu Pro His
325 330 335
Pro Arg Met Leu Met Met Pro Gly Gly Ile Glu Arg His Leu Glu Gly
340 345 350
Ser Glu Glu Glu Lys Ser Glu Ser Phe Val Pro Ser Asn Thr Arg Met
355 360 365
Leu Val Pro Pro Ser Lys Ser Cys Ser Ser Pro Arg Lys Pro Pro Phe
370 375 380
Lys Phe Leu Ile Tyr Gly Lys Thr Gly Trp Ile Gly Gly Val Leu Gly
385 390 395 400
Lys Leu Cys Glu Lys Gln Gly Ile Pro Phe Glu Tyr Gly Lys Gly Arg
405 410 415
Leu Gln Asp Arg Ser Ser Leu Leu Ala Asp Ile Gln Asn Val Arg Pro
420 425 430
Thr His Val Phe Asn Ala Ala Gly Val Thr Gly Arg Pro Asn Val Asp
435 440 445
Trp Cys Glu Ser His Lys Ala Glu Thr Ile Arg Thr Asn Val Ala Gly
450 455 460
Thr Leu Thr Leu Ala Asp Val Cys Arg Glu His Gly Leu Leu Met Met
465 470 475 480
Asn Phe Ala Thr Gly Cys Ile Phe Glu Tyr Asp Ala Lys His Pro Glu
485 490 495
Gly Ser Gly Val Gly Phe Thr Glu Glu Asp Lys Pro Asn Phe Phe Gly
500 505 510
Ser Phe Tyr Ser Lys Thr Lys Ala Met Val Glu Glu Leu Leu Lys Glu
515 520 525
Phe Asp Asn Val Cys Thr Leu Arg Val Arg Met Pro Ile Ser Ser Asp
530 535 540
Leu Asn Asn Pro Arg Asn Phe Ile Thr Lys Ile Ser Arg Tyr Asn Lys
545 550 555 560
Val Val Asn Ile Pro Asn Ser Leu Thr Ile Leu Asp Glu Leu Leu Pro
565 570 575
Ile Ser Ile Glu Met Ala Lys Arg Asn Leu Arg Gly Ile Trp Asn Phe
580 585 590
Thr Asn Pro Gly Val Val Ser His Asn Glu Ile Leu Glu Met Tyr Lys
595 600 605
Gln Tyr Ile Asp Pro Lys Phe Thr Trp Ala Asn Phe Thr Ile Glu Glu
610 615 620
Gln Ala Lys Val Ile Val Ala Ala Arg Ser Asn Asn Glu Met Asp Ala
625 630 635 640
Ser Lys Leu Lys Lys Glu Phe Pro Glu Leu Leu Pro Val Lys Glu Ser
645 650 655
Leu Ile Lys Tyr Val Phe Glu Pro Asn Lys Thr Asn Ser Thr Gln
660 665 670
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Unknow)
<400> 5
atggctactg cgtataaacc g 21
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Unknow)
<400> 6
tcagccagaa aatgccttct 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Unknow)
<400> 7
atgggttcat atacccccaa 20
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Unknow)
<400> 8
ttattgtgtg gagtttgtct t 21
<210> 9
<211> 37
<212> DNA
<213> 人工序列(Unknow)
<400> 9
taaggcctct gtcgacatgg ctactgcgta taaaccg 37
<210> 10
<211> 36
<212> DNA
<213> 人工序列(Unknow)
<400> 10
cagaattcgc aagctttcag ccagaaaatg ccttct 36
<210> 11
<211> 36
<212> DNA
<213> 人工序列(Unknow)
<400> 11
taaggcctct gtcgacatgg gttcatatac ccccaa 36
<210> 12
<211> 37
<212> DNA
<213> 人工序列(Unknow)
<400> 12
cagaattcgc aagcttttat tgtgtggagt ttgtctt 37

Claims (4)

1.两种催化桃UDP-鼠李糖生物合成的基因，其特征在于，所述两种基因为PpRHM1和PpRHM2，其核苷酸序列如SEQ:NO.1和SEQ:NO.2所示。
2.根据权利要求1所述的两种催化桃UDP-鼠李糖生物合成的基因，其特征在于，所述两种基因PpRHM1和PpRHM2编码蛋白的氨基酸序列如SEQ:NO.3和SEQ:NO.4所示。
3.两种重组质粒，其特征在于，所述重组质粒为将权利要求1两种催化桃UDP-鼠李糖合成的基因PpRHM1和PpRHM2连接到pET-28a载体的多克隆位点中构建获得，命名为pET-28a-PpRHM1和pET-28a-PpRHM2。
4.根据权利要求1或2所述的两种催化桃UDP-鼠李糖生物合成的基因在植物UDP-鼠李糖生物合成调控和商业化合成中的应用，其特征在于，所述应用包括所述基因编码的蛋白，通过在大肠杆菌中的表达pET-28a-PpRHM1或pET-28a-PpRHM2，得到重组蛋白，将UDP-葡萄糖转化为UDP-鼠李糖。
CN201910060742.6A 2019-01-21 2019-01-21 两种催化桃udp-鼠李糖生物合成的基因及编码蛋白和应用 Pending CN109609523A (zh)

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CN112852843A (zh) * 2021-02-02 2021-05-28 浙江大学 黄酮醇3-o-半乳糖基转移酶基因及其编码蛋白和应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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