CN109679972B - 一种催化杨梅udp-鼠李糖生物合成的基因及编码蛋白和应用 - Google Patents
一种催化杨梅udp-鼠李糖生物合成的基因及编码蛋白和应用 Download PDFInfo
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- CN109679972B CN109679972B CN201910053714.1A CN201910053714A CN109679972B CN 109679972 B CN109679972 B CN 109679972B CN 201910053714 A CN201910053714 A CN 201910053714A CN 109679972 B CN109679972 B CN 109679972B
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Abstract
本发明公开了一种催化杨梅UDP‑鼠李糖生物合成的基因及编码蛋白和应用,是一种来源于杨梅的基因MrRHM1,该是从杨梅果实中分离并克隆获得的分别具有如SEQ:NO.1核苷酸序列,SEQ:NO.2所示的氨基酸序列。本发明首次克隆并验证了杨梅UDP‑鼠李糖合成相关的MrRHM1的功能。通过构建重组质粒,实现MrRHM1在大肠杆菌中的重组表达,并得到纯化的重组蛋白。在体外,重组蛋白可以将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
技术领域
本发明属于植物分子生物技术和基因工程领域,涉及一种催化杨梅UDP-鼠李糖生物合成的基因及其编码蛋白和应用。
背景技术
杨梅(Morella rubra)属于我国特色水果,含有较高含量的黄酮类化合物,具有很好的医药学活性,而黄酮醇是杨梅中主要的黄酮类化合物,通常以糖苷衍生物形式存在于液泡中。大量研究报道了黄酮醇抗氧化、抗肿瘤、预防心血管疾病、消炎等医药学活性。黄酮醇鼠李糖苷是黄酮醇糖苷的重要组成部分,而UDP-鼠李糖是黄酮醇鼠李糖苷合成的一种重要前体物质,在黄酮醇鼠李糖苷的生物合成中有着不可替代的作用。
由于植物体内UDP-鼠李糖含量低、纯化难度大且效率低,而利用现代化学合成手段合成成本高、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,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一种催化杨梅UDP-鼠李糖生物合成的基因及其编码蛋白,所述基因为MrRHM1,其CDS序列如SEQ:NO.1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.2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基因MrRHM1及其编码蛋白在植物UDP-鼠李糖的生物合成中的应用,具体是在植物UDP-鼠李糖的生物合成调控和商品化合成中的应用。将上述基因MrRHM1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-MrRHM1。在大肠杆菌中表达pET-28a-MrRHM1,得到重组蛋白,可将UDP-葡萄糖转化为UDP-鼠李糖。
本发明提供了一种催化UDP-鼠李糖生物合成的基因MrRHM1及其编码蛋白及应用。首次克隆并验证了杨梅UDP-鼠李糖合成相关MrRHM1基因的功能。本发明还提供了含有MrRHM1基因的重组质粒pET-28a-MrRHM1,为通过生物工程方法大量合成UDP-鼠李糖,进一步开展黄酮醇鼠李糖苷生物合成调控研究奠定基础。
附图说明
图1:杨梅MrRHM1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MrRHM1重组蛋白。
图2:MrRHM1重组蛋白催化酶活产物结果图;其中,A:未加MrRHM1重组蛋白空白对照;B:MrRHM1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:杨梅MrRHM1基因克隆
以‘荸荠’杨梅果实为材料,取果实果肉组织,迅速用液氮冻透,然后放至-80℃冰箱中保存。利用CTAB法提取杨梅果肉的RNA,按照PrimeScriptTMRT reagent Kit with gDNAEraser(Takara)试剂说明书操作合成cDNA。
以反转录产物cDNA为模板,用SEQ:NO.3和SEQ:NO.4所示引物进行PCR扩增MrRHM1,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。获得与转录组数据库相匹配的基因序列SEQ:NO.1。
实施例2:MrRHM1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,MrRHM1引物序列如SEQ:NO.5和SEQ:NO.6所示。
以测序正确返还质粒为模板,用SEQ:NO.5和SEQ:NO.6所示引物进行PCR扩增MrRHM1,PCR反应体系为50μL,组分分别为:2×Phanta Max Buffer 25μL,dNTP Mix(10mMeach)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-MrRHM1重组质粒。
将pET-28a-MrRHM1重组质粒转化到大肠杆菌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,500mL收集1管,加入20mL1×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所示。
图1中可看出,pET-28a-MrRHM1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Clontech HisTALON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:MrRHM1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的磷酸缓冲液(pH=9.0)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,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30min,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2所示,可以看出MrRHM1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> 一种催化杨梅UDP-鼠李糖生物合成的基因及编码蛋白和应用
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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 Met Asp
260 265 270
Ala Glu Lys Ser 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 Asn Leu Gly Trp Ser Glu
290 295 300
Arg Thr Thr Trp Glu Glu Gly Leu Lys Lys Thr Met Glu Trp Tyr Ile
305 310 315 320
Asn Asn Pro Asp Trp Trp Gly Asp Val Thr Gly Ala Leu Leu Pro His
325 330 335
Pro Arg Met Leu Met Met Pro Gly Gly Ile Glu Arg His Phe Asp Gly
340 345 350
Ser Glu Glu Asp Lys Ser Ala Ser Tyr Val Ser Ser Asn Thr Arg Met
355 360 365
Leu Val Pro Pro Thr Lys Ser Ile Gly Ser Pro Arg Lys Pro Ser Leu
370 375 380
Lys Phe Leu Ile Tyr Gly Arg Thr Gly Trp Ile Gly Gly Leu Leu Gly
385 390 395 400
Lys Ile Cys Glu Lys Glu Gly Ile Ala Phe Glu Phe Gly Arg Gly Arg
405 410 415
Leu Glu Asp Arg Ser Ser Leu Met Ser Asp Met Gln Asn Val Lys 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 Thr 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 Ala His Pro Glu
485 490 495
Gly Ser Gly Ile Gly Tyr Lys Glu Glu Asp Lys Pro Asn Phe Thr Gly
500 505 510
Ser Phe Tyr Ser Lys Thr Lys Ala Met Val Glu Glu Leu Leu Lys Glu
515 520 525
Tyr 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 Ser Lys
545 550 555 560
Val Val Asn Ile Pro Asn Ser Val Thr Val 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 Gly Phe Lys Trp Val Asn Phe Thr Leu Glu Glu
610 615 620
Gln Ala Lys Val Ile Val Ala Pro Arg Ser Asn Asn Glu Met Asp Ala
625 630 635 640
Ser Lys Leu Lys Asn Glu Phe Pro Glu Leu Leu Ser Ile Lys Glu Ser
645 650 655
Leu Val Lys Tyr Val Phe Glu Leu Asn Lys Lys Asn Ile Ala Ala
660 665 670
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Unknow)
<400> 3
atggctagtt ataccccaaa g 21
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Unknow)
<400> 4
ttatgctgca atgtttttct 20
<210> 5
<211> 37
<212> DNA
<213> 人工序列(Unknow)
<400> 5
taaggcctct gtcgacatgg ctagttatac cccaaag 37
<210> 6
<211> 36
<212> DNA
<213> 人工序列(Unknow)
<400> 6
cagaattcgc aagcttttat gctgcaatgt ttttct 36
Claims (4)
1.一种催化杨梅UDP-鼠李糖生物合成的基因,其特征在于,所述基因为MrRHM1,其核苷酸序列如SEQ:NO.1所示。
2.一种催化杨梅UDP-鼠李糖生物合成的基因,其特征在于,所述基因MrRHM1编码蛋白的氨基酸序列如SEQ:NO.2所示。
3.一种重组质粒,其特征在于,所述重组质粒为将权利要求1所述的催化杨梅UDP-鼠李糖生物合成的基因MrRHM1连接到pET-28a载体的多克隆位点中构建获得,命名为pET-28a- MrRHM1。
4.根据权利要求1或2所述的一种催化杨梅UDP-鼠李糖生物合成的基因在植物UDP-鼠李糖的生物合成调控和商品化合成中的应用。
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