CN107828804B - 芒果hmgr基因、用于克隆芒果hmgr基因的引物及其克隆方法 - Google Patents
芒果hmgr基因、用于克隆芒果hmgr基因的引物及其克隆方法 Download PDFInfo
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Abstract
本发明涉及生物技术领域，具体涉及一种芒果HMGR基因、用于克隆芒果HMGR基因的引物及其克隆方法，该基因的核苷酸序列为SEQ ID NO：1；克隆步骤：对芒果的总RNA进行提取和逆转录合成cDNA第一链；芒果的HMGR基因5′‑RACE和3′‑RACE扩增；芒果的HMGR基因全长cDNA扩增；PCR克隆产物的纯化与筛选；本发明方法首次获得芒果HMGR基因的cDNA序列全长，同时通过一种克隆芒果HMGR基因的引物组和克隆方法，它能快速准确地克隆芒果HMGR基因的编码序列，为后续芒果HMGR基因分子研究提供了理论基础。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种芒果HMGR基因、用于克隆芒果HMGR基因的引物及其克隆方法。
背景技术
芒果（Mangifera indica L.）为漆树科芒果属热带常绿果树，原产印度及马来西亚等地区，在我国主要集中分布于海南、广东、广西、云南等南方省区。近年来全球芒果年产量约2700万吨，已经成为世界许多地区的主要产业经济支柱，我国芒果产量一直位居世界前列，并呈现增长的趋势（李日旺等，2013）。芒果色、香、味俱佳，深受消费者青睐，其中香气这一品质特征越来越受到消费者以及品质育种学家的重视，因此香气成分成为评价芒果果实品质的重要指标之一（郭安，2006）。先前的研究发现，芒果果实中存在至少300多种香气成分，主要有单萜、倍半萜、醇类、酸类、酮类、酯类、酸类等，其中，萜类物质是芒果主要的风味物质，往往达到总挥发性香气物质的70%-90%左右，根据单萜与倍半萜的优势关系又分为单萜主导型和倍半萜主导型（张劲，2011；Jorge等，2005）。许多萜类被认为是通过甲羟戊酸途径（Mevalonate pathway，MVA pathway）合成的（马靓等，2006）。
3-羟基-3-甲基戊二酸单酰辅酶A还原酶（3-hydroxy-3-methylglutarylcoenzyme A reductase, HMGR）属于MVA途径中的一个关键酶，催化HMG-CoA产生甲羟戊酸（mevalonic acid, MVA），这步反应是不可逆的，因此HMGR被认为是MVA途径中第一个重要的限速酶，也是MVA途径的重要调控点（Chappell等，1995）。研究表明，HMGR基因在很多植物中以基因家族的形式存在（Choi等，1992；Yang等，1991），目前在苹果、番茄、菠萝、荔枝等植物中克隆出该基因，但是在芒果中HMGR基因的研究至今还未发现有报道。因此，克隆芒果HMGR基因是相当有必要的，为后续深入研究芒果HMGR基因在果实成熟期间香气形成的特异性表达提供基础。
发明内容
本发明的发明目的在于提供一种芒果HMGR基因、用于克隆芒果HMGR基因的引物及其克隆方法。
为了实现上述发明目的，本发明采用的技术方案如下：
芒果HMGR基因，该基因的核苷酸序列为SEQ ID NO：1所示。
进一步说明，所述芒果HMGR基因编码的蛋白质，其氨基酸序列如SEQ ID NO：2所示。
进一步说明，所述芒果HMGR基因的克隆引物组引物序列如下：
HMGR-F：5′-aatgccgtcttcttcaccgtcttc-3′，
HMGR-R：5′-ttccttgatgctgctgcctacttg-3′；
UPM：5′-ctaatacgactcactatagggcaagcagtggtatcaacgcagagt-3′；
UPM short：5′-ctaatacgactcactatagggc-3′；
5′-HMGR-1R：5′-cgtcagtaacaagagctggtggcggcgaagt-3′；
5′-HMGR-2R：5′-gacagtgaagaagacggtgaagaaga-3′；
3′-HMGR-1F：5′-cgtggagagttctcactgtatcaccatgatggaattgg-3′；
3′-HMGR-2F：5′-cttcatgtctctgtcacaatgccttct-3′。
一种克隆芒果HMGR基因的方法，该方法包括以下步骤：
步骤一：对芒果的总RNA进行提取和逆转录合成cDNA第一链；
步骤二：芒果的HMGR基因5′RACE和3′RACE扩增，
步骤三：芒果的HMGR基因全长cDNA扩增；
步骤四：PCR克隆产物的纯化与筛选。
进一步说明，步骤二的具体操作方法为：将参考芒果的转录组基因注释结果（GenBank登录号：GBCV01017877.1），根据序列信息设计引物，以逆转录合成的cDNA第一链为模板，利用HMGR-F、HMGR-R引物进行PCR扩增，得到芒果HMGR基因保守区片段；以芒果HMGR基因保守区片段序列设计5′-RACE和3′-RACE引物，以逆转录后的cDNA为模板分别进行5′-RACE和3′-RACE反应，PCR扩增得到5′端和3′端的侧翼片段；其中，
HMGR-F引物序列：5′-aatgccgtcttcttcaccgtcttc-3′，
HMGR-R引物序列：5′-ttccttgatgctgctgcctacttg-3′；
5′-RACE引物序列包括：
UPM：5′-ctaatacgactcactatagggcaagcagtggtatcaacgcagagt-3′；
UPM short：5′-ctaatacgactcactatagggc-3′；
5′-HMGR-1R：5′-cgtcagtaacaagagctggtggcggcgaagt-3′；
5′-HMGR-2R：5′-gacagtgaagaagacggtgaagaaga-3′；
3′-RACE引物序列包括：
UPM：5′-ctaatacgactcactatagggcaagcagtggtatcaacgcagagt-3′；
UPM short：5′-ctaatacgactcactatagggc-3′；
3′-HMGR-1F：5′- cgtggagagttctcactgtatcaccatgatggaattgg-3′；
3′-HMGR-2F：5′-cttcatgtctctgtcacaatgccttct-3′。
由于采用了上述技术方案，本发明的有益效果是：
本发明方法首次获得芒果HMGR基因的cDNA序列全长，同时通过一种克隆芒果HMGR基因的引物组和克隆方法，它能快速准确地克隆芒果HMGR基因编码序列，为后续芒果HMGR基因分子研究提供了理论基础。
附图说明
图1是芒果总RNA琼脂糖凝胶电泳图；
图2是芒果HMGR基因保守区扩增琼脂糖凝胶电泳图，其中M是Marker分子量标准，条带从上至下依次为2000bp、1000bp、750bp、500bp、250bp、100bp；
图3是RACE法分别扩增获得5′和3′末端序列扩增琼脂糖凝胶电泳图，其中M是Marker分子量标准，条带从上至下依次为2000bp、1000bp、750bp、500bp、250bp、100bp；
图4是单菌落进行PCR鉴定琼脂糖凝胶电泳图，其中M是Marker分子量标准，条带从上至下依次为2000bp、1000bp、750bp、500bp、250bp、100bp，编号1-10从左至右分别是菌液3-1、3-2、3-3、3-4、3-5、5-1、5-2、5-3、5-4、5-5的DNA琼脂糖凝胶电泳图；
图5是芒果HMGR基因cDNA全长中包含的一个1671 bp完整的开放阅读框；
图6是本申请的芒果MiHMGR与其他植物来源HMGR的多重序列比对图，
说明：图中CmHMGR来源于Cucumis melo，GenBank登录号为BAA36291.1；DlHMG2来源于Dimocarpus longan，GenBank登录号为AET72045.1；AcHMGR来源于Ananas comosus，GenBank登录号为AIT52532.1；MdHMG1来源于Malus domestica，GenBank登录号为AAK95406.1；三角形符号表示活性中心的催化氨基酸；2标注的方框表示底物结合口袋；1标注的方框表示NADP(H)结合区；
图7是本申请的芒果MiHMGR蛋白与其他植物HMGR蛋白家族成员的进化树分析图；
图8是本申请的芒果MiHMGR蛋白的同源建模图的正面图；
图9是本申请的芒果MiHMGR蛋白的同源建模图的侧面图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
实施例1：
1. 实验方法
1.1芒果RNA提取及逆转录
取芒果果肉100 mg，液氮研磨，参照RNAprep Pure多糖多酚植物总RNA提取试剂盒（天根）说明书提取总RNA，提取后进行电泳检测见图1。
采用HiScript II Q RT SuperMix for qPCR (+gDNA wiper) 试剂盒（诺维赞）逆转录合成第一链cDNA并用于后续的RACE法扩增实验。先将RNA模板、RNase-Free Water、4×gDNA wiper Mix混匀，42℃孵育2分钟，然后再加入5×HiScript II qRT SuperMix II轻轻混匀。逆转录PCR反应：25℃ 10 min，50℃ 30 min，85℃ 5 min。反应结束后，短暂离心，置于冰上冷却备用。
表1 PCR反应体系试剂及用量
| 试剂 | 10 μl反应体系 |
| RNA Template | 1 pg-500 ng（终浓度） |
| 4×gDNA wiper Mix | 2 μl |
| 5×HiScript II qRT SuperMix II | 2 μl |
| RNase-Free Water | to 10 μl |
1.2 芒果HMGR基因保守区片段的扩增
参考芒果的转录组基因注释结果（GenBank登录号：GBCV01017877.1），根据序列信息设计引物，以逆转录后的cDNA为模板，利用表3中的HMGR-F/HMGR-R引物进行PCR扩增，其反应如下：94℃预变性2 min；94℃变性30 sec，56℃退火30 sec，72℃延伸1 min，35个循环；72℃延伸2 min。芒果HMGR基因保守区片段的扩增的琼脂糖凝胶电泳图见图2。
表2
| 试剂 | |
| cDNA Template | 1 μl |
| 2×Es Taq MasterMix | 25 μl |
| <i>HMGR</i>-F 10 µM | 1 µl |
| <i>HMGR</i>-R 10 µM | 1 µl |
| ddH<sub>2</sub>O | 22 μl |
1.3 芒果HMGR基因全长的扩增
根据上一步获得的已知序列分别设计5′-RACE和3′-RACE引物（见表3），UniversalPrimer Mix (UPM)和UPM short分别为第一轮和第二轮RACE反应中的通用引物。
以逆转录后的cDNA为模板分别进行5′-和3′-RACE反应，PCR扩增得到5′端和3′端的侧翼片段，见图3。
RACE法参照SMARTer RACE 5'/3' Kit (Clontech)进行。PCR反应如下：94℃30sec，68℃30 sec，72℃ 3 min，25个循环。
5′端和3′端的侧翼片段经琼脂糖凝胶回收后，连接pMD-19T载体(TaKaRa)，转化E.coli DH5α感受态，挑取白色单菌落进行PCR验证，初步鉴定片段大小（图4）并测序（挑选菌液3-1、5-1测序）。根据测序结果拼接得到HMGR基因的全长cDNA序列。
其中：
RACE法PCR反应体系（50 μl）：
cDNA Template 2.5 μl
10×UPM (or UPM short) 2.5 μl
5′ or 3′ HMGR引物 1 μl
2×SeqAmp Buffer 25 μl
SeqAmp DNA Polymerase 1 μl
ddH2O to 50 μl
连接pMD-19T载体的反应体系（10 μl）：
回收纯化的PCR扩增基因片段 4 μl
Solution I 5 μl
载体 0.3 μl
灭菌水 0.7 μl
配制好体系后16℃连接过夜。
转化的具体操作是：取出-80℃保存的感受态细胞（DH5α），置冰上融化；加入连接产物，轻旋转离心管以混匀内容物，冰浴30 min；将离心管置于42℃热击60-90 s，然后迅速冰浴2-3 min；向每个离心管中加入500 μl的LB培养基(不含抗生素)，混匀后置于37℃摇床振荡培养45 min（150 rpm/min）；将离心管内容物混匀，吸取100 μl加至含有氨苄抗性的LB培养基上，均匀涂开。将平板置室温至液体全部被吸收，倒置平板，37℃培养过夜。
挑取白色单菌落进行PCR鉴定，其反应体系和反应程序如下：
2×Es Taq MasterMix 25 µl
M13-47 (10 µM) 1 µl
M13-48 (10 µM) 1 µl
单菌落
ddH2O 23 µl
反应程序：94℃ 2 min；94℃ 30 sec，56℃ 30 sec，72℃ 30 sec，35个循环；72℃2 min。
表3
| Name of primers | Primers sequences (5′-3′) |
| <i>HMGR</i>-F | aatgccgtcttcttcaccgtcttc |
| <i>HMGR</i>-R | ttccttgatgctgctgcctacttg |
| UPM | ctaatacgactcactatagggcaagcagtggtatcaacgcagagt |
| UPM short | ctaatacgactcactatagggc |
| 5′-<i>HMGR</i>-1R | cgtcagtaacaagagctggtggcggcgaagt |
| 5′-<i>HMGR</i>-2R | gacagtgaagaagacggtgaagaaga |
| 3′-<i>HMGR</i>-1F | cgtggagagttctcactgtatcaccatgatggaattgg |
| 3′-<i>HMGR</i>-2F | cttcatgtctctgtcacaatgccttct |
1.4 芒果HMGR基因的生物信息学分析
将测序获得的序列与保守序列用DNAMAN进行拼接，得到完整的序列。利用NCBIORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)查找开放阅读框；利用CD-Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)分析结构域；利用在线软件ExPASyProtParam (http://web.expasy.org/protparam/)分析基因编码的氨基酸，蛋白质的分子量及等电点等特性；利用在线软件SignalP 4.1 (http://www.cbs.dtu.dk/services/SignallP/)分析其信号肽序列；利用BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/)查找其相似序列；利用TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/)分析跨膜结构；使用ClustalX 1.83软件进行多序列比对分析；使用在线软件Phyre (http://www.sbg.bio.ic.ac.uk/phyre/html/index.html)对基因编码的氨基酸序列进行结构预测。
2. 实验结果
2.1 芒果HMGR基因的扩增
PCR反应完毕后进行琼脂糖凝胶电泳检测，结果显示在1.7 kb左右有一条特异性DNA条带（图2），经测序鉴定发现该基因片段与参考的基因片段相似，BLAST比对结果表明与其他植物HMGR基因有较高的同源性，因此，获得了芒果HMGR基因的部分片段。
利用RACE法分别扩增获得5′和3′末端序列（图3），根据测序结果拼接末端序列后，得到芒果HMGR基因cDNA全长（2088 bp）。将整段序列进行比对查找开放阅读框（ORF），结果表明芒果HMGR基因cDNA全长包含一个1671 bp完整的ORF（图5），命名为MiHMGR。
2.2芒果HMGR基因的序列分析
基因MiHMGR编码556个氨基酸，无信号肽序列。TMHMM Server v.2.0预测结果表明，该基因的编码蛋白MiHMGR具有两个跨膜区，分别位于29-51 AA之间以及72-94 AA之间。通过ExPASy中的Protparam预测MiHMGR蛋白的理化性质，该蛋白的分子量及理论等电点pI值分别为59.18 kDa和7.83；原子组成为C2620H4206N706O787S31，总原子数为8350；不稳定系数为30.64，脂溶指数为93.13，亲水性平均系数为0.133，是一种稳定的疏水性蛋白。
根据NCBI Conserved-Domain数据库的预测分析结果发现MiHMGR含有1个HMG-CoA_reductase_class I（cd00643）结构域（143-546AA），包括4个关键的催化位点（catalytic residues），分别为Glu235、Lys367、Asp443、His541；多个NADP(H)结合位点；多个底物结合位点；多个抑制剂结合位点以及多个多肽结合位点。其中，NADP(H)结合区GDAMGMNMVS和VGTVGGGT，以及2个底物结合口袋EGCLVASTNRG和SLMSALAAGQLVKSHMKY（图6），与之前报道的植物HMGR蛋白序列具有高度的保守性。
将该基因的氨基酸序列在NCBI数据库中进行比对，BLAST分析结果表明，该序列与已报道的龙眼HMG2蛋白（GenBank登录号：AET72045.1）具有最高的一致性，为77%。其多重序列比对结果如图6。将MiHMGR与其他植物HMGR蛋白家族中的成员进行序列分析并构建系统进化树（如图7），结果发现MiHMGR与荔枝和龙眼的HMGR亲缘关系较近。
通过ExPASy中的SOPMA对芒果HMGR蛋白进行在线二级结构预测，结果表明：该蛋白由α-螺旋（Alpha helix，37.95%）、延伸链（Extended strand，20.68%）、β-转角（Beta turn，9.89%）和无规卷曲（Random coil，31.47%）四种结构组成，其中以α-螺旋和无规卷曲为主。由Phyre对芒果HMGR基因编码的氨基酸序列进行在线三级结构预测（图8和图9）。
以上分析结果均表明本研究克隆得到的基因MiHMGR属于HMGR基因家族中的一员。
本发明并不局限于前述的具体实施方式。本发明扩展到任何在本说明书中披露的新特征或任何新的组合，以及披露的任一新的方法或过程的步骤或任何新的组合。
序列表
<110> 广西壮族自治区农业科学院农产品加工研究所
<120> 芒果HMGR基因、用于克隆芒果HMGR基因的引物及其克隆方法
<141> 2017-11-13
<160> 10
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acatggggac accgaacttc atatcgatac caaatccaat atcgtaatcg tttccttttt 60
tctacaatcc cagattcagt ttcttcaccg accgccgtct atggaggctc gccggaaatc 120
tcttcactcc tcaaaaacct tccaccatga acctagttca aaaaaacctt cctgtgatgc 180
cgtttctctc cctttgtgcg tagcaaatgc cgtcttcttc accgtcttct tcactgtcgt 240
ttattattta ttatctcatt ggcgcgagaa gatccgtacc tccacgcctc ttcatctcgt 300
tactctctcc gagatgatcg cacttgtcgc cttctttgct tcctgtatct acttgttggg 360
attcttcggt atcgggtttt ttcactcttt tgttcttaaa gaggatgatg acagcaacga 420
cgtcgttctt aaacacaatt ctggaaagtt aactccttgc gctcaagctc ttgattgcac 480
tattgtcact tcgccgccac cagctcttgt tactgacgga gatgacgagg atataattaa 540
gtcagttgtg gacggaaaga cgccgtctta tgctttggaa tcaaaattgg gggattgcaa 600
gagggccgct tcgattaggc gtgaagcgtt ggagagaatc accggcaagt ctttgacggg 660
tttgccctta gaggggtttg attatgactc cattttaggg cagtgttgtg aattgccggt 720
tggttatgtt cagattccag tgggaatcgc cgggcctttg ttgctcaacg gaaacgagtt 780
ttcggttcct atggcgacta ctgaaggttg cttggtggct agcactaaca gaggctgtaa 840
agctattcat ttgtctggtg gggcaactag tgttttgttg aaggatggga tgactagagc 900
tccagttgta aggtttggta gtgctgaaca agctgctgag ttgaagttct tcttggagaa 960
tccagccaat tttggaaaat tgtctgcagt tttttacaag tctagcagat ttggcaggct 1020
tcaaagtatt aaatgtgcaa ttgctggtaa aaatctctac ttgagattca catgcggtac 1080
tggtgatgct atggggatga acatggtctc taagggtgtt caaaatgttc ttgaatttct 1140
ccaggacaag ttccctgaca tggatattat tggtatttct ggtaacttct gttcggataa 1200
gaagcctgca gctgtgaact ggatagaggg acggggaaag tctgtggttt gtgaggctgt 1260
aattaagggt gacgttgtgc agaaggtgtt gaagactagt gtggaagctt tggtggagct 1320
taacatgctc aagaacctga ctggttctgc tatggctgga gctcttggtg gcttcaatgc 1380
ccatgccagt aatattgtta ctgccattta cctagccact ggtcaagatc ccgctcaaaa 1440
cgtggagagt tctcactgta tcaccatgat ggaattggta aatgatggca aagatcttca 1500
tgtttctgtc acaatgcctt ctattgaggt cggtactgtt ggaggtggca cacagcttgc 1560
atctcagtca gcatgcttga atctgcttgg ggtgaaaggt gccagcaaag agacaccggg 1620
agcaaatgca aggctgttgg ctaccattgt agctggttct gttcttgcag gagagctgtc 1680
tctaatgtct gctcttgcag ctggacagct tgttaagagc cacatgaagt ataatagatc 1740
aaacaaagac aatactaagg ttccttcata ggaaaaatta aaatctcaag catggaaaat 1800
cctcagaatc tggtgtagaa ggcagaatct atgatataca cagaacttca agtaggcagc 1860
agcatcaagg aaattgtttc tctttatttc atgtgttttt tttttccttt ggtttcaatc 1920
aacgttagta ctttcaaatt ctgtttgtgc atgatgaaag ctttcaatct gttcatttcc 1980
tctgtgaaat gactcgacgt cataagtcaa agctagtact tgaatccatt gtaccattta 2040
tttacttatc aatgaaaaca acattcaggg caaaaaaaaa aaaaaaaa 2088
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Leu Glu Arg Ile Thr Gly Lys Ser Leu Thr Gly Leu Pro Leu Glu Gly
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Claims (3)
1.一种克隆芒果HMGR基因的方法，其特征在于：该方法包括以下步骤：
步骤一：对芒果的总RNA进行提取和逆转录合成cDNA第一链；
步骤二：芒果的HMGR基因5′-RACE和3′-RACE扩增，
步骤三：芒果的HMGR基因全长cDNA扩增；
步骤四：PCR克隆产物的纯化与筛选；
所述芒果HMGR基因的克隆引物组引物序列如下：
HMGR-F：5′-aatgccgtcttcttcaccgtcttc-3′；
HMGR-R：5′-ttccttgatgctgctgcctacttg-3′；
UPM：5′-ctaatacgactcactatagggcaagcagtggtatcaacgcagagt-3′；
UPM short：5′-ctaatacgactcactatagggc-3′；
5′-HMGR-1R：5′-cgtcagtaacaagagctggtggcggcgaagt-3′；
5′-HMGR-2R：5′-gacagtgaagaagacggtgaagaaga-3′；
3′-HMGR-1F：5′-cgtggagagttctcactgtatcaccatgatggaattgg-3′；
3′-HMGR-2F：5′-cttcatgtctctgtcacaatgccttct-3′；
所述PCR反应体系：
cDNA Template 2.5 μl
10×UPM 2.5 μl
5′ or 3′ HMGR引物 1 μl
2×SeqAmp Buffer 25 μl
SeqAmp DNA Polymerase 1 μl
ddH2O to 50 μl；
所述芒果HMGR基因的核苷酸序列为SEQ ID NO：1所示。
2.根据权利要求1所述的一种克隆芒果HMGR基因的方法，其特征在于：所述芒果HMGR基因编码的蛋白质，其氨基酸序列如SEQ ID NO：2所示。
3.根据权利要求1所述的一种克隆芒果HMGR基因的方法，其特征在于：
步骤二的具体操作方法为：将参考芒果的转录组基因注释结果GenBank登录号：GBCV01017877.1，根据序列信息设计引物，以逆转录合成的cDNA第一链为模板，利用HMGR-F、HMGR-R引物进行PCR扩增，得到芒果HMGR基因保守区片段；以芒果HMGR基因保守区片段序列设计5′-RACE引物和3′-RACE引物，以逆转录后的cDNA为模板分别进行5′-RACE和3′-RACE反应，PCR扩增得到5′端和3′端的侧翼片段；其中，
HMGR-F引物序列：5′-aatgccgtcttcttcaccgtcttc-3′；
HMGR-R引物序列：5′-ttccttgatgctgctgcctacttg-3′；
5′-RACE引物序列包括：
UPM：5′-ctaatacgactcactatagggcaagcagtggtatcaacgcagagt-3′；
UPM short：5′-ctaatacgactcactatagggc-3′；
5′-HMGR-1R：5′-cgtcagtaacaagagctggtggcggcgaagt-3′；
5′-HMGR-2R：5′-gacagtgaagaagacggtgaagaaga-3′；
3′-RACE引物序列包括：
UPM：5′-ctaatacgactcactatagggcaagcagtggtatcaacgcagagt-3′；
UPM short：5′-ctaatacgactcactatagggc-3′；
3′-HMGR-1F：5′- cgtggagagttctcactgtatcaccatgatggaattgg-3′；
3′-HMGR-2F：5′-cttcatgtctctgtcacaatgccttct-3′。
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