CN109082432B - 一种青稞阿魏酰酪胺酰基转移酶基因及其用途 - Google Patents
一种青稞阿魏酰酪胺酰基转移酶基因及其用途 Download PDFInfo
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Abstract
本发明提供了一种阿魏酰酪胺酰基转移酶基因HVUL4H11714，本发明还提供了包含该基因的重组载体、重组菌、转基因植物的制备方法，本发明还提供了阿魏酰酪胺酰基转移酶及其制备方法。本发明阿魏酰酪胺酰基转移酶可以合成中药单体阿魏酰酪胺，应用前景优良。
Description
技术领域
本发明涉及基因工程领域，具体涉及一种青稞阿魏酰酪胺酰基转移酶基因的应用。
背景技术
中药活性成分阿魏酰酪胺具有清除自由基、抗氧化作用，同时还具有抑菌、抑制黑色素合成、刺激胰岛素分泌等活性。目前阿魏酰酪胺主要由石柑子、地骨皮等中药中提取。由于中药自然资源有限，仅仅依赖直接提取不能满足人们长期的需求。
青稞，青稞英文名：hullessbarley是禾本科大麦属的一种禾谷类作物，因其内外颖壳分离，籽粒裸露，故又称裸大麦、元麦、米大麦。主要产自中国西藏、青海、四川、云南等地，是藏族人民的主要粮食。青稞在青藏高原上种植约有3500年的历史，从物质文化之中延伸到精神文化领域，在青藏高原上形成了内涵丰富、极富民族特色的青稞文化，有着广泛的药用以及营养价值，已推出了青稞挂面、青稞馒头、青稞营养粉等青稞产品。
未见青稞中含有阿魏酰酪胺的报道。
发明内容
本发明通过对青稞的研究，发现了一种新的青稞阿魏酰酪胺酰基转移酶基因，其可以有效将阿魏酰基辅酶A转化为阿魏酰酪胺。
本发明提供了一种基因(阿魏酰酪胺酰基转移酶基因HVUL4H11714)，其核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种重组载体，它包括SEQ ID NO.1所示的核苷酸序列。
前述的重组载体，所述重组载体是重组pGEX-6P-1(Novagen)。
前述的重组菌，它包含前述的重组载体。
前述的重组菌，所述重组菌为Transetta(DE3)。
本发明还提供了一种蛋白，其氨基酸序列如SEQ ID NO.2所示。
前述蛋白的方法，它是采用前述重组菌发酵制备。
前述蛋白的方法，步骤如下：
(1)取前述重组菌，活化，得菌液；
(2)将菌液接种到LB培养基中培养，3～4h后，加入IPTG诱导剂诱导培养，收集菌体；
(3)破菌，离心，取上清，纯化，即可。
步骤(1)中，所述菌液的浓度为1×106～107cfu/ml；优选地，所述活化是采用含有Amp的LB培养基培养培养，培养时间是6-10小时，培养温度为37℃；
和/或，步骤(2)中，所述菌液的接种比例为1:50；所述培养的温度是37℃，转速为200rpm；所述IPTG的浓度为1mol/L，加入量为培养液的1/100000；诱导培养的温度是20℃，转速为160rpm；
和/或，步骤(3)中，所述分离纯化的方法为GST标签融合蛋白纯化方法；优选地，所述纯化方法为：取上清，上树脂柱，穿流2次；采用Lysis buffer冲洗树脂去除杂蛋白；用15mmol/L还原型谷胱甘肽溶液洗脱，得蛋白；进一步优选地，所述树脂是GlutathioneSepharoseTM 4B树脂；所述还原型谷胱甘肽溶液是还原型谷胱甘肽溶于Lysis buffer得到的溶液。
前述的基因片段、重组载体、重组菌、蛋白在制备阿魏酰酪胺方面的用途。
本发明还提供了一种制备阿魏酰酪胺的方法，它是采用前述的基因片段、重组载体、重组菌、蛋白，以阿魏酰基辅酶A作为酰基供体，酪胺作为供体，制备阿魏酰酪胺。
本发明还提供了一种生产阿魏酰酪胺和香豆酰酪胺的转基因植物的构建方法，其特征在于：取前述的基因，转入植物中，获得表达前述蛋白的植株，即可。
前述的构建转基因烟草的方法，其特征在于：所述转入植物的方法是农杆菌法、基因枪法、电转法、PEG介导法、脂质体法和磷酸钙-DNA共沉淀法中的一种；本发明转基因植物为转基因烟草。
本发明还提供了前述的基因片段、重组载体、重组菌在制备高产阿魏酰酪胺和香豆酰酪胺青稞品种的用途。
本发明发现了青稞中的一种新的基因：青稞阿魏酰酪胺酰基转移酶基因，该基因表达的蛋白——青稞阿魏酰酪胺酰基转移酶，可以将阿魏酰基辅酶A转化为阿魏酰酪胺，提高青稞的保健价值；本发明还以该基因片段进行体外表达，获得了青稞阿魏酰酪胺酰基转移酶，在体外反应中，成功地将以阿魏酰基辅酶A作为酰基供体，酪胺作为供体，制备得到了阿魏酰酪胺；本发明还将该基因转移到烟草中，使得烟草植株中也表达阿魏酰酪胺酰基转移酶，进一步产生阿魏酰酪胺，提高其价值。本发明提供的新的基因，及其重组载体、重组菌、转基因植物均具有良好的应用前景。
本发明的基因片段和重组载体，可以用来提高青稞对阿魏酰酪胺和香豆酰酪胺的合成，实现青稞的定向改良。
本发明的转基因烟草构建方法，为青稞的定向改良提供了重要的参考。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为HVUL4H11714蛋白的SDS-PAGE电泳图，Marker：100,70,55,40,35,25KDa。
图2为体外催化反应的LC-MS图：tyramine，酪胺；N-feruloyl tyramine，N-阿魏酰酪胺。
图3为转基因植物提取物质谱图。
具体实施方式
实施例1HVUL4H11714基因的分离以及原核表达
本实施例主要是HVUL4H11714基因获得、载体构建及表达的方法。
(1)基因片段和载体的构建
称取1克青稞新鲜叶片，提取青稞RNA，用Thermo Fisher公司的M-MLV ReverseTranscriptase合成cDNA，设计引物，如下：
F:CGCGGATCCATGGAGATGACGAGCAGCACG
R:CCGGAATTCTCAATCCAACGAGTAGCAGATCTGC
进行PCR扩增，得到目的条带大小的片段。PCR产物用胶回收试剂盒(GelExtraction Kit D2500-02，OMEGA)纯化。
扩增得到的目的片段HVUL4H11714基因的核苷酸序列(SEQ ID NO.1)为：
ATGGAGATGACGAGCAGCACGATGGTGAAGCCGGCGTACGCCGTGCCGCACCCGCTCGTCGGCGAGAAGGTCCCGCTGACCGTCTTCGACCGCGCCGCCCTGGACATCTTCGTCCCCACCGTGCTCGCCTACCCCGCGCCGGCGCCGTCCAACGAGGCGCTCAGGGAAGGGCTCCTCAAGGCCGTCGCGCCGTACCCTCACCTGGCGGGCCGCCTCGCCCTCGACGACCTCGGCCGGCGCTTCCTCCACGTGAACAACGAGGGCGTGCTCCTGATCGAGGCCACCGTCCCGGCCGACCTGGCGGACGTGCTCGTCGACGGCCGCATGGCCGCTGGCGTCGACGACCTCTACCCGGCGATACCGGAGGAGAACATTGGGGCGGCGCTGCTGCAGATCCAGCTCAACAGGTACAAGTGCGGCGGCCTCGTGGTCGGCATAAGCTGCCACCACCACACCGCCGACGGCCATTCCATGAGCATGTTCTTCACCGCGTGGGCGACGGCGGTCCGCGAGGGTAAGGACTTCACCACGCCGACCCCGTTCCTCGACCGTGCGAGAACCGCGGTGCCTCGAAGCACGCCGACGCCGGTGTTCGACCACCGGTCACGCGAGTTCACTAGTGGAGACGGAGGAGACTCCTATGCCGTCGTCCCCATGGACAGGATCAAGAACCTCACCCTGCACTTCACGGCCGAGTTCGTCGCCGACCTCAAATCCCTCGTTGGCACCCGTTGCAGCACGTTCCAGTGCCTACTAGCGCACGTCTGGAAGAAGCTCACGGCGGCGCGGGACCTGAAGCCGGAGGAGTTCACCAAGGTGAGGCTCGCCGTGAACTGCAGGGGCAGGGCCGACCCTCCTGTGCCCATGGACTTCTTCGGGAACATGGTGCTCTGGGCTTTCCCAAGGCTTCAGGTCCGGGACATGCTGGACTCGAGCCACGGCAGCGTGGTCAGCGTCATCCGCGACGCCGTGGCGCGCATCGACGACGAGTACGTGCAGTCCTTCGTCGACTTCGGCGGAGTGGCGGACGCGAACGGGGAGGAGCTCGTCGCGACGGCGGCCGCTGCCGGCACGATGTTCTGCCCGGACGCGGAGGTGGACAGCTGGCTGGGATTCAGGTTCCATCAGCTCGATTTTGGCACCGGCGCACCGTCCGCTTTCGTGCCGCCGGACCTGCCTTTCGAGGGGCTCATGATCTTCATGCCATCGCGCAAGGCCAACGGCGGCGTCGACCTCTTCATGGCCGTCGCGGAGGAGCACGTCGCGGCGTTCGAGCAGATCTGCTACTCGTTGGATTGA
前述核苷酸序列所述的HVUL4H11714基因可以通过采用上述方法获得，也可以直接合成。
得到的基因片段转入载体pGEX-6P-1中，后重组载体转入Transetta(DE3)菌株中，得到含有目的片段的重组菌株。
(2)基因的表达
1.PCR检测阳性克隆，抽提质粒测序。
2.将测序正确的质粒载体热击转化大肠杆菌transeta(DE3)，抗性CN。
3.早上9点左右随机挑取2个正常大小克隆于5mL含有Amp的LB培养基中，37℃摇至下午4点左右。选其中一个，将4mL活化菌液(浓度为1×106～107cfu/ml)按照1:50比例转接到200mL大瓶LB培养基中，大摇床37℃培养，转速200rpm。3～4小时后，200mL培养基中加入2uL 1M IPTG诱导剂。20℃，160rpm条件下诱导过夜。剩余1mL菌液用来保菌。
4.第二天早上8点收集菌体。500mL离心瓶，4000rpm离心10min即可。
5.50mL Lysis buffer重悬菌体，涡旋混匀，转入50mL离心管中，分别加入50uLPMSF,10uLβ-巯基乙醇，混匀放置冰上。
6.采用高压破碎仪进行大肠杆菌细胞破碎实验。
7.破碎完成后样品，取20ul作为总蛋白样品。再取1mL样品4℃，13000rpm离心10min，取20uL上清作为上清液样品。加入等体积2*Loading buffer，煮沸5min,SDS-PAGE电泳检测蛋白表达情况。剩余上清可暂冻存于-20℃冰箱。剩余未离心样品可冻存于-80℃冰箱。
8.SDS-PAGE电泳完毕，加入考马斯亮蓝染色液，微波炉煮沸1min后染色半小时，加入脱色液脱色。每隔1h换一次脱色液，直至蛋白条带清晰，转至清水中。
9.GST标签融合蛋白纯化。将破碎未离心的所有样品离心，上清与1mL树脂在4℃混匀仪上混合3h。混匀结束后，将混合液穿过层析柱，穿流2次效果更佳。先用预冷的Lysisbuffer冲洗树脂(Glutathione SepharoseTM 4B，GE公司)，同时流出液用Bradford Assay检测，直至不变蓝色表示杂蛋白冲洗干净。然后，用15mmol/L还原型谷胱甘肽溶液(0.09g溶解于20mL lysis buffer)洗脱目的蛋白，每次加入1mL,层析柱底部用1.5mL的离心管收集，每管约1mL，分别记为E1、E2、E3、E4、E5、E6，直至Bradford Assay检测洗脱液中不含蛋白。未用完的还原型谷胱甘肽溶液继续全部洗脱树脂，然后分别用Lysis buffer，ddH2O 20％乙醇冲洗，保存于20％乙醇中。
10.收集的蛋白用SDS-PAGE检测，得到73kDa的条带(图1)，GST标签的分子量为26kDa，剩余目的蛋白的分子量为47kDa，与氨基酸计算的分子量相同，说明本发明制备得到了带有GST标签的目的蛋白。
得到的目的片段HVUL4H11714蛋白的氨基酸序列(SEQ ID NO.2)为：MEMTSSTMVKPAYAVPHPLVGEKVPLTVFDRAALDIFVPTVLAYPAPAPSNEALREGLLKAVAPYPHLAGRLALDDLGRRFLHVNNEGVLLIEATVPADLADVLVDGRMAAGVDDLYPAIPEENIGAALLQIQLNRYKCGGLVVGISCHHHTADGHSMSMFFTAWATAVREGKDFTTPTPFLDRARTAVPRSTPTPVFDHRSREFTSGDGGDSYAVVPMDRIKNLTLHFTAEFVADLKSLVGTRCSTFQCLLAHVWKKLTAARDLKPEEFTKVRLAVNCRGRADPPVPMDFFGNMVLWAFPRLQVRDMLDSSHGSVVSVIRDAVARIDDEYVQSFVDFGGVADANGEELVATAAAAGTMFCPDAEVDSWLGFRFHQLDFGTGAPSAFVPPDLPFEGLMIFMPSRKANGGVDLFMAVAEEHVAAFEQICYSLD
实施例2转基因烟草的构建
①将含有目的基因的瞬时表达载体(George Lomonossoff教授提供，John InnesCentre)转化农杆菌(EHA105)；
②挑取阳性农杆菌克隆于500ul含有相应抗生素(kn)的LB中，培养20-24小时；
③转接200ul于5ml含有相应抗生素(kn)的LB中，28℃摇床220rpm至OD＝2.0左右。
④10000rpm常温离心2min收集菌体，用提前配制转化缓冲液进行菌体的重悬，摇床震荡3h；缓冲液工作液成分以及浓度如下：10mM MES(pH5.7)，10mM MgCl2，100μM乙酰丁香酮(acetosyringone,AS)。
⑤拿准备好的1ml的注射器，去掉针头，选取出口光滑的注射器吸入菌液，取叶片长至眼镜片大小的本生烟草(Nicotiana benthamiana)，用手按住叶片，从叶片反面注射，使农杆菌渗透进去。给每株打过的烟草做好标记，可在叶片上圈出农杆菌渗透的区域，并选取转化缓冲液打烟草做对照。
⑥注射了农杆菌的烟草黑暗培养24h，然后移至烟草培养箱中光照培养24-48小时即可取样(注意打过的烟草不可直接在叶片上喷洒水)。
以下通过试验例的方式来说明本发明的有益效果：
试验例1 HVUL4H11714蛋白的酶活检测
1.方法
1.1HVUL4H11714蛋白的获取
实施例1方法制备得到的分子量73kDa的、带有GST标签的目的蛋白。1.2酶活检测
在Tris-HCl缓冲液(100mM，pH 7.4)中，以含有50μM阿魏酰基辅酶A作为酰基供体、100μM酪胺为酰基受体和500ng纯化蛋白的总体积100μl进行体外酰基转移酶测定。孵育10min后，加入300μL冰冷甲醇，停止反应。然后将反应混合物通过0.2μm的过滤器(微孔)过滤，然后用于LC-MS分析。
2.结果
HVUL4H11714可以催化阿魏酰基辅酶A和酪胺反应，生成阿魏酰酪胺(图2)。
试验例2转基因烟草生产
1.方法
1.1转基因烟草的构建
同实施例2。
1.2产物收集和纯化
剪取农杆菌渗透区域的叶片，放在已称重的装有钢珠的EP管中，做好标记，迅速放置液氮中，进行冻干。冻干后的样本，利用研磨仪(MM 400,Retsch)在30Hz条件下研磨60s，将研磨好的样本粉末装入2mlEP管内。用电子天平称取每个EP管的重量并记录；将已研磨好的样本取适量(范围30-60mg)于EP管中，称量并记录，算出所有EP管中样本的净重。已知每份样本的净重，按体积V＝样本净重(mg)*12μL/mg在4℃冰上操作加入70％MeOH溶液。混匀，涡旋15s，每隔半小时涡旋一次，共涡旋4次，放4℃冰箱内提取12h以上。后离心。先将离心机开机预冷到4℃，设置时间10min和转速12000rpm，将样本涡旋后放入离心，使用离心机注意对称平衡，离心后吸取上清液。将上清液用微孔滤膜(0.22μm pore size)过滤，装入上样瓶中，准备LC-MS检测。
1.3目的产物检测
将装有样本提取液的进样瓶放入自动进样器内的样品盘，并记录每个进样瓶编号所对应的进样孔位置。同时打开软件Analyst Software，双击Hardware Configuration，选择LCMS-V(有切换阀模式)，点击Activate Profile，并选择Acquire Mode模式，点击Acquire，点击图上方的Equilibrate键，一般设定时间为3min，此操作目的是预热仪器，使高压输液泵、色谱柱、柱温箱、离子源温度等达到方法中设置的条件。待各仪器部件状态Ready后，功能区Start Sample键成为可点击状态，此时表明仪器正常，分析条件正常，然后点击Start Sample开始跑样，首次跑样前先提交4针空白样。
2.结果
阿魏酰酪胺的峰度值为7.0E+05(图3)，表明本发明基因HVUL4H11714表达的阿魏酰酪胺酰基转移酶蛋白的活性高。
实验结果说明，本发明将基因HVUL4H11714转移到烟草中，使得烟草植株可以表达阿魏酰酪胺酰基转移酶，并催化生成阿魏酰酪胺，提高烟草的应用价值。
序列表
<110> 西藏自治区农牧科学院农业研究所
西藏自治区农牧科学院
<120> 一种青稞阿魏酰酪胺酰基转移酶基因及其用途
<130> GY462-18P1448
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1299
<212> DNA
<213> Hordeum vulgare
<400> 1
atggagatga cgagcagcac gatggtgaag ccggcgtacg ccgtgccgca cccgctcgtc 60
ggcgagaagg tcccgctgac cgtcttcgac cgcgccgccc tggacatctt cgtccccacc 120
gtgctcgcct accccgcgcc ggcgccgtcc aacgaggcgc tcagggaagg gctcctcaag 180
gccgtcgcgc cgtaccctca cctggcgggc cgcctcgccc tcgacgacct cggccggcgc 240
ttcctccacg tgaacaacga gggcgtgctc ctgatcgagg ccaccgtccc ggccgacctg 300
gcggacgtgc tcgtcgacgg ccgcatggcc gctggcgtcg acgacctcta cccggcgata 360
ccggaggaga acattggggc ggcgctgctg cagatccagc tcaacaggta caagtgcggc 420
ggcctcgtgg tcggcataag ctgccaccac cacaccgccg acggccattc catgagcatg 480
ttcttcaccg cgtgggcgac ggcggtccgc gagggtaagg acttcaccac gccgaccccg 540
ttcctcgacc gtgcgagaac cgcggtgcct cgaagcacgc cgacgccggt gttcgaccac 600
cggtcacgcg agttcactag tggagacgga ggagactcct atgccgtcgt ccccatggac 660
aggatcaaga acctcaccct gcacttcacg gccgagttcg tcgccgacct caaatccctc 720
gttggcaccc gttgcagcac gttccagtgc ctactagcgc acgtctggaa gaagctcacg 780
gcggcgcggg acctgaagcc ggaggagttc accaaggtga ggctcgccgt gaactgcagg 840
ggcagggccg accctcctgt gcccatggac ttcttcggga acatggtgct ctgggctttc 900
ccaaggcttc aggtccggga catgctggac tcgagccacg gcagcgtggt cagcgtcatc 960
cgcgacgccg tggcgcgcat cgacgacgag tacgtgcagt ccttcgtcga cttcggcgga 1020
gtggcggacg cgaacgggga ggagctcgtc gcgacggcgg ccgctgccgg cacgatgttc 1080
tgcccggacg cggaggtgga cagctggctg ggattcaggt tccatcagct cgattttggc 1140
accggcgcac cgtccgcttt cgtgccgccg gacctgcctt tcgaggggct catgatcttc 1200
atgccatcgc gcaaggccaa cggcggcgtc gacctcttca tggccgtcgc ggaggagcac 1260
gtcgcggcgt tcgagcagat ctgctactcg ttggattga 1299
<210> 2
<211> 432
<212> PRT
<213> Hordeum vulgare
<400> 2
Met Glu Met Thr Ser Ser Thr Met Val Lys Pro Ala Tyr Ala Val Pro
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His Pro Leu Val Gly Glu Lys Val Pro Leu Thr Val Phe Asp Arg Ala
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Ala Leu Asp Ile Phe Val Pro Thr Val Leu Ala Tyr Pro Ala Pro Ala
35 40 45
Pro Ser Asn Glu Ala Leu Arg Glu Gly Leu Leu Lys Ala Val Ala Pro
50 55 60
Tyr Pro His Leu Ala Gly Arg Leu Ala Leu Asp Asp Leu Gly Arg Arg
65 70 75 80
Phe Leu His Val Asn Asn Glu Gly Val Leu Leu Ile Glu Ala Thr Val
85 90 95
Pro Ala Asp Leu Ala Asp Val Leu Val Asp Gly Arg Met Ala Ala Gly
100 105 110
Val Asp Asp Leu Tyr Pro Ala Ile Pro Glu Glu Asn Ile Gly Ala Ala
115 120 125
Leu Leu Gln Ile Gln Leu Asn Arg Tyr Lys Cys Gly Gly Leu Val Val
130 135 140
Gly Ile Ser Cys His His His Thr Ala Asp Gly His Ser Met Ser Met
145 150 155 160
Phe Phe Thr Ala Trp Ala Thr Ala Val Arg Glu Gly Lys Asp Phe Thr
165 170 175
Thr Pro Thr Pro Phe Leu Asp Arg Ala Arg Thr Ala Val Pro Arg Ser
180 185 190
Thr Pro Thr Pro Val Phe Asp His Arg Ser Arg Glu Phe Thr Ser Gly
195 200 205
Asp Gly Gly Asp Ser Tyr Ala Val Val Pro Met Asp Arg Ile Lys Asn
210 215 220
Leu Thr Leu His Phe Thr Ala Glu Phe Val Ala Asp Leu Lys Ser Leu
225 230 235 240
Val Gly Thr Arg Cys Ser Thr Phe Gln Cys Leu Leu Ala His Val Trp
245 250 255
Lys Lys Leu Thr Ala Ala Arg Asp Leu Lys Pro Glu Glu Phe Thr Lys
260 265 270
Val Arg Leu Ala Val Asn Cys Arg Gly Arg Ala Asp Pro Pro Val Pro
275 280 285
Met Asp Phe Phe Gly Asn Met Val Leu Trp Ala Phe Pro Arg Leu Gln
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Val Arg Asp Met Leu Asp Ser Ser His Gly Ser Val Val Ser Val Ile
305 310 315 320
Arg Asp Ala Val Ala Arg Ile Asp Asp Glu Tyr Val Gln Ser Phe Val
325 330 335
Asp Phe Gly Gly Val Ala Asp Ala Asn Gly Glu Glu Leu Val Ala Thr
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Ala Ala Ala Ala Gly Thr Met Phe Cys Pro Asp Ala Glu Val Asp Ser
355 360 365
Trp Leu Gly Phe Arg Phe His Gln Leu Asp Phe Gly Thr Gly Ala Pro
370 375 380
Ser Ala Phe Val Pro Pro Asp Leu Pro Phe Glu Gly Leu Met Ile Phe
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Met Pro Ser Arg Lys Ala Asn Gly Gly Val Asp Leu Phe Met Ala Val
405 410 415
Ala Glu Glu His Val Ala Ala Phe Glu Gln Ile Cys Tyr Ser Leu Asp
420 425 430
Claims (5)
1.一种生产阿魏酰酪胺的转基因植物的构建方法，其特征在于：取核苷酸序列如SEQID NO.1所示的基因片段，转入植物中，获得表达氨基酸序列如SEQ ID NO.2所示的蛋白的植株，所述植物为烟草。
2.根据权利要求1所述的构建方法，其特征在于：所述转入植物的方法是农杆菌法。
3.基因片段、重组载体或重组菌在制备生产阿魏酰酪胺青稞品种中的用途，其中，所述基因片段的核苷酸序列如SEQ ID NO.1所示，所述重组载体包括SEQ ID NO.1所示的核苷酸序列，所述重组菌包含该重组载体。
4.根据权利要求3所述的用途，其特征在于：所述重组载体是重组pGEX-6P-1。
5.根据权利要求3所述的用途，其特征在于：所述重组菌为 Transetta(DE3)。
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