CN107418970B - 通过下调uxt基因提高植物糖化效率的方法及其应用 - Google Patents

通过下调uxt基因提高植物糖化效率的方法及其应用 Download PDF

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CN107418970B
CN107418970B CN201710674469.7A CN201710674469A CN107418970B CN 107418970 B CN107418970 B CN 107418970B CN 201710674469 A CN201710674469 A CN 201710674469A CN 107418970 B CN107418970 B CN 107418970B
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吴蔼民
赵先海
尚娜
邓小梅
刘念
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Abstract

本发明公开一种通过下调UXT基因提高植物糖化效率的方法及其应用，属于生物质能源领域。本发明通过使植物的UXT基因功能缺失或下调，来提高植物的糖化效率。通过本发明的方法使得木糖含量降低，相应地增加了纤维素相对含量，木糖含量的降低又导致突变体材料的糖化效率提高，降低了材料处理成本。因此，本发明从根本上改变了材料的生物质特性，使糖化效率有了根本上的提高，可以节约能源，降低劳动强度。通过本发明的方法获得的材料可以应用在生物质转化、纸浆和造纸工业中。

Description

通过下调UXT基因提高植物糖化效率的方法及其应用
技术领域
本发明属于生物质能源领域，具体涉及利用生物技术下调植物体内尿苷二磷酸木糖转运蛋白(UDP-Xylose transporters，UXTs)的含量，从而使植物获得更高的糖化效率。本发明涉及UXTs下调植物在各种领域的应用如生物质转化、纸浆和造纸工业中的应用；特别涉及一种通过下调UXT基因提高植物糖化效率的方法及其应用。
背景技术
生物质能为世界第4大能源，仅次于石油、煤炭与天然气。由于其来自于植物的光合作用，可在固态、液态和气态中转化，可谓取之不尽用之不竭，是可再生能源并具有环境友好的特性。当今世界的主要使用的仍为传统能源，储存有限、不可再生和环境危害严重，重重压力下全球注意力逐步在转移至可再生能源。如何高效合理地开发生物质能对经济和社会的可持续发展有着重要意义，也已成为大势所趋的热点。
木质纤维素作为重要的生物质能源，是生产生物乙醇的主要原料。木质纤维素中的纤维素和半纤维素经水解糖化成简单糖类，最后被发酵成乙醇，糖化效率是影响生物质产量的重要因素，木质纤维素的组成与交联方式直接影响糖化效率。被子植物次生壁的半纤维素主要为木聚糖(Xylan)，木聚糖的含量与组成直接影响植物的抗逆能力、工业应用价值等。木聚糖在植物次生壁中通过与纤维素、木质素交联来提高植物的韧性、抗逆能力等，但木聚糖的存在也对木质纤维素的工业应用带来了很大的影响，木聚糖在纤维素周围形成的交联网络结构阻止纤维素分解酶进入，影响糖化效率。木聚糖合成的底物尿苷二磷酸木糖(UDP-Xylose，UDP-Xyl)主要由尿苷二磷酸葡萄糖醛酸脱羧酶(UDP-Xylose synthases，UXSs)不可逆催化尿苷二磷酸葡萄糖醛酸(UDP-GlcA)脱羧而来，现已发现胞质中合成的UDP-Xyl对细胞壁木聚糖的合成更为重要(Kuang,B.,Zhao,X.,Zhou,C.,Zeng,W.,Ren,J.,Ebert,B.,Beahan,C.T.,Deng,X.,Zeng,Q.,Zhou,G.,Doblin,M.S.,Heazlewood,J.L.,Bacic,A.,Chen,X.,and Wu,A.M.(2016).Role of UDP-Glucuronic Acid Decarboxylasein Xylan Biosynthesis in Arabidopsis.Mol Plant 9,1119-1131；Zhong,R.,Teng,Q.,Haghighat,M.,Yuan,Y.,Furey,S.T.,Dasher,R.L.,and Ye,Z.H.(2016).Cytosol-Localized UDP-Xylose Synthases Provide the Major Source of UDP-Xylose for theBiosynthesis of Xylan and Xyloglucan.Plant&Cell Physiology.)，胞质中的UDP-Xyl合成后需要经由尿苷二磷酸木糖转运蛋白(UDP-Xylose transporters,UXTs)将之转运至高尔基体(Ebert,B.,Rautengarten,C.,Guo,X.,Xiong,G.,Stonebloom,S.,Smith-Moritz,A.M.,Herter,T.,Chan,L.J.,Adams,P.D.,and Petzold,C.J.(2015).Identification andCharacterization of a Golgi-Localized UDP-Xylose Transporter Family fromArabidopsis.Plant Cell 27,1218.)，再在高尔基体内合成木聚糖，最终转运到细胞壁上。
发明内容
为了克服现有技术的缺点与不足，本发明的目的在于提供一种通过下调UXT基因提高植物糖化效率的方法。
本发明通过改变植物木聚糖含量，从而使植物糖化效率提高。基于发明人的工作经验和当前生物质材料改良的需求，发明人认为下调木聚糖合成底物UDP-Xyl的转运蛋白UXTs可直接导致木聚糖含量的降低，从而改变木质纤维素的组成与结构。通过对拟南芥基因组中所有UXT的分析可知，拟南芥基因组中共有3个UXT，发明人从ABRC(ArabidopsisBiological Resource Center)订购到了这3个UXT的突变株，这3个突变株没有表现出明显的不良表型，进而对它们进行了杂交，最终得到了3个UXT基因都突变的突变株uxt1uxt2uxt3，发明人的研究工作正是针对这个突变体开展的。
uxt1uxt2uxt3三突变体生长矮小，叶片深绿，果荚结种量非常低；对之进行基部茎段切片分析发现，uxt1uxt2uxt3三突变体木质部导管坍塌，在扫描电镜下进行观察发现，uxt1uxt2uxt3三突变体的木纤维细胞壁明显变薄；单糖含量测定发现，uxt1uxt2uxt3三突变体中的木糖含量明显降低；木聚糖免疫分析发现，uxt1uxt2uxt3三突变体中的木聚糖含量明显降低；提取木聚糖进行酶解后进行1H-NMR分析发现，uxt1uxt2uxt3三突变体木聚糖侧链葡萄糖醛酸(Glucuronic acid，GlcA)的甲基化程度明显升高；对细胞壁进行糖化效率分析，发现uxt1uxt2uxt3三突变体的糖化效率明显高于野生型。以上实验结果表明，阻止胞质中的UDP-Xyl向高尔基体的转运会给植物带来较大的影响，能使植物木聚糖的分子量降低从而导致糖化效率提高。
本发明的另一目的在于提供上述方法获得的UXT下调植物的应用。
本发明的目的通过下述技术方案实现：
一种通过下调UXT基因提高植物糖化效率的方法，通过使植物的UXT基因功能缺失或下调，来提高植物的糖化效率。
所述的UXT基因功能下调优选为通过RNA干涉技术(RNAi)或者基因组编辑技术(CRISPRI-CAS9)来下调植物体内的UXT活性，达到提高糖化效率的目的。
所述的植物优选为拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)、杨树(Populus)、桉树(Eucalyptus)、水稻(Oryza sativa)、松树(Pinus)或麻疯树(Jatrophacarcas L.)等；更优选为拟南芥。但同时可以衍生到其它植物，通过RNA干涉技术(RNAi)或者基因组编辑技术(CRISPRI-CAS9)来下调该植物体内的UXT活性，达到提高糖化效率的目的。
获得uxt1uxt2uxt3三突变体。订购到3个UXT单突变体后，进行两两杂交，得到双基因突变纯合体，进一步对双基突变纯合体进行杂交得到三基因突变纯合体。
对uxt1uxt2uxt3三突变体进行表型分析。uxt1uxt2uxt3三突变体生长矮小，叶片深绿，果荚结种量非常低；对之进行基部茎段切片分析发现，uxt1uxt2uxt3三突变体木质部导管坍塌，在扫描电镜下进行观察发现，uxt1uxt2uxt3三突变体的木纤维细胞壁明显变薄。
对uxt1uxt2uxt3三突变体进行化学免疫分析。对茎部切片进行化学免疫(抗体：LM10)，uxt1uxt2uxt3三突变体的LM10荧光非常弱，表明木聚糖在uxt1uxt2uxt3三突变体中的含量非常低。
对uxt1uxt2uxt3三突变体的木聚糖结构进行分析。用KOH法提取突变体材料的半纤维素木聚糖(Zhao X,Ouyang K,Gan S,Zeng W,Song L,Zhao S,Li J,Doblin MS,BacicA,Chen XY,Marchant A,Deng X,Wu AM(2014)Biochemical and molecular changesassociated with heteroxylan biosynthesis in Neolamarckia cadamba(Rubiaceae)during xylogenesis.Front Plant Sci 5:602)，并用凝胶渗透色谱(Gel PermeationChromatography，GPC)测定分子量，1N KOH提取到的uxt1uxt2uxt3木聚糖重均分子量和数均分子量(51.3kDa，3.52kDa)皆小于野生型材料(76.9kDa，51.7kDa)，同样，4N KOH提取到的uxt1uxt2uxt3木聚糖重均分子量和数均分子量(48.9kDa，28kDa)皆小于野生型材料(85.6kDa，57.1kDa)。在大部分木聚糖相关的突变体中，葡萄糖醛酸(GlcA)的甲基化程度都会提高，即甲基化葡萄糖醛酸(MeGlcA)含量升高(Brown DM,Goubet F,Wong VW,GoodacreR,Stephens E,Dupree P,Turner SR(2007)Comparison of five xylan synthesismutants reveals new insight into the mechanisms of xylan synthesis.Plant J52:1154-1168；
Figure BDA0001373923020000041
MJ,Zhong R,Zhou GK,Richardson EA,O'Neill MA,Darvill AG,YorkWS,Ye ZH(2007)Arabidopsis irregular xylem8and irregularxylem9:implicationsfor the complexity of glucuronoxylan biosynthesis.Plant Cell19:549-563；Wu AM,Rihouey C,Seveno M,Hornblad E,Singh SK,Matsunaga T,Ishii T,Lerouge P,MarchantA(2009)The Arabidopsis IRX10and IRX10-LIKE glycosyltransferases are criticalfor glucuronoxylan biosynthesis during secondary cell wall formation.Plant J57:718-731)，为探明UXT突变体是否对MeGlcA的含量有影响，对提取到的木聚糖酶解并按照以前的报道的方法进行1H-NMR(Zhong,R.,Pena,M.J.,Zhou,G.K.,Nairn,C.J.,Wood-Jones,A.,Richardson,E.A.,Morrison,W.H.,3rd,Darvill,A.G.,York,W.S.,and Ye,Z.H.(2005).Arabidopsis fragile fiber8,which encodes a putativeglucuronyltransferase,is essential for normal secondary wall synthesis.PlantCell 17,3390-3408)，结果表明，uxt1uxt2uxt3三突变体GlcA的甲基化程度明显高于野生型(79.1％)。上述结果表明，uxt1uxt2uxt3三突变体木聚糖的分子量变小，GlcA甲基化程度升高，木聚糖的结构发生了较大变化。
对uxt1uxt2uxt3三突变体的糖化效率进行分析。木聚糖交联于纤维素和木质素，阻碍纤维素分解酶的进入影响糖化效率，得到的突变体木聚糖结构发生了很大变化，因此突变体糖化效率也会发生较大变化。在对突变体进行纤维素混合酶酶解后，测定得到的单糖含量以计算糖化效率，uxt1uxt2uxt3三突变体的糖化效率皆有很大提高。
另外，在杨树，桉树，水稻，松树，麻疯树等植物中也存在着UXT，它们的多突变体也有可预期的糖化效率提高的结果。
一种UXT下调植物，通过上述制备方法制得得到。
所述的UXT下调植物在生物质转化、纸浆和造纸工业中的应用。
本发明相对于现有技术，具有如下的优点及效果：
生物质能源的利用效率很大程度上取决于原材料的好坏，提高原材料的可利用成分，降低原材料的处理成本皆可以提高生物质能源利用效率，本发明的落脚点就在于这两个方面，得到的突变体材料木糖含量降低，相应地增加了纤维素相对含量，木糖含量的降低又导致突变体材料的糖化效率提高，降低了材料处理成本。现有方法大部分集中在改变材料的预处理方法上，对糖化效率的提高程度有限，本发明从根本上改变了材料的生物质特性，使糖化效率有了根本上的提高，可以节约能源，降低劳动强度。
附图说明
图1是60天大的野生型Col-0和uxt1uxt2uxt3三突变体植株及其果荚。
图2是野生型Col-0和uxt1uxt2uxt3三突变体植株茎基部横切切片的光学显微镜及扫描电镜观察。
图3是野生型Col-0和uxt1uxt2uxt3三突变体植株茎基部横切切片的LM10抗体免疫观察。
图4是野生型Col-0和uxt1uxt2uxt3三突变体植株茎的单糖含量分析。
图5是野生型Col-0和uxt1uxt2uxt3三突变体植株木聚糖的分子量分析。
图6是野生型Col-0和uxt1uxt2uxt3三突变体植株木聚糖的1H-NMR分析。
图7是野生型Col-0和uxt1uxt2uxt3三突变体植株细胞壁糖化效率分析。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的实验方法，通常按照常规实验条件或按照制造厂商所建议的实验条件。
拟南芥中AtUXT1、AtUXT2、AtUXT3的氨基酸序列如SEQ ID NO：2、4、6所示；cDNA序列如SEQ ID NO：1、3、5所示。
实施例1
AtUXT基因突变的T-DNA Line的获得。从ABRC(Arabidopsis BiologicalResourceCenter)订购到3个AtUXT的突变体：uxt1(SALK_086773)，uxt2(SALK_091753)和uxt3(SALK_079036.37.50.x)。在SALK(Salk InstituteGenomic Analysis Laboratory，http://signal.salk.edu)网站合成相应的T-DNA Line鉴定引物，如表1所示。将这3个突变体进行杂交，首先获得双突变体uxt1uxt3和uxt2uxt3，用这2个双突变体进行杂交获得三突变体uxt1uxt2uxt3。uxt1uxt2uxt3三突变体生长矮小，叶片深绿，果荚结种量非常低(图1)。
表1 T-DNA Line鉴定引物
突变体 正向引物F(5'-3') 反向引物R(5'-3')
uxt1 CTTCAGCTCAATTTTGTTGGC AGAAGAGAAAATGCCCATTGG
uxt2 AGAAGTTGGGTGGATGAAACC TGGTTTGATTCCACAGGAAAC
uxt3 GTTCTGATTGGTTAGGAGCCC CGAGAATTGCCTTCATGATTG
实施例2
拟南芥茎段切片制作与观察。选取生长6～7周的植株主茎，在土壤表层以上3cm取1cm茎段，用3％(w/v)的琼脂糖包埋茎段，在Leica VT1000S震动切片机上切片，厚度40μm，甲苯氨蓝染色1～2min，置于载玻片上于光学显微镜下观察拍照。野生型Col-0的木纤维细胞壁明显厚于uxt1uxt2uxt3三突变体，且野生型Col-0的木质部导管形态正常(图2)，uxt1uxt2uxt3三突变体的导管形态不规则，坍塌比例高(图2)。进而对切片进行扫描电镜观察，将切片置于样品台上镀金后，在Hitachi Ion Sputter E-1010(HITACHI)扫描电镜下观察。发现，uxt1uxt2uxt3三突变体木纤维的细胞壁厚度明显薄于野生型Col-0。
实施例3
对实施例2中用于光学显微镜观察的切片进行化学免疫观察。在培养皿中铺2层滤纸，用去离子水浸湿，其上放置一层Parafilm膜，将切片置于Parafilm膜上。然后按照如下步骤进行化学免疫染色：用0.1M磷酸盐缓冲液(PhosphateBuffer Solution，PBS；1L：0.218g KH2PO4，1.463g K2HPO4，29.22g NaCl，pH 7.2)洗涤切片5～10min；用新鲜的3％(w/v)脱脂牛乳浸泡切片1h，期间不断吹打脱脂牛乳；去除脱脂牛乳，并用PBS缓冲液洗涤切片5min；一抗孵育：用稀释20倍的大鼠抗木聚糖抗体LM10(Plantprobes)孵育切片1h，用PBS缓冲液洗涤切片10次以洗净未结合的一抗；二抗孵育：用稀释50倍的FITC-羊抗大鼠抗体(Zomanbio，Cat.Z1319)孵育1h，用PBS缓冲液洗涤切片10次以洗净未结合的二抗，将切片固定于载玻片上。将制备好的样品置于激光共聚焦显微镜(Zeiss LSM710，495nm)下观察。发现，uxt1uxt2uxt3三突变体的荧光强度明显弱于野生型Col-0(图3)。
实施例4
细胞壁单糖成分分析。收集茎段材料进行粉碎，用95％乙醇洗至没有绿色，最后用丙酮洗一次后风干。风干得到的细胞壁材料用2N三氟乙酸在120℃下水解1h，在高效阴离子交换色谱仪Dionex ICS 3000上进行单糖测定，色谱柱为CarboPac PA20阴离子交换柱(3×150mm；Dionex)。分析结果如图4，uxt1uxt2uxt3三突变体的木糖含量明显降低。
实施例5
凝胶渗透色谱(Gel Permeation Chromatography，GPC)测定木聚糖分子量。收集茎段材料进行粉碎，用95％乙醇洗至没有绿色，最后用丙酮洗一次后风干。将1g细胞壁材料置于50mL螺帽离心管，加入10mL 1N NaOH(含0.1％NaBH4)或4N NaOH(含0.1％NaBH4)，室温震荡过夜，10,000rpm离心10min，取上清至新的离心管，用HCl中和至无气泡产生。用分子量截留～3kDa的透析袋对之进行除盐，最后置于冷冻干燥机上冻干，得到固态的木聚糖。以1mg/mL的比例将木聚糖溶解于0.005M磷酸盐缓冲液中(pH7.5，含0.02N NaCl)，用GPC法测定木聚糖的分子量，仪器为Agilent 1200型HPLC，色谱柱为PLaquagel-OH 50colume(300)，流动相为磷酸盐缓冲溶液(pH7.5，含0.02N NaCl)，等度洗脱，流速为0.5mL min-1，测定结果如图5和表2所示，Mw表示重均分子量，Mn表示数均分子量，Mw/Mn表示分子量分散度，uxt1uxt2uxt3三突变体木聚糖的分子量明显小于野生型Col-0。
表2木聚糖分子量
Figure BDA0001373923020000071
实施例6
葡萄糖醛酸甲基化分析。对用4N KOH提取到的木聚糖用endo-β-xylanase M6(Megazyme)酶解，酶解产物进行1H-NMR分析(布鲁克)分析结果见图6。在野生型Col-0样品的共振峰上标注了质子归属糖残基，带有支链和不带有支链的β-Xyl来源于木聚糖主链，α-GlcA和Me-α-GlcA为木聚糖侧链，α-GalA和α-Rha来源于木聚糖的还原末端，G表示带有α-GlcA侧链的木糖残基，M表示带有Me-α-GlcA侧链的木糖残基。由分析结果可知，uxt1uxt2uxt3三突变体的α-GlcA和带有GlcA侧链的β-Xyl信号值消失了，只有Me-α-GlcA和带有Me-GlcA的β-Xyl信号，此结果说明，uxt1uxt2uxt3三突变体木聚糖的GlcA甲基化程度升高。
实施例7
糖化分析。称取～5mg材料(记录样品精确质量)，置于2mL螺帽离心管，加入1mL0.1M乙酸钠溶液(pH 7)，室温震荡1h，在120℃预处理1h；待样品冷却后加相应降解酶，木聚糖分解酶采用Xylanase(endo-1,4-β-Xylanase M1，Megazyme)，每个样品中加入1U，纤维素分解酶采用Cellulase(Cellulase from Aspergillus sp.，Sigma)，每个样品中加入0.01U。37℃振荡处理过夜。离心取上清液，利用硫酸苯酚法对上清中的糖浓度进行测定。测定结果见图7，由结果可知，uxt1uxt2uxt3三突变体在经过纤维素分解酶和木聚糖分解酶处理后，释放的糖都要多于野生型Col-0，说明uxt1uxt2uxt3三突变体的糖化效率提高。
实施例8
硫酸苯酚法测定总糖。标准曲线绘制：精确配制浓度分别为0，10，20，40，60，80，100，150，200μg mL-1的葡萄糖溶液，每个标准溶液和样品取300μL，加入150μL新配制的5％苯酚溶液，加入1.5mL浓硫酸，立即混匀，室温放置1h后用紫外可见光分光光度计(上海美谱达UV-1200)在490nm波长下测量吸光值，绘制标准曲线，根据标准曲线求得各样品的糖含量。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 华南农业大学
<120> 通过下调UXT基因提高植物糖化效率的方法及其应用
<130> 1
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1029
<212> DNA
<213> Artificial Sequence
<220>
<223> 拟南芥AtUXT1的cDNA序列
<400> 1
atgggagaga tgaagagtat gcaaatgggt gtgattggag cattgtttct atcagttgca 60
tcttctgtct ccattgtcat ttgcaacaaa gctttgatga ccaatcttgg tttccctttt 120
gcaacaacac ttactagttg gcatttgatg gtaacatatt gcacacttca tgtggcatat 180
aaactaaact tctttgagaa taaaccaata gacatgagaa ctgttgttct ctttggcctc 240
ctcaatggca tttccattgg tctcctcaat ctcagcctcg gctttaattc catcggcttc 300
tatcaaatga ccaaacttgc tatcatacca ttcactgttc ttttagaaac cctcttcctc 360
aacaagaagt tcagccagaa gataaaattc tcattgtttt tgctgctagt tggggttggt 420
atagcatcaa tcacagatct tcagctcaat tttgttggct ctgttctatc tctccttgcc 480
attgccacaa cttgcgtcgg ccaaattcta acaaacacta tccagaagag actgaacgtg 540
acatcaacac aactcctata ccaatcagcc ccgttccaag ctgctatact cttcgtctcg 600
ggtccttttg tcgacaaata cctcactagc ctcaatgttt tctccttcca ttactctccc 660
atcgtcgtgg ggttcataac gttgtcgtgt ttgatagcgg tttcggttaa cttcagtacg 720
tttttagtga ttggaaagac atctccggtg acataccaag tcttgggaca tctcaaaacg 780
tgtttggtgc tcgcatttgg ttacactctc cttcacgatc ccttcactcc tcgtaatatc 840
gcaggaatac tcatcgctgt cctcgggatg cttctctact cctacttctg ctcagttgct 900
tctaaatcca aacaagcctc ctccgattcc acttttcttg ggaaagatag agatacgacg 960
ccgcttttgg gacaagaaaa tgagaaccat catgaagcca agaaattaga caagcattct 1020
ccagtatga 1029
<210> 2
<211> 342
<212> PRT
<213> Artificial Sequence
<220>
<223> 拟南芥AtUXT1的氨基酸序列
<400> 2
Met Gly Glu Met Lys Ser Met Gln Met Gly Val Ile Gly Ala Leu Phe
1 5 10 15
Leu Ser Val Ala Ser Ser Val Ser Ile Val Ile Cys Asn Lys Ala Leu
20 25 30
Met Thr Asn Leu Gly Phe Pro Phe Ala Thr Thr Leu Thr Ser Trp His
35 40 45
Leu Met Val Thr Tyr Cys Thr Leu His Val Ala Tyr Lys Leu Asn Phe
50 55 60
Phe Glu Asn Lys Pro Ile Asp Met Arg Thr Val Val Leu Phe Gly Leu
65 70 75 80
Leu Asn Gly Ile Ser Ile Gly Leu Leu Asn Leu Ser Leu Gly Phe Asn
85 90 95
Ser Ile Gly Phe Tyr Gln Met Thr Lys Leu Ala Ile Ile Pro Phe Thr
100 105 110
Val Leu Leu Glu Thr Leu Phe Leu Asn Lys Lys Phe Ser Gln Lys Ile
115 120 125
Lys Phe Ser Leu Phe Leu Leu Leu Val Gly Val Gly Ile Ala Ser Ile
130 135 140
Thr Asp Leu Gln Leu Asn Phe Val Gly Ser Val Leu Ser Leu Leu Ala
145 150 155 160
Ile Ala Thr Thr Cys Val Gly Gln Ile Leu Thr Asn Thr Ile Gln Lys
165 170 175
Arg Leu Asn Val Thr Ser Thr Gln Leu Leu Tyr Gln Ser Ala Pro Phe
180 185 190
Gln Ala Ala Ile Leu Phe Val Ser Gly Pro Phe Val Asp Lys Tyr Leu
195 200 205
Thr Ser Leu Asn Val Phe Ser Phe His Tyr Ser Pro Ile Val Val Gly
210 215 220
Phe Ile Thr Leu Ser Cys Leu Ile Ala Val Ser Val Asn Phe Ser Thr
225 230 235 240
Phe Leu Val Ile Gly Lys Thr Ser Pro Val Thr Tyr Gln Val Leu Gly
245 250 255
His Leu Lys Thr Cys Leu Val Leu Ala Phe Gly Tyr Thr Leu Leu His
260 265 270
Asp Pro Phe Thr Pro Arg Asn Ile Ala Gly Ile Leu Ile Ala Val Leu
275 280 285
Gly Met Leu Leu Tyr Ser Tyr Phe Cys Ser Val Ala Ser Lys Ser Lys
290 295 300
Gln Ala Ser Ser Asp Ser Thr Phe Leu Gly Lys Asp Arg Asp Thr Thr
305 310 315 320
Pro Leu Leu Gly Gln Glu Asn Glu Asn His His Glu Ala Lys Lys Leu
325 330 335
Asp Lys His Ser Pro Val
340
<210> 3
<211> 1062
<212> DNA
<213> Artificial Sequence
<220>
<223> 拟南芥AtUXT2的cDNA序列
<400> 3
atgagcgatg cccagaagtt ccagcttgga acaatcggcg ctttgagttt atccgttgtg 60
tcctctgttt cgatcgtgat ctgtaacaag gcccttatta gcacccttgg tttcacattt 120
gcaactactt tgacaagctg gcatcttttg gtgacatttt gttcacttca tgtggcatta 180
tggatgaagt tttttgagca caagcctttt gatccacgag ctgtcctggg atttggtgta 240
ttaaatggta tatccattgg attattaaat ctcagcttgg gttttaattc tgttggtttt 300
taccagatga caaaacttgc aatcatcccc tgtactgttg tcttagagac catcttcttc 360
agaaagatgt tcagtcgaaa aatccagttt tcattagtca tccttctcct tggtgttgga 420
attgcaaccg taacagatct ccagcttaat atgctgggtt ctgtcttgtc gctactggct 480
gttatcacaa cttgtgttgc ccaaattatg accaatacga tccagaagaa atataaggtt 540
tcatccaccc aacttctgta tcagtcttgc ccatatcaag caatcacact ttttgttact 600
ggcccatttt tagatggtct cttaaccaac cagaacgtgt ttgctttcaa atacacgtct 660
caagttgtgt tcttcatcgt cttgtcctgc ctcatatctg tttcagtaaa cttcagcacg 720
tttctcgtca ttggaaagac atctcctgtc acttatcagg ttctaggaca tctaaaaaca 780
tgcttagtgt tagcatttgg gtatcttttg ctgaaagacg cattcagctg gcgcaacatt 840
cttggtattc ttgtcgccgt gattggaatg gtgctttatt cctattactg cacactcgaa 900
acccaacaga aggccacaga aacatcaact caattgcctc agatggatga aaacgagaaa 960
gatccgctag ttagtgcgga aaacgggagc ggattgatat cagacaatgg agtgcaaaag 1020
caggatcctg tatggaattc aaacaaagat tttcaagcgt ag 1062
<210> 4
<211> 353
<212> PRT
<213> Artificial Sequence
<220>
<223> 拟南芥AtUXT2的氨基酸序列
<400> 4
Met Ser Asp Ala Gln Lys Phe Gln Leu Gly Thr Ile Gly Ala Leu Ser
1 5 10 15
Leu Ser Val Val Ser Ser Val Ser Ile Val Ile Cys Asn Lys Ala Leu
20 25 30
Ile Ser Thr Leu Gly Phe Thr Phe Ala Thr Thr Leu Thr Ser Trp His
35 40 45
Leu Leu Val Thr Phe Cys Ser Leu His Val Ala Leu Trp Met Lys Phe
50 55 60
Phe Glu His Lys Pro Phe Asp Pro Arg Ala Val Leu Gly Phe Gly Val
65 70 75 80
Leu Asn Gly Ile Ser Ile Gly Leu Leu Asn Leu Ser Leu Gly Phe Asn
85 90 95
Ser Val Gly Phe Tyr Gln Met Thr Lys Leu Ala Ile Ile Pro Cys Thr
100 105 110
Val Val Leu Glu Thr Ile Phe Phe Arg Lys Met Phe Ser Arg Lys Ile
115 120 125
Gln Phe Ser Leu Val Ile Leu Leu Leu Gly Val Gly Ile Ala Thr Val
130 135 140
Thr Asp Leu Gln Leu Asn Met Leu Gly Ser Val Leu Ser Leu Leu Ala
145 150 155 160
Val Ile Thr Thr Cys Val Ala Gln Ile Met Thr Asn Thr Ile Gln Lys
165 170 175
Lys Tyr Lys Val Ser Ser Thr Gln Leu Leu Tyr Gln Ser Cys Pro Tyr
180 185 190
Gln Ala Ile Thr Leu Phe Val Thr Gly Pro Phe Leu Asp Gly Leu Leu
195 200 205
Thr Asn Gln Asn Val Phe Ala Phe Lys Tyr Thr Ser Gln Val Val Phe
210 215 220
Phe Ile Val Leu Ser Cys Leu Ile Ser Val Ser Val Asn Phe Ser Thr
225 230 235 240
Phe Leu Val Ile Gly Lys Thr Ser Pro Val Thr Tyr Gln Val Leu Gly
245 250 255
His Leu Lys Thr Cys Leu Val Leu Ala Phe Gly Tyr Leu Leu Leu Lys
260 265 270
Asp Ala Phe Ser Trp Arg Asn Ile Leu Gly Ile Leu Val Ala Val Ile
275 280 285
Gly Met Val Leu Tyr Ser Tyr Tyr Cys Thr Leu Glu Thr Gln Gln Lys
290 295 300
Ala Thr Glu Thr Ser Thr Gln Leu Pro Gln Met Asp Glu Asn Glu Lys
305 310 315 320
Asp Pro Leu Val Ser Ala Glu Asn Gly Ser Gly Leu Ile Ser Asp Asn
325 330 335
Gly Val Gln Lys Gln Asp Pro Val Trp Asn Ser Asn Lys Asp Phe Gln
340 345 350
Ala
<210> 5
<211> 1074
<212> DNA
<213> Artificial Sequence
<220>
<223> 拟南芥AtUXT3的cDNA序列
<400> 5
atgagcgagg gccagaagtt ccagttggga acaatcggcg ctttgagttt gtccgttgtg 60
tcatctgtct cgattgtgat ctgtaacaaa gcgcttatta gcacccttgg cttcacattc 120
gcgactactt tgacaagttg gcatcttttg gtcacatttt gttcccttca tgtggcatta 180
tggatgaaga tgttcgaaca caagcctttt gatccacgag ctgtgatggg atttggcata 240
ttgaatggga tatccatagg actattgaac ctcagcctgg gctttaattc tgtcggtttt 300
taccagatga ctaaactagc tatcatcccc tgcactgttc tcttggagac cctcttcttc 360
aggaaaaagt tcagtcgaaa aatccagttt tcattaacca tccttctcct tggtgttgga 420
attgcaaccg tcacagatct tcaacttaat atgctgggtt ctgtcttgtc gctgctggct 480
gttgtcacaa cttgtgttgc tcaaattatg acaaatacca tccagaagaa gttcaaagtt 540
tcatccacgc agcttctgta tcagtcttgc ccatatcaag caatcactct tttcgtcact 600
gggccatttt tagatgggct cctaaccaat cagaacgtgt ttgctttcaa gtacacttct 660
caagtagtgt tcttcatcgt cctgtcttgc ctcatatcag tctctgtaaa cttcagcaca 720
tttcttgtca ttggaaaaac atctcctgtc acatatcagg ttctaggaca tctcaaaaca 780
tgcctggttc tagcatttgg ctatgtgttg ctgcgagacc cattcgactg gcgcaacatt 840
ctcggtattc tagtggctgt gattggaatg gttgtttatt cctattactg ctcgattgag 900
actcagcaga aggcaagtga aacctcaact cagttgcctc agatgaaaga gagcgagaag 960
gatccgctaa tagcagctga aaatggaagc ggagtgttat cagatggcgg cgggggagtg 1020
cagcaaaaga cggtggctcc tgtatggaac tcaaataaag attttcaagc ctaa 1074
<210> 6
<211> 357
<212> PRT
<213> Artificial Sequence
<220>
<223> 拟南芥AtUXT3的氨基酸序列
<400> 6
Met Ser Glu Gly Gln Lys Phe Gln Leu Gly Thr Ile Gly Ala Leu Ser
1 5 10 15
Leu Ser Val Val Ser Ser Val Ser Ile Val Ile Cys Asn Lys Ala Leu
20 25 30
Ile Ser Thr Leu Gly Phe Thr Phe Ala Thr Thr Leu Thr Ser Trp His
35 40 45
Leu Leu Val Thr Phe Cys Ser Leu His Val Ala Leu Trp Met Lys Met
50 55 60
Phe Glu His Lys Pro Phe Asp Pro Arg Ala Val Met Gly Phe Gly Ile
65 70 75 80
Leu Asn Gly Ile Ser Ile Gly Leu Leu Asn Leu Ser Leu Gly Phe Asn
85 90 95
Ser Val Gly Phe Tyr Gln Met Thr Lys Leu Ala Ile Ile Pro Cys Thr
100 105 110
Val Leu Leu Glu Thr Leu Phe Phe Arg Lys Lys Phe Ser Arg Lys Ile
115 120 125
Gln Phe Ser Leu Thr Ile Leu Leu Leu Gly Val Gly Ile Ala Thr Val
130 135 140
Thr Asp Leu Gln Leu Asn Met Leu Gly Ser Val Leu Ser Leu Leu Ala
145 150 155 160
Val Val Thr Thr Cys Val Ala Gln Ile Met Thr Asn Thr Ile Gln Lys
165 170 175
Lys Phe Lys Val Ser Ser Thr Gln Leu Leu Tyr Gln Ser Cys Pro Tyr
180 185 190
Gln Ala Ile Thr Leu Phe Val Thr Gly Pro Phe Leu Asp Gly Leu Leu
195 200 205
Thr Asn Gln Asn Val Phe Ala Phe Lys Tyr Thr Ser Gln Val Val Phe
210 215 220
Phe Ile Val Leu Ser Cys Leu Ile Ser Val Ser Val Asn Phe Ser Thr
225 230 235 240
Phe Leu Val Ile Gly Lys Thr Ser Pro Val Thr Tyr Gln Val Leu Gly
245 250 255
His Leu Lys Thr Cys Leu Val Leu Ala Phe Gly Tyr Val Leu Leu Arg
260 265 270
Asp Pro Phe Asp Trp Arg Asn Ile Leu Gly Ile Leu Val Ala Val Ile
275 280 285
Gly Met Val Val Tyr Ser Tyr Tyr Cys Ser Ile Glu Thr Gln Gln Lys
290 295 300
Ala Ser Glu Thr Ser Thr Gln Leu Pro Gln Met Lys Glu Ser Glu Lys
305 310 315 320
Asp Pro Leu Ile Ala Ala Glu Asn Gly Ser Gly Val Leu Ser Asp Gly
325 330 335
Gly Gly Gly Val Gln Gln Lys Thr Val Ala Pro Val Trp Asn Ser Asn
340 345 350
Lys Asp Phe Gln Ala
355
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> uxt1-F
<400> 7
cttcagctca attttgttgg c 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> uxt1-R
<400> 8
agaagagaaa atgcccattg g 21
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> uxt2-F
<400> 9
agaagttggg tggatgaaac c 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> uxt2-R
<400> 10
tggtttgatt ccacaggaaa c 21
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> uxt3-F
<400> 11
gttctgattg gttaggagcc c 21
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> uxt3-R
<400> 12
cgagaattgc cttcatgatt g 21

Claims (3)

1.一种通过下调UXT基因提高植物糖化效率的方法，其特征在于：通过使拟南芥的UXT基因功能缺失或下调，来提高拟南芥的糖化效率；
所述的UXT基因为AtUXT1、AtUXT2和AtUXT3。
2.根据权利要求1所述的通过下调UXT基因提高植物糖化效率的方法，其特征在于：
所述的UXT基因功能下调为通过RNA干涉技术或者基因组编辑技术来下调拟南芥体内的UXT活性，达到提高糖化效率的目的。
3.通过权利要求1～2任一项所述的通过下调UXT基因提高植物糖化效率的方法获得的UXT下调植物在生物质转化、纸浆和造纸工业中的应用。
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