CN116622661B - 一种紫花苜蓿尿苷二磷酸糖基转移酶基因及应用 - Google Patents
一种紫花苜蓿尿苷二磷酸糖基转移酶基因及应用 Download PDFInfo
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- CN116622661B CN116622661B CN202310487420.6A CN202310487420A CN116622661B CN 116622661 B CN116622661 B CN 116622661B CN 202310487420 A CN202310487420 A CN 202310487420A CN 116622661 B CN116622661 B CN 116622661B
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Abstract
本发明公开了一种尿苷二磷酸糖基转移酶基因。所述基因编码序列含有1440个碱基,具体编码序列如SEQ ID NO:2所示。该基因表达的尿苷二磷酸糖基转移酶含有479个氨基酸,氨基酸序列如SEQ ID NO:1所示。将本发明得到的尿苷二磷酸糖基转移酶基因转入紫花苜蓿中,获得了过表达尿苷二磷酸糖基转移酶的紫花苜蓿,提高了紫花苜蓿的品质相关性状。
Description
技术领域
本发明属于分子生物学和植物基因工程领域,具体涉及一种紫花苜蓿尿苷二磷酸糖基转移酶基因及应用。
背景技术
植物尿苷二磷酸糖基转移酶(Uridine diphosphate glycosyltransferase,UGT)属于糖基转移酶一号家族(GT family 1),主要以植物中最常见的尿苷二磷酸(UDP)-糖类为活化糖基分子供体,以类黄酮、酚酸、萜类以及植物激素等小分子化合物为糖基受体分子。UGT参与调节植物多种次生代谢物质的合成积累。例如尿苷二磷酸糖基转移酶能够在体外合成肉桂酸葡萄糖缀合物,糖基化十字花科植物中主要的苯丙素类物质-芥子酸,合成芥子酸酯和木质素,影响植物细胞壁的形成,进而降低拟南芥中木质素的含量。缺失尿苷二磷酸糖基转移酶编码基因的拟南芥突变体茎部木质化加剧,表现出生长受阻、茎尖花青素积累等表型;回补该基因后植株次生细胞壁发育和木质化表型恢复正常,表明尿苷二磷酸糖基转移酶具有降低木质素含量的作用。利用尿苷二磷酸糖基转移酶糖基化调节木质素稳态,影响木质素的合成和含量,可以达到改善植物品质的作用。
紫花苜蓿(Medicago sativa L.)起源于中亚地区,是目前美国西部陆地面积最大的作物,也是世界范围种植最广泛的多年生饲草作物。紫花苜蓿产量高、品质优良、适应性强,同时还具有生物固氮的功能,被广泛应用于饲料产业及其相关加工产业链。饲料作物的质量评价指标主要包含蛋白质含量和纤维的含量,其水平高低对家畜饲料的适口性、消化率与营养物质的摄入量存在至关重要的作用。家畜动物对饲料的消化能力取决于含有营养成分的组成比例,低水平的非消化成分如木质素和纤维素是作为优质饲料作物的必要因素。提高粗蛋白,降低木质素含量是改良苜蓿品质和提升消化率的重要途径。因此,研究紫花苜蓿中调控关键品质性状的UGT基因,对利用分子生物学手段推进紫花苜蓿育种进程具有重要意义。
发明内容
为了提高和改善紫花苜蓿品质,本发明提供了一种新的技术方法。
第一方面,本发明提供了一种尿苷二磷酸糖基转移酶,该酶蛋白具有SEQ ID NO:1所示的氨基酸序列,或者该酶蛋白具有将SEQ ID NO:1所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加后仍具有尿苷二磷酸糖基转移酶活性的氨基酸序列。
第二方面,本发明提供一种尿苷二磷酸糖基转移酶基因,其编码第一方面所述的尿苷二磷酸糖基转移酶。
优选地,所述基因具有SEQ ID NO:2所示的核苷酸序列,
更优选地,所述基因具有SEQ ID NO:2所示的编码序列。
第三方面,本发明提供一种感受态细胞,所述感受态细胞含有第二方面所述的尿苷二磷酸基转移酶基因。
优选地,所述感受态细胞选自DH5α感受态细胞、Trans-T1感受态细胞,进一步优选为DH5α感受态细胞。
第四方面,本发明提供一种重组载体,所述重组载体含有第二方面所述的尿苷二磷酸糖基转移酶基因。
优选地,所述重组载体选自pCAMBIA3301、pCAMBIA1302,进一步优选为pCAMBIA3301。
第五方面,本发明提供一种重组基因工程菌,所述工程菌含有第二方面所述的尿苷二磷酸糖基转移酶基因。
优选地,所述工程菌采用第四方面所述的重组载体转化得到。
优选地,所述工程菌选自农杆菌EHA105、GV3101,进一步优选为农杆菌EHA105。
第六方面,本发明提供一种转基因细胞,所述转基因细胞含有第二方面所述的尿苷二磷酸糖基转移酶基因。
优选地,所述转基因细胞为植物转基因细胞,例如:紫花苜蓿转基因细胞、蒺藜苜蓿转基因细胞、大豆转基因细胞,进一步优选为苜蓿转基因细胞,更优选为紫花苜蓿转基因细胞。
第七方面,本发明提供一种培育紫花苜蓿的方法,所述方法具体包括以下步骤:
步骤1,以紫花苜蓿的基因组为模板,通过引物设计、基因扩增、序列测定,获得了一种尿苷二磷酸糖基转移酶的基因。
优选地,所述尿苷二磷酸糖基转移酶基因具有SEQ ID NO:2所示的核苷酸序列,
更优选地,所述尿苷二磷酸糖基转移酶基因具有SEQ ID NO:2所示的编码序列。
优选地,所述引物包括正向引物P1和反向引物P2,P1的序列为:5′
-ATGGAAATCTCAAAACAAGAACC-3′(SEQ ID NO:3);P2的序列为:5'-TTAATTTATTACAGTGGATTTGTTG-3'(SEQ ID NO:4)。
优选地,采用PCR方法进行所述的基因扩增。进一步优选所述PCR条件为:
95℃5min,95℃30s,46.5℃30s,72℃90s,共27个循环。
在本发明的一个具体实施方式中,所述的基因扩增还包括PCR产物转化感受态细胞,培养转化的感受态细胞、筛选阳性克隆,测序。
优选地,所述感受态细胞选自DH5α感受态细胞、Trans-T1感受态细胞,进一步优选为DH5α感受态细胞。
优选地,所述得细胞培养基为含氨苄抗生素的LB,进一步优选氨苄抗生素的浓度为100μg/mL。
优选地,将筛选得到的阳性克隆送生物公司测序。
步骤2,质粒提取、构建重组载体、转化获得转基因植物细胞、组织培养获得过表达植物株系。
优选地,提取步骤1中测序正确的质粒,然后采用表达载体PCR扩增。
优选地,PCR扩增的引物为P3和P4,进一步优选P3的序列为:5′-gagaacacgggggactcttgaccATGGAAATCTCAAAACAAGAACC-3′(SEQ ID NO:5)。P4的序列为:5'-gaaatttaccctcagatctaccatATTTATTACAGTGGATTTGTTG-3'(SEQ ID NO:6)。
优选地,所述质粒的重组载体选自pCAMBIA3301、pCAMBIA1302,进一步优选为pCAMBIA3301。
优选地,所述PCR的反应条件为:95℃5min,95℃30s,64.5℃30s,72℃90s,共27个循环。
在本发明的一个具体实施方式中,将PCR产物与pCAMBIA3301进行连接,然后转化感受态细胞、培养转化的感受态细胞、筛选阳性克隆,测序。
优选地,所述感受态细胞选自DH5α感受态细胞、Trans-T1感受态细胞,进一步优选为DH5α感受态细胞。
优选地,所述得细胞培养基为含氨苄抗生素的LB,进一步优选氨苄抗生素的浓度为100μg/mL。
优选地,将筛选的阳性克隆送生物公司测序。
在本发明的一个具体实施方式中,提取测序正确的质粒,转化获得重组工程菌,然后将工程菌转入植物细胞获得转基因细胞,在进行组织培养获得过表达尿苷二磷酸糖基转移酶的株系。
优选地,所述质粒转化的表达载体选自pCAMBIA3301、pCAMBIA1302,进一步优选为pCAMBIA3301。
优选地,所述工程菌选自农杆菌EHA105、GV3101,进一步优选为农杆菌EHA105。
优选地,所述转基因细胞为植物转基因细胞,例如:紫花苜蓿转基因细胞、蒺藜苜蓿转基因细胞、大豆转基因细胞,进一步优选为苜蓿转基因细胞,更优选为紫花苜蓿转基因细胞。
第八方面,本发明提供第一方面所述的尿苷二磷酸糖基转移酶、第二方面所述的基因、第三方面所述的感受态细胞、第四方面所述的重组载体、第五方面所述的重组基因工程菌、第六方面所述的转基因细胞在培育过表达尿苷二磷酸转移酶的植物中的应用。
优选地,所述植物为紫花苜蓿。
本发明的有益效果:
一、本发明以紫花苜蓿“中苜一号”基因组为模板,通过设计引物、基因扩增、序列测定,获得了一种全新的尿苷二磷酸糖基转移酶的编码基因序列,该基因序列长1440bp,编码479个氨基酸。
二、本发明将获得的尿苷二磷酸糖基转移酶基因转入紫花苜蓿细胞中,获得了过表达尿苷二磷酸糖基转移酶的紫花苜蓿,提高和改善了紫花苜蓿的品质。
附图说明
图1为本发明的尿苷二磷酸糖基转移酶基因的编码序列(SEQ ID NO:2);
图2为本发明的尿苷二磷酸糖基转移酶的氨基酸序列(SEQ ID NO:1);
图3为本发明的尿苷二磷酸糖基转移酶基因PCR检测结果;
图4为本发明构建的尿苷二磷酸糖基转移酶基因的表达载体pCAMBIA3301-UGT验证结果;
图5为本发明中植物表达载体pCAMBIA3301-UGT转化农杆菌EHA105的检测结果;
图6为本发明中用农杆菌侵染法转化紫花苜蓿的组织培养过程;
图7为本发明中获得的过表达紫花苜蓿的DNA水平验证结果,其中6、8、10、14、28、29、30表示7株阳性过表达紫花苜蓿材料,WT为中苜一号野生型紫花苜蓿,CK+为pCAMBIA3301-UGT质粒;
图8为本发明中获得的过表达紫花苜蓿的RNA水平验证结果;
图9为本发明中过表达紫花苜蓿品质测定结果柱状图,包含木质素(Lignin),酸性洗涤纤维(Acid detergent fiber,ADF),中性洗涤纤维(Neutral detergent fiber,NDF),粗蛋白(Crude protein,CP),粗脂肪(Crude Fat,CF),灰分(ASH),磷(P)、钙(Ca)和钾(K)。
具体实施方式
下面结合实施例和附图来进一步描述本发明的技术方案,本发明的优点和特点将会随着描述而更为清楚。但是应当理解,实施例仅是示例性的,不对本发明的范围构成限制。
本发明试验过程中使用的培养基如下:
LB培养基
YEB培养基
SH3a培养基
MSBK培养基
SH9a培养基
实施例1:紫花苜蓿尿苷二磷酸糖基转移酶基因(UGT)的获取:
1.1引物设计
以紫花苜蓿“中苜一号”基因组为DNA模板,从CDS两端设计2对特异性引物包括正向引物P1和反向引物P2,P1和P2的序列如下:
P1:5′-ATGGAAATCTCAAAACAAGAACC-3′(SEQ ID NO:3)。
P2:5'-TTAATTTATTACAGTGGATTTGTTG-3'(SEQ ID NO:4)。
1.2PCR扩增
1.2.1cDNA的合成
选取紫花苜蓿“中苜一号”为提取材料,取0.1g紫花苜蓿放入液氮中,利用EastepSpuer总RNA提取试剂盒进行RNA的提取。然后采用分光光度计测定样品RNA的浓度和质量,确定不存在降解与污染问题。再采用PrimeScriptTMRT reagent Kit(Perfect Real Time)反转录试剂盒将RNA样品合成cDNA。
1.2.2 PCR扩增
利用TaKaRa试剂盒进行PCR扩增试验。扩增体系为50μL:正向引物2.5μL、反向引物2.5μL、cDNA模板1μL、Ex Taq酶0.5μL、Buffer 5μL、dNTP 4μL、ddH2O 34.5μL。扩增程序为:在95℃初始变性5 min,95℃变性30 s,46.5℃退火30 s,72℃延伸90 s,共27个循环,得到PCR产物。
1.2.3 PCR产物的检测
用1%的琼脂糖凝胶电泳对1.2.2的得到的PCR产物进行检测,能够观察到清晰且单一的1440bp左右的DNA条带(见图3),用Quick Gel Extraction Kit试剂盒进行PCR产物的切胶回收试验。
1.2.4 PCR产物的测序
将1.2.3回收的PCR产物与pTOPO-TA sample Vector于37℃下反应15min,连接产物转化DH5α感受态细胞,然后将感受态细胞在37℃下用含氨苄抗生素(100μg/mL)的选择性LB平板培养12 h,挑选单菌落,接种含氨苄抗生素(100μg/mL)的LB液体培养基,在37℃下震荡培养6 h,选择阳性菌液送生物公司测序。测序结果(SEQ ID NO:2)如图1所示。
图1表明,紫花苜蓿尿苷二磷酸糖基转移酶基因(UGT)的序列由1440个核苷酸组成。
实施例2:紫花苜蓿尿苷二磷酸糖基转移酶基因(UGT)真核表达载体的构建及鉴定
2.1 PCR扩增
将测序正确菌液提取质粒,用带有pCAMBIA3301表达载体接头的引物P3和P4进行PCR扩增,其中P3和P4的序列如下:
P3:5′-gagaacacgggggactcttgaccATGGAAATCTCAAAACAAGAACC-3′(SEQ ID NO:5)。
P4:5'-gaaatttaccctcagatctaccatATTTATTACAGTGGATTTGTTG-3'(SEQ ID NO:6)。
具体PCR扩增条件为:在95℃初始变性5min,95℃变性30s,64.5℃退火30s,72℃延伸90s,共27个循环,得到PCR产物。
2.2PCR产物的测序
用1%的琼脂糖凝胶电泳对2.1的PCR产物进行检测,能够观察到清晰且单一的1440bp左右的DNA条带(见图4),用Quick Gel Extraction Kit试剂盒进行PCR产物的切胶回收试验。在37℃条件下,将PCR产物与和pCAMBIA3301载体用无缝连接酶进行1h连接反应,连接产物转化DH5α感受态细胞。然后在37℃下用含卡那霉素抗生素(50μg/mL)的选择性LB平板培12h,挑选单菌落,接种于含卡那霉素抗生素(50μg/mL)的LB液体培养基,37℃下震荡培养6h,阳性菌液送生物公司测序,测序结果(SEQ ID NO:2)见图1,其与紫花苜蓿尿苷二磷酸糖基转移酶基因(UGT)的序列完全一致。
实施例3:pCAMBIA3301-UGT表达载体转化农杆菌EHA105
3.1质粒转化
将2.2中测序正确的菌液提取质粒,将1ng质粒加入50μL放置在冰上刚刚融化的农杆菌EHA105,冰浴30min,加入液氮中速冻5min,37℃热激5min,冰浴5min后在超净工作台中加入不含抗生素的YEB液体培养基,用锡箔纸包裹完整。
3.2阳性菌的筛选
将3.1接种的YEB培养基在28℃,200rpm条件下培养90min,用含有卡那霉素抗生素(50μg/mL)和利福平抗生素(25μg/mL)的选择性培养基YEB平板28℃培养2d,用锡箔纸将平板包裹完整。挑选单菌落,接种含卡那霉素抗生素(50μg/mL)和利福平抗生素(25μg/mL)的YEB液体培养基,28℃震荡培养6h,用1%的琼脂糖凝胶电泳进行检测,能够观察到清晰且单一的1440bp左右的DNA条带(见图5)的阳性菌液,用终浓度为20%的甘油液体储存(-80℃低温保存箱)。
实施例4紫花苜蓿过表达株系的获得
利用农杆菌转化法将构建的pCAMBIA3301-UGT表达载体转入到紫花苜蓿叶片中,进行植物组织培养试验。试验历时周期为7个月,包括以下阶段:愈伤组织的诱导、胚状体的诱导、胚性愈伤组织的分化、生根培养阶段。具体培养过程如下:
先将农杆菌转化后的紫花苜蓿叶片外植体移至SH3a无抗生素的固体培养基中,在人工培养箱中黑暗避光放置48h后,将叶片转移至含有抗生素的SH3a培养基中继续避光培养,每两周更换一次培养基。6~8周后将蓬松的愈伤组织更换至MSBK培养基中,进行正常光照条件继续培养(16h光照/8h黑暗),2周后将愈伤组织转移至SH9a固体培养基中诱导根芽分化,持续培养至长出幼苗与根系。然后将幼苗转移至生长空间较充足的1/2SH9a瓶装培养基(无抗生素)中,直至再分化出完整的苜蓿再生植株,将植物移入土壤生长环境中培养(营养土:蛭石=1:2)。所有抗生素培养基均含4mg/L草铵膦、400mg/L头孢霉素用于筛选阳性植株。试验结果见图6。
图6的结果表明:经过头孢霉素和草铵膦抗生素筛选后,试验获得了紫花苜蓿过表达株系。
实施例5紫花苜蓿过表达株系的鉴定
将经过组织培养试验筛选获得的35株紫花苜蓿过表达材料培养至生根后转移至土壤基质(营养土:蛭石=1:2)中继续培养。利用TIANGEN试剂盒(DP305)提取紫花苜蓿叶片组织的DNA,以该DNA为模板,用带有表达载体接头的特异性引物P3和P4进行PCR扩增。
具体PCR扩增条件为:在95℃初始变性5min,95℃变性30s,64.5℃退火30s,72℃延伸90s,共27个循环,得到PCR产物。
将PCR产物用1%的琼脂糖凝胶电泳对PCR产物进行检测,观察到清晰且单一的1440bp左右的DNA条带为阳性材料,共7株(见图7);弃掉其余28株没有目的条带的假阳性材料,留下阳性苗进行后续RNA水平的鉴定试验。
选取野生型紫花苜蓿“中苜一号”(WT)和鉴定为阳性株系的7株紫花苜蓿叶片为材料,提取RNA。剪取0.1g紫花苜蓿放入1.5mL离心管中,液氮速冻,利用Eastep Spuer总RNA提取试剂盒进行RNA的提取。采用分光光度计测定样品RNA的浓度和质量,确定不存在降解与污染问题。再采用PrimeScriptTMRT reagent Kit(Perfect Real Time)反转录试剂盒将RNA样品逆转录为cDNA。以该cDNA为模板,选取在组织叶片中表达相对稳定的Ms-ACTIN(P5和P6)和该尿苷二磷酸糖基转移酶基因序列上设计的特异性引物(P7和P8),利用2×PCR SuperMix(+dye)试剂盒进行RT-qPCR实时荧光定量试验,检测过表达紫花苜蓿材料中糖基转移酶基因的转录水平。以野生型紫花苜蓿中苜一号cDNA的相对表达水平为对照组,过表达紫花苜蓿株系中cDNA的相对表达水平为试验组。经RT-qPCR分析过表达紫花苜蓿与对照组该基因的表达水平,结果见图8。
图8结果表明:过表达材料OE8、OE10和OE29的表达量分别为对照WT的2.9、5.4和3.2倍,选择OE8、OE10和OE29开展后续功能验证研究。
P5、P6、P7、P8四种引物的序列如下:
P5:5′-CAAAAGATGGCAGATGCTGAGGAT-3′(SEQ ID NO:7)。
P6:5'-CATGACACCAGTATGACGAGGTCG-3'(SEQ ID NO:8)。
P7:5′-CCAAAGTGTCCTTCGCAACG-3′(SEQ ID NO:9)。
P8:5'-TCCAATTTGACCTACTTCCCTGT-3'(SEQ ID NO:10)。
实施例6紫花苜蓿过表达株系的品质测定
收集生长状况良好的过表达紫花苜蓿植株OE8、OE10和OE29株系的地上部分组织,放于纸质信封中,在70℃烘箱中烘干至恒重。将干样粉碎后过筛(1mm),利用近红外光谱分析仪(FOSS NIRSTM DS2500)测定紫花苜蓿品质相关性状,其中影响苜蓿品质的9个重要指标为:紫花苜蓿的木质素(Lignin)、酸性洗涤纤维(Acid detergent fiber,ADF)、中性洗涤纤维(Neutral detergent fiber,NDF)、粗蛋白(Crude protein,CP)、粗脂肪(Crude Fat,CF)、灰分(ASH)、磷(P)、钙(Ca)和钾(K)的含量。检测结果见表1和图9。
表1紫花苜蓿植株OE8、OE10和OE29的品质测定结果
由表1的结果可以看出:
1、在粗蛋白(CP)含量方面,OE10、OE8、OE29中的CP含量均显著大于对照材料(WT)中的CP含量,且四株紫花苜蓿中CP含量大小的顺序依次为OE8、OE10、OE29、WT。与WT相比,OE8的CP含量增加了12%,OE10的CP含量增加了9.5%,OE29的CP含量增加了6.6%。
2、在粗脂肪(CF)含量方面,OE10、OE8、OE29中的CF含量均大于对照材料(WT)中的CF含量,且四株紫花苜蓿中CF含量大小的顺序依次为OE29、OE10、OE8、WT。与WT相比,OE29的CF含量增加了8.2%,OE10的CF含量增加了3.4%,OE8的CF含量增加了3.2%。
3、在灰分(ASH)含量方面,OE10、OE8、OE29中的ASH含量均显著小于对照材料(WT)中的ASH含量,且四株紫花苜蓿中ASH含量大小的顺序依次为WT、OE8、OE29、OE10。与WT相比,OE8的ASH含量降低了9.9%,OE29的ASH含量降低了19.5%,OE10的ASH含量降低了20.4%。
4、在酸性洗涤纤维(ADF)含量方面,OE10、OE8、OE29中的ADF含量均显著小于对照材料(WT)中的ADF含量,且四株紫花苜蓿中ADF含量大小的顺序依次为WT、OE29、OE10、OE8。与WT相比,OE29的ADF含量降低了5.1%,OE10的ADF含量降低了6.0%,OE8的ADF含量降低了11.1%。
5、在中性洗涤纤维(NDF)含量方面,OE10、OE8、OE29中的NDF含量均大于对照材料(WT)中的NDF含量,且四株紫花苜蓿中NDF含量大小的顺序依次为OE10、OE29、OE8、WT。与WT相比,OE10的NDF含量增加了12.4%,OE29的NDF含量增加了2.5%,OE8的NDF含量增加了1.3%。
6、在木质素(Lignin)含量方面,OE10、OE8、OE29中的Lignin含量均显著小于对照材料(WT)中的Lignin含量,且四株紫花苜蓿中Lignin含量大小的顺序依次为WT、OE29、OE8、OE10。与WT相比,OE29的Lignin含量降低了32%,OE8的Lignin含量降低了48.3%,OE10的Lignin含量降低了53.1%。
7、在磷(P)含量方面,OE10、OE8、OE29中的NDF含量均显著大于对照材料(WT)中的P含量,且四株紫花苜蓿中P含量大小的顺序依次为OE29、OE8、OE10、WT。与WT相比,OE29的P含量增加了18.2%,OE8的P含量增加了6.0%,OE10的P含量增加了3.0%。
8、在磷(Ca)含量方面,OE10、OE8、OE29中的Ca含量均显著大于对照材料(WT)中的Ca含量,且四株紫花苜蓿中Ca含量大小的顺序依次为OE29、OE10、OE8、WT。与WT相比,OE29的Ca含量增加了12.5%,OE10的Ca含量增加了6.6%,OE8的Ca含量增加了5.9%。
9、在钾(K)含量方面,OE10、OE8、OE29中的K含量均显著大于对照材料(WT)中的K含量,且四株紫花苜蓿中K含量大小的顺序依次为OE29、OE8、OE10、WT。与WT相比,OE29的K含量增加了38.9%,OE8的K含量增加了14.2%,OE10的K含量增加了8.5%。
图9结果显示,与对照材料(WT)相比,过表达紫花苜蓿材料的粗蛋白(CP)、粗脂肪(CF)、中性洗涤纤维(NDF)、磷(P)、钙(Ca)和钾(K)的含量明显提高。而灰分(ASH)、酸性洗涤纤维(ADF)和木质素(Lignin)的含量显著下降。
表1和图9的结果说明过表达紫花苜蓿的品质比野生型紫花苜蓿中苜一号(WT)明显提高,过表达紫花苜蓿材料更有利于改善和提高家畜动物的采食量和消化率。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
Claims (8)
1.一种尿苷二磷酸糖基转移酶,其特征在于:所述尿苷二磷酸糖基转移酶的氨基酸序列如SEQ ID NO:1所示。
2.一种尿苷二磷酸糖基转移酶基因,其特征在于:所述基因编码权利要求1所述尿苷二磷酸糖基转移酶。
3.根据权利要求2所述的基因,其特征在于:所述基因的编码序列如SEQ ID NO:2所示。
4.一种重组载体,其特征在于:所述重组载体含有权利要求2或3所述的基因。
5.一种重组基因工程菌,其特征在于:所述工程菌采用权利要求4所述的重组载体转化得到。
6.一种培育紫花苜蓿的方法,其特征在于:所述方法包括将权利要求2或3所述的基因导入紫花苜蓿细胞中,得到超表达尿苷二磷酸糖基转移酶的紫花苜蓿细胞及转基因植株。
7.权利要求1所述的尿苷二磷酸糖基转移酶、权利要求2所述的基因、权利要求4所述的重组载体、权利要求5所述的重组基因工程菌在培育超表达尿苷二磷酸转移酶的植物中的应用。
8.根据权利要求7所述的应用,其特征在于:所述植物为紫花苜蓿。
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