CN114686497B - 一种改良桑树DNJ生物合成的基因MnGutB1及其应用 - Google Patents
一种改良桑树DNJ生物合成的基因MnGutB1及其应用 Download PDFInfo
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Abstract
本发明提供一种改良桑树DNJ生物合成的基因MnGutB1及其应用,所述基因的核苷酸序列如SEQ ID No.1所示。其编码的蛋白质氨基酸序列如SEQ ID No.2所示。本发明通过构建过表达载体pZYGC‑MnGutB1在桑树中过表达MnGutB1,从而获得高丰度DNJ的桑树材料。通过构建病毒干涉载体pBinplus‑2mDNA1‑MnGutB1侵染桑树,获得低丰度DNJ桑树材料。本发明提供的基因表达对植物中DNJ含量调节变化显著,并且其过表达不影响桑树正常生长;其转化获得的桑树发根生长快速,可作为DNJ大规模植物生产的来源。
Description
技术领域
本发明涉及功能基因和植物基因育种领域,具体涉及一种改良桑树DNJ生物合成的基因MnGutB1及其应用。
背景技术
桑叶含有丰富的生物活性化合物,包括酚类、多糖和生物碱,特别是1-脱氧野尻霉素(DNJ)。由于是葡萄糖类似物,DNJ被认为是调节血糖水平的最有效的葡萄糖苷酶抑制剂。因其具有促进健康的作用而受到越来越多的关注,包括抗糖尿病、抗肥胖、抗病毒。先前的研究报道,不同品种桑树的干叶中DNJ的浓度为0.1341-1.472mg/g。高丰度DNJ的桑树资源大多为一些较难推广栽培的品种。
关于桑叶或其他植物中DNJ的生物合成基因的研究很少。为了阐明DNJ在高等植物中的生物合成,有学者在鸭跖草(Commelina communis)中进行了13C葡萄糖喂养实验,并采用13C NMR光谱分析来确定13C在植物代谢物中的分布,提出了DNJ的生物合成途径,从葡萄糖分子的亚胺化开始,然后经过还原、氧化和独特的C1/C5环化形成野尻霉素,然后经过脱水和还原产生DNJ。微生物中报道了一种还原酶GutB1能够催化葡萄糖衍生物形成DNJ。
川桑(Morus notabilis Schneid.)全基因组解析,获得了大规模的桑树基因资源,为功能基因组的研究奠定了基础,推进了桑树改良的研究进程。毛状根过表达技术和病毒介导的基因干涉技术已经运用于桑树基因功能的研究,例如桑树白藜芦醇调控基因功能的研究,桑树抗灰霉病基因的鉴定等等,这些都为DNJ生物合成相关基因的研究提供了有效的方法。但对DNJ生物合成相关基因的研究仍然有很多未知领域。
发明内容
有鉴于此,为了克服现有技术的不足,本发明通过大量的实验研究,提供了一种改良桑树DNJ生物合成的基因MnGutB1。
本发明提供的改良桑树DNJ生物合成基因MnGutB1的核苷酸序列如SEQ ID No.1所示。
本发明还提供上述基因MnGutB1编码的蛋白质,其氨基酸序列如SEQ ID No.2所示。
本发明还提供用于克隆获取如上所述基因MnGutB1的引物对,该引物对的引物序列为:
F:5’-atggcaaaatcaccagaaga-3’;(SEQ ID No.3)
R:5’-ctactgtgataaagagttccccac-3’。(SEQ ID No.4)
本发明还提供过表达基因MnGutB1的植物过表达载体,所述植物过表达载体为包含MnGutB1部分特异性序列的pZYGC植物过表达载体,命名为pZYGC-MnGutB1,扩增所述MnGutB1部分特异性序列的引物序列为:
F:5’-atttcatttggagaggacagATGGCAAAATCACCAGAAGA-3’;(SEQ ID No.5)
R:5’-tcctccaccggttctagaatgcatCTGTGATAAAGAGTTCCCCAC-3’;(SEQ ID No.6)
其中,小写字母为载体同源臂。
本发明还提供下调基因MnGutB1表达的病毒干涉载体,所述病毒干涉载体为包含MnGutB1部分特异性序列的pBinplus-2mDNA1载体,命名为pBinplus-2mDNA1-MnGutB1病毒干涉载体,扩增所述MnGutB1部分特异性序列的引物序列为:
F:5’-cgcggatccattgacacagtgtctgcagttcatc-3’;(SEQ ID No.7)
R:5’-ctagtctagagatcacaaaccggtacctaacatcg-3’。(SEQ ID No.8)
本发明还提供基因MnGutB1的蛋白表达载体,所述蛋白表达载体为包含MnGutB1部分特异性序列的pColdTF载体,扩增所述MnGutB1部分特异性序列的引物序列为:
F:5’-ggcatatggagctcggtaccATGGCAAAATCACCAGAAGA-3’;(SEQ ID No.9)
R:5’-attacctatctagactgcagCTACTGTGATAAAGAGTTCCCCAC-3’(SEQ ID No.10)
其中,小写字母为载体同源臂。
本发明还提供上述基因MnGutB1在改良桑树DNJ生物合成中的应用,所述应用包括通过植物过表达载体pZYGC-MnGutB1在桑树中过表达MnGutB1,从而获得高丰度DNJ的桑树材料。
所述改良桑树DNJ生物合成指的是调控桑树DNJ生物合成过程中GutB1基因表达。
本发明还提上述基因MnGutB1在下调桑树DNJ生物合成中的应用,所述应用包括通过病毒干涉载体pBinplus-2mDNA1-MnGutB1侵染桑树,获得的MnGutB1基因低表达的低丰度DNJ桑树材料。
MnGutB1是定位于川桑染色体Chr3上的基因。它的以下特征促使研究者将其作为桑树高丰度DNJ性状改良以及合成生物学生产DNJ的重要功能基因:①芽、嫩叶和根特异性表达:MnGutB1表达量随着叶位下降而下降,在愈伤组织中几乎不表达,叶片是DNJ合成的场所,DNJ含量随着叶位下降而下降,愈伤组织中不含DNJ;②MnGutB1表达产物作为桑树DNJ生物合成限速酶的作用:叶片干涉MnGutB1基因的表达显著减少了叶片中DNJ含量;桑树毛状根中过表达MnGutB1基因显著增加了根中DNJ含量。这些特征为利用桑树MnGutB1基因用于桑树高丰度DNJ性状改良以及合成生物学生产DNJ提供了理论支持。
与现有技术相比,本发明的有益效果在于:
1.本发明提供的基因MnGutB1的表达对植物中DNJ含量调节变化显著:与对照组相比,过表达MnGutB1基因的毛状根中DNJ含量显著提高;MnGutB1基因通过病毒干涉载体在叶片中表达受干涉,叶片中DNJ含量显著降低。
2.本发明提供的MnGutB1基因在桑树发根内过表达,不影响桑树正常生长;
3.本发明提供的MnGutB1基因其转化获得的桑树发根生长快速,可作为DNJ大规模植物生产的来源。
附图说明
图1为包含MnGutB1基因的植物过表达载体pZYGC-MnGutB1和pZYGC对照载体,箭头表示转录起始位点与转录方向;
图2为转基因发根的荧光观察后的灰度处理照片,a.转pZYGC载体发根(白光下)b.转pZYGC载体发根(荧光下)c.转pZYGC-MnGutB1载体发根(白光下)d.转pZYGC-MnGutB1载体发根(荧光下);
图3为转pZYGC-MnGutB1桑树发根的MnGutB1表达量分析,OX-4,OX-11,OX-17和OX-21为鉴定到的转基因发根株系;
图4为转pZYGC-MnGutB1桑树发根的DNJ含量测定,OX-4,OX-11,OX-17和OX-21为鉴定到的转基因发根株系。
图5为pBinplus-2mDNA1-MnGutB1病毒干涉载体介导的下调(基因沉默)桑叶中MnGutB1表达量分析,i17,i18和i26为鉴定到的三株MnGutB1下调最显著的株系。
图6为pBinplus-2mDNA1-MnGutB1病毒干涉载体介导的下调(基因沉默)桑叶中DNJ含量测定,i17,i18和i26为鉴定到的三株MnGutB1下调最显著的株系。
具体实施方式
实施例1:桑树MnGutB1基因获得
MnGutB1基因的获得是以川桑幼嫩叶片cDNA为模板进行PCR扩增,根据设计的MnGutB1基因上下游引物来扩增得到。游引物为:5’-atggcaaaatcaccagaaga-3’,下游引物为:5’-ctactgtgataaagagttccccac-3’,扩增条件为94℃预变性4分钟;94℃变性30秒,55℃退火30秒,72℃延伸80秒,共30个循环;最后72℃延伸10分钟,4℃保存。
川桑幼嫩叶片cDNA中扩增获得的MnGutB1基因经克隆和测序验证,其序列长度为1080bp。其DNA序列见SEQ ID NO:1所示。
实施例2:构建含桑树MnGutB1基因的重组载体pMD19-MnGutB1
如SEQ ID NO:1所示序列的桑树MnGutB1基因与载体pMD19-simple进行TA克隆,获得重组载体pMD19-MnGutB1。
实施例3:桑树过表达DNJ的重组载体pZYGC-MnGutB1的构建
本发明利用的pZYGC植物过表达载体由本实验室吕志远博士以商业化pCAMBIA系列载体为骨架改造而来(《核地杖菌漆酶基因ShLAC1和桑树几丁质识别受体的功能研究》,吕志远博士毕业论文,2018)。植物过表达载体pZYGC-MnGutB1是利用Seamless Cloning and Assembly Kit同源重组法构建的:将实施例所得,即如SEQ ID NO:1所示的桑树MnGutB1基因与载体pZYGC进行同源重组后得到的重组表达载体,即pZYGC-MnGutB1,具体为:以pMD19-MnGutB1质粒为模板进行PCR扩增,上游引物为:5’-atttcatttggagaggacagatggcaaaatcaccagaaga-3’,下游引物为:5’-tcctccaccggttctagaatgcatctgtgataaagagttccccac-3’,扩增条件为94℃预变性4分钟;94℃变性30秒,60℃退火30秒,72℃延伸80秒,共30个循环;最后72℃延伸10分钟,4℃保存,回收MnGutB1基因片段,KpnI和BamHI双酶切pZYGC质粒,回收pZYGC载体片段,将MnGutB1基因与pZYGC载体片段用Seamless Cloning and Assembly Kit同源重组酶连接,获得重组载体pZYGC-MnGutB1。其结构如图1所示。
实施例4:桑树MnGutB1基因的病毒干涉载体的制备
以pMD19-MnGutB1质粒为模板进行PCR扩增,上游引物为:5’-cgcggatccattgacacagtgtctgcagttcatc-3’,下游引物为:5’-ctagtctagagatcacaaaccggtacctaacatcg-3’,扩增条件为94℃预变性4分钟;94℃变性30秒,60℃退火30秒,72℃延伸80秒,共30个循环;最后72℃延伸10分钟,4℃保存,回收MnGutB1基因片段用限制性内切酶XbaI和BamHI双酶切,将切下的MnGutB1片段连接到内切酶XbaI和BamHI双酶切的pBinplus-2mDNA1载体,构建得到pBinplus-2mDNA1-MnGutB1病毒干涉载体。
实施例5:桑树MnGutB1基因的蛋白表达载体的制备
以pMD19-MnGutB1质粒为模板进行PCR扩增,上游引物为:5’-ggcatatggagctcggtaccatggcaaaatcaccagaaga-3’,下游引物为:5’-attacctatctagactgcagctactgtgataaagagttccccac-3’,扩增条件为94℃预变性4分钟;94℃变性30秒,60℃退火30秒,72℃延伸80秒,共30个循环;最后72℃延伸10分钟,4℃保存,回收MnGutB1基因片段通过Seamless Cloning and Assembly Kit同源重组连接到用内切酶XbaI和SacI双酶切的pColdTF载体,构建得到pColdTF-MnGutB1蛋白表达载体。
通过上述蛋白表达载体获得基因MnGutB1编码的蛋白质,其氨基酸序列如SEQ IDNo.2所示。
实施例6:桑树MnGutB1基因在发根中提高DNJ的运用
将构建好的植物过表达载体pZYGC-MnGutB1转入发根农杆菌K599,采用针刺法,注射菌液至四叶期桑苗的子叶节处诱导产生转化毛状根。筛选出特异激发绿色荧光的转基因阳性发根,如图2所示,其中21个株系中有16个筛选出了阳性个体,阳性率为76.2%。
挑选4个进行阳性株系通过RT-PCR检测MnGutB1基因在mRNA水平的表达变化。收获阳性株系发根,在液氮中瞬时冷却后放入-80℃冰箱备用。抽提所取材料的总RNA,反转录合成cDNA,分别采用MnGutB1基因特异引物进行RT-PCR检测,以MnRPL15(LOC21406832)(Cloning and expression analyses of the anthocyanin biosynthetic genes inmulberry plants,MOLECULAR GENETICS AND GENOMICS,2014)作为内参基因。引物序列为:5'-GGCTATGTGATTTACCGTGTT-3',5'-TTGGTCCAGTATGAGTTGAGAA-3',反应条件为:94℃预变性4min;94℃变性15s,55℃退火30s,72℃延伸1min,共24个循环;最后72℃延伸10min。结果详见图3,发现相对于空载转化的发根,阳性株系中MnGutB1基因表达水平显著提升。
通过HPLC-MS/MS检测转基因发根中DNJ的含量。取在液氮中瞬时冷却后的桑树发根进行组织液氮研磨后溶于70%甲醇,4℃过夜抽提,离心后取上清,测定DNJ的含量。结果表明在相对于空载,阳性株系的桑树发根DNJ相对含量显著提高,详见图4,该结果说明,MnGutB1基因可以显著提高转基因桑树发根DNJ的含量。
实施例7:下调桑树中DNJ的生物合成
将桑树MnGutB1基因的病毒干涉载体转入根癌农杆菌GV3101,注射菌液至二叶期桑苗子叶,1个月后收获全株叶片,在液氮中瞬时冷却后放入-80℃冰箱备用。抽提所取材料的总RNA,反转录合成cDNA,分别采用MnGutB1基因特异引物进行RT-PCR检测,以MnRPL15(LOC21406832)作为内参对照。引物序列为:5'-GGCTATGTGATTTACCGTGTT-3',5'-TTGGTCCAGTATGAGTTGAGAA-3',反应条件为:94℃预变性4min;94℃变性15s,55℃退火30s,72℃延伸1min,共24个循环;最后72℃延伸10min。结果详见图5,其中29个株系中有12个株系表现出了不同程度的基因表达水平下调。
通过HPLC-MS/MS检测MnGutB1基因下调桑树叶片中DNJ的含量变化。挑选3个MnGutB1基因下调最显著的株系叶片,在液氮中瞬时冷却后进行组织液氮研磨,然后溶于70%甲醇,4℃过夜抽提,离心后取上清,测定DNJ的含量。结果见图6,表明在相对于空载,当桑叶中MnGutB1基因被下调表达时,DNJ含量显著降低。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离所附权利要求书所限定的本发明的精神和范围。
SEQUENCE LISTING
<110> 西南大学
<120> 一种改良桑树DNJ生物合成的基因MnGutB1及其应用
<130> 《核地杖菌漆酶基因ShLAC1和桑树几丁质识别受体的功能研究》,吕志远博
士毕业论文,2018
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<213> Artificial Sequence
<220>
<223> 引物序列R
<400> 10
attacctatc tagactgcag ctactgtgat aaagagttcc ccac 44
Claims (2)
1.一种高丰度DNJ桑树毛根材料的制备方法,其特征在于:所述高丰度DNJ桑树毛根材料中过表达MnGutB1,所述基因MnGutB1的核苷酸序列如SEQ ID No .1所示;
所述方法包括:1)将构建好的植物过表达载体pZYGC- MnGutB1转入发根农杆菌 K599,采用针刺法,注射菌液至四叶期桑苗的子叶节处诱导产生转化毛状根;
2)筛选出特异激发绿色荧光的转基因阳性发根;
3)收获阳性株系发根,抽提所取材料的总RNA,反转录合成cDNA,分别采用MnGutB1基因特异引物进行RT-PCR检测,以MnRPL15作为内参基因;引物序列为:5'-GGCTATGTGATTTACCGTGTT-3',5'- TTGGTCCAGTATGAGTTGAGAA-3',反应条件为:94 ℃预变性4 min;94 ℃变性15 s,55 ℃退火30 s,72 ℃延伸1 min,共24个循环;最后72 ℃延伸10min;筛选步骤2)中转基因阳性发根中MnGutB1基因表达水平相对空载转化显著提升;
4)通过HPLC-MS/MS进一步验证步骤3)获得的转基因阳性发根中DNJ的含量相对空载转化显著提升,获得高丰度DNJ的桑树毛根材料。
2.根据权利要求1所述高丰度DNJ桑树毛根材料的制备方法,其特征在于:所述植物过表达载体pZYGC- MnGutB1为包含MnGutB1部分特异性序列的pZYGC植物过表达载体,扩增所述MnGutB1部分特异性序列的引物序列为:
F:5’- atttcatttggagaggacagATGGCAAAATCACCAGAAGA-3’;
R:5’- tcctccaccggttctagaatgcatCTGTGATAAAGAGTTCCCCAC-3’;
其中,小写字母为载体同源臂。
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