CN117025628A - 大豆抗病调控基因GmMYC2及其应用 - Google Patents
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Abstract
本发明公开一种大豆抗病调控基因GmMYC2及其应用,所述基因GmMYC2对大豆疫霉菌具有正调控作用,其序列如SEQ ID NO.1所示,编码的蛋白序列如SEQ ID NO.2所示。本发明所述的基因GmMYC2能够显著提高大豆对大豆疫霉根腐病的抗性。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一个大豆抗病调控相关基因GmMYC2及其应用。
技术背景
大豆疫霉根腐病是大豆生产上最严重的病害之一[1],由大豆疫霉菌引起,可在大豆的任何生育期发病,每年因大豆疫霉根腐病造成的经济损失高达数亿美元[2]。大豆疫霉菌可在大豆的任何生育期对其进行侵染[3],其主要危害部位是大豆根系。大豆疫霉菌侵染大豆时,会在潮湿的环境下形成游动孢子。游动孢子可以借助自身鞭毛在水中游动,主动趋向寄主[4]。菌丝在侵染寄主时可在细胞间延伸扩展,也可在细胞内延伸扩展。胞间菌丝侵入寄主皮层细胞后可形成吸器,以便从寄主细胞中不断地吸收营养成分[5]。
近十几年来,大豆疫霉菌引起的大豆疫霉根腐病在我国大豆主产区分布广泛,对我国大豆的产量和品质都造成严重影响。因此,对疫霉根腐病的防治是我国大豆生产中的重要工作,通过科学技术培养抗性品种,降低大豆根腐疫霉病对大豆生产造成的损失,是目前最好的解决策略,而抗性基因的鉴定及其调控通路的研究是大豆疫霉根腐病抗性遗传改良的关键[3]。
Zhang H P,Guo N,Niu J P,et al.Genetic Analysis of Resistance toPhytophthora sojae and Mapping of Resistance Gene in Soybean CultivarZheng97196[J].Soybean Science,2016.
Tyler B M.Phytophthora sojae:Root rot pathogen of soybean and modeloomycete[J].Molecular Plant Pathology,2007,8(1):1-8.
Dorrance AE,McClure S A,St.Martin S K.Effect of partial resistance onPhytophthora stem rot incidence and yield of soybean in Ohio[J].PlantDisease,2003,87(3):308-312.
Tyler B M.Molecular basis of recognition between Phytophthorapathogens and their hosts[J].Annual Review of Phytopathology,2002,40(1):137-167.
Enkerli K,Mims C W,Hahn M G.Ultrastructure of compatible andincompatible interactions of soybean roots infected with the plant pathogenicoomycete Phytophthora sojae[J].Canadian Journal of Botany,1997,75(9):1493-1508.
发明内容
本发明提供一种大豆抗病调控基因GmMYC2及其应用,其对大豆疫霉菌具有正调控作用。
本发明所述的大豆抗病调控基因GmMYC2,其序列如SEQ ID NO.1所示。
本发明还提供所述的基因GmMYC2编码的蛋白,序列如SEQ ID NO.2所示。
本发明还提供一种包含所述基因GmMYC2的重组表达载体。
所述表达载体可以为本领域的常规载体连接本发明所述的基因GmMYC2形成,例如使用限制性酶Kpn I和BamH I酶切载体pBinGFP4插入基因GmMYC2后形成的重组表达载体。
本发明还提供一种转化体,由包含所述基因GmMYC2的重组表达载体导入宿主细胞所得,所述的宿主细胞优选大肠杆菌细胞或农杆菌细胞。
本发明提供扩增本发明所述基因的引物。
本发明扩增基因GmMYC2的引物可以按照本领域的常规方法进行设计,在一种实施例中,为:
F:TGGCCGCCAGAACAAACA
R:GCTTCCCAAATCAAATTACCTCAA。
本发明还提供所述大豆抗病调控基因GmMYC2、重组表达载体、转化体、或者引物在提高大豆抗病性中的应用。更具体的,是在提高大豆抗疫霉菌导致的病害中的应用。
本发明还提供一种提高大豆抗大豆疫霉菌导致的病害的方法,将基因GmMYC2导入大豆进行过表达。
本发明所述的基因GmMYC2能够显著提高大豆疫霉菌导致的病害。
附图说明
图1:克隆条带大小和根茎叶子叶组织表达量;A:GmMYC2的克隆;B:根茎叶子叶组织表达量。
图2:实验组接种疫霉前后GmMYC2的表达模式。
图3:GmMYC2的亚细胞定位。
图4:GmMYC2过表达发状根和空载对照受到疫霉侵染后分子量的变化(EV:空载发状跟;OE:过表达发状根),其中:A:空载和过表达发根GmMYC2基因表达水平;B:空载和过表达发根疫霉侵染24小时后疫霉积累量;C:空载和过表达发根疫霉侵染24小时后茉莉酸信号通路防御基因GmPDF1.2表达量变化。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
以下实施例所用的大豆疫霉菌为大豆疫霉菌P6497,由南京农业大学植物保护学院王源超教授惠赠,现存于本课题组,本课题组承诺永久向公众开放提供。
农杆菌菌株K599、GV3101均由本实验室保存并提供,本课题组承诺永久向公众开放提供。
亚细胞定位所用pBinGFP4载体由南京农业大学植保学院窦道龙教授惠赠,本课题组承诺永久向公众开放提供。
所用核定位信号NLS-mCherry载体由南京农业大学农学院丁承强副教授惠赠,本课题组承诺永久向公众开放提供。
大肠杆菌(E.coli)Trans 5α感受态细胞,DL5000 Plus DNA Marker,Taq酶,高保真酶(Max Super-Fidelity DNA Polymerase)和质粒提取试剂盒均购自南京诺唯赞生物科技股份有限公司;DNA/RNA试剂提取盒购于上海浦迪生物科技有限公司;DNA凝胶回收试剂盒(GE706)购于北京金沙生物科技有限公司;同源重组试剂盒购于南京巨匠生物科技有限公司;逆转录和荧光定量试剂盒购于上海翊圣生物科技有限公司。
实施例1
以大豆品种Williams 82叶片的cDNA为模板(SEQ ID NO.1),使用GmMYC2的特异引物(F:TGGCCGCCAGAACAAACA;R:GCTTCCCAAATCAAATTACCTCAA)扩增出长度为1965bp的DNA片段(图1)。对幼苗期大豆的根、茎、叶和子叶四个组织进行取样,分析GmMYC2在不同组织中的表达模式。发现GmMYC2的表达量在根中最高,在茎和叶中的表达量次之,在子叶中的表达量则是最低(图1)。
对大豆进行接种大豆疫霉菌P6497。发现与未接种时相比,在接种疫霉菌时GmMYC2的转录水平显著提高(图2),可见基因GmMYC2与大豆疫霉菌抗性相关,基因GmMYC2核苷酸序列如SEQ ID NO.1所示,编码的蛋白序列如SEQ ID NO.2所示。
实施例2蛋白的亚细胞定位分析
1、瞬时表达载体的构建
使用Vazyme公司CE Design V1.04软件设计含有酶切位点(Kpn I和BamH I位点)的引物(F:atttacgaacgatagggtaccATGACCGAGTACCGGATGAACC;
R:gcccttgctcaccatggatccTCGTAGTTCATCGCCAACTTT),扩增GmMYC2基因序列(SEQID NO.1)。同时使用限制性酶Kpn I和BamH I酶切载体pBinGFP4。PCR结束后通过琼脂糖凝胶电泳检测条带大小,并进行产物纯化。使用TaKaRa公司的QuickCut限制酶Kpn I和BamH I对pBINGFP4表达载体进行酶切反应,随后扩增片段和酶切载体重组形成重组质粒pBin-GmMYC2-GFP4。
2、农杆菌注射烟草
(1)移液枪吸取10μL含pBin-GmMYC2-GFP4重组质粒的农杆菌放入4mL含利福平和卡那抗生素的YEP培养液中,28℃200rpm摇床中培养12-16h。
(2)从培养好的菌液中吸取200μL加入30mL含利福平和卡那抗生素的YEP培养液中(使用50mL离心管),28℃培养箱中过夜培养。
(3)配置渗透液试剂如表1。
表1渗透液配置
(4)将OD600值达到0.5-0.7的菌液4500×g离心5分钟。倒掉上清液后使用渗透液清洗三次,后加入渗透液混匀,OD600值到达0.5-0.7之间即可使用。
(5)将含有空载pBinGFP4的重悬液与核定位信号NLS-mCherry重悬液等体积混合作为对照组,含有pBin-GmMYC2-GFP4目的质粒的重悬液和NLS-mCherry重悬液等体积混合作为实验组。
(6)选择生长周期为4周且长势良好的烟草进行注射,注射时用1mL注射器吸取菌液,翻转叶片,将注射器口垂直压在叶片背面,轻轻用力推压注射器,观察菌液在烟草叶片中的扩散,直至整个叶片均受到侵染。
(7)注射后的烟草植株黑暗保湿48小时,使用激光共聚焦显微镜观察GmMYC2定位。
结果如图3:GmMYC2扩增后同源重组至pBinGFP4载体获得构建pBin-GmMYC2-GFP4融合载体,利用农杆菌介导的瞬时表达体系注射烟草观察GmMYC2的定位情况并发现GmMYC2蛋白定位在细胞核。
实施例3农杆菌介导的大豆遗传转化
1.表达载体的构建
使用Vazyme公司CE Design V1.04软件设计含有酶切位点的引物(Sal I和Sma I位点)扩增GmMYC2基因(SEQ ID NO.1,引物F:aggcctgacctgcaggtcgacATGACCGAGTACCGGATGAACC;R:tcgagctctccctgacccgggCTATCGTAGTTCATCGCCAACTTT),将GmMYC2重组至pCB301GFP表达载体,获得pCB301-GFP-GmMYC2重组质粒。
2.大豆转基因发状根的获得
2.1发根及筛选
(1)农杆菌K599培养及重悬液配置
从电转后获得的包含pCB301-GFP-GmMYC2重组质粒的K599农杆菌吸取10μL放入4mL含有50mg/L利福平和50mg/L卡那霉素的YEP液体培养基中,28℃200rpm摇床中培养过夜。菌液培养12~16h后,吸取500μL涂于含利福平和卡那霉素的YEP平板上,于28℃培养箱中培养。48h之后用灭菌的勺子将平板上的菌层轻轻刮到加入激素的CCM液体培养基中,混匀使其OD600值在0.6-1.0之间,并将得到的重悬液分装到培养皿中待用。
(2)大豆种子的灭菌
灭菌时挑选无病斑、种皮完好、饱满均一的Williams种子,将其放置在干净的玻璃皿中,灭菌大豆用量为4皿(大约200粒大豆种子)。将玻璃皿放入干燥器中,并将玻璃皿的盖子45°斜放在玻璃皿上。灭菌方法为氯气灭菌,灭菌后取出玻璃皿,立即放入超净工作台上中吹风并用保鲜膜密封。
(3)萌发
将灭菌完的种子播种在萌发培养基GM中,并用保鲜膜密封放到组培室中黑暗过夜,使种子充分吸胀10-16h。
(4)侵染及外植体的共培养
将过夜充分吸胀的豆子挑到空的培养皿中,用灭菌后的手术刀沿着大豆种子的胚根将大豆切分为两半,即为两个外植体,并将外植体浸泡在已经配置好的重悬液中侵染30min。侵染完成后用镊子将外植体从重悬液中取出,并去掉外面包裹的种皮。用镊子将外植体曲面在下放置于空皿中,组培室黑暗放置5天。
(5)毛状根的诱导
黑暗培养5天后,切掉外植体伸长的胚根,保留约2-3mm的长度,并将外植体的切面朝上斜插入White培养基中,放在植物组织培养室中培养2-3周直至诱导出适量毛状根。
2.2鉴定阳性根
将发状根放置于荧光体视镜下查看,发出绿色荧光的根则为阳性根。检测基因在表达水平上的变化,对阳性根进行取样提取总RNA,检测目的基因的表达水平。
结果如图4A所示,发现过表达发状根中GmMYC2的转录水平与对照组相比显著上调,上调倍数可达40倍,说明GmMYC2过表达成功。
实施例4探究GmMYC2是否能够参与大豆与大豆疫霉菌的互作
将实施例3过表达发状根和空载发状根中选取长势一致、粗细均匀且幼嫩的根尖,并浸泡在大豆疫霉菌P6497游动孢子液中。24小时之后测定过表达发状根和空载发状根中疫霉生物积累量和下游抗病基因GmPDF1.2的转录水平。结果发现,接种后过表达发状根中的疫霉生物积累量显著低于空载发状根中疫霉生物积累量量(图4B)。未接种时下游抗病基因GmPDF1.2转录水平在pCB301GFP-GmMYC2过表达发状根及空载发状根中无明显差异,但在接种大豆疫霉菌24小时后下游抗病基因GmPDF1.2转录水平在过表达发状根中显著高于空载发状根(图4C)。
以上结果说明GmMYC2的过表达可能会激活JA信号通路,进一步激活下游防御基因的表达,从而正调控大豆对大豆疫霉菌的抗性。而GmMYC2的沉默并没有显著影响大豆对大豆疫霉菌的抗性。
Claims (10)
1.大豆抗病调控基因GmMYC2,其序列如SEQ ID NO.1所示。
2.权利要求1所述的基因GmMYC2编码的蛋白,序列如SEQ ID NO.2所示。
3.包含权利要求1所述基因GmMYC2的重组表达载体。
4.根据权利要求3所述的重组表达载体,其特征在于,使用限制性酶Kpn I和BamH I酶切载体pBinGFP4插入基因GmMYC2后形成。
5.一种转化体,由包含权利要求1所述基因GmMYC2的重组表达载体导入宿主细胞所得。
6.根据权利要求5所述的转化体,其特征在于,所述的宿主细胞为大肠杆菌细胞或农杆菌细胞。
7.扩增权利要求1所述的基因GmMYC2的引物。
8.根据权利要求7所述的引物,其特征在于,具体为
F:TGGCCGCCAGAACAAACA
R:GCTTCCCAAATCAAATTACCTCAA。
9.权利要求1所述的基因GmMYC2、权利要求2所述的蛋白、权利要求3或4所述的重组表达载体、权利要求5或6所述的转化体、权利要求7或8所述的引物在提高大豆抗病性中的应用;优选的,在提高大豆抗大豆疫霉菌导致的病害中的应用。
10.一种提高大豆抗大豆疫霉菌导致的病害的方法,将权利要求1所述的基因GmMYC2导入大豆进行过表达。
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