CN116497054B - GmSGT1基因在控制大豆结瘤中的应用 - Google Patents
GmSGT1基因在控制大豆结瘤中的应用 Download PDFInfo
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Abstract
本发明公开一种GmSGT1基因在控制大豆结瘤中的应用,属于农业种植的技术领域。为了控制大豆的结瘤能力,在降低人工人本的前提下,减少根瘤的数量,解决了很难控制大豆根瘤数量的技术问题。本发明提供GmSGT1基因在大豆中超表达或者突变后获得的大豆毛状根后,接种根瘤菌后,获得的的大豆植株的结瘤数量会相应的减少或者增加,达到控制大豆根瘤数量的效果。为后续研究大豆‑根瘤菌共生体系形成提供了基础,为提高大豆与根瘤菌的共生效率提供了可能性。
Description
技术领域
本发明属于农业种植的技术领域,具体涉及GmSGT1基因在控制大豆结瘤中的应用。
背景技术
大豆是重要的经济作物和油料作物,能够为人类和动物提高大量优质的植物蛋白及油脂。氮肥的施用对全球作物的高产起到了关键作用,但是目前每年农业消耗的1.09亿吨氮肥中,只有不到一半被农作物吸收。过多施用的氮肥会造成土壤养分失衡,导致土壤中钙、磷和镁等其他重要矿物质的枯竭,甚至会导致土壤酸化。过量施用的氮肥会导致一氧化二氮(N2O)的排放,这是一种导致全球变暖的强有力的温室气体。氮肥与周围环境和地下水有着密切的联系,当河流和溪流中的氮水平增加时,会导致藻类过度生长,藻类的生长、死亡和分解消耗大量氧气,导致河水中氧含量下降,造成鱼、蟹和其他水生生物的死亡。过量施用的氮肥还对人类健康构成了严重威胁,氮肥的过量施用导致叶类蔬菜和根类蔬菜中硝酸盐的积累,食用硝酸盐含量高的饮食会导致甲状腺疾病、人类各种癌症、神经管缺陷和糖尿病等疾病。由此可见缩减氮肥的施用量早已变成全世界亟需破解的难题。
豆科植物与根瘤菌能够建立共生关系,根瘤菌将大气中的N2转化为植物新陈代谢所需的铵态氮,植物向根瘤菌提供光合产物作为碳源。这种相互作用固氮过程具有重要的经济和生态效益。根瘤菌的III型分泌系统(T3SS)对于建立和调控豆科植物与根瘤菌的共生关系极为关键。解析T3SS分泌的III型效应因子在共生固氮过程中的功能,阐明根瘤菌III型效应因子调控大豆固氮的分子机制,可以为从基因工程和分子育种手段解决该问题奠定一定的理论基础。
根瘤是植物和根瘤菌互作产生的特化共生器官,根瘤菌在其中可以将大气中的氮气转化为植物可吸收利用的铵态氮,宿主植物也要为根瘤菌光合产物保证其生存繁衍。结瘤和固氮过程都高度耗能,过多的结瘤会抑制宿主的生长发育。
发明内容
本发明的目的是为了控制大豆的结瘤能力,在降低人工人本的前提下,减少根瘤的数量,解决了很难控制大豆根瘤数量的技术问题。
本发明提供一种GmSGT1基因在抑制大豆结瘤中的应用。
进一步地限定,所述大豆结瘤是减少根瘤数目和根瘤干重。
进一步地限定,GmSGT1基因序列的登录号为Glyma.01g222400。
本发明提供一种培育结瘤数少的大豆植株的方法,具体的步骤如下:构建过表达GmSGT1基因的重组载体,将重组载体转入发根农杆菌中,利用发根农杆菌侵染大豆,经鉴定后获得阳性的大豆毛状根材料,然后接种根瘤菌HH103,获得结瘤数少的大豆植株。
进一步地限定,重组载体的出发载体为pSoy1。
本发明提供一种培育结瘤数多的大豆植株的方法,具体的步骤如下:构建突变的GmSGT1基因的重组载体,将重组载体转入发根农杆菌中,利用发根农杆菌侵染大豆,经鉴定后获得阳性的大豆毛状根材料,然后接种根瘤菌HH103,获得结瘤数多的大豆植株。
进一步地限定,突变的GmSGT1基因的序列如SEQ ID NO.14所示。
进一步地限定,构建突变GmSGT1基因的重组载体的方法如下:构建GmSGT1基因的sgRNA序列如SEQ ID NO.12和SEQ ID NO.13所示,将sgRNA序列连接到pYLsgRNA-AtU3b载体上,获得靶标sgRNA表达盒。
本发明提供一种减少大豆结瘤的方法,所述的方法如下:将大豆的GmSGT1基因进行超表达,获得该基因超表达的大豆转基因毛状根,接种根瘤菌。
本发明提供一种增加大豆结瘤的方法,将大豆的GmSGT1基因进行突变,获得该基因超表达的大豆转基因毛状根,接种根瘤菌。
有益效果:对过表达GmSGT1的毛状根及GmSGT1敲除转基因大豆的结瘤鉴定表明,接种根瘤菌HH103后,过表达GmSGT1毛状根的根瘤数目和根瘤干重相较于野生型受体大豆都显著降低,而GmSGT1基因敲除突变体大豆的根瘤数目和根瘤干重相较于野生型受体大豆都显著增加,GmSGT1是激活NLR蛋白介导的免疫的核心调节因子,并影响宿主免疫,从而影响大豆与根瘤菌共生的建立。通过对GmSGT1基因过表达和敲除的植物材料的结瘤鉴定,表明GmSGT1对大豆结瘤起负调控作用。为后续研究大豆-根瘤菌共生体系形成提供了基础,为提高大豆与根瘤菌的共生效率提供了可能性。
附图说明
图1为大豆GmSGT1基因与部分植物基因系统进化分析;
图2为GmSGT1基因结构分析;
图3为GmSGT1基序分析;
图4为GmSGT1基因在大豆组织中的表达模式;qRT-PCR检测中以GmUKN1作为内参基因使用2^(-ΔCT)计算相对表达量。各组织表达量数据均为3次生物学重复的均值±标准差。
图5为pSoy1-GmSGT1载体构建;M:Trans 2K Plus DNA marker;1:阳性对照;2:以水为模板的阴性对照;3-7:单克隆菌液。
图6为超表达GmSGT1转基因根毛bar试纸条检测。
图7为GmSGT1基因转化DN50的遗传转化过程。
图8为Bar试纸条检测T0代转基因植株。
图9为D-1株系的T2代植株的PCR检测;1-34:T2代植株;M:2K plus DNA Marke。
图10为GmSGT1超表达和突变的材料的结瘤鉴定。
具体实施方式
东农50(Dongnong50,DN50)是黑龙江常见的品种,来自东北农业大学大豆生物学教育部重点实验室。拟南芥在23℃长日照条件恒温培养箱生长(16hr光照/8hr黑暗)。
快生型费氏中华根瘤菌HH103(源于西班牙塞维利亚大学Francisco Javier López-Baena实验室,记载在Weidner S,Becker A,Bonilla I,et al.Genome Sequence ofthe Soybean Symbiont Sinorhizobium fredii HH103[J].Journal of Bacteriology,2012,194(6):1617.)。
大肠杆菌(Escherichia coli)菌株DH5α;根癌农杆菌(AgrobacteriumTumefaciens)菌株EHA105;发根农杆菌(Agrobacterium rhizogenes)菌株K599;快生费氏中华根瘤菌(Sinorhizobium fredii)菌株HH103,均是常见的菌种。
2×TY液体培养基:胰蛋白胨(bacto-tryptone)16g、酵母提取物(bacto-yeastextract)10g、NaCl 5g、水1000mL,pH7.2,121℃灭菌30min.
植物表达载体pSoy1由本实验室保存提供;多靶点pYLsgRNA-AtU3b和pYLCRISPR/Cas9载体由华南农业大学刘耀光教授馈赠,载体构建过程参考Tan,J.,Zhao,Y.,Wang,B.,Hao,Y.,Wang,Y.,Li,Y.,Luo,W.,Zong,W.,Li,G.,Chen,S.,Ma,K.,Xie,X.,Chen,L.,Liu,Y.-G.and Zhu,Q.(2020)Efficient CRISPR/Cas9-based plant genomic fragmentdeletions by microhomology-mediated end joining.Plant Biotechnol.J.,https://doi.org/10.1111/pbi.13390。
下表中的载体来自于文章Lu M,Cheng Z,Zhang X M,et al.Spatial Divergenceof PHR-PHT1 Modules Maintains Phosphorus Homeostasis in Soybean Nodules[J].Plant Physiology,2020.文章的编号在论文中的参考文献中都有对应。
构建载体名称 | 载体骨架 | 构建方法 | 载体来源 |
pGWC-GmSGT1 | pGWC | TA克隆 | Chen et al.,2006 |
pSoy1-GmSGT1 | pSoy1 | LR反应 | Fu Lab Unpublished |
实施例1.构建GmSGT1基因过表达载体
大豆材料在25℃的长日照条件下(16hr光照/8hr黑暗)下恒温生长。
1.转录组测序及分析:根瘤菌的接种与取材:28℃,200rpm活化根瘤菌活化后的菌液接种于50mL的TY(胰蛋白胨5g·L-1、酵母粉3g·L-1、氯化钙0.4g·L-1)液体培养基中,28℃,200rpm培养至OD600=0.6后,5000rpm、8min弃上清并用10mM的MgSO4重旋菌液2次。加入无菌10mM的MgSO4,至菌液OD600为0.6,每棵苗接种2mL菌液;自接种开始按照0hpi.、24hpi和48hpi对大豆植株根部从上至下3cm左右进行取材,用水将根部洗净,吸干水分后锡纸包裹后放入液氮,完成取材并交予百迈客公司进行测序。
2.GmSGT1基因家族序列比对及生物信息学分析:大豆GmSGT1相关基因的获取
从phytozome(https://phytozome-next.jgi.doe.gov/)下载大豆GmSGT1蛋白序列,将该序列作为查询序列,与拟南芥、苜蓿等进行在线比对。在基于E值(E<1×10-5)筛选高度匹配的序列。使用InterProScan分析GmSGT1蛋白质序列的保守结构域。
GmSGT1系统进化分析:从phytozome(https://phytozome-next.jgi.doe.gov/)BLAST获得不同种属植物的同源蛋白序列,包括拟南芥、菜豆、鸡血藤、苜蓿、小豆以及向日葵等。使用MEGA软件进行系统进化分析。
GmSGT1启动子元件分析:选取Gm基因组序列上游2000bp作为该基因的启动子区域,利用Plant CARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)网站对该基因的启动子进行元件分析。
结果:(1)GmSGT1的系统发育:以大豆GmSGT1的蛋白质序列在phytozome上BLAST比对不同种属植物的同源蛋白序列,包括拟南芥、菜豆、鸡血藤、苜蓿、小豆以及向日葵等。使用MEGA软件进行系统进化分析(图1)。结果表明GmSGT1蛋白根据进化关系的远近可以聚类成4个亚家族,其中菜豆与鸡血藤的GmSGT1与大豆亲缘关系较近,同属于豆科,在根瘤菌与大豆共生建立时可能具有类似的功能。
(2)GmSGT1基因结构及编码蛋白质结构域分析:为了分析GmSGT1以及同源基因的基因结构,从Phytozome数据库下载GmSGT1的Gff3文件,使用TBtools分析GmSGT1以及同源基因的基因结构,得到基因结构模式构图(图2)结果表明通过基因结构可以表明GmSGT1基因家族进化上是保守的。
为分析GmSGT1基因编码蛋白质的保守基序,通过phytozome数据库下载蛋白序列,使用TBtools将结果进行可视化,所有蛋白均具有相同的基序结构(图3),表明这些蛋白可能行使相似的功能。
(3)GmSGT1基因在大豆中的表达模式分析
利用引物qGmSGT1-F和qGmSGT1-R(在表1中),为分析GmSGT1在不同组织的表达情况,本研究利用qRT-PCR检测大豆根、茎、叶、根瘤、花以及种子六个组织中GmSGT1的基因表达水平,如图(图4)所示,GmSGT1在大豆花组织表达水平最低,在根部、茎部表达量较高,这与phytozome公共数据库转录组数据相似。
3.基因克隆及载体构建
GmSGT1过表达载体构建:将GmSGT1的编码区(SEQ ID NO.1所示,Glyma.01g222400)
ATGGCTACGGCGCTGGAGAAGAAAGCGAAGGAGGCATTCTTCGACGACGAATTCGGTCTGGCCGTCGATCTCTACTCTGAAGCCATACGATTGGATCCGAACGACGCCAATCTCTTCGCGGACAGAGCCCAAGCCCATATCAAACTCAACGCTTTCACTGAAGCCGTTTCCGATGCCAACAAATCCATCCAATTAAACCCTTCCTTGCCCAAGGCCTATCTTCGTAAAGCCACCGCTTGTATCAAACTCCAAGAATACCACACCGCCAAGGTAGCGCTTCAAAACGGTGCCGCTTTTGCCCAAGACGATTCAAGATTCGCCAACTTGATTCAACAATGTGATCGCTGCATTGCAGAGGAATCAAGTGGCCTTACTAGCACCTTGTCTTCCCATTTGAGCAATAGAAATAATGGGATGACTAAAGAAGAAGCTGAAGGAGACAGTTTGCTTTCCCAAAAGAATGAAGCCACACTAAACAGACCAAAATACAGGCATGAATACTACCAGAAGCCAGAAGAAGTGGTTGTGACAATATTTGCAAAAGGAATATCAGCAAAAGATGTGGTTGTTGACTTTGGTGAACAGATTCTTAGTGTTACTATTGACGTTCCTGGCCAAGATGCCTATCATTATCAACCTCGATTGTTTGGAAAGATCATTCCCAACAACTGCAGAGTTGAGGTGTTGTCAACCAAAATTGAAATCCGTCTTGCAAAAGCTGAAGCTATTAATTGGACATCTCTGGAATATGGCAAGAATACACTTCCCCCAATAATAAATAGGCCGATAGTTCAATCTGAAAGGGCTTCATACCCATCACCAAAACCAAGGACAAAAGATTGGGATAAGTTGGAAGCTCAAGTGAAAAAAGAGGAGAAAGAAGAAAAGCTTGATGGTGATGCTGCTTTGAATAAATTGTTCCGTGATATTTACCAAAATGCAGATGAGGACATGAGGAGAGCAATGAGCAAGTCTTTCTTGGAGTCAAATGGAACAGTGCTATCAACTGATTGGAAAGAAGTGGGATCAAAGAAGGTGGAAGGCAGTCCCCCAGAAGGTATGGAGTTGAAAAAATGGGAGTATTGA。
用GmSGT1-F和GmSGT1-R(在表1中)分别扩增,将GmSGT1的CDS通过酶切连接至入门载体pGWC,然后通过LR反应将入门载体pGWC上的GmSGT1的编码区转移至真核表达载体pSoy1上获得pSoy1-GmSGT1。该部分载体用于大豆GmSGT1过表达的毛状根转化。pSoy1载体的改造过程记载在专利申请号为CN114989278A的专利文献中。
为了验证GmSGT1对共生结瘤的影响,本研究通过构建植物过表达载体用以验证过表达GmSGT1调控大豆与根瘤菌共生体系建立。使用GmSGT1-F和GmSGT1-R(在表1中)对重组载体进行菌液PCR验证,见图(图5)。测序成功后命名为pSoy1-GmSGT1。
2.毛状根转化:
挑取饱满的大豆种子使用氯气熏蒸法灭菌8hr后取出置于超净台内吹净氯气,用灭菌的超纯水浸泡16hr至胚根伸长。
将含有目标载体的K599农杆菌,挑取单克隆鉴定(转化及PCR鉴定方法同2.2.5.1),活化后用YEP培养基(5g·L-1酵母提取物;10g·L-1蛋白胨;5g·L-1NaCl;pH=7.0)振荡培养至菌液OD600值在1.0左右,RCF=1700g离心20min,并重悬于LCCM液体培养基(1/10×Gamborg B5盐;30g·L-1蔗糖;3.9g·L-1MES;pH=5.4;灭菌后加入40mg·L-1乙酰丁香酮);
将浸泡好的大豆胚根部分切下,保留靠近子叶节的下胚轴和子叶,将外植体浸于重悬菌液中浸泡20min完成侵染。侵染后将外植体放于滤纸上吸干浸染液,而后转移至共培养基(1/10×Gamborg B5盐;30g·L-1蔗糖;3.9g·L-1MES;4.25g·L-1琼脂;pH=5.4;灭菌后加入400mg·L-1Cysteine;154.2mg·L-1Dithiothrietol;40mg·L-1乙酰丁香酮),暗培养48hr。
将共培养后的大豆外植体的下胚轴垂直插入含有毛状根诱导培养基(1×GamborgB5盐;30g·L-1蔗糖;0.59g·L-1MES;7g·L-1琼脂;pH=5.7;灭菌后加入100mg·L-1
Timentin;100mg·L-1Cefotaxime)的组培瓶中,16hr光照、8hr黑暗下培养14d。
待毛状根生长至2cm时,从培养基上移出,使用荧光体式显微镜筛选阳性根,剪去非阳性根,获得转基因毛状根用于后续实验。
将表达载体pSoy1-GmSGT1导入发根农杆菌K599菌株中进行大豆毛状根转化,获得转基因的毛状根。
3.过表达阳性毛状根的Bar试纸条检测:提取GmSGT1过表达毛状根,将其溶解在200μL所提供的缓冲液中,然后用研磨棒捣碎,将试纸条插入其中,1min之后观察条带的数目。若Bar试纸条上面只显示一条带,说明此转基因植株是阴性的;若Bar试纸条均显示2条带,说明此转基因植株是阳性植株。见图(图6)。
实施例2.构建GmSGT1基因敲除载体
1.GmSGT1的CRISPR-Cas9载体构建:以GmSGT1为靶基因,通过CRISPR-P工具(http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR)设计靶基因的sgRNA,每个靶基因挑选2个评分较高,距离较近,均在外显子区域的靶序列为CRISPR-Cas9的编辑位点,然后合成两个靶序列片段
序列1:GGTAGAAGTGGGGTTCTTTC(SEQ ID NO.12);
序列2:GCAAAGCGTGTTTGGGTACG(SEQ ID NO.13);
分别用引物对GmSGT1-gRT1/GmSGT1-AtU3bT1和GmSGT1-gRT2/GmSGT1-AtU3bT2(在表1中)扩增构建gRNA表达盒(序列1和序列2),并将Bsa I酶切线性化的载体pYLCRISPR/Cas9-DB与构建好的gRNA表达盒进行连接转化,并筛选阳性克隆,获得CRISPR-Cas9的基因编辑载体。pYLsgRNA-AtU3b用于构建靶标表达盒,pYLCRISPR/Cas9-DB是载体,两者共同组成了pYLCRISPR/Cas系统。
将CRISPR-Cas9的基因编辑载体导入到农杆菌的感受态中,获得含有目的基因的农杆菌用于转化大豆使用。
2.大豆遗传转化:
(1)灭菌:挑取饱满的大豆种子使用氯气熏蒸法灭菌14hr后取出置于超净台内吹净氯气。
(2)萌发:用灭菌的超纯水浸泡18-24hr至胚根伸长。
(3)预培养:取出浸泡好的种子,在超净台上剥掉种皮,把两片子叶从胚轴上剥离下来,并去掉刚刚萌动的胚尖上的原叶,将胚尖的胚根朝下垂直接种至预培养培养基P1中,但是在培养基中胚尖的间隔要适度,不宜过密,于25~28℃,16hr光照培养1d。
(4)农杆菌菌液准备:从划线的含有目的基因的农杆菌的平板中挑取1~2个单菌落,接种到YEB液体培养基(含50mg/LKan)中,于恒温水浴摇床中,28℃、避光、120r/min条件下振荡培养至对数生长期(OD600=0.5)。本步骤应在侵染共培养之前完成。
(4)侵染共培养:将活化好的农杆菌菌液50ml,于4000r/min,4℃离心10min并弃上清液,用等体积的侵染培养基P2(液体)将离心沉淀的菌体重悬。将预培养后的胚尖外植体浸泡在重悬备用菌液中28℃,120r/min暗培养20h,侵染结束后用无菌滤纸擦干菌液。遗传转化过程见图(图7)。
3.基因编辑效率检测:
Bar试纸条检测:选取四分之一幼嫩的转基因植株叶片,将其溶解在200μL所提供的缓冲液中,然后用研磨棒捣碎,将试纸条插入其中,1min之后观察条带的数目。若Bar试纸条上面只显示一条带,说明此转基因植株是阴性的;若Bar试纸条均显示2条带,说明此转基因植株是阳性植株。见图(图8)。
选取GmSGT1基因编辑T2代植株叶片,液氮磨碎分装0.2g于1.5ml离心管中,进行大豆DNA提取。DNA提取方法如下:
(1)分装样品的1.5ml离心管加入小钢珠,震荡研磨仪60%额定功率研磨3min;
(2)加入CTAB提取液500μL,用涡旋振荡器摇匀,使组织粉末完全分散在CTAB提取中;
(3)将1.5mL离心管置于烘箱中,65℃处理一个小时,期间轻摇数次;
(4)上述反应液室温,RCF=12000g离心20min;
(5)离心完成后,取上清液,加入600μL氯仿,离心机离心,RCF=12000g,离心20min;
(6)取上清液,加入200μL的-20℃预冷异丙醇;
(7)离心机离心RCF=12000g,离心20min弃上清,分别加入无水乙醇和75%乙醇洗涤DNA沉淀两次;
(8)DNA晾干后,加入灭菌水,待DNA完全溶解后,-20℃保存。
分别提取突变体转基因株系D-1和野生型受体大豆东农50的基因组,使用Cas-F/R对其进行PCR扩增(图9)并测序,说明此转基因植株是阳性植株突变的GmSGT1基因的序列如SEQ ID NO.14所示:
atggctacggcgctggagaagaaaGAAGGGGACGCTTTTTTAGGAGACATTCGGTCTGGCCGTCGATCTCTACTCTGAAGCCATACGATTGGATCCGAACGACGCCAATCTCTTCGCGGACAGAGCCCAAGCCCATATCAAACTCAACGCTTTCACTGAAGCCGTTTCCGATGCCAACAAATCCATCCAATTAAACCCTTCCTTGCCCAAGGCCTATCTTCGTAAAGCCACCGCTTGTATCAAACTCCAAGAATATCACACCGCCAAGTAGCGCTTCAAAACGGTGCCGCTTTTGCCCAAGACGATTCAAGATTCGCCAACTTGATTCAACAATGTGATCGCTGCATTGCAGaggaatcaagtggccttactagcaccttgtcttcccatttgagcaatagaaataatgggatgactaaagaagaagctgaaggagacagtttgctttcccaaaagaatgaagccacactaaacagaccaaaatacaggcatgaatactaccagaagccagaagaagtggttgtgacaatatttgcaaaaggaatatcagcaaaagatgtggttgttgacggtgaacagattcttagtgttactattgacgttcctggccaagatgcctatcattatcaacctcgattgtttggaaagatcattcccaacaactgcagagttgaggtgttgtcaaccaaaattgaaatccgtcttgcaaaagctgaagctattaattggacatctctggaatatggcaagaatacacttcccccaataataaataggccgatagttcaatctgaaagggcttcatacccatcaccaaaaccaaggacaaaagattgggataagttggaagctcaagtgaaaaaagaggagaaagaagaaaagcttgatggtgatgctgctttgaataaattgttccgtgatatttaccaaaatgcagatgaggacatgaggagagcaatgagcaagtctttcttggagtcaaatggaacagtgctatcaactgattggaaagaagtgggatcaaagaaggtggaaggcagtcccccagaaggtatggagttgaaaaaatgggagtattga。
用到的引物序列如表1所示。
1引物信息
利用以下实验验证实验效果:
1.大豆结瘤试验:大豆于双层钵中培养至第一片三出复叶展开时(大豆毛状根移栽至双层钵后缓苗2-3天)分别接种快生根瘤菌(Sinorhizobium fredii)HH103以及HH103ΩNopM,放置在温室中正常培养。培养根瘤菌HH103的TY培养基配方为:3g·L-1酵母提取物,5g·L-1蛋白胨,0.4g·L-1氯化钙pH=7.0。植物低氮营养液配方:0.5μM硫酸锌、0.25μM硫酸镁、2μM硼酸、1mM氯化钙、1μM硫酸锰、0.25μM硫酸钾、0.2μM硫酸铜、0.1μM硫酸钴、100μM磷酸二氢钾、10μM柠檬酸铁、0.1μM钼酸钠以及1μM硝酸钾,pH调至5.8。
结果:将携带有pSoy1-GmSGT1的真核表达载体的发根农杆菌K599进行大豆毛状根转化,并使用pSoy1空载作为阴性对照。
筛选阳性根后分别接种根瘤菌HH103接种28d后统计根瘤表型,结果如表2所示,过表达GmSGT1的植株(GmMIP-OE)在接种HH103根瘤数以及根瘤干重显著减少,GmSGT1突变体转基因大豆(Gmmip)的结瘤数以及根瘤干重显著增加(图10)。过表达GmSGT1抑制结瘤发生。
表2
筛选阳性根后分别接种根瘤菌HH103以及NopM突变体(根瘤菌HH103ΩNopM),接种28d后统计根瘤表型,,过表达GmSGT1的植株在接种HH103和NopM突变体后根瘤菌(根瘤菌HH103ΩNopM)根瘤数以及根瘤干重显著减少,而GmSGT1突变体大豆的结瘤数以及根瘤干重显著增加。使用引物对qGmSGT1-F/R(表1)对接种两种不同根瘤菌的大豆根部进行qRT-PCR扩增,检测接种野生型根瘤菌及NopM突变体根瘤菌HH103ΩNopM后受体大豆DN50中GmSGT1表达情况,结果发现,分别接种两种根瘤菌后0-48h,GmSGT1的表达趋势完全相反,接种根瘤菌HH103后12h,GmSGT1的表达量最高;而接种NopM突变体(根瘤菌HH103ΩNopM)后24h,GmSGT1的表达量最高。
综合结果说明在根瘤菌侵染前期GmSGT1蛋白参与了大豆对根瘤菌的免疫反应从而抑制共生关系的建立,导致过表达GmSGT1的植株在接种HH103和NopM突变体后根瘤菌HH103ΩNopM根瘤数以及根瘤干重显著减少,而GmSGT1突变体大豆的结瘤数以及根瘤干重显著增加。综上,GmSGT1负调控共生结瘤。
Claims (8)
1.GmSGT1基因在抑制大豆结瘤中的应用,其特征在于,GmSGT1基因序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于,所述大豆结瘤是减少根瘤数目和根瘤干重。
3.一种培育结瘤数少的大豆植株的方法,其特征在于,具体的步骤如下:构建过表达GmSGT1基因的重组载体,将重组载体转入发根农杆菌中,利用发根农杆菌侵染大豆,经鉴定后获得阳性的大豆毛状根材料,然后接种根瘤菌HH103,获得结瘤数少的大豆植株;GmSGT1基因序列如SEQ ID NO.1所示。
4.根据权利要求3所述的方法,其特征在于,重组载体的出发载体为pSoy1。
5.一种培育结瘤数多的大豆植株的方法,其特征在于,具体的步骤如下:构建对GmSGT1基因进行突变的载体,将重组载体转入发根农杆菌中,利用发根农杆菌侵染大豆,经鉴定后获得阳性的大豆毛状根材料,然后接种根瘤菌HH103,获得结瘤数多的大豆植株;GmSGT1基因序列如SEQ ID NO.1所示。
6.根据权利要求5所述的方法,其特征在于,突变的GmSGT1基因的序列如SEQ ID NO.14所示。
7.一种减少大豆结瘤的方法,其特征在于,所述的方法如下:将大豆的GmSGT1基因进行超表达,获得该基因超表达的大豆转基因毛状根,接种根瘤菌;GmSGT1基因序列如SEQ IDNO.1所示。
8.一种增加大豆结瘤的方法,其特征在于,将大豆的GmSGT1基因进行突变,获得该基因突变的大豆转基因毛状根,接种根瘤菌;GmSGT1基因序列如SEQ ID NO.1所示。
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