CN117904175A - GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用 - Google Patents
GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用 Download PDFInfo
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Abstract
本发明提供了GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用，属于生物育种技术领域。本发明的GmLecRLK基因是从大豆胞囊线虫抗源材料中克隆的，能够编码一种G型凝集素类受体激酶。接种大豆胞囊线虫后，植物体内GmLecRLK表达水平显著上调，过表达该基因显著增强了大豆对胞囊线虫的抗性水平。
Description
技术领域
本发明涉及生物育种技术领域，尤其涉及GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用。
背景技术
大豆胞囊线虫病(Soybean cyst nematode，SCN)是危害大豆生产最严重的病害之一，一般造成大豆产量损失10％～30％，严重时甚至绝产。SCN具有分布范围广、传播途径多、胞囊存活时间长等特点，采用化学杀线虫剂、轮作等措施控制SCN发生效果有限。抗线虫大豆品种的培育是控制SCN发生最为经济有效的措施。SCN生理小种分化明显，不同小种致病性存在很大差异。国际上已鉴定的SCN小种有16种，其中以1、3和4号小种分布最为广泛。目前生产上利用的抗胞囊线虫大豆品种主要来源于PI 88788、Peking、PI 437654等少数抗源，其遗传基础极为狭窄，品种抗性单一。由于自然条件下SCN群体的多样性和高度变异性，极易造成SCN小种突破现有品种抗性，进化出毒性更强的SCN群体，并导致大豆大幅减产。
在大豆—SCN协同进化中，SCN通过食道腺分泌的效应子与寄主靶蛋白相互作用，抑制寄主大豆的免疫防御反应，并诱导取食位点的形成与维持，帮助线虫完成寄生生活。效应子蛋白也是SCN逃避或抑制寄主防御，造成寄主大豆抗性丧失的一个主要原因。为抵御SCN的侵染和寄生，大豆进化出不同的抗性机制以抑制SCN取食位点的形成及线虫生长发育和繁殖。目前在大豆基因组中已定位的SCN抗性位点达160多个，包括隐性抗病基因rhg1、rhg2、rhg3，显性抗病基因Rhg4、Rhg5等(http://www.soybase.org)。其中较为明确的是由rhg1和Rhg4位点介导的SCN抗性机制。rhg1位点为一段长度为31kb的多基因重复序列，编码3个抗病相关基因(AAT、α-SNAP、WI12)。与典型NBS-LRR类抗病基因不同，rhg1位点涉及一种多拷贝抗性机制。Rhg4编码丝氨酸羟甲基转移酶(SHMT)，调控细胞内丝氨酸和甘氨酸互变，是一种由一碳代谢参与的SCN抗性机制。大豆与SCN之间的互作关系十分复杂，不同大豆抗源对不同SCN生理小种的抗性存在明显差异。作为SCN鉴别寄主，大豆抗源Peking或PI437654(携带rhg1a和Rhg4位点)对1、2、3和5号SCN小种表现为高抗或免疫，而PI 88788(携带rhg1b位点)则对3号和14号小种表现高抗，但上述三个大豆主要抗源对SCN 4号强致病力小种并不具有抗性。而少数地方品种如灰皮支黑豆、赤不流黑豆、应县小黑豆等则对4号小种表现为高抗，暗示在这些大豆抗源中可能存在不同于rhg1和Rhg4介导的SCN抗性机制。揭示SCN抗性机制，开发更多SCN抗性品种，对大豆种植产业发展具有重要意义。
发明内容
本发明的目的在于提供GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用。本发明的GmLecRLK基因是从大豆胞囊线虫抗源材料中克隆的，能够编码一种G型凝集素类受体激酶。接种大豆胞囊线虫后，植物体内GmLecRLK表达水平显著上调，过表达该基因显著增强了大豆对胞囊线虫的抗性水平。
为了实现上述目的，本发明提供以下技术方案：
本发明提供了GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用。
优选的，所述植物包括大豆。
优选的，所述GmLecRLK基因的cDNA核苷酸序列如SEQ ID NO.5所示。
优选的，所述GmLecRLK基因编码蛋白的氨基酸序列如SEQ ID NO.6所示。
优选的，所述GmLecRLK基因cDNA的扩增引物对的序列如SEQ ID NO.3和SEQ IDNO.4所示。
优选的，所述增强植物对胞囊线虫病抗性的方法，包括如下步骤：
(1)将所述GmLecRLK基因的cDNA与启动子相连，得到重组表达载体；
(2)将所述重组表达载体转导入植物中，得到转基因植物。
优选的，所述启动子为组成型强启动子CaMV35S或根特异性启动子GmRoot7。
优选的，所述根特异性启动子GmRoot7的序列如SEQ ID NO.7所示。
优选的，所述转导的方法为农杆菌转化法。
本发明提供了GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用。发明基于全基因组关联和转录组联合分析，从大豆抗源-灰皮支黑豆中鉴定出1个可能参与抗SCN 4号小种的基因GmLecRLK。研究表明，接种SCN后GmLecRLK表达水平显著上调，同时参与细胞壁形成相关基因和具有抗虫活性的次生代谢产物-硫代葡萄糖苷及萜类物质合成路径的基因显著上调。转基因验证表明，过表达GmLecRLK显著增强了大豆对SCN 4号小种的抗性，表明GmLecRLK在SCN 4号小种的抗性应答中具有重要的作用。不同于rhg1或Rhg4介导的SCN抗性机制，GmLecRLK编码一种G型凝集素类受体激酶。该类受体激酶主要通过与胞外配体识别并激活其胞内激酶结构域，参与植物对内源生长发育或外源侵染信号的响应。因此，GmLecRLK可能涉及一种新的不同于rhg1和Rhg4介导的SCN抗性机制。本发明为克服现有大豆胞囊线虫抗性资源匮乏，开发SCN抗病靶标和培育抗SCN大豆品种提供新思路，具有重要的育种应用价值。
附图说明
图1为侵染胞囊线虫4号小种不同天数的大豆中GmLecRLK基因表达水平。
图2为不同大豆材料中GmLecRLK基因的PCR检测结果，其中，Ctl+代表阳性对照pTF101-GmRoot7-GmLecRLK，Nt代表野生型大豆，Bk代表空白对照ddH2O，1#、3#、4#、5#、6#、8#均为T3代转基因大豆植株。
图3为接种胞囊线虫4号小种35d后，不同大豆材料根部的胞囊数量，其中，Nt为野生型大豆，3#、4#和8#均为转基因大豆。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
本发明实施例中涉及的大豆胞囊线虫4号小种为我国黄淮海大豆主产区广泛分布的生理小种，可通过商业途径或自然界分离获得。
本发明实施例中涉及的大豆品种如灰布支、Williams 82为大豆种质资源，公众可以从国家大豆种质资源库或吉林省农业科学院获得。
本发明实施例中涉及的组成型强启动子CaMV35S、植物表达载体pTF101和农杆菌EHA101可通过常规市售获得。涉及的大豆根特异性启动子GmRoot7(Glyma20g36300.1)可以从大豆基因组中扩增获得。
实施例1
分别提取侵染了胞囊线虫4号小种0d、3d、9d、17d的灰布支黑豆根部组织样品总RNA，并反转录为cDNA。利用特异性引物GmLecRLK-F1和GmLecRLK-R1进行实时定量PCR检测。检测仪器为ABIPRISM 7500 Fast Real-Time PCR System(Applied Biosystems,CA,USA)，内标基因为GmACT6(GenBank No.NM_001289231)，采用2-ΔΔCT(Livak法)检测基因的相对表达量。
GmLecRLK-F1(SEQ ID NO.1)：5'-CTACGCAACGACGGACACCATC-3'
GmLecRLK-R1(SEQ ID NO.2)：5'-TGATTGGGTTGTCACGGTTGGC-3'
RT-qPCR程序如下：50℃，2min；95℃，10min；95℃，2min；62℃，30sec；72℃，30sec；45个循环；72℃，10min。
检测结果如图1所示，结果表明，相较于未接种对照(0d)，接种3d、9d和17d后，GmLecRLK基因表达水平分别上调1.87倍、3.29倍和2.38倍，表明GmLecRLK可能是参与大豆胞囊线虫抗性的重要候选基因。
实施例2
以侵染大豆胞囊线虫4号小种9d后的大豆根部组织为材料提取总RNA(EasyPurePlantRNA Kit)，并反转录为cDNA(One-Step gDNARemoval and cDNASynthesis SuperMix)，利用GmLecRLK特异性引物GmLecRLK-F2和GmLecRLK-R2，扩增获得GmLecRLK基因cDNA。
GmLecRLK-F2(SEQ ID NO.3)：5'-ggatccATGGCCATTCC
TCTTGTAATCCTAC-3'
GmLecRLK-R2(SEQ ID NO.4)：5'-gagctcTTATCTAGCATTTA
AAAGTGAGATAGATAC-3'
(小写字母为BamH和SacI酶切位点)。
PCR扩增程序为：95℃，3min；95℃，30sec；60℃，30sec；72℃，2min；29个循环；72℃，10min。
将PCR扩增产物在1％琼脂糖凝胶上电泳分离。将含有目的片段的琼脂糖凝胶切胶后收集至离心管，使用Omega Gel Extraction Kit试剂盒回收。经测序验证，GmLecRLK基因的cDNA核苷酸序列如下(SEQ ID NO.5)：
ATGGCCATTCCTCTTGTAATCCTACTTATTTGCAAGCTATTATCACTCTTCTCTCAAATCTGCTACGCAACGACGGACACCATCACCAAGGGTCAGCCACTCCCTGATGATGGCAACACCTTGCTTTCCAAGGACGGAACCTTTGAGTTGGGTTTCTTCAACCCCGGGAGTTCAAATAACCGCTATGTCGGAATTTGGTACAAAAACATTGTGGTCAAAACAGTGGTTTGGATCGCCAACCGTGACAACCCAATCAGAAACAACTCAAGCAAGTTGGTCATAAGCCAAGACGGAAACCTCGTTCTTCTTAGCCAGAACGAGTCTCTTATTTGGACAACAAATGCATCATCATCAGAAGTTTCGAGTTCGAGTCCAATTGTGCAGCTCTTGGATACTGGAAACTTGGTAATTAAAGATGGTAATGACAAAGAGAGTGTTTTTCTGTGGCAAAGCTTTGACTATCCTTGTGACACACTATTGCCAGGAATGAAGTTTGGGTGGGACTTGAGAACTGGGCTTAACCGGCGTCTTACTTCTTGGAAAAGCTGGGATGATCCATCTTCAGGGGACTTTACTTGGGGGGTGGAAATAGGAAGCAATCCTGACATTGTGATGTGGAAGGGTAACGTTGAGTACTTTAGGACGGGGCCTTATACTGGGAACATGTTCAGTGGAGTATATGGGCCCAGGAATAACCCACTTTATGATTACAAGTTCGTCAATAACAAAGACGAAGTGTATTACCAATACACCCTCAAGAATAGTTCGGTGATTACTATGATTGTTATGAACCAAACACTTTATCTTCGTCACCGCCTTACATGGATTCCCGAGGCCAAATCTTGGACTGTTTACCAATCATTGCCACGAGATAGTTGCGACGTTTACAACACTTGTGGTCCCAATGGAAATTGCATCATTGCTGGGTCTCCAATTTGCCAGTGCTTAGATGGGTTCGAGCCGAAATCGCCCCAACAATGGAACGTGATGGATTGGAGGCAAGGGTGTGTGCGCAGTGAAGAGTGGAGTTGTGGGGTAAAAAATAAAGATGGTTTTCGGAGATTTGCTAGCATGAAATTGCCAAACACTACGTTTTCTTGGGTTAATGAAAGTATGACACTTGAGGAGTGCAGGGCAAAATGTTTGGAAAATTGTTCGTGCAAGGCCTATTCAAACTTGGACACGAGGGGAGGAGGTAATGGGTGTTCCATTTGGGTTGGTGACCTAGTTGATTTAAGAGTTATAGAAAGTGGGCAAGATTTGTATGTTCGAATGGCCACTTCGGACATGGATGGTAAACACGAGCATCGGAGAAAGGTAGTCTTGGTGGTTTCAACTATAGCATCGTTGGTGCTTGTGATGCTAGTAGCATTCTGCATTTACATGATCAAAAAAATATATAAAGGGAAAACAAAAACAAGAATGTCAAGAGAGGATAAAGACGAAGGCAGACAAGAAGATTTGGAGCTTCCTTTCTTTGATCTTGCTACAATAGTTAATGCCACTAATAATTTTTCAATCGAAAATAAGCTAGGTGAAGGTGGTTTTGGTCCTGTATACAAGGGTACATTAGTAAATGGACAAGAAATTGCAATCAAAAGGCTTTCACGAAGTTCAGGACAAGGATTGAAAGAATTTAGAAATGAAGTTATATTGTGTGCCAAATTACAACACCGAAATCTTGTCAAGGTTTTGGGCTATTGCATTCAAGGAGAGGAGAAAATGTTACTTTATGAATACATGCCCAACAAAAGTCTCGATTTATTTCTTTTTGATTCGGAGCAAAGTAAATTCTTAAATTGGCCAGTGCGCTTTAACATTTTGAATGCGATTGCTCGGGGACTTCTTTATCTCCATCAGGATTCTAGATTAAGGATCATACACAGAGATCTAAAGGCAAGTAATATTTTATTAGACAATAATATGAATCCAAAAATTTCAGATTTTGGCCTTGCAAGAATGTGTGGAAGTGATCAAGTTGAAGGGAGTACAAGCATCATAGTTGGGACACACGGTTACATGGCACCTGAATATGCCATTGATGGATTATTCTCTACAAAATCAGATGTGTTCAGCTTTGGAGTTTTATTGCTAGAAATTATTAGTGGAAAGAAAAACAGAGCATTTACCTACCAAGACAACGATCATAATCTTATTGATCATGCATGGAGACTGTGGAAAGAGGGTACTCCAGAGCGATTGACTGATGCACATTTGGCAAACTCATGTAATATATCTGAAGTCATACGGTGCATTCAAATTAGTCTTCTGTGTCTACAACATCATCCAGATGATAGGCCAAACATGACATCAGTTGTTGTGATGTTGACCAGTGAAAATGCTTTACACGAACCTAAGGAACCTGGTTTTTTAATTAGAAGAGTTTCAAACGAAGGAGAACAATCTTCTAATAGGCAGACATCTTCATTCAATGAAGTATCTATCTCACTTTTAAATGCTAGATAA。
GmLecRLK基因编码蛋白的氨基酸序列如下(SEQ ID NO.6)：
MAIPLVILLICKLLSLFSQICYATTDTITKGQPLPDDGNTLLSKDGTFELGFFNPGSSNNRYVGIWYKNIVVKTVVWIANRDNPIRNNSSKLVISQDGNLVLLSQNESLIWTTNASSSEVSSSSPIVQLLDTGNLVIKDGNDKESVFLWQSFDYPCDTLLPGMKFGWDLRTGLNRRLTSWKSWDDPSSGDFTWGVEIGSNPDIVMWKGNVEYFRTGPYTGNMFSGVYGPRNNPLYDYKFVNNKDEVYYQYTLKNSSVITMIVMNQTLYLRHRLTWIPEAKSWTVYQSLPRDSCDVYNTCGPNGNCIIAGSPICQCLDGFEPKSPQQWNVMDWRQGCVRSEEWSCGVKNKDGFRRFASMKLPNTTFSWVNESMTLEECRAKCLENCSCKAYSNLDTRGGGNGCSIWVGDLVDLRVIESGQDLYVRMATSDMDGKHEHRRKVVLVVSTIASLVLVMLVAFCIYMIKKIYKGKTKTRMSREDKDEGRQEDLELPFFDLATIVNATNNFSIENKLGEGGFGPVYKGTLVNGQEIAIKRLSRSSGQGLKEFRNEVILCAKLQHRNLVKVLGYCIQGEEKMLLYEYMPNKSLDLFLFDSEQSKFLNWPVRFNILNAIARGLLYLHQDSRLRIIHRDLKASNILLDNNMNPKISDFGLARMCGSDQVEGSTSIIVGTHGYMAPEYAIDGLFSTKSDVFSFGVLLLEIISGKKNRAFTYQDNDHNLIDHAWRLWKEGTPERLTDAHLANSCNISEVIRCIQISLLCLQHHPDDRPNMTSVVVMLTSENALHEPKEPGFLIRRVSNEGEQSSNRQTSSFNEVSISLLNAR。
实施例3
将实施例2扩增得到的GmLecRLK基因的cDNA，采用BamH/SacI双酶切，与大豆根特异性启动子GmRoot7连接，并连接至植物表达载体pTF101中，得到重组表达载体pTF101-GmRoot7-GmLecRLK。
其中，GmRoot7(SEQ ID NO.7)：ATATATTATACGCAATTTTTCAT
TAGTCCATCTAATACTTATGCGTATCAAACATCATACAGTAAAAGAAAAATATTTGCGTTGTATTTCAATTTCAAAATAATAGACTGATCAAGGTGTGCTATTACTCAACTATACCATACAGTATTACAGTCCTGGAGTGCATATTCTATTTCTCAATAAACAACAAACGTAGTTGTTGGTTTTGAGATTTTGAATATTATATGAATTTTCCTATTCGCCTAAGACTATGGAGGGGAAAATAAATGTATGCACTTTCTGTTTTCCCGAATACCTACTGCTTAAGATATTAAGAGTGGGTATAATATTTGTCCAGAAGGGTAAAGATAAAATAAAACTCGTTATGCTTATGATTTCGCAATTGTAAAATAGAACAGGGGTGGAGAAGGATCACAATTTTTCCAGTGGATATTCCAAGTTTCTAAAACTAACTCAGATTCATAAATTTTGAAGTCATTGTCATTAAAGTCTTTAAATACCTGCTTTGCAACTCTTCTTTTCGGTACAAGTCTTTTCTTACCCTTATCCTATTGCATGGGTAGAAAAAAAAAAGATAAGGAAACAAATAAAAGAAAAAAGACAAAATGATAGATATGATAACTGATTTGATGAAAAATGAAAAGAAGAAATAATAAGAAAAATAGAATAGAAATGAAATGGAAGTGAAAGAAAATGTATGATAATGTATATGTGTGGTAATTTCTACAAAATCTGATATCTTTCTTGGGTAGGAACCATTCATTTACGTCACTTGTGTGAGTGAGGCATCTTCAAACACTGTAGTTCGTCTGAAATCCTTGGGTAGTGTAAGAAAATAAATCAAGGATGCAACTAAGTTATTGCCTGGATAGAGATCTCAGATCAGAGACTTTGACGACAAGATAAATATGCAATAGTGGACACTCGTTTCTTTCAAAATATAATTACGGGTTGATACTTGGAAGAAAATTTAATCAATTTTTTGCCTTAAATAATTTCCATTATCCTCCACTTTCTCTATACGATATCTATACCGTAACTTGAAGAACCAATTAAAGACTTAGGCTAAAATAAAATAAAACCATTGAGGTGTAGCACTAGTTCTGGTCTTCTAAATGCTCAGAAGATTTTAATTAGCCGCCAGGTCTCAAAACAAACATGCTCTAAATCATACACCTGCTCATATTATAAAAGTAATATTTCAAGCACAGGTAGTGGATACGTACAATGTCGAGTGTACATGGTAATACTGAAAAAATCATCATGACGATGATGTGGGGCATCTTAATCTAATATTTTGACAAGCGTGCAGACATTAAGAATGTGCTTCCACCATCAAATATTCTAGAAATGCTAAAATATCTATAGCAATGGCTCCGTGCCCTATAAATAGCTAGTTTCCCATGCTGCAAAATACAAGTCTCACACACTTAGAATTAAGTTAGTAGCGAGAGGGAGAACCATGGAGGTTTTTT。
测序验证无误后，将重组表达载体pTF101-GmRoot7-GmLecRLK导入农杆菌EHA101。采用农杆菌介导法进行大豆转化，转化品种为Williams82。具体转化流程参照Yang等(YangX,et al.Plant Cell Rep,2018,37:103-114)，获得的转基因植株在温室中生长结实，用于后续检测鉴定。
利用特异性检测引物GmLecRLK-F3和GmLecRLK-R3，对T1、T2和T3代转基因大豆以及野生型大豆Williams82中的GmLecRLK基因进行PCR检测。同时，以重组表达载体pTF101-GmRoot7-GmLecRLK为阳性对照，ddH2O为空白对照进行PCR检测。检测结果如图2所示，其中，Ctl+代表阳性对照pTF101-GmRoot7-GmLecRLK，Nt代表野生型大豆，Bk代表空白对照ddH2O，1#、3#、4#、5#、6#、8#均为T3代转基因大豆植株。然后结合除草剂Basta(500mg/L)筛选，获得纯合转基因大豆后进行抗性鉴定。最终得到3个纯合转基因大豆，编号为3#、4#和8#。
GmLecRLK-F3(SEQ ID NO.8)：5'-CTAAATCATACACCTGCTCATATTA-3'
GmLecRLK-R3(SEQ ID NO.9)：5'-TCCTGGGCCCATATACTCCACT-3'
试验病土采自山西省连作3年的大豆地块，采用Golden法确定病土中胞囊线虫小种类型为4号生理小种。将病土与细沙等比例混匀后，置于15cm×30cm塑料盆。将转基因大豆材料和野生型大豆Williams 82分别种植于上述塑料盆中，于温室中生长(25℃，16h光照/8h黑暗)。在接种35d以后，大豆根部出现肉眼可见的白色雌虫，取出大豆根部组织，调查每株胞囊数量(个/株)。
调查结果如图3所示，结果表明，野生型大豆Williams 82(Nt)胞囊数量为64.46个/株，而3个转基因大豆(3#、4#和8#)胞囊数量仅为3.5～5.7个/株，较对照减少91.5～94.6％，表明GmLecRLK过表达显著增强了大豆对SCN 4号小种的抗性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.GmLecRLK基因在增强植物对胞囊线虫病抗性中的应用。
2.根据权利要求1所述的应用，其特征在于，所述植物包括大豆。
3.根据权利要求2所述的应用，其特征在于，所述GmLecRLK基因的cDNA核苷酸序列如SEQ ID NO.5所示。
4.根据权利要求3所述的应用，其特征在于，所述GmLecRLK基因编码蛋白的氨基酸序列如SEQ ID NO.6所示。
5.根据权利要求4所述的应用，其特征在于，所述GmLecRLK基因cDNA的扩增引物对的序列如SEQ ID NO.3和SEQ ID NO.4所示。
6.根据权利要求5所述的应用，其特征在于，所述增强植物对胞囊线虫病抗性的方法，包括如下步骤：
(1)将所述GmLecRLK基因的cDNA与启动子相连，重组于表达载体上，得到重组表达载体；
(2)将所述重组表达载体转导入植物中，得到转基因植物。
7.根据权利要求6所述的应用，其特征在于，所述启动子为组成型强启动子CaMV35S或根特异性启动子GmRoot7。
8.根据权利要求7所述的应用，其特征在于，所述根特异性启动子GmRoot7的序列如SEQID NO.7所示。
9.根据权利要求8所述的应用，其特征在于，所述表达载体包括pTF101载体。
10.根据权利要求9所述的应用，其特征在于，所述转导的方法为农杆菌转化法。
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