2024, Vol. 40中国科学院微生物研究所、中国微生物学会主办
文章信息
- 钟晨丽, 王文絮, 廖莉娜, 刘建中
- ZHONG Chenli, WANG Wenxu, LIAO Lina, LIU Jianzhong
- 沉默大豆GmWRKY33B基因导致大豆抗病性降低
- Silencing GmWRKY33B genes leads to reduced disease resistance in soybean
- 生物工程学报, 2024, 40(1): 163-176
- Chinese Journal of Biotechnology, 2024, 40(1): 163-176
- 10.13345/j.cjb.230226
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文章历史
- Received: March 26, 2023
- Accepted: May 26, 2023
- Published: June 2, 2023
引用本文
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2. 浙江师范大学生命科学学院 浙江省特色经济植物生物技术重点实验室, 浙江 金华 321004
2. Zhejiang Provincial Key Laboratory of Biotechnology on Specialty Economic Plants, College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号转导途径在植物免疫反应的调控中起着重要作用[1-3]。植物细胞膜表面受体激酶或类受体蛋白在识别病原菌相关分子模式(pathogen-associated molecular patterns, PAMPs)或刺激信号后，激活MAPK级联反应[1-2]。MAPK激酶信号转导途径可将无数外部刺激转化成细胞内的应答反应。WRKY是植物特有的转录因子家族，可以特异性结合靶基因启动子区域的W-box顺式元件(TTGACC/T)[4-5]以调控基因表达，在植物防御反应中起着重要作用[6-9]。在拟南芥中，某些WRKY成员是MPK3/MPK6激酶的靶蛋白，WRKY蛋白的磷酸化可以改变其结合W-box的能力从而影响其调控下游基因表达[10]。在真菌侵染条件下，MPK3/MPK6对WRKY33的磷酸化是其激活植保素(camalexin)生物合成途径中编码关键酶基因所必需的[11-12]。与此类似，MPK3/MPK6还可通过磷酸化ERF6、MYB41和MYB44等转录因子分别调控抗真菌、抗盐以及种子萌发等[2, 13-14]。另外，拟南芥MPK4可以通过磷酸化MKS1 (MAP kinase 4 substrate 1)[15]而解除其对WRKY33的抑制效应[16]，从而使WRKY33激活PAD3等基因的表达，继而增强植保素的生物合成[16-17]。WRKY在大豆防御中也起着重要作用[18-20]。但这些WRKY是否受MAPK激酶途径调控以及是否参与大豆植保素的生物合成仍有待研究。
葡糖异硫氰酸盐(glucosinolates, GSs)包括吲哚葡糖异硫氰酸盐(indole glucosinolates, IGSs)，是十字花科植物中重要的次生代谢物，在免疫中起着重要作用[19, 21]。MYB转录因子参与调控IGS生物合成途径中基因的表达[21-22]。MPK3/MPK6可调控MYB51与MYB122编码基因的表达；MPK3/MPK6可通过其底物ERF6调控CYP81F2及IGMT1/IGMT2编码基因的表达，CYP81F2及IGMT1/IGMT2是将吲哚-3-基甲基硫代葡萄糖苷(indole-3-yl-methylglucosinolate, I3G)转化成具抑菌效应的4-甲氧基吲哚-3-基甲基硫代葡萄糖苷(4-methoxyindole-3-yl-methylglucosinolate, 4MI3G)的关键酶[23]。此项研究表明MPK3/MPK6可通过调控下游2个转录因子协同完成植保素IGS的生物合成。
与拟南芥不同，大豆中的主要植保素是大豆抗毒素(glyceollin)。大豆抗毒素是一组拥有紫檀碱(pterocarpan)骨架及不同C5基团经异戊烯基化修饰的次生代谢物[24]，经不同异戊烯基转移酶(prenyltransferase, PT)催化而成[24]。源于同一前体物的大豆抗毒素衍生物可分为Ⅰ−Ⅴ类，但Ⅰ−Ⅲ类是主要的植保素[25]。不同病原菌侵染可诱导大豆合成大豆抗毒素[26]。通过转基因策略提高病原菌诱导的大豆抗毒素合成可增强大豆的抗病性[27-31]。但大豆抗毒素生物合成的调控机理仍不清楚。MAPK途径是否参与其合成的调控、哪个(些)转录因子参与其生物合成途径中关键基因的表达调控也未见报道。
通过菜豆斑驳病毒(bean pod mottle virus, BPMV)介导的病毒诱导基因沉默(virus-induced gene silencing, VIGS)技术同时沉默两个GmWRKY33B，导致大豆对大豆花叶病毒及大豆斑点病菌[丁香假单胞菌pv.甘氨酸(Pseudomonas syringae pv. glycinea, Psg)]抗性降低、flg22诱导的GmMPK6激活程度降低以及Psg诱导的负责大豆抗毒素生物合成的异戊烯基转移酶PT基因表达水平的显著降低，说明GmWRKY33B为大豆免疫反应的正调控因子。
1 材料与方法 1.1 材料 1.1.1 大豆本研究中使用的大豆(Glycine max)为Williams 82。大豆植株在温室或生长室中保持在22 ℃下，光周期为16 h。
1.1.2 菌种大肠杆菌(Escherichia coli) DH5α/TOP10、Pseudomonas syringae pv. glycinea (Psg) R4菌株以及SMV-N-GUS[32]。
1.1.3 flg22源自Psg的flg22小肽由杭州华安生物技术有限公司合成[33-34]。
1.2 方法 1.2.1 BPMV介导的VIGS技术-载体构建、基因枪法侵染及沉默效果验证对于BPMV-VIGS载体系统以及如何使用PDS-1000/He (Bio-Rad实验室)基因枪法将BPMV接种到大豆幼苗上在此前已有描述[35-36]。通过在Phytozome基因组数据库(https://phytozome-next.jgi.doe.gov/Phytozome)中进行关键词搜索，确定了2个大豆GmWRKY33B同源基因：Glyma.09G280200以及Glyma.18G208800。首先用Glyma.09G280200-F与Glyma.09G280200-R这对引物(表 1)从大豆cDNA中扩增出315 bp的片段，接着用BamH I与Kpn I对该扩增片段进行双酶切，然后连接至经同样双酶切的BPMV2载体中，构建成可同时沉默Glyma.09G280200和Glyma.18G208800的BPMV沉默载体BPMV2-GmWRKY33B。
| Primer name | Primer sequence (5ʹ→3ʹ) | Size (bp) |
| Construction of primers for BPMV-VIGS vector | ||
| Glyma.09G280200-F | AAAGGATCCATAAAATATGGACAGAAACAAGTGA | 34 |
| Glyma.09G280200-R | AAAGGTACCCTTCCTCTCCAACAGAAGCTG | 30 |
| Primers for silencing examination | ||
| Glyma.09G280200-F | ACCTTTCAACCAATAATAAACAA | 23 |
| Glyma.09G280200-R | ATTAGAAAGAGCATAGTAGAAATAG | 25 |
| Glyma.18G208800-F | ACCTTCTTTCCATCAATAATAAAG | 24 |
| Glyma.18G208800-R | TAAAAGTAGAAATTGCCAAAAC | 22 |
| Primers for RT-PCR | ||
| Glyma.10G295300.1-F | TGTGCTGCCTCAATTGTTTA (G4DT) | 20 |
| Glyma.10G295300.1-R | TGCAAGAGCCCAAGTAGAA | 19 |
| Glyma.01G134600.1-R | AATGTAATTGGCAATACAAATG | 22 |
| Glyma.13G097800-F | TAACAACGCTTCTTCTCTCG (HPT) | 20 |
| Glyma.13G97800-R | AAGGAATTCTTGACAGAGTCC | 21 |
| Glyma.10G070200-F | CATAATCATTGCTGTGAGTACTG (C4DT) | 23 |
| Glyma.10G070200-R | CAAGAACCTATAACCAAGCTAAG | 23 |
| GmELF1b-F | GAGCTATGAATTGCCTGATGG | 21 |
| GmELF1b-R | CGTTTCATGAATTCCAGTAGC | 21 |
| Primers for RT-qPCR | ||
| Glyma.10G295300-F | TCTACTCCTTGGGTTTGG | 18 |
| Glyma.10G295300-R | CCCGTTTCTGACCTAGAC | 18 |
| Glyma.10G070200-F | AAGCAGCCCATGCTATAC | 18 |
| Glyma.10G070200-R | CCAGTACAAAGCAACCAAG | 19 |
| Glyma.01G134600-F | ACCTCATACTTCCAATCCTC | 20 |
| Glyma.01G134600-R | GCAAGGAGAGACGAAGAA | 18 |
| Glyma.13G097800-F | GCTTCATGGCACAATAGG | 18 |
| Glyma.13G097800-R | CATGTACACCCTCCTTCA | 18 |
| GmELF1b-F | GTTGAAAAGCCAGGGGACA | 19 |
| GmELF1b-R | TCTTACCCCTTGAGCGTGG | 19 |
| The bold sequences are BamH Ⅰ and Kpn Ⅰ restriction sites, for cloning PCR fragments into BPMV-VIGS. | ||
载体构建成功后，将BPMV2空载体和BPMV2-GmWRKY33B质粒分别与BPMV1质粒混合包被在1.6 μm金粉颗粒上，利用PDS-1000/He基因枪将上述金粉颗粒分别轰至生长7 d左右大豆幼苗两片展开的真叶上；3周左右，侵染成功的大豆植株的上部系统叶片便会出现病毒症状。在有症状的系统叶片打孔取样后提取RNA进行沉默效果的验证。验证成功后，将所有具病毒症状的系统叶片收集后在磷酸缓冲液中研磨，离心后含大量病毒的汁液‒80 ℃储存，用于后续大规模摩擦接种。
1.2.2 RT-PCRcDNA的合成：总RNA的提取按生产商提供的方法进行(Invitrogen)；cDNA的合成按厂商提供的方法(ReverTra Ace qPCR RT Kit, TOYOBO)进行；逆转录反应为：将RNA在65 ℃热变性5 min，置于冰上。然后在离心管里顺次加入：2 μL 5×RT Buffer；0.5 μL Primer Mix；0.5 μL RT Enzyme Mix；1.0 μg RNA；最后用不含核酸酶的水将终体积调至10.0 μL；逆转录反应在37 ℃下进行15 min；98 ℃变性5 min；将cDNA ‒20 ℃储存备用。
PCR反应：PCR反应按Tian等[34]所述方法进行。PCR扩增程序为：95 ℃预变性2 min；95 ℃ 15 s，56 ℃ 30 s，72 ℃ 20 s，35个循环；72 ℃延伸5 min。用SYBR green qPCR RT试剂盒(TOYOBO)进行实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)，qPCR反应按生产商提供的操作手册进行。qPCR扩增所用仪器为Applied Biosystems Quantstudio 1 (Thermofisher)。qPCR扩增程序为：95 ℃预变性2 min；95 ℃ 15 s，52 ℃ 20 s，72 ℃ 30 s，共40个循环；72 ℃延伸5 min。
1.2.3 沉默效果验证分别用Glyma.09G280200与Glyma.18G208800的沉默验证引物(表 1)对从沉默植株中提取的RNA所合成的cDNA进行RT-PCR分析。所用正向与反向沉默引物分别位于构建沉默载体所用插入片段上、下游200 bp左右的位置。
1.2.4 接种大豆斑点病病菌及菌落单位计数分析Psg的培养、接菌及菌落计数分析按文献中所描述方法进行[34, 37]。
1.2.5 SMV-N-GUS接种、GUS染色和GUS病灶测量SMV-N-GUS的侵染、GUS (β-glucuronidase)染色和GUS病灶测量已有描述[32]。通过基因枪法将与GUS报告基因融合的SMV-N菌株(SMV-N-GUS)分别接种到空载体对照植株(BPMV-0)与BPMV-GmWRKY33B沉默植株离体叶片中[33, 38-40]；接着将被侵染的离体叶片置于铺有湿润滤纸的培养皿中于室温下培养3 d后进行GUS染色。使用显微镜(Olympus)对GUS染色情况进行观察并拍照；并用Image J软件测量GUS病斑直径。
1.2.6 免疫印迹法分析检测磷酸化GmMPK3/6从大豆叶片组织中提取蛋白质按以前发表的方法进行[33-34, 38, 40]。蛋白提取液组分为：50 mmol/L Tris-MES (pH 8.0)，0.5 mol/L sucrose，1 mmol/L MgCl2，10 mmol/L EDTA，5 mmol/L DTT，另加蛋白酶抑制剂混合液(protease inhibitor cocktail, Sigma-Aldrich)。将从flg22处理后的叶片中提取的蛋白通过SDS-PAGE胶进行分离，然后用半干电转移装置(Bio-Rad)将胶中的蛋白转移至PVDF膜上(Millipore)；将转移后的PVDF膜在TBST缓冲液中封闭，然后用1:3 000稀释的p44/p42抗体(cell signaling technology)进行孵育；一抗孵育后的膜用TBST清洗3次，然后用1:7 500稀释的二抗进行孵育。最后，用化学发光的辣根过氧化物酶(horseradish peroxidase, HRP)底物，即过氧化物溶液和增强型鲁米诺溶液的混合液(Millipore)通过曝光检测条带。
2 结果与分析 2.1 大豆GmWRKY33同源基因基之间同源性关系分析利用拟南芥WRKY33基因编码区序列对大豆基因组进行BLAST (phytozome 13)，结果表明大豆基因组中有6个WRKY33同源基因：Glyma.02G232600、Glyma.11G163300、Glyma.14G200200、Glyma.18G056600、Glyma.09G280200以及Glyma.18G208800。对这6个GmWRKY33同源基因编码序列的进化分析表明，这6个基因可分为3组(图 1)，组内2个基因之间同源性高达91%以上。由于前4个基因之间的同源性高达84%，因此将前4个基因统称为GmWRKY33A，而将后2个同源性高达95%但却与GmWRKY33A不同成员间同源性仅为58%‒68%的基因统称为GmWRKY33B[41] (表 2)。
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| 图 1 大豆6个GmWRKY33基因编码区序列的进化聚类分析 Fig. 1 Phylogenetic analysis of the sequences of the coding regions of six GmWRKY33 genes discovered in soybean. The Arabidopsis WRKY33 gene (At2G38470) was used as a reference gene. The software used for phylogenetic analysis was MEGA 5.10. |
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| Gene ID | Glyma. 02G232600 | Glyma. 14G200200 | Glyma. 11G163300 | Glyma. 18G056600 | Glyma. 09G280200 | Glyma. 18G208800 |
| Glyma.02G232600 silencing fragment (%) | 100 | 97 | 85 | 85 | 69 | 70 |
| Glyma.09G280200 silencing fragment (%) | 67 | 68 | 58 | 59 | 100 | 95 |
| The first four genes are GmWRKY33A, and the last two genes are GmWRKY33B. | ||||||
为了探讨GmWRKY33B的功能，构建了携带Glyma.09G280200与Glyma.18G208800保守区315 bp片段的BPMV-VIGS载体，以期同时沉默2个GmWRKY33B基因。用上述构建好的GmWRKY33B沉默载体侵染大豆幼苗。侵染后15 d左右便会观察到病毒症状。接种BPMV-0空载体的对照植株与BPMV-GmWRKY33B沉默植株在表型上无显著差异(图 2A)，说明同时沉默这2个基因对大豆的生长发育并无影响。RT-PCR验证分析表明，GmWRKY33B沉默植株中Glyma.09G280200与Glyma.18G208800这2个基因的转录水平较BPMV-0空载体对照植株均显著降低(图 2B)，说明这2个基因被同时沉默，进一步证实了BPMV-VIGS在克服基因功能冗余的优越性[42-43]。
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| 图 2 同时沉默2个GmWRKY33B基因未导致大豆表型变化 Fig. 2 Silencing 2 GmWRKY33B genes simultaneously does not result in morphological changes in soybean. A: Comparisons of the morphological phenotypes between the GmWRKY33B-silenced plants and the empty vector plants. B: The transcript levels of the two GmWRKY33B genes were significantly reduced in GmWRKY33B-silenced plants relative to empty vector control plants. |
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为了检测同时沉默2个GmWRKY33B基因对Psg抗性的效应，用Psg R4菌株对空载体对照植株及GmWRKY33B沉默植株系进行菌落计数分析。结果表明，沉默植株叶片上的病症均较BPMV-0叶片上严重(图 3A)；与发病症状结果相一致，接菌不同时间后GmWRKY33B沉默植株叶片中Psg繁殖数显著高于BPMV-0叶片(图 3B)，说明同时沉默GmWRKY33B增加了大豆对Psg侵染的敏感性。
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| 图 3 BPMV-0和GmWRKY33B沉默株系接种Psg后的表型及菌落形成单位比较 Fig. 3 Comparison of the symptom severity and the growth rates between the BPMV-0 plants and the GmWRKY33B-silenced plants infected by Psg R4 strain. A: The bacterial symptoms on the leaves of the GmWRKY33B-silenced and BPMV-0 plants 8 days post Psg inoculation. B: The bacterial growth assays on the BPMV-0 and the GmWRKY33B-silenced plants after Psg inoculation for the indicated time. CFU represents colony forming unit. The unit for Y axis means the log10 number of colonies replicated in 1 cm2 leaf area. Standard deviation, ***: P < 0.001 (Student's t-test). |
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钟晨丽[41]证明同时沉默4个GmWRKY33A基因可降低大豆中对SMV的抗性。为了检测同时沉默2个GmWRKY33B基因对SMV抗性的效应，将SMV-N-GUS (与报告基因GUS融合的SMV N毒株)分别接种至BPMV-0与GmWRKY33B沉默植株的离体叶片上，在室温下培养3 d后进行GUS染色。染色后蓝色斑点的出现意味着侵染成功，而GUS斑点的大小代表病毒复制和/或运动的程度。结果表明GmWRKY33B沉默植株系叶片中的GUS斑点的直径的大小及染色强度均显著高于空载体植株(图 4)，说明同时沉默2个GmWRKY33B同源基因可显著降低大豆对SMV-N-GUS的抗性。
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| 图 4 同时沉默2个GmWRKY33B基因导致大豆对SMV-N-GUS抗性的降低 Fig. 4 Silencing 2 GmWRKY33B genes simultaneously leads to the reduced resistance of soybean to SMV-N-GUS. A: Comparisons of the GUS foci on the leaves of the BPMV-0 and the BPMV-GmWRKY33B plants under a microscopy. Bar=5 mm. B: Comparisons of the diameters of the SMV-N-GUS foci on the leaves of BPMV-0 and BPMV-GmWRKY33B plants. The error bars indicate that the standard deviation calculated by measuring at least 30 lesions (≥120 lesions) in 4 independent leaves; ***: P < 0.001 (Student's t-test). |
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Flg22为细菌鞭毛蛋白N端的含22个氨基酸的小肽，其作为PAMP可被定位于质膜上的受体激酶或受体蛋白识别而激活免疫反应[44]。激活的受体可通过磷酸化激活MAPK信号转导途径，而激活的MPK3/6可通过磷酸化WRKY等转录因子以激活防御相关基因[2, 12, 45]。前期研究表明源自Psg与Pst (Pseudomonas syringae pv. tomato, DC3000)的flg22均可诱导大豆GmMPK3/6的激活[34]。为了检测大豆中flg22诱导的GmMPK3/6的激活是否依赖于GmWRKY33B，用10 μmol/L源自Psg的flg22分别处理BPMV-0空载体与GmWRKY33B沉默株叶片，在侵染不同时间取样并提取蛋白，并用专一性识别磷酸化MPK3/4/6的抗体Phospho-p44/42 MAP Erk1/2进行激酶分析。结果表明沉默GmWRKY33B显著降低了flg22对GmMPK6的激活程度(图 5)。虽然GmMPK3在BPMV-0空载体植株与GmWRKY33B沉默植株中激活水平均很低，但在GmWRKY33B沉默植株中激活程度略低于BPMV-0空载体植株(图 5)，表明GmMPK6而非GmMPK3的诱导激活依赖于GmWRKY33B。
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| 图 5 沉默GmWRKY33B同源基因降低了大豆应对flg22诱导的GmMPK3/6激酶的激活程度 Fig. 5 The activation of GmMPK3 and GmMPK6 in response to flg22 is significantly reduced in GmWRKY33B-silenced plants. Leaf discs from the BPMV-0 and GmWRKY33B-silenced plants were incubated on a moist filter paper for 24 h to allow recovery from wounding before being treated with 10 μmol/L flg22 derived from Psg over the indicated time points. The kinase activities were detected by Western blotting using Phosphp44/p42 MAP Erk1/2 antibody, which specifically recognizes phosphorylated MPK3/4/6 across the kingdoms. Coomassie blue stained gel (CBS) was used as loading controls. The Arabidopsis sample treated with flg22 for 10 min was used as a positive control. |
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大豆中主要植保素为大豆抗毒素[25]，而拟南芥中的主要植保素为亚麻荠素(camalexin)[11, 23]，它们的生物合成途径不同。大豆异黄酮类特异性异戊烯基转移酶基因家族在大豆抗毒素的生物合成中起着关键作用[25]。对与大豆抗毒素生物合成密切相关的11个异黄酮类特异性异戊烯基转移酶基因[25]启动子区1.5 kb左右的序列分析，发现这些基因的启动子区含有1‒5个W-box序列(TTGACC/T)，暗示这些基因的表达可能受GmWRKY调控。通过RT-PCR和RT-qPCR分析比较BPMV-0空载体对照植株与GmWRKY33B沉默植株叶片受Pst侵染48 h后GmHPT、GmG2DT、GmG4DT和GmIDT3的诱导表达情况。结果表明，沉默GmWRKY33B导致上述基因诱导表达水平显著降低(图 6A、6B)，说明GmWRKY33B基因家族参与调控这些基因的诱导表达，进而参与大豆抗毒素的生物合成。
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| 图 6 细菌侵染对GmPT基因表达效应的分析 Fig. 6 RT-PCR and RT-qPCR analysis of the GmPT genes in response to Psg infection. The BPMV-0 and GmWRKY33B-silenced plants were infected with Psg for 48 hours, and the RNA was subsequently isolated from the infected leaves. RT-PCR (A) and RT-qPCR (B) were performed to monitor the expression of prenyltransferases genes. GmElF1b was used as an internal reference gene. The name, Glyma number, protein function and the number of W-box presented in the promoter region for each gene are listed on the right. The enzyme names and abbreviations encoded by the PT genes are: G2DT: Glycinol 2-dimethylallyltransferase; C4DT: Glycinol 4-dimethylallyltransferase coumestrol 4-dimethylallyltransferase; HPT: Homogentisate phytyltransferase. ** and *** represent P < 0.01 and P < 0.001, respectively, by Student's t-tests. |
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WRKY是高等植物特有的转录因子，在植物免疫反应中起着重要作用[12, 46-47]。大豆是古四倍体植物，75%以上的基因存在功能冗余的情况[48]，用传统筛选突变体的方法不适用于大豆基因功能的研究。而VIGS技术恰好可弥补这一缺陷。VIGS技术可以同时沉默同源性高达85%以上的基因，可以克服大豆基因功能冗余的障碍[42-43]。通过BPMV-VIGS技术利用单个沉默载体同时沉默了2个GmWRKY33B基因(图 2)。抗病性鉴定结果表明沉默GmWRKY33B可降低大豆对Psg及SMV-N-GUS的抗性(图 3，图 4)，说明GmWRKY33B基因家族在大豆免疫反应中起着重要作用。通过在保守区构建携带单一Glyma.02G232600片段的BPMV-VIGS载体成功同时沉默GmWRKY33A的4个同源基因，并证明其在大豆防御中起着正调控作用[41]。拟南芥WRKY33通过调控植保素生物合成关键基因的表达而参与大豆植保素的生物合成[12]；在油菜中过表达BnWRKY33可增强对Sclerotinia sclerotiorum的抗性[49]，说明不同植物的WRKY33在抗性方面的功能是保守的。
当拟南芥受到病原体侵害时，其表面模式识别受体可识别PAMPs并被激活，激活的受体复合体可将磷酸化信号传递给MKK4/5-MPK3/6，然后通过MPK3/6磷酸化WRKY33，磷酸化后的WRKY33能够启动下游植保素合成相关基因的表达[12]。激酶分析结果表明在大豆中沉默GmWRKY33B显著降低了flg22诱导的GmMPK6的激活程度(图 5)，说明GmWRKY33B的功能是GmMPK3/6激活所必需的，亦即GmWRKY33B是大豆PAMP触发的免疫力(PAMP-triggered immunity, PTI)途径的必要组分。有研究表明在胁迫条件下，拟南芥MPK3/MPK6的激活可诱导ACC合成酶与WRKY的表达，而诱导表达的WRKY又可上调MPK3/MPK6的表达，从而形成一个正反馈环调控ACC合成酶的表达及乙烯的合成[50]。另外，MPK3/MPK6通过磷酸化ERF转录因子调控防御基因的表达及对真菌的抗性[45]。与此相类似，推测GmMPK6可以激活GmWRKY33B的转录活性；反过来，GmWRKY33B又可调控GmMPK6基因的表达，二者之间形成一个正反馈调控环路促进防御相关基因的表达。
异黄酮是大豆中广泛存在的黄酮类次生代谢物，而异黄酮通过异戊烯基转移酶羟基化以及进一步环化形成含有一个或多个C5或C10芳香环的大豆抗毒素[51-52]，目前有11个特异性的异戊烯基转移酶编码基因已被证实参与大豆抗毒素的生物合成[25]。发现这11个基因启动子区含有1‒5个W-box，暗示这些基因可能受WRKY调控。比较了在Psg侵染条件下PT基因在GmWRKY33B沉默植株与BPMV-0空载体植株间诱导表达的差异，结果发现沉默这些GmWRKY33B显著降低了大豆抗毒素生物合成途径中这4个关键酶编码基因的表达(图 6)，说明GmWRKY33B可通过调控大豆抗毒素的生物合成而参与防御反应。接下来通过高效液相色谱测定在病原菌侵染条件下GmWRKY33B沉默植株与BPMV-0空载体植株中的抗毒素含量便可验证推测。如果测定结果如所期，将创制过表达GmWRKY33B的转基因株系，为抗性育种提供材料。
致谢: 感谢美国艾奥瓦州立大学(Iowa State University)的Steve A. Whitham和John Hill教授提供的BPMV-VIGS系统、Pseudomonas syringae pv. glycinea R4菌株及SMV-N-GUS质粒。
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