柱花草SgMPK6互作蛋白的筛选与验证

doi:10.11686/cyxb2023273

摘要/Abstract

摘要：

丝裂原活化蛋白激酶（MAPK）级联反应在调节植物免疫中起关键作用。柱花草炭疽病是危害柱花草生产的严重病害，柱花草SgMPK6基因具有抗炭疽菌的功能。为探究响应胶孢炭疽菌侵染的柱花草SgMPK6下游互作蛋白，本研究采用酵母双杂交技术，以SgMPK6激酶结构域作为诱饵蛋白，筛选柱花草cDNA文库，共获得74个与SgMPK6激酶结构域潜在的互作蛋白，并通过酵母双杂交点对点试验，验证了候选互作蛋白SgbHLH32、SgbHLH33、SgbHLH44与SgMPK6激酶结构域间的互作关系。磷酸化位点预测显示，3个bHLH转录因子均具有MAPKs潜在的磷酸化位点。柱花草响应炭疽菌侵染的qRT-PCR分析表明，SgbHLH32、SgbHLH33、SgbHLH44转录因子均显著上调，预示互作蛋白可能作为SgMPK6的底物调控柱花草对炭疽病的抗性。本研究为进一步解析SgMPK6响应炭疽菌侵染的分子机制提供了试验依据。

关键词: 柱花草, 酵母双杂交, MPK6, 炭疽病, bHLH转录因子

Abstract:

The mitogen-activated protein kinase （MAPK） cascade plays a key role in regulating plant immunity. Anthracnose is a serious disease affecting the production of Stylosanthes （Stylo）. The SgMPK6 gene of Stylo has been shown to function in resistance against anthracnose， which is caused by Colletotrichum gloeosporioides. To investigate the interacting proteins downstream of SgMPK6 in the response to C. gloeosporioides infection， a yeast two-hybridization technique was used to screen the cDNA library of Stylo using the SgMPK6 kinase domain as the bait. Seventy-four potential interacting proteins were obtained. The interactions between the SgMPK6 kinase domain and the bHLH transcription factors SgbHLH32， SgbHLH33， and SgbHLH44 were verified in yeast two-hybrid experiments. Phosphorylation site analysis revealed that all three transcription factors contained MAPK phosphorylation sites. The results of qRT-PCR analyses showed that SgbHLH32， SgbHLH33， and SgbHLH44 were significantly up-regulated in Stylo by C. gloeosporioides infection. Together， these results suggest that SgbHLH32， SgbHLH33， and SgbHLH44 are substrates of SgMPK6 and may be involved in resistance against anthracnose. The results of this study provide a basis for further analysis of the role of SgMPK6 in resistance to C. gloeosporioides infection.

Key words: Stylosanthes, yeast two-hybrid, MPK6, anthracnose, bHLH transcription factors

王芳, 张世子, 戴镕徽, 杨丽云, 罗丽娟, 蒋凌雁. 柱花草SgMPK6互作蛋白的筛选与验证[J]. 草业学报, 2024, 33(7): 84-93.

Fang WANG, Shi-zi ZHANG, Rong-hui DAI, Li-yun YANG, Li-juan LUO, Ling-yan JIANG. Screening and verification of SgMPK6-interacting proteins of Stylosanthes[J]. Acta Prataculturae Sinica, 2024, 33(7): 84-93.

图/表 11

表1 试验所用引物序列

Table 1 Primer sequences used in the experiment

引物名称 Primer names	序列 Sequences （5′-3′）
pGBKT7-SgMPK6-F	CATGGAGGCCGAATTCATGGAAGGAGGAGCTGC
pGBKT7-SgMPK6-R	GCAGGTCGACGGATCCACTGATATTCCGGGTTAAATGCC
pGBKT7-SgMPK6-KD-F	CATGGAGGCCGAATTCTTCGAAGTCACCGCCAAATAC
pGBKT7-SgMPK6-KD-R	GCAGGTCGACGGATCCACGGGTTAAATGCCAGAGACT
pGADT7-SgbHLH32-F	GGAGGCCAGTGAATTCATGGGGTGCAACAAAGGG
pGADT7-SgbHLH32-R	CGAGCTCGATGGATCCATTATTGGAACTGTACCAACTCACC
pGADT7-SgbHLH33-F	GGAGGCCAGTGAATTCATGGAGAGAGACATGCTTCC
pGADT7-SgbHLH33-R	CGAGCTCGATGGATCCATCACATCATGTCATGGATCCC
pGADT7-SgbHLH44-F	GGAGGCCAGTGAATTCATGCATATGAGAGGTTTGTTTCC
pGADT7-SgbHLH44-R	CGAGCTCGATGGATCCACTATGAATGGGTGTTGGATTCAG
qRT-SgbHLH32-F	AAATCAGGAGCTTGAATCACGT
qRT-SgbHLH32-R	CACATGCAAATCCACCATCCTC
qRT-SgbHLH33-F	CGTACCCACTACTGCTGCTG
qRT-SgbHLH33-R	TGTCAGGTCCGAAATCGAAGG
qRT-SgbHLH44-F	GTCGAGCAGGGTGAGGAAGTA
qRT-SgbHLH44-R	CATGGCTGCTCCAACATCTTC
UBCE1-F	CAGATCAAGCTGCTGACGAA
UBCE1-R	GAACAAGCGATCATCAGGTTT

图1 pGBKT7-SgMPK6菌落PCR鉴定M：DNA分子标记 DL2000 plus；1~4：单菌落。下同。 M： DNA Marker DL2000 plus； 1-4： Single colony. The same below.

Fig.1 Colony PCR identification of pGBKT7-SgMPK6

图2 pGBKT7-SgMPK6自激活检测阳性对照Positive control： pGBKT7-53+pGADT7-largeT；阴性对照Negative control： pGBKT7-Lam+pGADT7-largeT； 100、10-1、10-2、10-3：稀释倍数Dilution factors. 下同The same below. 自激活检测组Self-activation detection group： pGBKT7-SgMPK6+pGADT7.

Fig.2 Self-activation detection of pGBKT7-SgMPK6

图3 3-AT抑制浓度的筛选

Fig.3 Screen of minimal inhibitory concentration of 3-AT

图4 SgMPK6保守结构域分析

Fig.4 Conservative domain analysis of SgMPK6

图5 pGBKT7-SgMPK6-KD诱饵载体构建和自激活检测A：菌落PCR筛选阳性克隆Screening for positive clones by colony PCR； B： pGBKT7-SgMPK6-KD自激活检测pGBKT7-SgMPK6-KDself-activation detection.

Fig.5 Construction of pGBKT7-SgMPK6-KD bait vector and self activation detection

图6 pGBKT7-SgMPK6-KD互作蛋白酵母菌落PCR鉴定1~41：酵母菌落的PCR产物PCR products of yeast colonies.

Fig.6 Identification of pGBKT7-SgMPK6-KD interacting proteins in yeast colony by PCR

表2 酵母阳性克隆序列部分结果分析

Table 2 Partial results of sequence analysis of yeast positive clones

基因ID Gene ID	拟南芥编号Arabidopsis number	蛋白质名称Protein name
Unigene15192_SG	AT1G74110.1	细胞色素P450家族蛋白 Cytochrome P450 family protein
CL672.Contig2_SG	AT1G12860.1	bHLH转录因子33 bHLH transcription factor 33
Unigene44350_SG	AT2G42380.2	bZIP转录因子家族蛋白 bZIP transcription factor family protein
CL7251.Contig3_SG	AT5G66540.1	U3小核仁核糖核蛋白 U3 small nucleolar ribonucleoprotein
Unigene29237_SG	AT5G17820.1	过氧化物酶超家族蛋白 Peroxidase superfamily protein
CL1493.Contig6_SG	AT3G25710.1	bHLH转录因子32 bHLH transcription factor 32
Unigene3271_SG	AT3G25230.1	FK506结合蛋白62 FK506 binding protein 62
CL3082.Contig4_SG	AT4G16190.1	半胱氨酸蛋白酶RD19C Cysteine protease RD19C
CL3756.Contig1_SG	AT1G18400.1	bHLH转录因子44 bHLH transcription factor 44

图7 SgbHLH32、SgbHLH33、SgbHLH44与SgMPK6-KD点对点验证互作互作组Interaction group： pGBKT7-SgMPK6-KD+pGADT7-bHLH32， pGBKT7-SgMPK6-KD+pGADT7-bHLH33， pGBKT7-SgMPK6-KD+pGADT7-bHLH44.

Fig.7 Verification of the interactions between SgbHLH32， SgbHLH33， SgbHLH44 and SgMPK6-KD

表3 MAPK底物磷酸化位点及得分统计

Table 3 Phosphorylation sites and score statistics of MAPK substrates

蛋白 Protein	磷酸化位点 Phosphorylation site	磷酸化肽段 Phosphorylation peptides	可信值Trusted value
SgbHLH32	124 S	LNAIS^* PQHV	0.506
SgbHLH33	393 T	LQPLT^* PTPQ	0.615
SgbHLH44	16 S	QLHNS^* PPPP	0.512
SgbHLH44	47 T	QRQQT^* PNPV	0.533

图8 SgMPK6、SgbHLH32、SgbHLH33、SgbHLH44在炭疽菌侵染后不同时间点的表达模式采用T-test方差分析，*与**分别表示柱花草未接种与接种炭疽菌后基因表达量在统计学上具有显著差异（P<0.05）或极显著差异（P<0.01）。Using T-test analysis of variance， * and ** respectively indicate that there is a statistically significant difference （P<0.05） or extremely significant difference （P<0.01） in gene expression between the uninoculated and inoculated stylo.

Fig.8 Expression analysis of SgMPK6， SgbHLH32， SgbHLH33， SgbHLH44 at different time points after Colletotrichum infection

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