大豆GmWRKY148的克隆与功能分析

doi:10.3864/j.issn.0578-1752.2018.18.002

摘要/Abstract

摘要：

【目的】WRKY转录因子与植物的生物和非生物胁迫应答密切相关。通过对大豆WRKY转录因子基因GmWRKY148的克隆及在大豆发状根中过表达后对疫霉根腐病抗性的分析,探究大豆与大豆疫霉菌互作的作用机理。【方法】以拟南芥的AtWRKY44序列为探针,利用同源克隆的方法在大豆Williams 82的根部组织中得到其同源基因Glyma.14G199800,命名为GmWRKY148。对GmWRKY148蛋白进行系统进化树分析;利用qRT-PCR方法分析该基因在大豆的根、茎、叶、子叶和接种大豆疫霉菌不同时间点的转录水平;利用双酶切的方法将GmWRKY148完整的CDS序列连接到植物过表达载体pBinGFP2中,利用基因枪法将重组质粒转化到洋葱表皮细胞中,进行亚细胞定位分析。利用发根农杆菌K599介导的遗传转化体系,经过GFP荧光筛选和qRT-PCR检测,获得大豆过表达GmWRKY148的阳性发状根（OE-GmWRKY148）和转入pBinGFP2空载体（EV）的阴性对照发状根。对过表达GmWRKY148发状根和阴性对照发状根接种大豆疫霉菌,统计病斑长度、疫霉积累量和卵孢子萌发情况。【结果】GmWRKY148的CDS序列全长为999 bp,编码332个氨基酸,等电点为7.61。系统进化分析发现,GmWRKY148与菜豆(Phaseolus vulgaris)、蒺藜苜蓿（Medicago truncatula）的WRKY转录因子亲缘关系十分相近,且与菜豆的亲缘关系最近。亚细胞定位结果显示,GmWRKY148定位在细胞核中。组织表达分析显示,GmWRKY148在根中的表达量最高,在茎和叶中次之,在子叶中最低。荧光定量PCR结果表明,接种大豆疫霉菌P6497后,在感病品种Williams和抗病品种Williams 82（含有Rps1k）中,GmWRKY148受诱导逐渐上调表达,在侵染24 h后表达水平均达到最高,但在Williams 82中的上调倍数更高。对过表达GmWRKY148和转空载体的大豆阳性发状根分别接种大豆疫霉菌P6497的菌丝块,比较接种24 h后的病斑长度和疫霉积累量,结果显示,与对照EV相比,过表达GmWRKY148大豆阳性发状根的病斑长度显著变短,疫霉积累量显著降低。对过表达GmWRKY148和EV的大豆阳性发状根分别接种疫霉菌P6497的游动孢子,并在接种后24、36和48 h用显微镜观察菌丝的侵染及卵孢子萌发数量,统计结果显示,过表达GmWRKY148的大豆阳性发状根与对照EV相比,菌丝的侵染率及卵孢子萌发率均显著降低。【结论】GmWRKY148参与调控大豆与大豆疫霉的互作,能够增强大豆对大豆疫霉菌的抗性。

关键词: 大豆, GmWRKY148, 大豆疫霉根腐病, 抗性

Abstract:

【Objective】WRKY transcription factor is closely related to the biological and abiotic stress responses of plants. In order to study the function of GmWRKY148, GmWRKY148 was cloned and its expression was analyzed, then the gene was over-expressed in soybean hairy roots for analyzing resistance to Phytophthora root rot.【Method】Based on the sequence of AtWRKY44, the homologous gene Glyma.14G199800 was obtained from the root tissue of soybean Williams 82 by method of homologous cloning, named GmWRKY148. Phylogenetic analysis was performed. The transcriptional level of GmWRKY148 under four tissues and P. sojae infection was analyzed by qRT-PCR. For subcellular localization analysis, the recombinant plasmid pBinGFP2-GmWRKY148 was constructed, and transformed into onion epidermal cells by gene gun method. Transgenic soybean hairy roots were obtained by using A. rhizogenes-mediated method. The transgenic over-expressing hairy roots and empty-vector hairy roots were obtained by GFP fluorescence screening and qRT-PCR. To confirm whether GmWRKY148 is involved in the interaction between soybean and P. sojae, the positive soybean hairy roots were inoculated by P6497, and measured the lesions length, accumulation of P. sojae and the germination of oospores.【Result】The CDS of GmWRKY148 is 999 bp, encoding 332 amino acids, and the isoelectric point is 7.61. Phylogenetic analysis indicated that the WRKYs from Phaseolus vulgaris and Medicago truncatula were highly similar to GmWRKY148. Subcellular localization results showed that GmWRKY148 was located in the nucleus. Tissues expression analysis showed that GmWRKY148 had the highest expression in roots, followed in stems and leaves, and lowest in cotyledons. qRT-PCR analysis showed that the expression level of GmWRKY148 was up-regulated upon infection by P. sojae both in resistant variety Williams 82 (containing the Rps1k) and susceptible variety Williams, which has the highest expression at 24 h post infection. The transcript of GmWRKY148 is higher in Williams 82 than that in Williams. Transgenic over-expressing hairy roots (OE-GmWRKY148) and empty-vector hairy roots (EV) were inoculated with P. sojae P6497 mycelium blocks, the lesions length and the accumulation of P. sojae were compared after 24 h inoculation. The lesion length in OE-GmWRKY148 soybean hairy roots was significantly shorter than that in the EV, and the accumulation of P. sojae was significantly reduced. The rate of mycelial infection and zoospores germination was observed by microscopy at 24, 36 and 48 h after inoculated with P. sojae zoospores. Compared to the control EV, the rate of mycelia infection and zoospores germination of over-expressing soybean hairy roots was significantly decreased.【Conclusion】GmWRKY148 is a positive regulator in the interaction between soybean and P. sojae.

Key words: Glycine max, GmWRKY148, Phytophthora root rot, resistance

王莎莎, 崔晓霞, 黄颜众, 轩慧冬, 郭娜, 邢邯. 大豆GmWRKY148的克隆与功能分析[J]. 中国农业科学, 2018, 51(18): 3445-3454.

ShaSha WANG, XiaoXia CUI, YanZhong HUANG, HuiDong XUAN, Na GUO, Han XING. Cloning and Functional Analysis of the GmWRKY148 in Soybean[J]. Scientia Agricultura Sinica, 2018, 51(18): 3445-3454.

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基因Gene	引物序列 Sequence of primer (5'-3')	引物用途 Function of primer
GmWRKY148	F：GCAGCTTCTAACTCTGGCGA	GmWRKY148克隆 Cloning of GmWRKY148
GmWRKY148	R：TTTATTATAAGTGATAAGATCTACAAGCAA
GmWRKY148-MQ	F：CGGGGTACCATGCCTTTCCGAAATTCCAATATGA
GmWRKY148-MQ	R：CGCGGATCCTCATATTTCATTTGGATATTGAGGA
GmWRKY148-2	F：ATGCACACAACCCACTTGCT	GmWRKY148实时荧光定量PCR qRT-PCR of GmWRKY148
GmWRKY148-2	R：CAGATGAAGAGTTTCTCTTC	GmWRKY148实时荧光定量PCR qRT-PCR of GmWRKY148
GmEF1β	F：GTTGAAAAGCCAGGGGACA	疫霉积累量实时荧光定量内参 Reference gene in qRT-PCR
GmEF1β	R：TCTTACCCCTTGAGCGTGG	疫霉积累量实时荧光定量内参 Reference gene in qRT-PCR
GmActin	F：GGTGGTTCTATCTTGGCATC	实时荧光定量PCR内参 Reference gene in qRT-PCR
GmActin	R：CTTTCGCTTCAATAACCCTA	实时荧光定量PCR内参 Reference gene in qRT-PCR
pBinGFP2	F：AAGACCCCAACGAGAAGC	pBinGFP2载体引物 Primer of vector
pBinGFP2	R：GAACCCTAATTCCCTTATCTG	pBinGFP2载体引物 Primer of vector

	24 hpi		36 hpi		48 hpi
	GFP	OE-GmWRKY148	GFP	OE-GmWRKY148	GFP	OE-GmWRKY148
被成功侵染的大豆发状根数 Number of roots infected	7/20	3/20	13/20	5/20	16/20	9/20
萌发卵孢子的大豆发状根数 Number of roots developed oospores	3/20	0/20	6/20	2/20	12/20	4/20