Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (18): 3445-3454.doi: 10.3864/j.issn.0578-1752.2018.18.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Functional Analysis of the GmWRKY148 in Soybean

ShaSha WANG(), XiaoXia CUI, YanZhong HUANG, HuiDong XUAN, Na GUO, Han XING()   

  1. College of Agriculture, Nanjing Agricultural University/National Center for Soybean Improvement/Key Laboratory for Biology and Genetic Improvement of Soybean (General), Ministry of Agriculture/National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 210095
  • Received:2018-04-11 Accepted:2018-06-19 Online:2018-09-16 Published:2018-09-16

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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

Fig. S1

Formation of mycelium of P6497a: Choose fresh mycelium edges, then cut them into small pieces, to carry on the propagation. b-f: Growth situation of P6497, after 2 to 6 days"

Fig. S2

Formation of zoospores of P6497 a: After 4 days of propagation, the growth situation of P6497. b: Choose fresh mycelium edges, cut them into tiny pieces, then put them into liquid V8 medium. c: Thoroughly clean the debris with sterile water. d: Growth situation of zoospores of P6497, 10h later. e: Strain the liquid through a piece of gauze, in order to get transparent zoospores fluid. f: Put positive soybean hairy roots in to zoospores fluid"

Fig. S3

GFP fluorescence in enlarged positive transgenic soybean hairy roots a, b, c: GFP Williams hairy roots. d, e, f : OE-GmWRKY148 Williams hairy roots"

Table S1

Primers used in this study"

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
R：TTTATTATAAGTGATAAGATCTACAAGCAA
GmWRKY148-MQ F：CGGGGTACCATGCCTTTCCGAAATTCCAATATGA
R：CGCGGATCCTCATATTTCATTTGGATATTGAGGA
GmWRKY148-2 F：ATGCACACAACCCACTTGCT GmWRKY148实时荧光定量PCR
qRT-PCR of GmWRKY148
R：CAGATGAAGAGTTTCTCTTC
GmEF1β F：GTTGAAAAGCCAGGGGACA 疫霉积累量实时荧光定量内参
Reference gene in qRT-PCR
R：TCTTACCCCTTGAGCGTGG
GmActin F：GGTGGTTCTATCTTGGCATC 实时荧光定量PCR内参
Reference gene in qRT-PCR
R：CTTTCGCTTCAATAACCCTA
pBinGFP2 F：AAGACCCCAACGAGAAGC pBinGFP2载体引物
Primer of vector
R：GAACCCTAATTCCCTTATCTG

Fig. 1

Phylogenetic tree analysis of GmWRKY148"

Fig. 2

Tissue-specific expression of GmWRKY148 Asterisks indicate statistically significant differences at P < 0.01. The same as below"

Fig. 3

Expression pattern of GmWRKY148 response to P. sojae in Williams and Williams82 A: Expression pattern of GmWRKY148 response to P. sojae in Williams. B: Expression pattern of GmWRKY148 response to P. sojae in Williams 82"

Fig. 4

Subcellular localization analysis of GmWRKY148 protein in onion epidermal cells a, b, c, d: 35S-GFP alone location; e, f, g, h: 35S-WRKY148::GFP location"

Fig. 5

GFP fluorescence in transgenic hairy roots a: GFP Williams hairy roots. b: OE-GmWRKY148 Williams hairy roots"

Fig. 6

Analysis of over expression efficiency in Williams transgenic soybean hairy roots"

Fig. 7

GmWRKY148 could improve the resistance to P. sojae in soybean Williams A：Disease phenotypes of GFP Williams hairy roots after inoculated with P6497 at 24 hpi. B：Disease phenotypes of OE-GmWRKY148 Williams hairy roots after inoculated with P6497 at 24 hpi. C: P6497 infected hairy roots 24h later, the lesion length of GFP Williams hairy roots and OE-GmWRKY148 Williams hairy roots. D: P. sojae biomass was determined by qPCR in inoculated Williams hairy roots OE-GmWRKY148 or EV at 24 and 36 hpi"

Fig. 8

Microscopic analysis of P. sojae infection in soybean Williams hairy roots"

Table S2

Table S2 Summary of the numbers of roots that were successfully infected by P. sojae or oospores development"

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
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