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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (8): 1158-1165.doi: 10.3724/SP.J.1006.2019.81017

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

Cloning and functional analysis of wheat receptor-like kinase gene TaPK3A

SU Qiang,RONG Wei,ZHANG Zeng-Yan()   

  1. National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-09-30 Accepted:2019-01-19 Online:2019-08-12 Published:2019-07-16
  • Contact: Zeng-Yan ZHANG E-mail:zhangzengyan@caas.cn
  • Supported by:
    This study was support by the National Natural Science Foundation of China(31771789)

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Abstract:

Wheat sharp eyespot has become an important limiting factor of wheat production in China. The precondition for wheat sharp eyespot resistant breeding is to study the molecular basis of wheat defense response and to identify effective resistance genes. In this study, a wheat receptor-like kinase (RLK) gene, named TaPK3A, was cloned from sharp eyespot-resistant wheat line CI12633, and the expression and function of the TaPK3A gene were analyzed. TaPK3A contains an open reading frame with 1983 bp length. It encodes a protein kinase that is consisted of 660 amino acids. RT-qPCR analysis showed that the expression of TaPK3A in sharp eyespot-resistant wheat line CI12633 was significantly induced by the pathogen of sharp eyespot (Rhizoctonia cerealis). The TaPK3A gene was expressed in all the tissues, with the highest expression level in the leaves. The expression of TaPK3A was significantly up-regulated by salicylic acid. By means of barley stripe mosaic virus (BSMV) based virus-induced gene-silencing (VIGS), TaPK3A was silenced in CI12633 plants. After R. cerealis (WK207) inoculation, TaPK3A-silenced CI12633 plants displayed a significant decrease in resistance to R. cerealis infection compared with BSMV: GFP-infected CI12633 plants (control). These results suggested that TaPK3A is required for wheat defense response to sharp eyespot.

Key words: receptor-like kinase TaPK3A, wheat sharp eyespot, resistance response, virus-induced gene silencing

Fig. 1

Transcriptional analyses of TaPK3A in RIL A: expression of TaPK3A in RNA-Seq data of resistant and susceptible materials. B: RT-qPCR verification of the transcription of TaPK3A. R stands for sharp eyespot resistant wheat lines in RIL. S stands for sharp eyespot susceptible wheat lines in RIL. R0/S0 stands for the ratio of TaPK3A expression in R and S at 0 h. R4/S4 stands for the ratio of TaPK3A expression in R and S at 4 d. R10/S10 stands for the ratio of TaPK3A expression in R and S at 10 d."

Fig. 2

Phylogenetic tree analysis of TaPK3A protein The phylogenetic tree of TaPK3A was analyzed by MEGA7. TaPK3A protein was tagged with star. The protein in the vertical lines belongs to the same subfamily. AtCERK1 (NP_566689.2), AtLecRK-VI.2 (Q9M021.1), AtLPK1 (NP_567233.1), AtPSKR1 (OAP07882.1), AtWAK1 (OAP13193.1), AtWAKL10 (NP_178086.1) are from Arabidopsis thaliana; OnN25-1 (AHW98543.1) is from Oryza nivara; NbLRK1 (BAG68210.1) is from Nicotiana benthamiana; OsWAK1 (BAG89532.1), OsCERK1 (A0A0P0XII1.1), OsXa26 (AAR08150.1) are from Oryza sativa; Sbds1 (BAM45642.1) is from Sorghum bicolor; SiBti9 (ADL16642.1) is from Setaria italic; TaLRK10 (AAC49629.1) is from Triticum aestivum L.; ZmPK3 (CAA09029.1) is from Zea mays L."

Fig. 3

Expression analysis of TaPK3A in wheat line CI12633 A: expression analysis of TaPK3A in resistant wheat line CI12633. 0 h, 2 d, 4 d, 7 d, 10 d, 14 d, and 21 d stand for the samples of wheat line CI12633 after inoculation of R. cerealis. B: expression analysis of TaPK3A induced by R. cerealis in the organs of wheat line CI12633. C: expression analysis of TaPK3A after SA, JA, ABA, and ET treatment. 0 h, 0.5 h, 1 h, 6 h, and 24 h stand for the samples of wheat line CI12633 after different phytohormones treatments, t-test was used to statistically analyze the significant difference of TaPK3A expression. * P < 0.05; ** P < 0.01."

Fig. 4

Expression analysis of CP and TaPK3A and the symptoms of BSMV and sharp eyespot in BSMV-VIGS wheat A: transcriptional expression of CP by semi-quantitative PCR, and the amplification was 26 cycles. B: RT-qPCR analysis of the relative transcript levels of TaPK3A in the wheat plants infected by BSMV: GFP or BSMV: TaPK3A at 10 dpi. **P < 0.01 (t-test). C: BSMV virus spots on wheat leaves 10 days later after inoculation. D: sharp eyespot symptoms on wheat stems 30 days after inoculation with R. cerealis. IT: infection type."

Table 1

R. cerealis responses in TaPK3A silenced and control wheat plants "

基因型
Genotype		 病级
Infection type		 病情指数
Disease index
BSMV:TaPK3A 2.31** 46.2**
BSMV:GFP 1.27 25.4
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