Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (12): 2021-2032.doi: 10.3864/j.issn.0578-1752.2019.12.001

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

Expression Profiling and Functional Characterization of Rice Transcription Factor OsWRKY68

Yue CHEN,TianXingZi WANG,Shuo YANG,Tong ZHANG,JinJiao MA,GaoWei YAN,YuQing LIU,Yan ZHOU,JiaNan SHI,JinPing LAN,Jian WEI,ShiJuan DOU,LiJuan LIU,Ming YANG,LiYun LI,GuoZhen LIU()   

  1. College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei
  • Received:2019-01-30 Accepted:2019-03-28 Online:2019-06-16 Published:2019-06-22
  • Contact: GuoZhen LIU E-mail:gzhliu@hebau.edu.cn

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Abstract:

【Objective】 There are nearly 100 WRKY transcription factor members in rice genome, many of them are involved in plant growth and development, biotic and abiotic stress responses. Molecular biology & bioinformatics lab identified that the expression of OsWRKY68 protein was induced after inoculation with Xanthomonas oryzae pv. oryzae (Xoo) in rice. The aim of this study is attempt to further explore the function of OsWRKY68. 【Method】Rice TP309 samples of different tissues at different developmental stages, including germination, seedling, tillering, booting and flowering stages of root, stem, leaf, sheath, cushion, panicle, anther, husk, seed, abiotic stress (4℃, 44℃, 48℃, submerge, NaCl, PEG, constant light, constant dark) and hormone treatments (abscisic acid, methyl jasmonate, salicylic acid, ethephon) were collected. Total protein were extracted and analyzed by Western blot (WB) systematically using OsWRKY68-specific antibody. The expression patterns of OsWRKY68 protein isolated from different tissues at different developmental stages, and tissues obtained from abiotic stresses and hormone treatments were investigated. RNA interfering vector was constructed and transformed to wildtype TP309 rice variety via Agrobacterium tumefaciens strategy. Identification of transgenic plants were carried out by PCR and WB. The phenotype of OsWRKY68 RNAi transgenic plants were monitored and plant height, tiller number, spike length, spikelet number and seed-setting rate were measured.【Result】By comparing the abundance of OsWRKY68 protein in different tissues, it was found that OsWRKY68 protein was expressed in a constitutive way during the normal growth and development of rice, the abundance of OsWRKY68 protein expressed among different tissues were not varied too much. However, different levels of OsWRKY68 were observed. The expression level of OsWRKY68 in anthers at flowering stage was higher than that in mature panicles, panicle axis and husk. It was not expressed in sheaths at tillering and booting stages, but it was expressed in sheaths at flowering stage. In panicles, the abundance of OsWRKY68 was decreased gradually along with the growth of the young panicle. By investigating the expression patterns of OsWRKY68 protein under abiotic stress and hormone treatments, it was found that the abundance of OsWRKY68 protein decreased steadily under salt stress. The expression of OsWRKY68 protein increased steadily at constant light treatment, a specific band (designated as OsWRKY68 +) with higher molecular weight appeared at three days and enhanced in the following timepoints. After methyl jasmonate (MeJA) and ethephon (ET) treatments, OsWRKY68 + band appeared also and its intensity increased as the treatments continues. Four homozygous OsWRKY68 RNAi transgenic lines (Y316, Y317, Y326 and Y337) were checked by PCR and WB analyses and verified at T3 generation. The abundance of OsWRKY68 protein in RNAi transgenic plants was lower than that in wildtype TP309. Phenotypic investigation revealed significant reduction in plant height, tiller number and seed setting rate in transgenic plants.【Conclusion】Rice OsWRKY68 protein plays an important role in the process of normal growth and development of rice. Knocking down the abundance of OsWRKY68 protein via RNAi affected the normal growth of rice. In addition, the data of expression patterns suggested that the function of OsWRKY68 protein may be involved with salt stress, light, MeJA and ethene-mediated signal transduction pathways.

Key words: rice, WRKY transcription factor, expression patterns, western blot, RNA interference

Fig. 1

Expression profiling of OsWRKY68 protein in different tissues at different developmental stagesWB conditions: Sample loading volume 10 μL. Electrophoresis: 80 V, 20 min, 160 V, 90 min, Gel concentration 10% tricine. Electrophoresis apparatus Mini PROTEAN Tetra cell. Membrane (PVDF) transfer: 100 V, 60 min, Primary Antibody: anti-OsWRKY68 polyclonal antibody (BPI, AbP80049-A-S), anti-HSP82 monoclonal antibody (BPI, AbM51099-31-PU), Secondary antibody: goat anti-rabbit secondary antibody (BPI, AbP-71001-D-HRP), goat anti-mouse secondary antibody (BPI, AbP-71003-D-HRP). HSP: Rice OsHSP82; WRKY68: Rice OsWRKY68. The same as below. An: Anther; Bt: Booting; Fw: Flowering; Low: Lower part of leave; Mid: Middle part of leave; MP: Mature panicle; PA: Panicle axis; Rt: Root; Ti: Tillering; Up: Upper part of leave"

Fig. 2

Expression profiling of OsWRKY68 protein under abiotic stressesA: Expression profiling of rice OsWRKY68 protein at seedling stage under 200 mmol·L-1 NaCl treatment; B: Expression profiling of rice OsWRKY68 protein under constant light in vitro. CK: Control; CL: Constant light"

Fig. 3

The expression of OsWRKY68 protein were induced by MeJA and ET treatments"

Fig. 4

The construction and verification of transformation plasmid for OsWRKY68 RNAiElectrophoresis conditions: 125 V, 250 mA, 30 min, 1% agarose gel. H: Hind III; P: PstⅠ; Sa: SacⅠ; M1: DL2000 plus Marker; M2: DL5000 plus Marker; M3: DL10000 plus Marker"

Fig. 5

The identification of OsWRKY68 RNAi transgenic lines"

Fig. 6

The phenotype and agronomic traits of OsWRKY68 RNAi transgenic lines A and B: The phenotype of transgenic plants; C: Plant height; D: Tiller number; E: Seed-setting rate; F: Spike length; G: Spikelet number; WT: Wild type"

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