Genome-wide identification and expression characteristic analysis of EIN3/ EIL transcription factor family in grape

Abstract:【Objective】Ethylene is a gaseous phytohormone which regulates the process of growth and development in plants. EIN3/EIL (Ethylene-insensitive3/EIN3-like) transcription factors are key genes in the pathway of ethylene signaling transduction. Bioinformatics and qRT-PCR were used to explore the family evolutionary characteristic and the expression level of responding to various environmental stresses in different tissues of grape, and to find out candidate genes.【Methods】Vitis vinifera‘Pinot Noir’in vitro propagated plantlets and field plants were used as the experiment materials. The stem-seg- ment with single bud of Pinot Noir in vitro propagated plantlet was subcultured on the medium (MS+0.2 mg·L^-1 IAA) for 30 d. The experimental materials were cultured in a growth chamber under the con-ditions of 25 ℃, 16 h light / 20 ℃, 8 h dark. After 30 d cultured, these plantlets were cultured on MS media with 10% PEG, 100 mmol · L^{- 1} ABA, 200 mmol · L^{- 1} NaCl, 100 mmol · L^{- 1} MeJA, respectively.Some plantlets were cultured in the growth chamber under 5 ℃ stress. Grape roots, stems and leaveswere collected at 0 h and 24 h and then stored in -80 ℃ for the extraction of total RNA. Meanwhile, the same volume of sterile water was used as a CK, each treatment was repeated three times. Field grape plants with consistent growth state were collected, then were sprayed with various concentrations of eth-ephon and 1-Methylcyclopropene on grape leaves and sterile water was used as a CK, each treatment was repeated three times. Ethephon treatment concentrations were 1000 mg·L-1, 500 mg·L^-1, 250 mg·L^-1.1-Methylcyclopropene treatment concentrations were 5.409 mg · L^-1, 2.705 mg · L^-1, 1.352 mg · L^-1. The grape leaves were collected at 0 h and 24 h. The EIN3/EIL gene family members were identified from grape genome using Blast and HMMER. The EIN3/EIL gene family was analyzed using bioinformatics,and the main contents included physicochemical property, Motif, subcellular location, phylogenetic rela-tionship, cis-acting element distribution and gene microarray expression profiles. The qRT-PCR was ad-opted to detect the expression level of VvEIL genes in different tissues under various environmental stresses.【Results】Six homogenous EIN3/EIL transcription factor members in grape had been acquired from grape genome. The EIL genes were mainly distributed on No.6 chromosomes of grape while the position of the VvEIL1 and VvEIL2 were unknown, the number of encoding amino acids was mainly be-tween 450 and 600 aa, molecular weight was 39.3-69.4 ku, isoelectric point was 5.99-6.57. The phyloge-netic analysis found that the VvEILs and AtEILs gene had highest homology, secondly to apple. Subcel-lular localization analysis indicated that the VvEIL transcription factors family were expressed mainly in nucleus. The analysis of secondary structure showed the VvEIL gene family consisted mainly of α-helix and irregular coil. The Motif 1, Motif 2 and Motif 3 conserved sequence existed in all transcription fac-tors member. The Cis-acting element analysis found that the promoter sequence of EIL genes contained a great deal of acting elements related to hormones response, stresses response, plant defense and plant growth. The promoter of all EIL genes included MYB binding site related to drought. The tissue-specif-ic expression profiles indicated that the expression level from low to high of EILs gene in different tis-sues were roots, leaves and stems, respectively. The VvEIL2 was highly expressed in all tissues. Under adversity stress, the VvEILs gene was highly expressed by induction of NaCl and the expression level of the VvEIL1 gene was highest in the roost, which was 18 times higher than that of the control group, and the expression level of the VvEIL6 gene in the leaves was 3.3 times higher than that of the control group. Under the treatment of 5 ℃ stress, compared with the control, the expression of the VvEIL1 gene in the roots, stems and leaves showed a significant down-regulation trend, while the expression of the VvEIL5 gene showed a significant up-regulation trend. The expression levels of the VvEIL2, VvEIL5 and VvEIL6 were higher in the leaves, among three genes the expression of the VvEIL2 was the highest,12 times higher than that of the control group. After PEG treatment, the expression of the VvEIL3 genes in the stems were significantly up-regulated, 7.92 times higher than those in the control group; the ex-pression of the VvEIL1 genes in the leaves were significantly up-regulated, 13 times higher than those in the control group. The expression of the VvEIL6 gene peaked at 250 mg·L^-1 ethephon for 24 h, which was 93 times higher than that of the control group. With the increase of treatment concentration, the ex-pression of the VvEIL2 and VvEIL5 genes began to increase significantly, on the contrary to the expres-sion trend induced by ethephon. The results showed that the VvEIL2 and VvEIL5 genes might be nega-tive regulators in the regulation of ethylene signaling.【Conclusion】The EIN3/EIL gene family mem-bers might play an important part in response to abiotic stresses in grape. The VvEILs might regulate plant growth by responding to low temperature, salt, ethylene, ABA and MeJA. The VvEIL2 and VvEIL5 genes may be a negative factor in ethylene signaling transduction.