Identification of WRKY gene in A.sativa
165 WRKY genes were identified from the whole genome of A.sativa , which were located on Chr1A-Chr7D and named AsWRKY1-AsWRKY165 respectively according to the sequence of their chromosome locations (TableS1). AsWRKY proteins ranged in length from 200 to 1509 bp, molecular weight between 14.6 and 168.2 kD. There was little change in the theoretical isoelectric point ,between 4.61 and 10.44, the average isoelectric point is 6.99. Information on the WRKY protein is provided in the tableS1.
WRKY gene family system in A.sativa
After comparing 90 A.thaliana , 128 Oryza sativa and 165 identified WRKY family sequences of A.sativa , MEGA6 was used to construct the evolutionary tree (FigureS1). According to the WRKY family information of A.thaliana and O.sativa, 165 WRKY protein sequences could be divided into seven subfamilies, named I, IIa, IIb, IIc, IId, IIe and IIf. Among them, IIe members were the least, accounting for only 4.18%. Among IId, members accounted for 9.40% of all genes, 2.87% of I, 7.31% of IIa, 6.0% of IIb, 8.36% of IIc and 4.50% of IIf. A.thaliana and O.sativa WRKY genes were also mainly concentrated in this group, and A.sativa and A.thaliana were closely related in this group.
Motif analysis of WRKY gene family in A.sativa
The analysis of motifs of 165 WRKY members showed that the A.sativa WRKY gene family identified 15 motifs (Figure2). Most of the members were arranged in the same sequence motif7-1-2 , most members in IIc were in the same sequence motif9-1-2. Motif1 and motif2 were present in all WRKY genes. Motif4 appears in group I , motif12 appeared in group IIe and group IIb, and motif (4/3/11/5/6/1/2/9) appear in group IIa . The same groups have the same structural distribution, the same motif sequences are included in the same group, which may be related to the molecular function of that group .
The gene structure of the WRKY gene family was analyzed. The result showed that the number of introns in 165 genes ranged from 1 to 19. The 146 genes contained only two introns, and 15 genes contained three introns, 4 genes contained four introns. WRKY exons is 1 to 20, the group I, IIb, IId, IIe exons ranged 3 to 4, and the group IIa, IIc, IIf exons ranged 4 to 20. Most genes in the same group have similar genetic machinery. Among them, AsWRKY87 has 19 introns and 20 exons . The group IIf gene is longest, and the group IIe gene is the shortest. The length of AsWRKY87 is 4527 . It is the longest in the group IIf .
Chromosome localization and gene replication analysis of WRKY gene in A.sativa
A total of 165 WRKY genes were found on 21 chromosomes of A.sativa (Figure 3). Among which Chr6D contains the least WRKY gene and Chr5A contains the most WRKY gene.The results showed that there are 82 WRKY genes participate in 144 segmental duplications. 102 WRKY genes were involved in 39 tandem replication pairs. There were 108 pairs of chromosomes replication relationships on chr1A ~1D, 137 pairs on chr2A~2C, and 51 pairs on chr2D~3A. The highest chromosome replication relationship was 105 pairs in group A, 92 pairs in group C and 100 pairs in group D.
Evolutionary analysis of WRKY genes in A.sativa
We analyzed collinearity diagrams between the WRKY genes in A.sativa , A.thaliana and O.sativa (Figure 4). The result showed that , there are 249 collinear gene pairs in A.sativa and O. sativa, 167 collinear gene pairs in A.sativa and A.thaliana. And mainly distributed on Chr1A, Chr1D and Chr2C chromosomes. Among them, ChrA and ChrD had the highest homology in A.thaliana, with 60 homologous genes. ChrC has the lowest homology, with 43 homologous genes. ChrA has the highest homology in O.sativa, with 96 homologous genes . ChrC has the lowest homology, with 70 homologous genes. ChrD has 81 homologous genes. Therefore, the WRKY gene exists earlier than the differentiation time of monocotyledonous plants and dicotyledons.
Cis-acting regulatory elements in the promoter of the WRKY gene in A.sativa
The promoter cis component analysis results are showed that AsWRKY contains multiple cis elements and 13 genes contain light responsive elements (G-box, MRE), 11 genes contain drought-induction elements, 14 genes contain gibberellin-responsive elements (GARE, P-box), 43 genes contain absclslc acld elements (ABRE) (FigureS2). The presence of these cis regulatory elements suggests that hormones have a potential regulatory effect on AsWRKY.
WRKY protein interaction analysis in A.sativa
We predicted the interaction between the WRKY genes and other proteins using the PAIR tool (Figure5). These WRKY genes are thought to interact with different proteins, there are 22 genes were interactions, including the VQ proteins (MKS, SIB), and MPK. Among these, 4 genes interacted with MKS, 8 genes with MPK, and 8 genes with SIB. WRKY33 interacts with WRKY48 / 70.
Expression pattern of WRKY genes in A.sativa under abiotic stress
We used qTR-PCR to select 20 genes to examine its expression under four stress conditions (Figure6). In cold stress, three genes (AsWRKY19 / 80 / 61) were upregulated, and it was the highest at 48h. (AsWRKY95 / 75 / 92) was upregulated at 12h and 24h, and (AsWRKY83 / 8 / 55) was downregulated at 6h. In low temperature stress , (AsWRKY19 / 80 / 61) was upregulated,and it was the highest at 48h. (AsWRKY26 / 79 / 70 / 92 / 45) were upregulated and had the highest multiple at 24h. In salt stress, the AsWRKY19 was upregulated at 48h. (AsWRKY83 / 63 / 90 / 45 / 56 / 55) the expression was all upregulated at 6h and 12h. In saline-alkali stress, AsWRKY19 was upregulated at 48h and AsWRKY100 was downregulated at 12h. (AsWRKY80 / 26) was only downregulated at 6h, ( AsWRKY90 / 19 / 79) were upregulated and significantly expressed at 24h.