Identification of CIPK Family in Rice and qRT-PCR Analysis on OsCIPK5 Induced by Magnaporthe oryzae
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摘要:目的 植物通过启动一系列信号传导过程来应对外部环境，这些过程通常涉及多种蛋白激酶，包括钙调神经磷酸酶B样蛋白互作激酶（calcineurin B-like protein-interacting protein kinases, CIPKs）。为更加清晰全面地了解水稻CIPK基因家族，本研究根据最新的基因组测序数据对水稻基因组中的CIPKs进行了鉴定。方法 通过探讨拟南芥和水稻中CIPKs蛋白家族的结构特点，结合生物信息学和qRT-PCR技术系统分析水稻中CIPKs家族蛋白的结构。结合转录组数据，比较了粳稻云引受稻瘟病菌诱导后的表达情况。结果 根据最新的水稻基因组数据，鉴定出31个水稻OsCIPK基因。系统发育树分析结果表明，31个OsCIPK基因可分为5个亚家族，这些亚家族具有不同的外显子-内含子和UTR的结构特点。从广谱抗稻瘟病品种粳稻云引受稻瘟病菌诱导的基因表达谱的趋势聚类中筛选出了OsCIPK5基因并对其进行了表达分析，结果表明，云引中OsCIPK5基因受稻瘟病菌的诱导表达。结论 内含子缺失和片段重复在水稻OsCIPK基因家族的扩展中起到重要作用，同时OsCIPK5受到稻瘟病菌的诱导表达。Abstract:Objective The family of calcineurin B-like protein-interacting protein kinase genes (CIPKs) in rice was studied using the latest sequencing data to further understand the signal transduction involving a variety of kinases in plants in respond to environmental changes.Methods The structures of CIPKs in Arabidopsis thaliana and rice were analyzed. Combining the bioinformatics and qRT-PCR technology, expressions of the CIPK familie and that of the genes induced by M. grisea in Japonica rice cv. 'Yunyin' were compared.Results A total of 31 OsCIPK genes were identified in the rice genome databank. The phylogenetic tree analysis showed that these 31 OsCIPKs could be divided into 5 subfamilies, which had different structural characteristics of exon-intron and UTR. The expression of OsCIPK5, which was selected from a trend cluster of the gene expressiong profile of 'Yunyin' induced by M. grisea, could be induced by M. grisea.Conclusions It appeared that the intron deletion and fragment duplication played an important role in the expansion of OsCIPK family in rice, and that OsCIPK5 expression was induced in 'Yunyin' by M. grisea.
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图 1 水稻CIPK基因染色体分布及染色体间关系
注：红色线表示直系同源的CIPK基因对。染色体数显示在每条染色体的底部
Figure 1. Chromosomal distribution and inter-chromosomal relationship of CIPKs in rice
Note: Red line indicates homology pair of CIPKs. Number of chromosomes is shown at bottom of each chromosome.
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图 2 水稻CIPK基因的系统发育分类、基因结构及保守蛋白基序的特征
注：① a为系统发育树，b为蛋白基序结构特征，c为基因结构；② OsCIPK系统发育树分为五组：A、B、C、D和E，用不同的颜色标出；③b图标尺为氨基酸长度，c图标尺为核苷酸长度；④绿色方框表示UTR，黄色方框表示CDS，黑线表示内含子，其他颜色方框代表保守结构域。
Figure 2. Phylogenetic relationships, gene structure and architecture of conserved protein motifs in CIPKs from rice
Note: ① a, phylogenetic tree; b, protein motif structure; and c, gene structure. ② OsCIPK phylogenetic tree was divided into 5 groups -A, B, C, D, and E- shown in different colors. ③ The ruler of b is for amino acid length; and the ruler of c is for nucleotide length. ④ Green box shows UTR; yellow box shows CDs; black line shows introns; and boxes of other colors show conservative domains.
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图 3 水稻和拟南芥中CIPK蛋白的系统发育分析
Figure 3. Phylogenetic trees constructed with 26 CIPKs from A. thaliana and 31 CIPKs from rice
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图 4 OsCIPK5在稻瘟病菌侵染后不同时间段的表达情况
Figure 4. Expressions of OsCIPK5 at times after M. grisea induction
表 1 OsCIPK5和Actin的引物序列
Table 1 The primer sequence of OsCIPK5 and Actin
基因 Genes q-F q-R OsCIPK5 CAGAGCGTCGCCATCAAGGTC GTGACAGAAGTCCACAGCCCCTATC Actin CCTCGTCTGCGATAATGGAACTG CCCTGGGCGCATCATCTC 
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表 2 水稻基因组CIPK家族的特征
Table 2 Characteristics of CIPK family in rice genome
基因名称
Name of gene转录本ID(RAP-DB)
Transcriptome染色体位置
Locus of chromosome氨基酸长度
Length of amino acid等电点(PI)
Isoelectric point分子量(WM)
Weight of molecular/kDaOsCIPK1 Os01t0292200-01 chr1:10622951-10627532 462 6.26 52.20 OsCIPK2 Os07t0678600-01 chr7:28726817-28729527 444 9.18 50.27 OsCIPK3 Os07t0687000-01 chr7:29191197-29194187 446 6.9 50.95 OsCIPK5 Os01t0206700-03 chr1:5809589-5811389 462 9.28 51.97 OsCIPK6 Os08t0441100-01 chr8:21467453-21468593 305 5.44 32.48 OsCIPK7 Os03t0634400-01 chr3:24226372-24227930 448 9.35 48.37 OsCIPK8 Os01t0536000-01 chr1:19469262-19477590 447 6.45 50.60 OsCIPK9 Os03t0126800-00 chr3:1515355-1518947 479 8.31 53.78 OsCIPK10 Os03t0339900-01 chr3:12630705-12635182 452 9.01 51..49 OsCIPK11 Os01t0824600-01 chr1:35228913-35231725 503 8.48 56.56 OsCIPK12 Os01t0759400-02 chr1:31943529-31948580 541 8.3 59.81 OsCIPK13 Os01t0206300-00 chr1:5799607-5801142 512 8.05 56.01 OsCIPK14 Os12t0113500-01 chr12:679079-682143 440 9.44 50.32 OsCIPK15 Os11t0113700-01 chr11:630585-633622 435 9.53 49.70 OsCIPK16 Os09t0418000-03 chr9:15009182-15010716 457 8.84 50.50 OsCIPK17 Os05t0136200-01 chr5:2113595-2117110 455 6.93 50.92 OsCIPK18 Os05t0332300-01 chr5:15556085-15560404 458 8.84 51.50 OsCIPK19 Os05t0514200-01 chr5:25489349-25491319 509 7.26 56.91 OsCIPK20 Os05t0208100-01 chr5:6701053-6702933 467 8.29 51.35 OsCIPK21 Os07t0637000-01 chr7:26464444-26468858 445 7.96 50.10 OsCIPK22 Os05t0334750-00 chr5:15643309-15644732 452 8.03 49.32 OsCIPK23 Os07t0150700-01 chr7:2643540-2647773 451 9.23 50.71 OsCIPK24 Os06t0606000-01 chr6:24038959-24044785 454 8.52 50.94 OsCIPK25 Os06t0543400-01 chr6:20490388-20492271 515 8.78 56.98 OsCIPK26 Os02t0161000-01 chr2:3283264-3286366 494 9.13 55.91 OsCIPK27 Os09t0418500-00 chr9:15027311-15034300 809 5.53 87.30 OsCIPK28 Os05t0476350-00 chr5:23430891-23432198 436 9.32 49.33 OsCIPK29 Os07t0678300-01 chr7:28710749-28712470 444 8.69 48.21 OsCIPK30 Os01t0759200-01 chr1:31938559-31940184 477 9.34 53.56 OsCIPK31 Os03t0319400-01 chr3:11526273-11530306 450 7.98 50.95 OsCIPK33 Os11t0134300-01 chr11:1605182-1609130 406 6.13 46.60
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表 3 水稻基因组中的直系同源CIPK基因对的Ka/Ks比值
Table 3 Ka/Ks value of orthologous CIPK gene pairs in rice genome
直系同源CIPK基因对
The homologous CIPK gene pairKa Ks Ka/Ks OsCIPK31/OsCIPK3 0.113 65 1.115 82 0.101 85 OsCIPK15/OsCIPK14 0.004 17 0.035 81 0.116 45 OsCIPK11/OsCIPK28 0.161 62 0.880 47 0.183 55 OsCIPK5/OsCIPK20 0.221 63 1.297 74 0.170 78 OsCIPK6/OsCIPK27 0.348 63 0.529 36 0.658 59
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