Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage
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摘要:
目的 研究OfNAC基因对桂花Osmanthus fragrans花开放的调控作用。 方法 从桂花品种‘堰虹桂’O. fragrans ‘Yanhonggui’转录组数据中，筛选获得相关OfNAC基因序列，分析预测其理化性质和结构，运用实时荧光定量PCR技术分析花开放过程的表达特性。 结果 筛选得到22条OfNAC序列。生物信息学分析发现：22条OfNAC转录因子均含有NAM结构域，氨基酸序列含有5个保守的亚结构域(A~E)，其保守性由强到弱依次为C、A、D、B、E；二级结构中不同结构的占比由大到小表现为无规则卷曲、α-螺旋、延伸链、β-折叠；亚细胞定位及跨膜结构预测表明：OfNAC17、OfNAC17-X2、OfNAC53、OfNAC91、OfNTM1-9在膜上表达，其他OfNAC在细胞核内发挥功能。在桂花花开放进程中，OfNAC100-2、OfNAC43、OfNAC73相对表达量在铃梗期(S4)到达顶峰，在此之后相对表达降低; OfNAC43在铃梗期(S4)骤然升高，并且在此时期相对表达最大；OfNAC71、OfNAC29-1、OfNAC21/22从起始期(S1)呈缓慢上升趋势，在顶壳期 (S3) 到达最高，随后整体呈现下降趋势；OfNAC29-2在圆珠期(S2)相对表达量陡然上升，在铃梗期(S4)相对表达最低。 结论 推测OfNAC100-2、OfNAC43、OfNAC73、OfNAC71、OfNAC29-1、OfNAC21/22、OfNAC29-2等成员极有可能参与调控桂花的花开放。图6表3参33 Abstract:Objective This study aims to investigate the regulation of OfNAC genes on flower opening of Osmanthus fragrans. Method From the transcriptome data of O. fragrans ‘Yanhonggui’, the related OfNAC g enes were screened and analyzed to predict the physicochemical properties and structure. Real-time fluorescence quantitative PCR was used to analyze the expression characteristics of the flower opening process. Result 22 OfNAC sequences were screened from transcriptome. Bioinformatics analysis showed that all of the 22 OfNAC transcription factors contained NAM domains, and the amino acid sequence contained 5 conserved subdomains (A−E). The sequence of conservatism ranging from strong to weak was C, A, D, B, and E. The proportion of different structures in the secondary structure from large to small was random coils, α-helices, extended strands, and β-sheets. Subcellular localization and transmembrane structure prediction showed that OfNAC17, OfNAC17-X2, OfNAC53, OfNAC91, OfNTM1-9 was on the membrane, while other OfNAC transcription factors functioned in nucleus. During the O. fragrans flowering process, the relative expression levels of OfNAC100-2, OfNAC43 and OfNAC73 reached the peak at the boll stem stage (S4), and then decreased. The relative expression of OfNAC43 was the highest at the boll stem stage (S4). The relative expression of OfNAC71, OfNAC29-1, and OfNAC21/22 increased slowly from the initial stage (S1), reached the highest at the apical shell stage, and then decreased as a whole. The relative expression of OfNAC29-2 increased sharply at the bead stage (S2) and reached the lowest at the boll stem stage (S4). Conclusion It is speculated that OfNAC100-2, OfNAC43, OfNAC73, OfNAC71, OfNAC29-1, OfNAC21/22, OfNAC29-2 may be involved in the regulation of O. fragrans flower opening. [Ch, 6 fig. 3 tab. 33 ref.] -
Key words:
- botany /
- Osmanthus fragrans /
- flower opening /
- NAC transcription factor /
- expression analysis
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图 1 桂花NAC的NAM保守功能域分析
绿色部分表示NAM保守功能域
Figure 1 NAM conserved functional domain about NAC
图 2 OfNAC转录因子氨基酸序列比对
黑色区域表示序列一致，红色区域保守性稍弱，蓝色区域保守性较差。字母A、B、C、D、E下面的方框部分为OfNAC转录因子N端保守区5个保守亚结构域
Figure 2 Sequence alignment of OfNAC transcription factor
图 3 桂花OfNAC转录因子进化分析及保守基序分析
Figure 3 Evolution analysis of 22 OfNAC transcription factors and analysis of conserved motif
图 4 桂花OfNAC蛋白质跨膜结构预测
Figure 4 Transmembrane structure prediction related to OfNAC protein
图 5 桂花OfNAC与拟南芥、水稻的系统进化树
标黄色阴影的是桂花OfNAC
Figure 5 Phylogenetic tree of OfNAC, Arabidopsis thaliana and Oryza sativa
图 6 22个OfNAC基因在不同花开放时期的表达结果
Figure 6 Expression results of 22 OfNAC genes in different flower opening periods
表 1 桂花OfNAC转录因子RT-PCR特异性引物
Table 1. Specific primers for RT-PCR of OfNAC in O. fragrans
基因名称 用于荧光定量的引物序列(5'→3') 基因名称 用于荧光定量的引物序列(5'→3') OfNAC100-1 F:TGAACAAGATTGAGCCTTGGG OfNAC104 F:TGCATTTTACATTGGTGAAGATGTC R:CCTTTCCTGTGGCTTTCCAG R:GCTCGTACACTTGACACACCA OfNAC53 F:AGATTGTGGGGATGAAGAAAA OfNAC92 F:TCCTAGTCGGAATGAAGAAAACTC R:CAACTCCATATCAGTAAGCCG R:ATGGCTTTCTAATCTGTATTCGTGC OfNTM1-9 F:GGTTGCTCTAATGCCCACTTC OfNAC72-1 F:GGAAAAGCCCCCAAAGGAAC R:CTGGTTCCGTAGCACGATACT R:CCCAATCATCCAACCTTGAGC OfNAC73 F:AGGCAAGGATGGCCAAATTC OfNAC72-2 F:ACGTAGGAAAAGCACCAAAAGG R:TTGTGCCATCTTGTTTCTCC R:AGCATCCAACCTTGCGCTTC OfNAC43 F:AGGCTACTGGTCGTGATAAAG OfNAC29-1 F:TTACAAGGGAAGGCCTCCAAAG R:GGGGTCATGAGTGTCGTCC R:TTGAGCCATTTTGCGTGTTAGG OfNAC91 F:TCTACAAAGGTCGTGCTCCG OfNAC29-2 F:CCCAAAGGGCGTCAAAACTG R:CCCTGACCAGGATAAGTGCC R:GCACACAATCATCCAACCTCA OfNAC50-X2 F:TGGCAAAGGGTATTGGAAAGC OfNAC71 F:CTATCGTGGAAGAGCACCACT R:TCGCCCACTATGGAAAACAAG R:TCCCTGAAATCTTGGGGTGTC OfNAC50 F:AAGCAACTGGAAAGGATCGC OfNAC2 F:TTGGGAATAAAGAAGGCTCTGGTG R:AATTCTGCATCGCAAAGCCTG R:ACACAACACCCAATCATCAAGCCTC OfNAC21/22 F:GAAGGGAAGCCTGGTTGGAAT OfNAC100-2 F:TCAGAGGAAAAATCCTCGTCGG R:CCCAATCCTCCTTGACAGATG R:TTTGGGAGGTTGTGGATCGAG OfNAC56 F:TCTATGGTGGAAAGCCTCCT OfNAC32 F:AAGCCTTGGTTTTCTATGCCG R:CATCAAGCCTTAAAGAGCCC R:AAGCTGTTGTTCTTGTTTCGA OfNAC17-X2 F:TCCTGTTGGGGTGAAGAAGA OfACT F:CCCAAGGCAAACAGAGAAAAAAT R:ATAGTCATCCTGTGCATCCTGC R:ACCCCATCACCAGAATCAAGAA OfNAC17 F:TGGTCTTCCATAAAGGTCGTGC R:TTGTACAGAGCATAGCAATCCCGTG 
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表 2 桂花OfNAC蛋白质理化性质及二级结构分析
Table 2. Physicochemical properties and secondary structure analysis of OfNAC of O. fragrans
序列名称 氨基酸序列长度/aa 相对分子量 电点 碱正 酸负 不稳定系数 脂溶性指数 总平均疏水值 亚细胞定位 位置 预测值/% OfNAC100-1 345 38986.11 8.69 40 36 40.06 61.01 −0.574 细胞核 61.54 OfNAC53 558 62807.47 4.60 51 88 38.87 69.37 −0.564 内质网 42.42 OfNTM1-9 606 68053.7 5.63 71 86 51.49 62.43 −0.725 细胞核 76.92 OfNAC73 300 33768.95 8.79 39 34 38.21 69.47 −0.754 细胞核 76.92 OfNAC43 399 45513.66 5.77 43 55 46.31 63.26 −0.752 细胞核 69.23 OfNAC91 609 67741.52 4.98 63 86 52.83 71.07 −0.593 细胞核 76.92 OfNAC50-X2 389 43765.29 5.29 46 58 47.83 69.43 −0.629 叶绿体 58.33 OfNAC50 399 44952.52 5.35 46 61 50.45 67.69 −0.652 叶绿体 46.15 OfNAC21/22 296 33613.15 6.54 33 34 51.26 66.52 −0.570 细胞核 71.43 OfNAC56 322 35946.51 8.62 37 34 39.48 64.81 −0.725 细胞核 92.30 OfNAC17-X2 573 64828.71 4.87 63 91 40.60 77.70 −0.530 细胞核 53.85 OfNAC17 606 68408.12 4.85 60 97 46.79 74.62 −0.571 细胞核 30.77 OfNAC104 186 21498.77 4.59 19 31 46.96 68.60 −0.695 细胞核 84.61 OfNAC92 325 36813.71 6.47 40 42 29.27 71.35 −0.579 细胞核 38.46 OfNAC72-1 339 38289.93 8.64 40 37 40.14 64.96 −0.671 细胞核 84.61 OfNAC72-2 342 38848.46 8.64 42 39 40.04 61.64 −0.762 细胞核 100 OfNAC29-1 280 32538.56 7.71 36 35 42.96 61.96 −0.816 细胞核 52.50 OfNAC29-2 275 31330.36 9.33 35 26 33.81 61.35 −0.741 细胞核 100 OfNAC71 303 34864.61 5.42 33 43 51.89 53.37 −0.795 细胞核 85.71 OfNAC2 296 34249.55 6.09 35 38 50.01 66.22 −0.735 细胞核 69.23 OfNAC100-2 334 37778.8 6.51 38 40 43.10 67.96 −0.540 细胞核 85.71 OfNAC32 263 30253.45 8.45 38 35 43.91 69.32 −0.635 细胞核 61.54 说明：不稳定系数大于40为不稳定序列，小于40为稳定序列
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表 3 桂花OfNAC蛋白质理化性质及二级结构分析
Table 3. Secondary structure analysis of OfNAC of O. fragrans
序列名称 二级结构占比 序列名称 二级结构占比 α-螺旋/% β-螺旋/% 延伸/% 无规则卷曲/% α-螺旋/% β-螺旋/% 延伸/% 无规则卷曲/% OfNAC100-1 15.07 3.19 14.78 66.96 OfNAC17 24.59 2.81 12.71 59.90 OfNAC53 21.33 3.76 14.52 60.39 OfNAC104 18.82 3.76 15.59 61.83 OfNTM1-9 23.27 3.14 9.74 63.86 OfNAC92 14.15 3.69 15.69 66.46 OfNAC73 13.00 3.67 20.00 63.33 OfNAC72-1 17.99 3.54 16.22 62.24 OfNAC43 21.55 4.26 14.29 59.90 OfNAC72-2 16.96 3.51 16.08 63.45 OfNAC91 20.36 2.63 10.51 66.50 OfNAC29-1 3.21 3.57 17.86 55.36 OfNAC50-X2 31.11 5.91 12.34 50.64 OfNAC29-2 15.64 4.36 16.73 63.27 OfNAC50 27.82 5.01 10.53 56.64 OfNAC71 16.17 4.95 14.52 64.36 OfNAC21/22 17.23 3.04 15.20 64.53 OfNAC2 21.62 4.05 11.82 62.50 OfNAC56 16.15 3.11 15.84 64.91 OfNAC100-2 16.47 3.59 14.07 65.87 OfNAC17-X2 25.83 2.97 12.91 58.29 OfNAC32 17.11 2.66 12.93 67.30
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