谷子SiSAP基因家族的鉴定与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0576

摘要/Abstract

摘要：

【目的】胁迫相关蛋白（stress associated protein, SAP）是一类具有A20/AN1锌指结构域的蛋白，在植物抵御非生物胁迫方面发挥重要作用，目前，谷子SiSAP基因家族的功能尚不明晰。对SiSAP基因家族进行鉴定和表达模式分析，为探究SiSAP基因的功能奠定基础。【方法】通过生物信息学方法对该基因家族进行鉴定及分析，经实时荧光定量PCR（RT-qPCR）验证SiSAP基因家族成员在非生物胁迫和激素处理下的表达模式。【结果】谷子SiSAP基因家族有17个成员，不均匀分布在6条染色体上，15个成员没有内含子，另外2个成员均含有1个内含子；氨基酸序列长度为150-291 aa，分子量介于15.05-32.12 kD，等电点为6.62-9.36；SiSAP基因家族成员与单子叶植物的亲缘关系更近；所有成员启动子序列均含有逆境胁迫和激素响应相关作用元件，以及ERF、Dof、C2H2等转录因子结合位点；转录组数据显示，SiSAP1、SiSAP2、SiSAP3、SiSAP4、SiSAP6、SiSAP7、SiSAP8、SiSAP11和SiSAP14等9个基因在各个组织器官中都有表达，其余8个基因几乎不表达；RT-qPCR结果证实，以上9个基因对低温、高盐和干旱3种非生物胁迫，以及脱落酸、茉莉酸甲酯、生长素和赤霉素4种激素处理的响应模式不同。【结论】SiSAP基因家族可能参与谷子抗旱、耐盐和耐低温胁迫的应答。

关键词: 谷子, SAP基因家族, 非生物胁迫, 激素响应, 表达分析

Abstract:

【Objective】Stress associated proteins（SAP）, a class of A20/AN1 zinc finger domain-containing proteins, play an critical role in plant resistance to abiotic stresses. However, the functions of SiSAP gene family in foxtail millet remain elusive. This study aims to identify the SiSAP gene family in foxtail millet and analyze its expression patterns, laying the foundation for exploring the function of SiSAP gene.【Method】The SiSAP gene family was identified and analyzed using bioinformatics approaches, and its expression levels under different abiotic stresses and hormone treatments were examined by real-time quantitative PCR（RT-qPCR）.【Result】The SiSAP gene family in foxtail millet（Setaria italica）consisted of 17 members, unevenly distributed on six chromosomes. Among these, 15 members had no introns, whereas the remaining contained one intron. The protein length, molecular weight, and isoelectric point ranged in 150-291 aa, 15.05-32.12 kD, and 6.62-9.36, respectively. Phylogenetic analysis revealed that the SiSAP gene family had a closer genetic relationship with monocots. The promoter regions of SiSAP gene family contained cis-acting elements involving in stresses and hormone responses, as well as transcription factor binding sites such as ERF, DOF, C2H2, etc. Transcriptome data indicated that nine genes, including SiSAP1, SiSAP2, SiSAP3, SiSAP4, SiSAP6, SiSAP7, SiSAP8, SiSAP11, and SiSAP14, were expressed in various tissues, while the remaining eight genes were almost not expressed. RT-qPCR analysis showed that the nine genes presented distinct response patterns to low temperature, high salt and drought stresses, as well as to treatments with abscisic acid, methyl jasmonate, auxin and gibberellin.【Conclusion】The SiSAP gene family may be involved in response to drought, salt and low temperature stresses in foxtail millet.

Key words: foxtail millet, SAP gene family, abiotic stress, hormone response, expression analysis

李禹欣, 李苗, 杜晓芬, 韩康妮, 连世超, 王军. 谷子SiSAP基因家族的鉴定与表达分析[J]. 生物技术通报, 2025, 41(1): 143-156.

LI Yu-xin, LI Miao, DU Xiao-fen, HAN Kang-ni, LIAN Shi-chao, WANG Jun. Identification and Expression Analysis of SiSAP Gene Family in Foxtail Millet（Setaria italica）[J]. Biotechnology Bulletin, 2025, 41(1): 143-156.

图/表 13

表1 RT-qPCR引物信息

Table 1 Primers for RT-qPCR in this study

基因名称Gene name	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
SiSAP1	CTGCTGCTGTAATCCCCAAG	AGTGGCAGTCATGTTTGTCG
SiSAP2	GTTCCTCGCAATTTTCTGGA	CCAAGTACGCGTTTGCATAG
SiSAP3	ACGGTATATCTTGCGCTCGT	GCACTGTTGCCGAGATGTAA
SiSAP4	GCAGATAGCCAAGCAGAACC	CCACCACCTGAAGGAACCTA
SiSAP6	GTCTCCTCTCGCTCATCACG	CCTCACGCTTCTTCTTCTGC
SiSAP7	AAGATTCCATCCAGCAGAGC	CATCACCGCCATCATTACAC
SiSAP8	ATCCGGTGTTGCTAGTGGAG	ATGGGCAAATTTGAAGTCCA
SiSAP11	ATACCGGACCCAAACAACTG	ACCGTCTCAAGACAGGTTCG
SiSAP14	AACCCATTTCCTCTCCCTCA	GGCTCCTCCACCTTCTTGTC
SiACTIN	TGATCTCACTGACAGTCTGATG	GATGTCTCTTACAATTTCCCGC

图1 谷子SiSAP基因家族成员在染色体上的定位

Fig. 1 Location of SiSAP family members in foxtail millet in chromosome

表2 SiSAP家族成员的鉴定

Table 2 Identification of SiSAP gene family members

基因Gene	基因号Gene ID	基因位置Gene location	蛋白长度Protein length/aa	分子量Molecular weight/kD	等电点pI	亲水性GRAVY
SiSAP1	Seita.1G049700	Chr.1:4 826 713-4828307	172	18.38	7.99	-0.216
SiSAP2	Seita.1G050100	Chr.1:4 844 574-4846624	172	18.21	8.28	-0.198
SiSAP3	Seita.1G179600	Chr.1:25 817 455-25 819 730	150	15.65	9.28	-0.289
SiSAP4	Seita.2G045100	Chr.2:3 644 125-3 646 735	165	18.09	8.02	-0.479
SiSAP5	Seita.2G188600	Chr.2:28 469 651-28 470 203	184	19.74	8.75	-0.526
SiSAP6	Seita.2G195200	Chr.2:29 175 966-29 176 521	185	19.76	9.04	-0.546
SiSAP7	Seita.2G252300	Chr.2:35 515 181-35 516 761	170	17.55	8.90	-0.234
SiSAP8	Seita.2G358600	Chr.2:43 671 478-43 674 297	291	32.12	8.66	-0.627
SiSAP9	Seita.5G232000	Chr.5:29 446 450-29 446 969	173	17.47	8.96	-0.097
SiSAP10	Seita.5G232100	Chr.5:29 462 246-29 462 780	178	17.86	9.06	-0.063
SiSAP11	Seita.5G297700	Chr.5:35 289 769-35 291 603	154	15.86	8.74	-0.352
SiSAP12	Seita.5G330400	Chr.5:37 812 781-37 813 282	167	17.11	8.76	-0.089
SiSAP13	Seita.6G159700	Chr.6:28 237 350-28 238 434	256	26.19	8.47	-0.190
SiSAP14	Seita.6G203700	Chr.6:32 355 017-32 361 096	179	18.82	9.14	-0.357
SiSAP15	Seita.7G243600	Chr.7:30 408 518-30 409 232	236	24.02	8.60	-0.373
SiSAP16	Seita.9G061100	Chr.9:3 504 533-3 504 971	146	15.05	9.36	-0.312
SiSAP17	Seita.9G316800	Chr.9:36 489 146-36 489 611	155	16.52	6.62	-0.408

图2 SiSAP基因家族进化树、基因结构及motifs分析 A：进化关系；B：基因结构；C：蛋白保守基序；motif2、motif14-motif7为A20；motif3-motif1、motif5-motif1、motif9-motif3-motif11为AN1

Fig. 2 Analysis of SiSAP gene family evolutionary tree, gene structure and motifs A: Phylogenetic relationship. B: Gene structure. C: Conserved motifs. Motif2 and motif14-motif7 are A20; motif3-motif1, motif5-motif1 and motif9-motif3-motif11 are AN1

图3 SiSAP蛋白序列比对分析蓝框代表A20保守结构域，红框代表AN1保守结构域

Fig. 3 Alignment and analysis of sequences of SiSAP protein A20 and AN1 are highlighted with blue and red squares, respectively

图4 SiSAP基因家族共线性分析

Fig. 4 Collinearity analysis of SiSAP gene family

表3 SiSAP家族片段重复基因对的Ka/Ks值

Table 3 Ka/Ks ratios of segmental duplicated gene pairs in the SiSAP gene family

同源基因 Homologous gene	非同义替换率（Ka） Non-synonymous substitution	同义替换率（Ks） Synonymous substitution	非同义替换率/同义替换率 Ka/Ks
SiSAP4-SiSAP16	0.41	1.21	0.34
SiSAP7-SiSAP14	0.20	0.47	0.42

图5 SiSAP基因家族成员与水稻和拟南芥SAP基因共线性分析

Fig. 5 Collinearity analysis of SiSAP gene family members and SAP genes in Oraza sativa and Arabidopsis thaliana

图6 植物SAP基因家族系统发育树红色五角星：谷子；橙色对钩：水稻；蓝色三角形：小麦；绿色圆形：大麦；黄色正方形：拟南芥；紫色方框：刚毛柽柳

Fig. 6 Phylogenetic tree of SAP gene family in plants Red stars: Setaria italica. Orange checks: Oraza sativa. Blue triangles: Triticum aestivum. Green circles: Hordeum vulgare. Yellow rects: Arabidopsis thaliana. Purple squares: Tamarix hispida

图7 谷子SiSAP基因家族的顺式作用元件及转录因子结合位点预测分析 A：SiSAP家族基因启动子序列顺式作用元件分析。网格中不同强度的颜色和数量表示SiSAP基因中的顺式作用元件数量；B：不同颜色的直方图表示每个类别中的顺式作用元件的总和；C：显示前15个与SiSAP基因启动子结合的转录因子家族；网格中不同强度的颜色和数量表示转录因子结合位点的数量

Fig. 7 Prediction of cis-acting element and transcription factor binding sites in the SiSAP gene family of foxtail millet A: Analysis of cis-acting element in promoter sequence of SiSAP family genes. The different intensity colors and numbers of the grid indicate the cis-acting element numbers in the SiSAP genes. B: The different colored histogram represents the sum of the cis-acting elements in each category. C: Only the top 15 TFs that bind to the promoter of SiSAP genes are shown here. The different intensity colors and numbers of the grid indicate the numbers of putative TF sites

图8 谷子SiSAP基因家族的组织表达模式分析 T1：未成熟种子S1；T2：未成熟种子S2；T3：未成熟种子S3；T4：未成熟种子S4；T5：未成熟种子S5；T6：萌发3 d的种子；T7：两叶一心幼苗；T8：灌浆期根；T9：灌浆期穗下节；T10：灌浆期顶端第2茎；T11：抽穗2 d后的新叶（顶2和3叶）；T12：灌浆期旗叶；T13：灌浆期顶端第4叶；T14：灌浆期旗叶叶鞘；T15：灌浆期顶端第4叶叶鞘；T16：分化初始阶段穗；T17：分化第3阶段穗；T18：未成熟小穗S2；T19：未成熟小穗S4

Fig. 8 Tissue expression pattern analysis of SiSAP gene family in foxtail millet T1: Immature seed at S1. T2: Immature seed at S2. T3: Immature seed at S3. T4: Immature seed at S4. T5: Immature seed at S5. T6: Seeds germinated for 3 d. T7: Plant of one-tip-two-leaf. T8: Root at filling stage. T9: Neck-panicle-internodes at filling stage. T10: Stem-top-second at filling stage. T11: Leaves after 2 d of heading（Leaf-top-2-3）. T12: Flag leaf at filling stage. T13: Leaf-top-fourth at filling stage. T14: Flag leaf sheath at filling stage. T15: Leaf-sheath-top-fourth at filling stage. T16: Primary panicle at differentiation stage. T17: Third panicle at differentiation stage. T18: Immature spikelet at S2. T19: Immature spikelet at S4

图9 非生物胁迫下SiSAP基因的表达模式分析横坐标表示各胁迫处理后0、1、3、6和12 h。*，**，***分别表示P<0.05，P<0.01，P<0.001。下同

Fig. 9 Expression patterns of SiSAP gene in response to abiotic stress Abscissa indicates 0, 1, 3, 6 and 12 h after treatment. *, **, *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively. The same below

图10 激素处理后SiSAP基因的表达模式分析

Fig. 10 Expression patterns of SiSAP gene in response to hormone treatment

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