林业科学 ›› 2022, Vol. 58 ›› Issue (2): 148-158.doi: 10.11707/j.1001-7488.20220215
李顺福,王慧敏,房丽莎,刘震*
收稿日期:
2021-02-03
出版日期:
2022-02-25
发布日期:
2022-04-26
通讯作者:
刘震
基金资助:
Shunfu Li,Huimin Wang,Lisha Fang,Zhen Liu*
Received:
2021-02-03
Online:
2022-02-25
Published:
2022-04-26
Contact:
Zhen Liu
摘要:
目的: 对短日照诱导的白花泡桐顶芽进行转录组测序分析,探究其顶芽死亡的分子机制,为解决泡桐“冠大干低”提供理论依据。方法: 在25 ℃恒温条件下,对白花泡桐1年生苗进行短日照(SD)处理,在高生长期(SDa)、高生长停止期(SDb)、顶芽死亡发生期(SDc)3个时期进行转录组测序,并利用荧光定量PCR(qRT-PCR)测定11个差异基因的表达量,同时结合转录组信息进行验证。结果: 白花泡桐顶芽3个时期转录组测序共得到57.47 Gb数据,使用DESeq2软件进行3个时期样品间比较,共筛选出差异基因44 397条,其中注释到7大数据库(NR、NT、GO、EggNOG、KEGG、UniProt、Pfam)的基因为37 076条(83.51%)。3个时期的差异基因在KEGG上主要富集在植物激素信号通路,对顶芽死亡发生的SDc时期植物激素信号通路上差异基因的分析得出:脱落酸(ABA)激素信号的响应因子ABF下调表达,乙烯(ETH)激素信号的响应因子ERF1上调表达,油菜素内酯(BR)激素信号上编码木葡聚糖内糖基转移酶的TCH4和细胞周期特异蛋白基因CYCD3上调表达,这些基因与大多数植物芽休眠过程中表达情况不同。qRT-PCR与转录组测序结果在基因表达量差异倍数上存在偏差,但整体趋势一致,可以证明转录组结果的可靠准确。结论: 白花泡桐顶芽死亡发生期的植物激素信号转导通路中ABF、ERF1、TCH4和CYCD3基因的表达情况,使其在高生长停止后顶端分生组织细胞分裂活动仍在继续进行,此时顶端分生组织分化的叶原基展开的幼叶脱落而无法形成芽鳞包裹,导致顶芽未向休眠芽方向发展而死亡。
中图分类号:
李顺福,王慧敏,房丽莎,刘震. 短日照诱导白花泡桐顶芽死亡过程相关基因的表达[J]. 林业科学, 2022, 58(2): 148-158.
Shunfu Li,Huimin Wang,Lisha Fang,Zhen Liu. Gene Expression Related to Terminal Bud Death Induced by Short-Day in Paulownia fortunei[J]. Scientia Silvae Sinicae, 2022, 58(2): 148-158.
表1
短日照诱导白花泡桐顶芽3个时期reads质量统计"
样品 Sample | 原始数据 Raw reads | 有效数据 Clean reads | 有效碱基数 Clean bases/Gb | Q30(%) | 有效读数比例 Clean reads ratio (%) |
SDa-1 | 47 170 900 | 46 155 682 | 6.45 | 93.51 | 97.85 |
SDa-2 | 47 296 568 | 46 028 158 | 6.43 | 93.84 | 97.32 |
SDa-3 | 47 694 770 | 46 368 244 | 6.48 | 93.96 | 97.22 |
SDb-1 | 48 454 868 | 47 249 798 | 6.60 | 92.85 | 97.51 |
SDb-2 | 45 454 586 | 44 452 908 | 6.21 | 93.76 | 97.80 |
SDb-3 | 45 592 110 | 44 391 996 | 6.20 | 93.77 | 97.37 |
SDc-1 | 49 038 356 | 47 567 810 | 6.64 | 94.58 | 97.00 |
SDc-2 | 48 460 682 | 46 921 468 | 6.55 | 93.50 | 96.82 |
SDc-3 | 44 069 318 | 42 734 802 | 6.01 | 93.71 | 96.97 |
图3
不同时期差异表达基因GO聚类分析 1-17:细胞组分;18-43:生物学功能;44-57:分子功能。1.胞外区;2.细胞;3.核;4.膜;5.细胞连接;6.膜封闭腔;7.大分子复合物;8.细胞器;9.其他组织部分;10.胞外区组分;11.细胞器组分;12.病毒组分;13.膜组分;14.突触组分;15.细胞成分;16.突触;17.超分子复合物;18.生殖;19.细胞杀伤;20.免疫系统过程;21.行为;22.代谢过程;23.细胞增殖;24.细胞过程;25.碳利用;26.氮利用;27.生殖过程;28.生物黏附;29.信号;30.多细胞器官过程;31.发育过程;32.生长;33.运动;34.色素沉积;35.节律过程;36.应激反应;37.定位;38.多组织过程;39.生物调控;40.组织或生物发生细胞组分;41.细胞聚集;42.降解;43.化学突触传递的突触前膜过程;44.催化活性;45.信号转导活性;46.结构分子活性;47.转运活性;48.结合活性;49.抗氧化活性;50.蛋白标签;51.翻译调节活性;52.营养库活性;53.分子转导活性;54.分子功能调节因子;55.劫持分子功能;56.分子载体活性;57.转录调控活性。"
表3
差异表达基因KEGG富集通路分析"
通路名称 Pathway | 上调基因数 UP regulated genes | 下调基因数 Down regulated genes | 校正P值 Corrected P value | 通路位置 Pathway ID |
SDb vs SDa | ||||
植物激素信号转导Plant hormone signal transduction | 36 | 59 | 1.06×10-10 | map04075 |
角质、木栓质和蜡的合成Cutin, suberine and wax biosynthesis | 19 | 7 | 2.56×10-7 | map00073 |
光合作用天线蛋白Photosynthesis-antenna proteins | 0 | 18 | 5.07×10-6 | map00196 |
苯丙烷类合成Phenylpropanoid biosynthesis | 36 | 20 | 8.72×10-6 | map00940 |
MAPK信号通路MAPK signaling pathway-plant | 46 | 21 | 9.74×10-5 | map04016 |
乙醛酸和二羧酸代谢Glyoxylate and dicarboxylate metabolism | 64 | 11 | 0.000 2 | map00630 |
光合作用Photosynthesis | 1 | 19 | 0.000 7 | map00195 |
碳代谢作用Carbon metabolism | 175 | 12 | 0.007 1 | map01200 |
SDc vs SDb | ||||
植物激素信号转导Plant hormone signal transduction | 26 | 84 | 0.000 2 | map04075 |
氨基酸合成Biosynthesis of amino acids | 197 | 76 | 0.001 5 | map01230 |
淀粉和蔗糖代谢Starch and sucrose metabolism | 95 | 58 | 0.001 9 | map00500 |
苯丙氨酸等合成Phenylalanine, tyrosine and tryptophan biosynthesis | 40 | 15 | 0.004 6 | map00400 |
SDc vs SDa | ||||
植物激素信号转导Plant hormone signal transduction | 59 | 93 | 3.94×10-11 | map04075 |
淀粉和蔗糖代谢Starch and sucrose metabolism | 146 | 67 | 1.25×10-10 | map00500 |
苯丙烷类合成Phenylpropanoid biosynthesis | 59 | 42 | 1.94×10-9 | map00940 |
MAPK信号通路MAPK signaling pathway-plant | 72 | 44 | 9.89×10-5 | map04016 |
氰氨基酸代谢Cyanoamino acid metabolism | 48 | 21 | 1.77×10-5 | map00460 |
吲哚生物碱生物合成Indole alkaloid biosynthesis | 9 | 13 | 5.27×10-5 | map00901 |
角质、木栓质和蜡的合成Cutin, suberine and wax biosynthesis | 17 | 14 | 0.000 2 | map00073 |
苯丙氨酸等合成Phenylalanine, tyrosine and tryptophan biosynthesis | 45 | 19 | 0.001 6 | map00400 |
乙醛酸和二羧酸代谢Glyoxylate and dicarboxylate metabolism | 103 | 19 | 0.001 9 | map00630 |
氨基糖核苷酸糖代谢Amino sugar and nucleotide sugar metabolism | 103 | 56 | 0.007 3 | map00520 |
表4
富集到植物激素信号转导KEGG通路上差异基因及功能描述①"
植物激素 Hormone | 基因编号 Gene number | KO ID | 基因功能描述 Function description |
生长素Auxin | DN80863_c0_g1 DN81206_c0_g2 DN64977_c0_g1 DN70584_c2_g2 DN77909_c1_g1 DN79226_c1_g1 DN67543_c0_g1 DN63863_c0_g1 | K13946 K14485 K14484 K14486 K14487 K14488 | 生长输入载体AUX1 Auxin influx carrier AUX1(AUX1) 转运响应抑制因子1 Transport inhibitor response 1 (TIR1) 生长素响应蛋白IAA Auxin responsive protein (IAA) 生长素响应因子 Auxin response factor (ARF) 生长素响应GH3基因家族 Auxin responsive GH3 gene family (GH3) SAUR家族蛋白SAUR family protein(SAUR) |
细胞分裂素Cytokinin | DN68870_c1_g5 | K14491 | 双组分响应调节器ARR-B家族 Two component response regulator ARR-B family (ARR-B) |
赤霉素Gibberellin | DN82220_c1_g1 DN82158_c1_g2 DN81860_c1_g1 DN81860_c1_g2 | K14493 K14494 | 赤霉素受体GID1 Gibberellin receptor GID1 (GID1) DELLA蛋白 DELLA protein (DELLA) |
脱落酸Abscisic acid | DN67794_c1_g1 DN59959_c0_g1 DN81200_c0_g1 DN79605_c0_g2 DN79605_c0_g3 DN83661_c2_g2 | K14496 K14497 K14498 K14432 | 脱落酸受体PYR/PYL蛋白家族Abscisic acid receptor PYR/PYL family (PYL) 蛋白磷酸酶2C Protein phosphatase 2C (PP2C) 蔗糖非酵解型蛋白激酶2 Sucrose non-fermenting1-related protein kinase 2 (SNRK2) ABA反应元件结合因子 ABA responsive element binding factor (ABF) |
乙烯Ethylene | DN75732_c0_g1 DN75455_c0_g1 DN82109_c0_g1 DN72495_c1_g1 DN72737_c2_g4 | K14510 K14513 K14514 K14516 | 丝氨酸/苏氨酸蛋白激酶CTR1 Serine/threonine-protein kinase CTR1 (CTR1) 乙烯不敏感蛋白2 Ethylene insensitive protein 2 (EIN2) 乙烯不敏感蛋白3 Ethylene insensitive protein 3 (EIN3) 乙烯应答转录因子1 Ethylene responsive transcription factor 1 (ERF1) |
油菜素内酯Brassinolide | DN80256_c0_g5 | K13416 | 油菜素内酯不敏感相关受体激酶1 Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) |
DN74700_c1_g1 | K14499 | BRI1激酶抑制子1 BRI1 kinase inhibitor 1 (BKI1) | |
DN80006_c0_g7 | K14500 | BR信号激酶BR signaling kinase (BSK) | |
DN78469_c0_g1 | K14503 | 油菜素内酯抗性因子1/2 Brassinosteroid resistant 1/2 (BZR1/2) | |
DN80006_c0_g7 | K14504 | 木糖葡萄糖基转移酶TCH4 Xyloglucosyl transferase TCH4 (TCH4) | |
DN78469_c0_g1 | K14505 | 细胞周期蛋白D3 Cyclin D3 (CYCD3) |
表5
选取qRT-PCR验证的基因编号和引物序列"
基因编号 Gene number | 基因功能描述 Function description | 引物序列 Primer sequence (5’- 3’) | KO ID |
DN82158_c1_g1 | 赤霉素受体GID1 Gibberellin receptor GID1 (GID1) | F: TGCGGGGTGAAAACAATAG R: TCAGAGTAACACGGGAAGAAAT | K14493 |
DN81200_c0_g1 | 蔗糖非酵解型蛋白激酶2 Sucrose non-fermenting1-related protein kinase 2 (SNRK2) | F: TGTTTTGCCTCTTTGATTGTTC R: CTCACCTTCTTGCCCCTCT | K14498 |
DN81546_c5_g3 | 光系统Ⅰ亚单位F Photosystem I subunit F (PSI-F) | F: AGTGAGGCAAACCATCAGAAC R: CGAGAAGCAGGAGATCAAGAA | K02694 |
DN67975_c4_g7 | 茉莉酸氨基合成酶JAR1 Jasmonic acid resistant 1 (JAR1) | F: GCTTGGCTGCTGCTTCTACA R: TCAAGGGTTCCACAACTCCAC | K14506 |
DN68587_c3_g1 | 采光复合物II叶绿素a/b结合蛋白2 Light harvesting complex II chlorophyll a/b binding protein 2 (LHCB2) | F: GCCTCCTCCAACTCTGTATCC R: CGTCTTCCCCGAAATCCT | K08913 |
DN74090_c1_g2 | 采光复合物II叶绿素a/b结合蛋白4 Light harvesting complex II chlorophyll a/b binding protein 4 (LHCB4) | F: CTGGATGGCTCCCTCGTT R: GCTGTAAGGCTGGAATGGC | K08915 |
DN68271_c1_g1 | 采光复合物II叶绿素a/b结合蛋白6 Light harvesting complex II chlorophyll a/b binding protein 6 (LHCB6) | F: GTTGCTTTTCCTACTCGGTTTA R: GCTTCTGGACTCTGGCACTAT | K08917 |
DN80256_c0_g5 | 油菜素内酯不敏感相关受体激酶1 Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) | F: CGCTGCCCTTCTATTTGCT R: GAACTTCGGGGTCCTCCTC | K13416 |
DN67597_c0_g3 | 光系统Ⅰ亚单位D Photosystem I subunit D (PSI-D) | F: GAGAATTGTGCAAGGGACTGT R: CCACCCCTCCGAACTCAT | K02692 |
DN68054_c0_g4 | TGA转录因子TGA transcription factor (TGA) | F: TGCTTGCTTTCGGGGAGT R: GCTGCTTGTGCCATTTTGTAG | K14431 |
DN81860_c1_g2 | DELLA蛋白DELLA protein (DELLA) | F: TTGCATATCTGCCTCCCTAAAT R: AATGAACCCGAAAATCGTGAC | K14494 |
18S rRNA | 18S核糖体RNA 18S ribosomal RNA | F: ACATAGTAAGGATTGACAGA R: TAACGGAATTAACCAGACA |
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