Bioinformatics Analysis of CbuDELLAs Gene Family and Functional Analysis of CbuGRAS9

doi:10.7525/j.issn.1673-5102.2024.01.016

Abstract

Abstract:

To provide theoretical basis for genetic improvement of reproductive regulatory traits of Catalpa bungei， DELLAs family genes were identified and the function of CbuGRAS9 was analyzed. Based on the genomic data of Catalpa bungei， five CbuDELLAs genes homologous to Arabidopsis thaliana were identified and cloned. ExPASy， SWISS-MODEL， Plant-mPloc， PlantCare and other online tools were used to predict the isoelectric point， protein structure， sub-cellular localization and promoter cis-acting elements of CbuDELLAs protein. The expression differences of CbuDELLAs were analyzed by using Catalpa ‘Bairihua’ and Catalpa bungei ‘Luoqiu No.1’ as materials， and the molecular function of CbuGRAS9 was confirmed by heterologus transformation in Arabidopsis thaliana， and the proteins interacted with CbuGRAS9 were screened by yeast two-hybrid library. The results showed that the amino acid number of the five CbuDELLAs proteins ranged from 455 to 588 aa， the relative molecular weight of the proteins ranged from 5.04 to 6.43 kDa， and the isoelectric point value ranged from 4.81 to 5.14. All CbuDELLAs proteins contained DELLA and GRAS conserved domains and are hydrophilic proteins. Sub-cellular localization prediction showed that CbuDELLAs protein was located in the nucleus. The analysis of promoter cis-acting elements showed that the five promoter regions of DELLAs all contained cis-acting elements involved in gibberellin reaction. The results of qRT-PCR showed that the expression of CbuDELLAs in Catalpa ‘Bairihua’ were significantly higher than that in Catalpa bungei ‘Luoqiu No.1’， and CbuGRAS9 was the most significantly gene， and the flowering time of CbuGRAS9 transgenic plants were significantly delayed. Proteins interacted with CbuGRAS9 were mainly concentrated in metabolic pathways such as ribosome， amino acid synthesis， secondary metabolism， photosynthesis and TCA cycle.

Key words: Catalpabungei, DELLAs, bioinformatics analysis, function analysis, interacting proteins

CLC Number:

Q949.777.9

Shanshan WANG, Rui WANG, Erqin FAN, Pengyue FU, Guanzheng QU, Nan WANG. Bioinformatics Analysis of CbuDELLAs Gene Family and Functional Analysis of CbuGRAS9[J]. Bulletin of Botanical Research, 2024, 44(1): 139-151.

Figures/Tables 10

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References 49

1	雍伟东，种康，许智宏，等.高等植物开花时间决定的基因调控研究［J］.科学通报，2000，45（5）：455-466.
	YONG W D， ZHONG K， XU Z H，et al.The regulation of flowering time determination in higher plants［J］.Chinese Science Bulletin，2000，45（5）：455-466.
2	孙昌辉，邓晓建，方军，等.高等植物开花诱导研究进展［J］.遗传，2007，29（10）：1182-1190.
	SUN C H， DENG X J， FANG J，et al.An overview of flowering transition in higher plants［J］.Hereditas（Beijing），2007，29（10）：1182-1190.
3	IZAWA T.What is going on with the hormonal control of flowering in plants？［J］.The Plant Journal，2021，105（2）：431-445.
4	周琴，张思思，包满珠，等.高等植物成花诱导的分子机理研究进展［J］.分子植物育种，2018，16（11）：3681-3692.
	ZHOU Q， ZHANG S S， BAO M Z，et al.Research of molecular mechanisms of flowering induction in higher plants［J］.Molecular Plant Breeding，2018，16（11）：3681-3692.
5	杨小凤，李小蒙，廖万金.植物开花时间的遗传调控通路研究进展［J］.生物多样性，2021，29（6）：825-842.
	YANG X F， LI X M， LIAO W J.Advances in the genetic regulating pathways of plant flowering time［J］.Biodiversity Science，2021，29（6）：825-842.
6	FUKAZAWA J， OHASHI Y， TAKAHASHI R，et al.DELLA degradation by gibberellin promotes flowering via GAF1-TPR-dependent repression of floral repressors in Arabidopsis ［J］.The Plant Cell，2021，33（7）：2258-2272.
7	YOSHIDA H， HIRANO K， SATO T，et al.DELLA protein functions as a transcriptional activator through the DNA binding of the indeterminate domain family proteins［J］.Proceedings of the National Academy of Sciences of the United States of America，2014，111（21）：7861-7866.
8	高秀华，傅向东.赤霉素信号转导及其调控植物生长发育的研究进展［J］.生物技术通报，2018，34（7）：1-13.
	GAO X H， FU X D.Research of gibberellin signal transduction and regulation of plants growth and development［J］.Biotechnology Bulletin，2018，34（7）：1-13.
9	BAO S J， HUA C M， SHEN L S，et al.New insights into gibberellin signaling in regulating flowering in Arabidopsis ［J］.Journal of Integrative Plant Biology，2020，62（1）：118-131.
10	GAO X H， HUANG X Z， XIAO S L，et al.Evolutionarily conserved DELLA-mediated gibberellin signaling in plants［J］.Journal of Integrative Plant Biology，2008，50（7）：825-834.
11	赵春丽.苋菜AtGAI基因克隆及功能分析［D］.福州：福建农林大学，2020.
	ZHAO C L.Cloning and functional analysis of AtGAI from Amaranth ［D］.Fuzhou：Fujian Agriculture and Forestry University，2020.
12	MURASE K， HIRANO Y， SUN T P，et al.Gibberellin-induced DELLA recognition by the gibberellin receptor GID1［J］.Nature，2008，456（7221）：459-463
13	李强，吴建明，梁和，等.高等植物赤霉素生物合成及其信号转导途径［J］.生物技术通报，2014，（10）：16-22.
	LI Q， WU J M， LIANG H，et al.Biosynthesis and signal transduction pathways of gibberellin in higher plants［J］.Biotechnology Bulletin，2014，（10）：16-22.
14	FUKAZAWA J， TERAMURA H， MURAKOSHI S，et al.DELLAs function as coactivators of GAI-ASSOCIATED FACTOR1 in regulation of gibberellin homeostasis and signaling in Arabidopsis ［J］.The Plant Cell，2014，26（7）：2920-2938
15	XUE H D， GAO X， HE P，et al.Origin，evolution，and molecular function of DELLA proteins in plants［J］.The Crop Journal，2022，10（2）：287-299.
16	岳川，曾建明，曹红利，等.高等植物赤霉素代谢及其信号转导通路［J］.植物生理学报，2012，48（2）：118-128.
	YUE C， ZENG J M， CAO H L，et al.Metabolism and signal transduction pathways of gibberellin in higher plants［J］.Plant Physiology Journal，2012，48（2）：118-128.
17	JIANG C F， FU X D.GA action：turning on de-DELLA repressing signaling［J］.Current Opinion in Plant Biology，2007，10（5）：461-465.
18	CONTI L， NELIS S， ZHANG C J，et al.Small ubiquitin-like modifier protein SUMO enables plants to control growth independently of the phytohormone gibberellin［J］.Developmental Cell，2014，28（1）：102-110.
19	ACHARD P， GENSCHIK P.Releasing the brakes of plant growth：how GAs shutdown DELLA proteins［J］.Journal of Experimental Botany，2009，60（4）：1085-1092.
20	GONÇALVES N M， FERNANDES T， NUNES C，et al.SUMOylation of rice DELLA SLR1 modulates transcriptional responses and improves yield under salt stress［J/OL］.bioRxiv（2020-03-10）［2023-05-02］..
21	DAVIÈRE J M， ACHARD P.A pivotal role of DELLAs in regulating multiple hormone signals［J］.Molecular Plant，2016，9（1）：10-20.
22	黄昊，宫铭，张金凤，等.珍珠黄杨矮化基因BsGAI2的克隆及功能分析［J］.植物遗传资源学报，2018，19（1）：150-156.
	HUANG H， GUO M， ZHANG J F，et al.Cloning and function analysis on BsGAI2 gene from Buxus sinica var.parvifolia M.Cheng［J］.Journal of Plant Genetic Resources，2018，19（1）：150-156.
23	王智.楸树开花突变体“百日花”的分子调控机制［D］.北京：中国林业科学研究院，2019.
	WANG Z.The mechanism of the flowering mutant “Bai ri hua” in Catalpa bungei ［D］.Beijing：Chinese Academy of Forestry，2019.
24	麻文俊，杨桂娟，王军辉，等.楸树不同单株花性状变异分析［J］.植物研究，2020，40（3）：386-393.
	MA W J， YANG J J， WANG J H，et al.Analysis of variation in flower traits of different individual of Catalpa bungei ［J］.Bulletin of Botanical Research，2020，40（3）：386-393.
25	赵鲲，王军辉，麻文俊，等.杂交新品种“百日花”楸树选育与栽培试验研究［J］.安徽林业科技，2020，46（6）：15-18.
	ZHAO K， WANG J H， MA W J，et al.Research on the breeding and cultivation experiment of the hybrid variety “Bai ri hua” Catalpa bungei ［J］.Anhui Forestry Technology，2020，46（6）：15-18.
26	WANG Z， MA W J， ZHU T Q，et al.Multi-omics sequencing provides insight into floral transition in Catalpa bungei.C.A.Mey［J］.BMC Genomics，2020，21（1）：508.
27	WANG Z， ZHU T Q， MA W J，et al.Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei ［J］.BMC Plant Biology，2020，20（1）：105
28	LANGMEAD B， SALZBERG S L.Fast gapped-read alignment with Bowtie 2［J］.Nature Methods，2012，9（4）：357-359.
29	WANG R， ZHANG Y， WANG C，et al.ThNAC12 from Tamarix hispida directly regulates ThPIP2；5 to enhance salt tolerance by modulating reactive oxygen species［J］.Plant Physiology and Biochemistry，2021，163：27-35.
30	KECHIN A， BOYARSKIKH U， KEL A，et al.cutPrimers：a new tool for accurate cutting of primers from reads of targeted next generation sequencing［J］.Journal of Computational Biology，2017，24（11）：1138-1143.
31	QUINLAN A R， HALL I M.BEDTools：a flexible suite of utilities for comparing genomic features［J］.Bioinformatics，2010，26（6）：841-842.
32	KANEHISA M， GOTO S， KAWASHIMA S，et al.The KEGG databases at GenomeNet［J］.Nucleic Acids Research，2002，30（1）：42-46.
33	于磊.水曲柳DELLA家族基因应答低温胁迫及参与被动休眠调控研究［D］.哈尔滨：东北林业大学，2021.
	YU L. DELLA family in Fraxinus mandshurica to low temperature stress and involvement in passive dormancy regulation［D］.Harbin：Northeast Forestry University，2021.
34	蔡健宇，余婧，张景云，等.烟草DELLA基因家族的克隆与表达模式分析［J］.烟草科技，2022，55（10）：10-18.
	CAI J Y， YU J， ZHANG J Y，et al.Expression pattern analysis of DELLA family in tobacco ［J］.Tobacco Science & Technology，2022，55（10）：10-18.
35	ZHONG G Y， YANG Y Z.Characterization of grape gibberellin Insensitive1 mutant alleles in transgenic Arabidopsis ［J］.Transgenic Research，2012，21（4）：725-741.
36	温玮.棉花DELLA蛋白的基因克隆与功能的初步鉴定［D］.海口：海南大学，2010.
	WEN W.Cloning and functional identification of DELLA protein in cotton［D］.Haikou：Hainan University，2010.
37	张晶星，马彦广，王辉丽，等.油松JAZ基因家族特征及其与DELLA蛋白互作的功能域鉴定［J］.北京林业大学学报，2022，44（12）：12-22.
	ZHANG J X， MA Y G， WANG H L，et al.Characterization of JAZ family of Pinus tabulaeformis and identification of functional domain interacting with DELLA Protein［J］.Journal of Beijing Forestry University，2022，44（12）：12-22.
38	周鹏，李钱峰，熊敏，等.DELLA蛋白介导的激素互作调控植物生长发育研究进展［J］.植物生理学报，2020，56（4）：661-671.
	ZHOU P，LI Q F，XIONG M，Advances in DELLA protein-mediated phytohormonal crosstalk in regulation of plant growth and development［J］.Plant Physiology Journal，2020，56（4）：661-671.
39	DAVIÈRE J M， ACHARD P.Gibberellin signaling in plants［J］.Development，2013，140（6）：1147-1151.
40	FENG S H， MARTINEZ C， GUSMAROLI G，et al.Coordinated regulation of Arabidopsis thaliana development by light and gibberellins［J］.Nature，2008，451（7177）：475-479.
41	CAO D N， CHENG H， WU W，et al.Gibberellin mobilizes distinct DELLA-dependent transcriptomes to regulate seed germination and floral development in Arabidopsis ［J］.Plant Physiology，2006，142（2）：509-525.
42	MOURADOV A， CREMER F， COUPLAND G.Control of flowering time：interacting pathways as a basis for diversity［J］.The Plant Cell，2002，14（）：S111-S130.
43	FUKAZAWA J， MIYAMOTO C， ANDO H，et al.DELLA-GAF1 complex is involved in tissue-specific expression and gibberellin feedback regulation of GA20ox1 in Arabidopsis ［J］.Plant Molecular Biology，2021，107（3）：147-158.
44	SUN T P.The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants［J］.Current Biology，2011，21（9）：R338-R345.
45	岳溶榕.梨单性结实相关DELLA基因的功能分析［D］.杨凌：西北农林科技大学，2019.
	YUE R R.Functional analysis of DELLA related to parthenocarpy in Pear［D］.Yangling：Northwest A&F University，2019.
46	YU S， GALVÃO V C， ZHANG Y C，et al.Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMOSA promoter binding-like transcription factors［J］.The Plant Cell，2012，24（8）：3320-3332.
47	段怡红，阎媛媛，陈丽婷，等.陆地棉开花时间相关基因GhMYB44的克隆与功能验证［J］.中国农业科技导报，2020，22（12）：29-38.
	DUAN Y H， YAN Y Y， CHEN L T，et al.Cloning and functional verification of flowering time related gene GhMYB44 in cotton［J］.Journal of Agricultural Science and Technology，2020，22（12）：29-38.
48	李巍.水稻MYB21-like基因Os01g45090和Os05g49310的功能研究［D］.哈尔滨：东北林业大学，2015.
	LI W.Functional studies on the MYB21-like genes Os01g45090 and Os05g49310 in rice［D］.Harbin：Northeast Forestry University，2015.
49	冯震.陆地棉开花相关基因GhAP1互作基因筛选及其功能鉴定［D］.北京：中国农业科学院，2021.
	FENG Z.Functional identification of GhAP1 interaction genes related to flowering in cotton ［D］.Beijing：Chinese Academy of Agricultural Sciences，2021.

拟南芥蛋白名称 Protein names in Arabidopsis thaliana	楸树蛋白名称 Protein names in Catalpa bungei	编码区长度 Encoding length/bp	氨基酸数目 Amino acid number	等电点 Isoelectric point	相对分子质量 Molecular weight/kDa	亚细胞定位 Sub-cellular localization
AtGAI（AT1G14920）	CbuGRAS22	1755	585	4.81	6.41	细胞核Nucleus
AtRGA1（AT2G01570）	CbuGRAS1	1767	588	4.99	6.43	细胞核Nucleus
AtRGL2（AT3G03450）	CbuGRAS9	1662	553	4.93	6.01	细胞核Nucleus
AtGAI（AT1G14920）	CbuGRAS2	1368	455	5.14	5.04	细胞核Nucleus
AtRGA1（AT2G01570）	CbuGRAS32	1755	584	5.08	6.40	细胞核Nucleus

楸树蛋白名称 Protein names in Arabidopsis thaliana	α螺旋 α-helix		β转角 β-sheet		无规则卷曲 Random coil		延伸链 Extended strand
楸树蛋白名称 Protein names in Arabidopsis thaliana	数量 Number	比例 Percent/%	数量 Number	比例 Percent/%	数量 Number	比例 Percent/%	数量 Number	比例 Percent/%
CbuGRAS1	265	45.07	28	4.76	240	40.82	55	9.35
CbuGRAS2	257	56.48	20	4.40	130	28.57	48	10.55
CbuGRAS9	273	49.37	26	4.70	197	35.62	57	10.31
CbuGRAS22	269	45.98	33	5.64	228	38.97	55	9.40
CbuGRAS32	269	46.06	28	4.79	235	40.24	52	8.90

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[2]	Denggao LI, Rui LIN, Qinghui MU, Na ZHOU, Yanru ZHANG, Wei BAI. Identification and Analysis of the Potato StCRKs Gene Family and Expression Patterns in Response to Stress Signals [J]. Bulletin of Botanical Research, 2022, 42(6): 1033-1043.
[3]	He CHENG, Shuanghui TIAN, Yang ZHANG, Cong LIU, De’an XIA, Zhigang WEI. Genome-wide Identification and Expression Analysis of nsLTP Gene Family in Populus trichocarpa [J]. Bulletin of Botanical Research, 2022, 42(3): 412-423.
[4]	Yuning Yang, Hao Dong, Shiwei Dong, Nairui Wang, Yue Song, Hanguo Zhang, Shujuan Li. Cloning and Expression Analysis of Transcription Factor LobHLH34 from Larix olgensis [J]. Bulletin of Botanical Research, 2022, 42(1): 112-120.
[5]	Jiaming Zhao, Erqin Fan, Yi Liu, Zhi Wang, Junhui Wang, Guanzheng Qu. Cloning and Bioinformatics Analysis of CbuATX1,CbuATX1-like and CbuATX2 Genes from Catalpa bungei [J]. Bulletin of Botanical Research, 2022, 42(1): 47-61.
[6]	Shu-Ping PENG, Cheng-Ming DONG, Yun-Hao ZHU. Cloning and Expression Analysis of Two Key Genes of Jasmonic Acid Synthesis in Response to Endophytic Infection from Rehmannia glutinosa [J]. Bulletin of Botanical Research, 2021, 41(2): 294-301.
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[9]	WANG Qi, XU Zhi-Ru, CHEN Jin-Yuan, ZHANG Shuang, HUANG Jia-Huan, LIU Guan-Jun. Identification and Expression Analysis of Heavy Metal-associated Isoprenylated Plant Proteins(HIPPs) Genes in Populus [J]. Bulletin of Botanical Research, 2019, 39(6): 935-946.
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