光皮桦BlBLH1基因的克隆、表达及互作蛋白筛选

doi:10.11707/j.1001-7488.20220307

Abstract

Abstract:

Objective: BLH (BEL1-like homeodomain) gene family belongs to a transcription factor widely existing in plant, and its interaction with KNOX and other transcription factors are considered to play an important regulatory role in the development of plant secondary cell wall (SCW). The aim of this study is to clone BlBLH1 gene of Betula luminifera, then analyze its expression pattern and identify proteins that can interact with BlBLH1. Method: The BlBLH1 sequence in the genome sequence of B. luminifera was first identified by blast and other software. After verification by cloning, further bioinformatic analyses, including multiple sequence alignment and phylogenetic analysis, were also carried out. Quantitative Real-time PCR (qRT-PCR) was used to analyze the expression pattern of BlBLH1 in different tissues, organs and tension wood (TW). Yeast two hybrid technology was used to screen the proteins interacting with BlBLH1, and bimolecular fluorescence complementary (BiFC) was used to verify its interactions with some identified proteins in Arabidopsis thaliana cells. Result: The full-length of BlBLH1 was 2 128 bp in length with an open reading frame (ORF) of 1 830 bp. It encoded 609 amino acids harboring three conserved domains of SKY, BEL and HD. Phylogenetic analysis showed that BlBLH1 had the closest homologous relationship with A. thaliana BLH1. Expression analysis showed that the expression level of BlBLH1 in male inflorescence was the highest, followed by that in lignified stem. During the induction of tension wood, the expression level of BlBLH1 in tension wood was significantly higher than that in control. Through yeast two hybrid, 47 proteins, including structural proteins, enzymes and transcription factors, with strong interaction with BlBLH1 were identified, and the interactions of BlBLH1 with BlKNOX4 and BlKNOX9 were also verified by BiFC in A. thaliana protoplasts. Moreover, the expression patterns of these three genes in stems and tension wood were similar. Conclusion: In this study, BlBLH1 gene is cloned from B. luminifera. Based on the results of bioinformatics, expression and protein interaction analysis, it is inferred that BlBLH1 may be involved in the process of wood formation in B. luminifera, and interacted with KNOX proteins.

Key words: BlBLH1, secondary cell wall, yeast two hybrid, bimolecular

CLC Number:

S718.46

Hebi Zhuang,Zicheng Yu,Erpei Lin,Huahong Huang. Cloning, Expression and Interaction Protein Screening of BlBLH1 Gene in Betula luminifera[J]. Scientia Silvae Sinicae, 2022, 58(3): 59-68.

Figures/Tables 8

Table 1

Fig.1

Fig.2

Fig.3

Fig.4

Table 2

The BLAST results of BlBLH1 yeast library hybridization positive clones"

编号 No.	基因编号 Gene_ID	同源基因序列号 Homolog No.	注释 Description
1	GPH67635-RA	XM_018972085.1	甲基转移酶PMT26 Methyltransferase PMT26
2	GPH79708-RA	DQ471302.1	可能的叶绿体叶绿素a/b结合蛋白 Putative chloroplast chlorophyll a/b-binding protein
3	GPH63128-RA	XM_018962628.1	F-box蛋白 F-box protein SKIP27-like
4	GPH00558-RA	XM_018953092.1	木聚糖内转葡萄糖基酶/水解酶蛋白6 Xyloglucan endotransglucosylase/hydrolase protein 6
5	GPH52393-RA	XM_018993881.1	β-半乳糖苷酶 Beta-galactosidase
6	GPH12478-RA	XM_018999934.1	L-抗坏血酸过氧化物酶6 L-ascorbate peroxidase 6
7	GPH50419-RA	XM_018990252.1	甘氨酸脱氢酶 Glycine dehydrogenase
8	GPH19320-RA	XM_018980394.1	超敏诱导反应蛋白1 Hypersensitive-induced response protein 1-like
9	GPH69606-RA	XM_024066525.1	甲酸脱氢酶 Formate dehydrogenase
10	GPH62533-RA	XM_024031608.1	过氧化物酶42 Peroxidase 42
11	GPH15474-RA	XM_018967583.1	肽基脯氨酰顺反异构酶 Peptidyl-prolyl cis-trans isomerase
12	GPH07814-RA	—	光皮桦KNOX4转录因子 BlKNOX4
13	GPH63909-RA	XM_018988520.1	丝氨酸羟甲基转移酶4 Serine hydroxymethyltransferase 4
14	GPH42621-RA	XM_018967753.1	蛋白磷酸酶2C 23 Protein phosphatase 2C 23
15	GPH08071-RA	XM_018969160.1	乙酰鸟氨酸脱乙酰酶 Acetylornithine deacetylase
16	GPH38087-RA	XM_018983680.1	组蛋白赖氨酸N-甲基转移酶2D Histone-lysine N-methyltransferase 2D
17	GPH33933-RA	XM_024050483.1	DNA导向的RNA聚合酶Ⅱ DNA-directed RNA polymerases Ⅱ
18	GPH07814-RA	XM_024065073.1	同源异型盒knotted-1样蛋白LET6 Homeobox protein knotted-1-like LET6
19	GPH77232-RA	KP711310.1	苯丙氨酸解氨酶2 Phenylalanine ammonia-lyase 2 (PAL2)
20	GPH25088-RA	XM_024029965.1	核糖核酸酶2 Ribonuclease 2
21	GPH67318-RA	XM_018996369.1	细胞色素b6-f复合铁硫亚基1 Cytochrome b6-f complex iron-sulfur subunit 1
22	GPH04217-RA	—	光皮桦KNOX9转录因子 BlKNOX9
23	GPH60229-RA	XM_018982373.1	α, α-海藻糖磷酸合酶6 Alpha, alpha-trehalose-phosphate synthase 6
24	GPH09215-RA	XM_024059006.1	EXORDIUM样蛋白 EXORDIUM-like protein
25	GPH57276-RA	XM_018982529.1	SAR1A样GTP结合蛋白 GTP-binding protein SAR1A-like
26	GPH08734-RA	XM_024042059.1	rac样GTP结合蛋白5 Rac-like GTP-binding protein 5
27	GPH77067-RA	XM_018990062.1	WD-40重复序列蛋白 WD-40 repeat-containing protein MSI4-like
28	GPH39769-RA	XM_018953914.1	65kDa微管相关蛋白8 65-kDa microtubule-associated protein 8-like
29	GPH26685-RA	XM_024016523.1	核苷水解酶15 Nudix hydrolase 15
30	GPH24400-RA	XM_018962128.1	40S核糖体蛋白 40S ribosomal protein S9-2-like
31	GPH75230-RA	—	光皮桦STM转录因子 BlSTM
32	GPH10713-RA	XM_018988639.1	类糊精硫醇蛋白酶 Thiol protease aleurain-like
33	GPH72582-RA	XM_018991497.1	天冬酰胺-tRNA连接酶 Asparagine-tRNA ligase
34	GPH50474-RA	XM_018984817.1	酮酸还原异构酶 Ketol-acid reductoisomerase
35	GPH00799-RA	XM_024032479.1	真核翻译起始因子3 Eukaryotic translation initiation factor 3
36	GPH41993-RA	XM_018966504.1	细胞色素P450 Cytochrome P450 84A1-like
37	GPH54999-RA	XM_024058733.1	钙调素 Calmodulin-7-like
38	GPH68179-RA	XM_019001046.1	sm样蛋白LSM8 Sm-like protein LSM8
39	GPH41167-RA	XM_024073889.1	脱水诱导样蛋白19 Protein DEHYDRATION-INDUCED 19-like
40	GPH04307-RA	XM_018957060.1	SWI/SNF复合亚基SWI3B SWI/SNF complex subunit SWI3B
41	GPH64726-RA	XM_024063283.1	LHCII型叶绿素a-b结合蛋白 Chlorophyll a-b binding protein of LHCII type 1-like
42	GPH19951-RA	XM_018964336.1	泛素蛋白连接酶E3 Ubiquitin-protein ligase E3
43	GPH32771-RA	XM_024035128.1	40S核糖体蛋白 40S ribosomal protein S2-3-like
44	GPH42243-RA	XM_006449315.2	休眠相关蛋白1 Dormancy-associated protein 1
45	GPH74993-RA	XM_018953684.1	外二元环裂解双加氧酶 Extradiol ring-cleavage dioxygenase-like
46	GPH43902-RA	XM_018973673.1	氨基酰化酶-1 Aminoacylase-1
47	GPH20575-RA	XM_024047220.1	GATA转录因子26 GATA transcription factor 26

Table 2

Fig.5

Fig.6

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引物名称 Primer name	引物序列(5′-3′) Primer sequence(5′-3′)	用途 Application
BlBLH1-FF	GCATCAGAATGGGATAAAGTC	基因克隆 Gene clone
BlBLH1-FR	CCATTTCCTTTTCGATCTTG	基因克隆 Gene clone
BlBLH1-F	ATGAATCCCAACTACGTGCCGTA	构建酵母杂交载体 Construction of yeast two hybrid plasmids
BlBLH1-R	GGCCACGAAATCTGGCAACAATTG
BlKNOX4-F	ATGGAAGGAGGAGTTGCTGGTAG
BlKNOX4-R	AAGGAGCATAGGGGTGCAATCCA
BlKNOX9-F	ATGCATTTGTATATCCTCATCCC
BlKNOX9-R	GTCATTCCTGAAAAGCGGTCCAT
BlSTM-F	ATGGAGAGTGGCTCTAATG
BlSTM-R	GAGCAGTGTGGGCGAAATAT
BlEF1α-QF	GAATAAGATGGATGAGCCTACCGT	荧光定量PCR Quantitative real-time PCR
BlEF1α-QR	CAGACCAGATATTGGCAGGAACT
BlBLH1-QF	ACCCCACAACCATCTCATTC
BlBLH1-QR	AGTTCTTCAATCAGGCCACG
BlKNOX4-QF	CATTGGAAGCCTTCAGAAGATATG
BlKNOX4-QR	TACAGTGACCAGGAAAATGTCATC
BlKNOX9-QF	GCTCAAGCTTCGATGGATGAACTG
BlKNOX9-QR	TGCAGAATTTCAGTCATTCCTGAG
BlBLH1-inF	AGATCTCGAGCTCAAGCTTCGATGAATCCCAACTACGTGCCG	构建BiFC载体 Construction of BiFC plasmids
BlBLH1-inR	GGTACCGTCGACTGCAGAATTTCAGGCCACGAAATCTGGCAAC
BlKNOX4-inF	AGATCTCGAGCTCAAGCTTCGATGGAAGGAGGAGTTGCTGGTAG
BlKNOX4-inR	GGTACCGTCGACTGCAGAATTTCAAAGGAGCATAGGGGTGCAAT
BlKNOX9-inF	AGATCTCGAGCTCAAGCTTCGATGGATGAACTGTATGGCATC
BlKNOX9-inR	GGTACCGTCGACTGCAGAATTTCAGTCATTCCTGAAAAGCG
BlSTM-inF	AGATCTCGAGCTCAAGCTTCGATGGAGAGTGGCTCTAATG
BlSTM-inR	GGTACCGTCGACTGCAGAATTTCAGAGCAGTGTGGGCGAAAT

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Cloning, Expression and Interaction Protein Screening of BlBLH1 Gene in Betula luminifera

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