Molecular Mechanism of Transcriptional Factor AtbHLH68 in Regulating Cell Wall Development by Transcriptome Analysis

doi:10.13560/j.cnki.biotech.bull.1985.2023-0091

Abstract

Abstract:

Cell wall is a plant-specific structure and plays important role in morphogenesis, water and nutrients transportation, and biotic and abiotic resistance. Previous studies have shown that AtbHLH68 as a member of the 10th bHLH protein subfamily, is expressed in the vascular tissue of Arabidopsis thaliana stem. To investigate its molecular mechanism of AtbHLH68 in cell wall development, the estradiol-induced pER8-AtbHLH68 expression system was established in Arabidopsis thaliana. Quantitative real-time PCR（RT-qPCR）results showed that 10 μmol/L estradiol treatment for 4 h can effectively induce the expression of AtbHLH68, and the expression of AtbHLH68 gradually increased with the prolongation of induction time, reaching 29 times after 8 h. The differentially expressed genes（DEGs）induced by AtbHLH68 in Arabidopsis stems were detected by transcriptome analysis. Compared with the control group, after 8 h of treatment with 10 μmol/L estradiol, a total of 2 334 DEGs were detected, among which 831 genes were significantly up-regulated and 1 503 genes were significantly down-regulated. The significantly enrichment GO terms were mainly related to cell wall components, defense response, pectin modification and degradation and hormone response. KEGG analysis showed that DEGs involved in the processes such as pectin modification and lignin biosynthetic metabolism. These results indicated that the DEGs involved in the above processes may be regulated by AtbHLH68 directly or indirectly. This study provided theoretical evidences to clarify the molecular mechanism of transcription factor AtbHLH68 in cell wall development.

Key words: induced expression, AtbHLH68, transcriptome, cell wall

LIN Hong-yan, GUO Xiao-rui, LIU Di, LI Hui, LU Hai. Molecular Mechanism of Transcriptional Factor AtbHLH68 in Regulating Cell Wall Development by Transcriptome Analysis[J]. Biotechnology Bulletin, 2023, 39(9): 105-116.

Figures/Tables 10

References 37

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基因ID Unigene ID	引物名称 Primer name	引物序列 Primer sequence（5'-3'）
AT3G18780	Actin2-F	CGTATGAGCAAGGAGATCAC
AT3G18780	Actin2-R	CACATCTGTTGGAAGGTGCT
AT4G29100	bHLH68-F	ACCACGGATGATGCCCACT
AT4G29100	bHLH68-R	CCATCATCAATCCACCCAAAA
AT1G56100	PMEI-F	TGGTTGCCTCGTTGGATG
AT1G56100	PMEI-R	TGTGTAGGTTGACCATTGATGTAAA
AT2G47030	VGD1-F	GCACAGTCCAGGTTGAGTCCGAAGG
AT2G47030	VGD1-R	GGTCTCCGTTCACTCTGATAGCCAC
AT4G34230	CAD5-F	GTTGCCTCGTTGGATGTTGC
AT4G34230	CAD5-R	CGCCTTGTGTAGGTTGACCATTGAT
AT5G62350	PMEI-F	ACTAAGCCGAGCCCAATCC
AT5G62350	PMEI-R	GTATCGTTCATCTCCTCGACGCAAT
AT3G56400	WRKY70-F	GCGATAGTCGGAAAAGATTGG
AT3G56400	WRKY70-R	TGGGAATTTGGCATTAAGAA
AT1G30760	BBE13-F	TCGGCGTAATACTTGCCTGGAAAAT
AT1G30760	BBE13-R	TATCAGCGACTTGTTGCCACTT
	YXM-pER8-bHLH68-F	GGTAATGCCATGTAATATGCTCG
	YXM-pER8-bHLH68-R	CATCATCAATCCACCCAAAA

基因ID Unigene ID	引物名称 Primer name	引物序列 Primer sequence（5'-3'）
AT3G18780	Actin2-F	CGTATGAGCAAGGAGATCAC
AT3G18780	Actin2-R	CACATCTGTTGGAAGGTGCT
AT4G29100	bHLH68-F	ACCACGGATGATGCCCACT
AT4G29100	bHLH68-R	CCATCATCAATCCACCCAAAA
AT1G56100	PMEI-F	TGGTTGCCTCGTTGGATG
AT1G56100	PMEI-R	TGTGTAGGTTGACCATTGATGTAAA
AT2G47030	VGD1-F	GCACAGTCCAGGTTGAGTCCGAAGG
AT2G47030	VGD1-R	GGTCTCCGTTCACTCTGATAGCCAC
AT4G34230	CAD5-F	GTTGCCTCGTTGGATGTTGC
AT4G34230	CAD5-R	CGCCTTGTGTAGGTTGACCATTGAT
AT5G62350	PMEI-F	ACTAAGCCGAGCCCAATCC
AT5G62350	PMEI-R	GTATCGTTCATCTCCTCGACGCAAT
AT3G56400	WRKY70-F	GCGATAGTCGGAAAAGATTGG
AT3G56400	WRKY70-R	TGGGAATTTGGCATTAAGAA
AT1G30760	BBE13-F	TCGGCGTAATACTTGCCTGGAAAAT
AT1G30760	BBE13-R	TATCAGCGACTTGTTGCCACTT
	YXM-pER8-bHLH68-F	GGTAATGCCATGTAATATGCTCG
	YXM-pER8-bHLH68-R	CATCATCAATCCACCCAAAA

样品名 Sample	原始序列数 Raw reads/M	高质量序列数 Clean reads/M	Q20/%	Q30/%	比对到参考基因组序列数 No. of alignment reference genome sequences/%	唯一参考序列数 No. of unique alignment sequences/%
4 h	47.33	45.22	98.02	94.51	96.83	95.08
6 h	45.57	43.95	97.83	93.99	97.12	95.35
8 h	45.57	44.01	97.78	93.86	96.74	94.97
Control	47.33	45	97.9	94.18	96.13	94.11

样品名 Sample	原始序列数 Raw reads/M	高质量序列数 Clean reads/M	Q20/%	Q30/%	比对到参考基因组序列数 No. of alignment reference genome sequences/%	唯一参考序列数 No. of unique alignment sequences/%
4 h	47.33	45.22	98.02	94.51	96.83	95.08
6 h	45.57	43.95	97.83	93.99	97.12	95.35
8 h	45.57	44.01	97.78	93.86	96.74	94.97
Control	47.33	45	97.9	94.18	96.13	94.11

基因表达Gene expression	基因号Gene ID	Log₂x	P值P Value	描述Description
下调基因	AT2G06850	-1.06394221	1.85E-50	Xyloglucan endotransglucosylase/hydrolase 4
Down-regulated gene	AT2G36870	-1.266714752	3.34E-19	Xyloglucan endotransglucosylase/hydrolase 32
	AT4G25820	-2.74819285	1.28E-06	Xyloglucan endotransglucosylase/hydrolase 14
	AT5G57530	-5.654911378	8.25E-28	Xyloglucan endotransglucosylase/hydrolase 12
	AT5G57540	-3.641717279	7.07E-17	Xyloglucan endotransglucosylase/hydrolase 13
	AT1G71695	-1.298988679	3.66E-22	Peroxidase superfamily protein
	AT1G30870	-3.50779464	6.16E-08	Peroxidase 7
	AT3G49110	-2.420843121	6.80E-11	Peroxidase 33
	AT3G50990	-2.306246473	8.33E-06	Peroxidase 36
	AT4G37980	-1.756033681	3.73E-18	Cinnamyl-alcohol dehydrogenase 7
	AT2G47030	-2.29336778	2.45E-142	Pectin methylesterase 4
	AT2G47040	-2.786313785	0	Vanguard1, VGD1
	AT3G10720	-2.090197809	2.92E-13	Pectin methylesterase 25
	AT3G62170	-3.528498693	4.35E-207	Pectin methylesterase 37
	AT4G15980	-2.788495895	1.82E-05	Pectin methylesterase 43
	AT5G07410	-4.717215431	2.64E-240	Pectin methylesterase 48
	AT3G05610	-2.626956589	1.03E-120	Pectin methylesterase 21
	AT2G26440	-2.370550991	3.78E-132	Pectin methylesterase 12
	AT3G06830	-2.258402957	2.42E-15	Plant invertase/pectin methylesterase inhibitor superfamily
	AT5G07420	-4.837943242	2.77E-08	Pectin lyase-like superfamily protein
	AT5G07430	-2.606610505	7.12E-169	Pectin lyase-like superfamily protein
	AT3G17060	-2.165538573	6.39E-22	Pectin lyase-like superfamily protein
	AT1G14420	-2.298373367	3.91E-46	Pectin lyase-like superfamily protein
	AT2G02720	-2.19730144	4.49E-24	Pectin lyase-like superfamily protein
	AT3G01270	-2.71241092	0	Pectin lyase-like superfamily protein
	AT5G15110	-2.336194664	2.35E-29	Pectate lyase family protein
	AT5G48900	-6.312882955	1.10E-22	Pectin lyase-like superfamily protein
	AT5G55720	-3.135159583	4.76E-11	Pectin lyase-like superfamily protein
	AT3G45860	-1.803254412	3.34E-18	Cysteine-rich RLK（receptor-like protein kinase）4
	AT4G23170	-1.533408039	1.62E-109	Cysteine-rich RLK（receptor-like protein kinase）9
	AT2G40750	-1.152515323	8.63E-64	WRKY DNA-binding protein 54
	AT2G46400	-1.299919474	3.63E-61	WRKY DNA-binding protein 46
	AT3G56400	-1.40629482	0	WRKY DNA-binding protein 70
	AT4G23810	-1.452162691	1.12E-65	WRKY DNA-binding protein 53
	AT5G24780	-1.294335074	0	Vegetative storage protein 1
	AT3G16470	-1.212665758	0	A JA-responsive gene that coordinates with GRP 7
	AT2G32690	-2.181267688	6.01E-32	Glycine-rich protein 23
	AT1G20510	-1.035196623	4.70E-93	OPC-8:CoA ligase involved in jasmonic acid biosynthesis
上调基因	AT1G11545	2.054076673	1.34E-06	Xyloglucan endotransglucosylase/hydrolase 8
Up-regulated gene	AT3G48580	3.262777493	3.34E-61	Xyloglucan endotransglucosylase/hydrolase 11
	AT4G14130	1.838579331	4.09E-18	Xyloglucan endotransglucosylase/hydrolase 15
	AT4G37800	2.408582477	8.79E-21	Xyloglucan endotransglucosylase/hydrolase 7
	AT1G30700	3.103198888	3.07E-254	FAD-binding berberine family protein, AtBBE 7
	AT1G30720	3.04026387	2.09E-22	FAD-binding berberine family protein, AtBBE 10
	AT1G30730	2.767165832	8.28E-09	FAD-binding berberine family protein, AtBBE 11
	AT4G20800	3.940837928	8.47E-14	FAD-binding berberine family protein, AtBBE 17
	AT5G44390	2.190683562	4.52E-05	FAD-binding berberine family protein, AtBBE 25
	AT5G44400	2.779652634	0	FAD-binding berberine family protein, AtBBE 26
	AT1G30760	1.250535773	3.26e-210	Berberine bridge enzyme-like 13, AtBBE 13
	AT2G34790	2.036462643	1.06E-182	Berberine bridge enzyme-like 15, AtBBE 15
	AT4G36430	2.677849798	3.42E-36	Peroxidase superfamily protein
	AT3G21770	1.766366511	5.33E-07	Peroxidase superfamily protein
	AT3G28200	1.80094768	1.06E-34	Peroxidase superfamily protein
	AT1G05260	1.600816452	2.62E-31	Peroxidase 3
	AT5G05340	2.40141963	1.48E-80	Peroxidase 52
	AT5G60020	1.132349101	5.96E-135	Laccase 17
	AT4G34230	1.168213629	0	Cinnamyl alcohol dehydrogenase 5
	AT4G37990	1.401205226	3.90E-06	Cinnamyl-alcohol dehydrogenase 8
	AT1G11590	2.950611973	1.48E-19	Pectin methylesterase 19
	AT5G09760	1.08363098	2.08E-22	Pectin methylesterase 15
	AT1G14890	1.984238146	0	Plant invertase/pectin methylesterase inhibitor superfamily protein
	AT5G62350	1.901654358	0	Plant invertase/pectin methylesterase inhibitor superfamily protein
	AT1G62770	1.620506618	3.20E-05	Pectin methyleseterase inhibitor 19
	AT1G56100	1.25	0	Plant invertase/pectin methylesterase inhibitor 14
	AT3G47380	1.787395021	2.75E-23	Plant invertase/pectin methylesterase inhibitor 11
	AT3G27400	2.985665084	5.10E-106	Pectin lyase-like superfamily protein
	AT3G53190	1.145912382	1.86E-14	Pectin lyase-like superfamily protein
	AT4G13710	1.186011986	2.50E-09	Pectin lyase-like superfamily protein
	AT4G24780	1.1622914	1.26E-84	Pectate lyase 19
	AT5G04310	1.318331844	6.35E-11	Pectate lyase like 12