Functional Study and Validation of Transcriptional Coactivator PwMBF1c in Picea wilsonii

doi:10.13560/j.cnki.biotech.bull.1985.2022-0812

Abstract

Abstract:

We cloned PwMBF1c, a candidate gene in response to drought stress based on the transcriptomes of Picea wilsonii under drought stress and verified its biological function in order to explore the drought tolerance mechanism of P. wilsonii and to provide genetic resources for breeding excellent resistant forest varieties. RT-qPCR was used to analyze the expression profile of PwMBF1c to drought, high temperature, low temperature and hormone treatments. Transiently transformed tobacco leaf and onion epidermal cells were used to detect the subcellular localization of PwMBF1c. The transcriptional activity of PwMBF1c was verified by yeast one-hybrid. The function of PwMBF1c was verified in Arabidopsis thaliana and potato systems. PwMBF1c was mainly expressed in mature leaves, and its transcription level was strongly up-regulated by drought, high temperature and salicylic acid（SA）treatment. The C-terminus of PwMBF1c showed transcriptional activation activity, but not for the N-terminus or full-length of PwMBF1c. PwMBF1c was localized in cell membrane, cytoplasm and nucleus, and overexpression of PwMBF1c significantly increased drought tolerance in both Arabidopsis and potato. Under drought stress, compared with the control（Col-0 and VC）, PwMBF1c-L1 and PwMBF1c-L2 lines showed higher survival rate, higher chlorophyll content and lower MDA content. After PEG treatment, PwMBF1c significantly increased seedling height compared with wild-type potato（Y5）. The expression of PwMBF1c significantly increased by multiple abiotic stresses such as drought. PwMBF1c is mainly localized in cell membrane, cytoplasm and nucleus, and the C-terminus of PwMBF1c showed transcriptional activation activity. PwMBF1c significantly enhanced the drought tolerance of A. thaliana and potato.

Key words: Picea wilsonii, MBF1c coactivator, stress response, gene expression, drought tolerance

LIU Kui, LI Xing-fen, YANG Pei-xin, ZHONG Zhao-chen, CAO Yi-bo, ZHANG Ling-yun. Functional Study and Validation of Transcriptional Coactivator PwMBF1c in Picea wilsonii[J]. Biotechnology Bulletin, 2023, 39(5): 205-216.

Figures/Tables 9

Table 1 Primer sequences

用途Use	引物名称Primer name	序列Sequence
RT-qPCR	AtActin-F	GGTAACATTGTGCTCAGTGGTGG
	AtActin-R	AACGACCTTAATCTTCATGCTGC
	PwEF1α-RT-F	AACTGGAGAAGGAACCCAAG
	PwEF1α-RT-R	AACGACCCAATGGAGGATAC
	PwMBF1c-RT-F	ATGCCGAGCCGAACGAA
	PwMBF1c-RT-R	AACCGCCTTGGGATCACG
载体构建 Vector construction	pEASY-T1-PwMBF1c-F	GAAGGATGCCGAGCCGAA
	pEASY-T1-PwMBF1c-R	GATGACGACAAGCAATTAGTGAGA
	pGBKT7-PwMBF1c-F	CCGGAATTCATGCCGAGCCGAACGAAC
	pGBKT7-PwMBF1c-R	TCCCCCGGGGGTGGACTGATGACGACAAGCAATTA
	pGBKT7-PwMBF1c-C-F	CCGGAATTCGATTCGACATGCTATACAGAAGGC
	pGBKT7-PwMBF1c-C-R	TCCCCCGGGGGTGGACTGATGACGACAAGCAATTA
	pGBKT7-PwMBF1c-N-F	CCGGAATTCATGCCGAGCCGAACGAAC
	pGBKT7-PwMBF1c-N-R	TCCCCCGGGGGGCCTTCTGTATAGCATGTCGAATC

Fig. 1 PwMBF1c protein sequence alignment and phylog-enetic tree

Fig. 2 Expressions of PwMBF1c under different stress conditions and hormone treatments

Fig. 3 Subcellular localization of PwMBF1c GFP: pCM1205 empty vector. PwMBF1c-GFP: Fusion protein of PwMBF1c and GFP tag. A: Subcellular localization of PwMBF1c in tobacco leaves. B: Subcellular localization of PwMBF1c in onion. The bar in the image: 50 µm

Fig. 4 Identification of PwMBF1c transcriptional activity

Fig. 5 Overexpression of PwMBF1c significantly improved the drought tolerance of Arabidopsis Col-0: Wild type; VC: empty vector; L1, L2: overexpressing lines of PwMBF1c. A: Phenotypes of different PwMBF1c transgenic lines under drought treatment. B: Relative expressions of PwMBF1c in different lines. C: Statistics of survival rates after drought treatment. D, E: MDA content and chlorophyll content of isolated leaves under drought treatment

Fig. 6 Germination rates of different lines of Arabidopsis under drought stress A: Germination status of different lines treated with 0/100/200/300 mmol/L mannitol. B-E: Germination rates of different lines treated with 0,100,200, and 300 mmol/L mannitol.

Fig. 7 Root length of overexpressing PwMBF1c in Arabidopsis A: Root length growths of different lines treated with mannitol. B: Root length statistics of different lines treated with mannitol

Fig. 8 Drought phenotypes of different potato lines Y5: Wild type. A: Growth status of different lines under PEG treatment. B: Seedling heights of different lines under PEG treatments

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