Cloning and Functional Identification of BnNF-YA1 in Brassica napus L.

doi:10.13560/j.cnki.biotech.bull.1985.2021-0959

Abstract

Abstract:

NF-YA is one of the three subunits of NF-Y（nuclear factor Y）transcription factors,which is involved in plant growth and development,stress response,and interaction with microorganisms in plants. The bioinformatics and expression pattern analysis of cloned BnNF-YA1 were carried out. The coding sequence of the BnNF-YA1 was isolated and cloned. The overexpression vector of the BnNF-YA1 was constructed and transformed into rapeseed. The positive transgenic plants were identified,and the performances of the transgenic plants overexpressing BnNF-YA1 under stress treatments were evaluated. The results showed that the length of open reading frame of BnNF-YA1 was 858 bp,encoding 285 amino acids. Tobacco-transient expression analysis uncovered that BnNF-YA1 protein was localized in the nucleus. Biochemical analysis using yeast system showed that there was no transactivation activity in BnNF-YA1 full-length protein. Expression analysis revealed that the transcript abundance of BnNF-YA1 was induced by PEG treatment,and BnNF-YA1 showed a relatively high expression in the seedling leaf,bud,and 20 DAP silique. The overexpression of BnNF-YA1 increased the tolerance of transgenic rapeseed plants to PEG and methyl viologen（MV）treatments,suggesting that BnNF-YA1 may be involved in regulating the responses of rapeseed to osmotic and oxidative stress.

Key words: Brassica napus L., transcription factor, NF-YA, overexpression, abiotic stress

LIN Ke-yun, DUAN Yu-jing, WANG Gao-sheng, SUN Nian-li, FANG Yu-jie, WANG You-ping. Cloning and Functional Identification of BnNF-YA1 in Brassica napus L.[J]. Biotechnology Bulletin, 2022, 38(4): 106-116.

Figures/Tables 8

Fig. 1 Analysis of hydrophilicity/hydrophobicity,phos-phorylation site,secondary structure and tertiary structure of BnNF-YA1 A：Hydrophilic/hydrophobic analysis of BnNF-YA1. B：Prediction of phosphorylation sites of BnNF-YA1. C：Prediction of secondary structure of BnNF-YA1. D：Prediction of the tertiary structure of BnNF-YA1

Fig. 2 Multiple sequence alignments of BnNF-YA1 and its homologs

Fig. 3 Phylogenetic analysis of NF-YA1 proteins

Fig. 4 Expression profile of BnNF-YA1 A：Changes of BnNF-YA1 expression in B. napus under PEG,cold,heat,and ABA treatments. B：Spatial-temporal expression profile of BnNF-YA1 in the different organs of B. napus

Fig. 5 Generation of BnNF-YA1-OE transgenic rapeseed plants A：Schematic diagram of the overexpresed vector backbone. B：PCR positive identification of overexpresed BnNF-YA1 in T0 generation. C：RNA detection of BnNF-YA1-OE transgenic positive plants. D：Reverse transcription detection of BnNF-YA1-OE transgenic positive plants. E：Expression of BnNF-YA1-OE in the T0 generation of overexpressed plants

Fig. 6 Phenotypic identification of T2 generation of BnNF-YA1-OE transgenic plants at the cotyledon stage A：Phenotype of T2 generation of BnNF-YA1-OE transgenic plants at the cotyledon stage. B：The hypocotyl length,root length and fresh weight of T2 generation of BnNF-YA1-OE transgenic plants. The error bars represent the standard deviation（SD）. * represents significant difference（P<0.05）,and ** represents extremely significant difference（P<0.01）

Fig. 7 Subcellular localization of BnNF-YA1 protein

Fig. 8 Transactivation activity analysis of BnNF-YA1 full-length protein

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