The Gene JrSnRK1α1.1 of Walnut Regulates Seed Oil Synthesis and Accumulation

doi:10.13560/j.cnki.biotech.bull.1985.2023-0043

Abstract

Abstract:

In order to investigate the regulation mechanism of SnRK1 protein kinase on seed oil synthesis and accumulation in walnut, the gene JrSnRK1α1.1, coding the α subunit of SnRK1 protein kinase, was cloned and isolated from cDNA of ‘Xiangling’ walnut kernel. The total length of its CDS sequence was 1 539 bp, encoding 512 amino acids. The overexpression recombinant vector PBI121-JrSnRK1α1.1 was constructed, transformed, and the transgenic Arabidopsis strains J-1, J-2, and J-3 were obtained. There was no significant difference between transgenic plants and wild-type during the process of plant growth and development. Some transgenic seeds were shrunk compared with the wild-type observed by digital microscope. The percentage of the shrunken seeds of J-1, J-2 and J-3 were 43.69%, 60.80% and 45.87% respectively, which were significantly higher than that of WT（8.43%）. The weight of 1 000 seeds decreased by 27.95% to 29.32% compared with the wild-type, with a significant difference. The average length and width of seeds also decreased significantly. Therefore, the gene JrSnRK1α1.1 may be involved in the development of Arabidopsis seeds. The crude fat content of transgenic Arabidopsis seeds decreased 9.66%, 23.86% and 17.61% respectively compared with WT. Fluorescence bimolecular assay showed that JrSnRK1α1.1 interacted with JrWRI1 of key transcription factor in oil synthesis. To sum up, over-expressing the walnut JrSnRK1α1.1 gene has no obvious effects on transgenic Arabidopsis plant growth and development, but significantly affects the development of seeds, and negatively regulates the synthesis and the accumulation of seed oil.

Key words: walnut, JrSnRK1α1.1, transgenosis, seed, oil, phenotype, thousand-seed weight

WANG Gui-fang, YAO Yuan-tao, XU Hai-feng, XIANG Kun, LIANG Jia-hui, ZHANG Shu-hui, WANG Wen-ru, ZHANG Ming-juan, ZHANG Mei-yong, CHEN Xin. The Gene JrSnRK1α1.1 of Walnut Regulates Seed Oil Synthesis and Accumulation[J]. Biotechnology Bulletin, 2023, 39(9): 183-191.

Figures/Tables 8

Table 1 Primer sequence of target gene JrSnRK1α1.1

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Uses
Jr SnRK1α1.1-F Jr SnRK1α1.1-R SNF1-2-BiFC-S SNF1-2-BiFC-A Jr SnRK1α1.1-A Jr SnRK1α1.1-B 18S-F 18S-R	GGGGTACCATGGATGGCTCAACTG CGGAATTCTTAAAGGACTCGGAGC GCGTCGACATGGATGGCTCAACTG GGGGTACCAAGGACTCGGAGCTGG CTTTGGGGATGAACCGACCA GCCAGGAAAGCAGCACAAAG ACAGGACCTCTCACGATCCA	JrSnRK1α1.1基因全长扩增 Full length amplification of the gene JrSnRK1α1.1 荧光双分子互作试验 Fluorescent bimolecular interaction test JrSnRK1α1.1基因表达荧光定量分析 Fluorescence quantitative analysis of JrSnRK1α1.1 expression
	CAGCAAATCCAGCACGCATT

Fig. 1 Phylogenetic analysis of the proteins of α subunit in SnRK1 protein kinase from different plant species

Fig. 2 Expressions of the JrSnRK1α1.1 gene in different growth stages of ‘Xiangling’ walnut kernel Different lowercase letters indicate significant difference（P<0.05）. The same below

Fig. 3 Seedling phenotype of wild-type（WT）and transgenic Arabidopsis thaliana lines（J-1, J-2 and J-3）

Fig. 4 Gel map of the JrSnRK1α1.1 gene amplified by PCR（A）and gene relative expressions of JrSnRK1α1.1（B）in transgenic A.thaliana lines M: DL2000 Marker; WT: wild type; Water: blank control; J-1-J-3: JrSnRK1α1.1 overexpressed transgenic A. thaliana lines. The same below

Fig. 5 Seed phenotypes of WT and transgenic A. thaliana lines（J-1, J-2 and J-3）

Fig. 6 Percents of shrunken seeds， size of a seed and the contents of crude fatty acid in the seeds of WT and transgenic A. thaliana transgenic lines（J-1, J-2 and J-3） A: Percent of shrunken seeds. B: 1000- seed weight. C: Seed length. D: Seed width. E: Ratio of seed length to width. F: Content of crude fat in dry seeds

Fig. 7 Bimolecular fluorescence complementation of walnut JrSnRK1α1.1 with JrWRI1, a key transcription factor for lipid synthesis（Scale bar=10 μm）

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