核桃JrSnRK1α1.1调控种子油脂合成与积累

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

生物技术通报 ›› 2023, Vol. 39 ›› Issue (9): 183-191.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0043

核桃JrSnRK1α1.1调控种子油脂合成与积累

王贵芳¹(), 姚元涛², 许海峰¹, 相昆¹, 梁家慧³, 张淑辉³, 王文茹³, 张明娟¹^,⁴, 张美勇¹(), 陈新¹()

1.山东省果树研究所国家核桃板栗种质资源圃农业农村部黄淮设施园艺工程重点实验室，泰安 271000
2.山东省农业科学院茶叶研究所，济南 250100
3.山东农业大学园艺科学与工程学院，泰安 271018
4.山东农业大学生命科学学院，泰安 271018

收稿日期:2023-01-18 出版日期:2023-09-26 发布日期:2023-10-24
通讯作者: 陈新，男，博士，研究员，研究方向：果树种质资源与育种；E-mail: sdaucx@163.com；
张美勇，男，研究员，研究方向：核桃栽培及育种；E-mail: zyj625@163.com
作者简介:王贵芳，女，博士，助理研究员，研究方向：核桃栽培及育种；E-mail: gfwang05@163.com
第一联系人：
姚元涛为本文共同第一作者
基金资助:
山东省自然科学基金面上项目(ZR2020MC161);山东省农业良种工程项目(2020LZGC009)

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

WANG Gui-fang¹(), YAO Yuan-tao², XU Hai-feng¹, XIANG Kun¹, LIANG Jia-hui³, ZHANG Shu-hui³, WANG Wen-ru³, ZHANG Ming-juan¹^,⁴, ZHANG Mei-yong¹(), CHEN Xin¹()

1. Shandong Institute of Pomology, National Walnut and Chestnut Germplasm Resource Nursery, Huanghuai Protected Horticulture Engineering Key Laboratory of Ministry of Agriculture and Rural Affairs, Tai'an 271000
2. Tea Research Institute of Shandong Academy of Agricultural Sciences, Jinan 250100
3. College of Horticultural Science and Engineering, Shandong Agricultural University, Tai'an 271018
4. College of Life Sciences, Shandong Agricultural University, Tai'an 271018

Received:2023-01-18 Published:2023-09-26 Online:2023-10-24

摘要/Abstract

摘要：

为探讨核桃SnRK1蛋白激酶对种子油脂合成与积累的调控机理，本研究以‘香玲’核桃种仁cDNA为模板，克隆分离得到SnRK1蛋白激酶α亚基的一个编码基因JrSnRK1α1.1，其CDS序列全长为1 539 bp，编码512个氨基酸。构建植物超表达重组载体PBI121- JrSnRK1α1.1，转化并获得了超表达核桃JrSnRK1α1.1基因的拟南芥株系J-1、J-2和J-3。与野生型拟南芥相比，转基因植株表型无明显差异。采用数码显微镜对转基因拟南芥株系种子进行观察，结果显示，相对于野生型，转基因拟南芥株系的种子瘪小、表皮皱缩；株系J-1、J-2和J-3瘪小种子占比分别为43.69%、60.80%和45.87%，显著高于野生型WT（8.43%）；种子的千粒重比野生型降低了27.95%-29.32%，差异显著；种子平均粒长和粒宽也显著变小；因此JrSnRK1α1.1可能参与了种子的发育进程。进一步对种子的粗脂肪含量进行测定，结果表明，与野生型相比，转基因拟南芥株系J-1、J-2和J-3的种子粗脂肪含量显著下降，分别比野生型下降9.66%、23.86%和17.61%。荧光双分子试验结果表明，核桃JrSnRK1α1.1与油脂合成关键转录因子JrWRI1互作。综上所述，超表达核桃JrSnRK1α1.1对拟南芥植株生长发育影响不明显，显著影响了拟南芥种子的发育，负调控种子油脂的合成与积累过程。

关键词: 核桃, JrSnRK1α1.1, 转基因, 种子, 油脂, 表型, 千粒重

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

王贵芳, 姚元涛, 许海峰, 相昆, 梁家慧, 张淑辉, 王文茹, 张明娟, 张美勇, 陈新. 核桃JrSnRK1α1.1调控种子油脂合成与积累[J]. 生物技术通报, 2023, 39(9): 183-191.

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.

图/表 8

表1 目的基因JrSnRK1α1.1的引物序列

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

图1 不同植物中SnRK1蛋白激酶α亚基蛋白进化树分析

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

图2 ‘香玲’核桃种仁不同生长阶段JrSnRK1α1.1基因表达水平不同小写字母表示差异显著（P<0.05）。下同

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

图3 野生型拟南芥（WT）和转基因拟南芥株系（J-1、J-2和J-3）幼苗表型

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

图4 转基因拟南芥株系中JrSnRK1α1.1基因PCR扩增凝胶图谱（A）及基因相对表达水平（B） M：DL2000 Marker；WT：野生型；水：空白对照；J-1-J-3：超表达转基因株系。下同

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

图5 野生型拟南芥（WT）和转基因拟南芥株系（J-1、J-2和J-3）种子表型

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

图6 野生型拟南芥（WT）和转基因拟南芥基因株系（J-1、J-2和J-3）瘪小种子百分比、种子大小和种子粗脂肪酸含量 A：瘪小种子百分比；B：种子千粒重；C：种子粒长；D：种子粒宽；E：种子长宽比；F：种子脂肪酸含量

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

图7 核桃JrSnRK1α1.1与油脂合成关键转录因子JrWRI1双分子荧光互补

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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核桃JrSnRK1α1.1调控种子油脂合成与积累

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

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