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作物学报 ›› 2019, Vol. 45 ›› Issue (11): 1638-1648.doi: 10.3724/SP.J.1006.2019.94003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生硬脂酰-ACP酸脱饱和基因FAB2表达的分子机制

刘浩,鲁清,李海芬,李少雄,陈小平,梁炫强(),洪彦彬()   

  1. 广东省农业科学院作物研究所 / 广东省农作物遗传改良重点实验室, 广东广州 510640
  • 收稿日期:2019-01-02 接受日期:2019-05-12 出版日期:2019-11-12 网络出版日期:2019-05-30
  • 通讯作者: 梁炫强,洪彦彬
  • 作者简介:E-mail: liuhao2054@stu.scau.edu.cn
  • 基金资助:
    本研究由国家自然科学基金项目(31771841);本研究由国家自然科学基金项目(31801401);广东省自然科学基金项目(2017A030311007);国家重点研发计划项目(2018YFD0201009-02);广东省农业科学院优秀博士计划联合资助

Molecular mechanism of stearoyl-ACP desaturase gene FAB2 expression in peanut

LIU Hao,LU Qing,LI Hai-Fen,LI Shao-Xiong,CHEN Xiao-Ping,LIANG Xuan-Qiang(),HONG Yan-Bin()   

  1. Crops Research Institute, Guangdong Academy of Agricultural Science / Guangdong Provincial Key Laboratory of Crop Genetic Improvement, Guangzhou 510640, Guangdong, China
  • Received:2019-01-02 Accepted:2019-05-12 Published:2019-11-12 Published online:2019-05-30
  • Contact: Xuan-Qiang LIANG,Yan-Bin HONG
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31771841);This study was supported by the National Natural Science Foundation of China(31801401);the Natural Science Foundation of Guangdong Province(2017A030311007);the National Key Research and Development Program(2018YFD0201009-02);Excellent PhD Program of Guangdong Academy of Agricultural Science

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摘要：

FAB2位于油酸合成通路的上游, 编码硬脂酰-ACP脱饱和酶, 调控硬脂酸(C18:0)向油酸(C18:1)转化。本研究发现高油酸品种开农176种子发育前期FAB2的表达量升高, 而成熟期油酸过量积累会抑制FAB2的表达。利用开农176与开农70构建F2杂交群体, 发现当植株油酸含量超过60%时会从整体水平上抑制FAB2的表达。种子发育前期, 油酸不断积累会导致过氧化物酶活性升高, 并且活性氧含量随之增加; 但是在种子发育后期均降低, 该结果与FAB2的表达量变化趋势相同。亚细胞定位结果表明, FAB2与FAD2分别定位于叶绿体与内质网。FAB2编码区序列多态性分析显示, 该蛋白序列氮端的氨基酸结构缺失可能会导致硬脂酸含量升高。FAB2启动子序列存在大量AT碱基的富集区域, 并且含有光响应、激素调控、转录因子结合的保守顺式元件。本研究发现过量积累的油酸会激活过氧化物酶介导的活性氧信号途径, 进而通过细胞核内的未知转录因子调节上游基因FAB2的表达量, 该结果不仅拓展了对FAB2的功能认知, 也为培育高油酸花生品种提供了相关的理论指导。

关键词: 花生, 高油酸, FAB2, ROS, 反馈调节

Abstract:

Stearoyl-ACP desaturase, encoded by FAB2, and located on the upstream of oleic acid biosynthesis pathway, modulates the conversion of stearic acid (C18:0) into oleic acid (C18:1). The expression of FAB2 was increased at the early stage of seed development in high-oleic variety Kainong 176, but over-accumulated oleic acid repressed the FAB2 expression at the period of seed maturation. An F2 hybrid population was constructed using Kainong 176 and Kainong 70, showing that the content of oleic acid up to 60% directly repressed the FAB2 expression on the whole. The activity of peroxidase (POD) and content of ROS were increased at the early stage of seed development with the oleic acid gradual accumulation, but decreased at the maturation stage. Subcellular localization analysis indicated that FAB2 and FAD2 were located on chloroplast and endoplasmic reticulum, respectively. Encoding sequence polymorphism analysis of FAB2 suggested that amino acid deficient at the N-terminal of FAB2 protein sequence probably induced the high content of stearic acid in peanut. Furthermore, the promotor sequence of FAB2 contained multiple AT-rich region, and possessed the light responsive, hormone regulation, and transcription factor binding site cis-regulating elements. Taken together, this study found that over-accumulated oleic acid activated the POD-induced ROS robust pathway, and then regulated the expression of FAB2 by unknown transcription factor in nucleus. The results extend the knowledge of FAB2 function, and provide a relevant theoretical guidance in future peanut breeding for high oleic acid.

Key words: peanut, high oleic acid, FAB2, ROS, feedback regulation

图1

花生油酸积累反馈调节FAB2的表达量 A: 油酸合成示意图; B: 开农176与开农70的植株照片; C: 种子发育的6个时期; D: FAB2在高、低油酸品种种子发育过程中的表达变化量; E: 开农176与开农70的油酸与亚油酸含量; F: 开农176与开农70种子发育阶段油酸与亚油酸含量变化。"

图2

花生FAB2表达受抑制的油酸积累值 A: F2群体单株的油酸与亚油酸含量。OA: oleic acid; LOA: linoleic acid。B: F2群体单株的FAB2表达量变化。F2-25位对照单株, 柱状图结果表明3次独立重复。"

图3

POD活性与H2O2含量变化 A: POD活性检测; B: H2O2含量检测; **代表P < 0.01, *代表P < 0.05。"

图4

FAB2与FAD2的亚细胞定位"

表1

种质资源农艺性状"

花生品种
Peanut variety		 蛋白质含量
Protein content
(%)		 脂肪含量
Fatty acid content
(%)		 油酸
Oleic acid
(%)		 亚油酸
Linoleic acid
(%)		 硬脂酸
Stearic acid
(%)		 植物学类型
Botanical classification
冀甜 Jitian 24.10 42.04 30.28 44.87 4.28 珍珠豆型 Spanish type
汕油523 Shanyou 523 25.46 51.98 26.48 46.90 3.47 珍珠豆型 Spanish type
贺油11 Heyou 11 25.11 50.47 29.90 44.69 3.09 珍珠豆型 Spanish type
粤油18 Yueyou 18 26.03 51.24 22.04 51.34 3.62 珍珠豆型 Spanish type
徐花13 Xuhua 13 25.89 52.02 39.61 37.76 3.73 珍珠豆型 Spanish type
湛油12号 Zhanyou 12 20.98 55.68 33.59 42.77 2.39 珍珠豆型 Spanish type
花育30 Huayu 30 24.05 52.51 36.04 40.89 3.78 珍珠豆型 Spanish type
粤北种 Yuebeizhong 44.95 26.27 31.76 46.50 2.98 珍珠豆型 Spanish type
ICGV87123 25.73 48.86 29.46 44.89 2.32 珍珠豆型 Spanish type
粤油35 Yueyou 35 21.87 49.91 39.66 39.08 0.88 珍珠豆型 Spanish type
小粒种 Xiaolizhong 24.12 53.45 40.54 37.16 0.92 珍珠豆型 Spanish type
G40423 30.13 50.03 36.64 41.70 2.09 珍珠豆型 Spanish type
湛油1号 Zhanyou 1 29.88 46.76 28.99 43.87 3.74 珍珠豆型 Spanish type
闽花6号 Minhua 6 24.16 51.64 32.94 43.06 2.84 珍珠豆型 Spanish type
狮头企 Shitouqi 28.22 49.53 28.32 43.99 3.54 珍珠豆型 Spanish type
大明勾鼻 Daminggoubi 28.37 46.47 17.16 52.16 4.88 珍珠豆型 Spanish type
大罗大粒 Daluodali 46.42 23.75 30.61 47.88 2.22 普通型 Normal type
仲恺花1号 Zhongkaihua 1 22.81 52.10 31.10 45.22 1.95 珍珠豆型 Spanish type
宛花8908 Wanhua 8908 22.97 53.39 34.66 40.38 3.24 珍珠豆型 Spanish type
粤油3粒白 Yueyou 3 libai 25.45 48.53 41.38 37.20 0.94 珍珠豆型 Spanish type
文摘墩 Wenzhandun 28.08 48.88 47.66 34.59 0.30 珍珠豆型 Spanish type
ICGV92267 23.25 53.00 46.26 29.66 2.05 珍珠豆型 Spanish type
白沙1016 Baisha 1016 25.24 51.67 44.60 32.11 3.65 珍珠豆型 Spanish type
早花生1 Zaohuasheng 1 42.64 27.90 33.71 45.73 2.48 普通型 Normal type
西藏红 Xizanghong 26.01 51.18 34.15 41.31 2.50 珍珠豆型 Spanish type
花生品种
Peanut variety		 蛋白质含量
Protein content
(%)		 脂肪含量
Fatty acid content
(%)		 油酸
Oleic acid
(%)		 亚油酸
Linoleic acid
(%)		 硬脂酸
Stearic acid
(%)		 植物学类型
Botanical classification
狮选3号 Shixuan 3 30.32 47.70 19.26 51.07 4.26 珍珠豆型 Spanish type
HZ-152 24.11 40.90 15.49 53.25 5.12 珍珠豆型 Spanish type
蚂花2号 Mahua 2 27.09 51.04 31.05 43.64 2.62 珍珠豆型 Spanish type
汕油3号 Shanyou 3 45.69 25.08 35.41 45.52 1.78 珍珠豆型 Spanish type
仲恺花2号 Zhongkaihua 2 25.46 49.71 36.82 42.34 1.95 珍珠豆型 Spanish type
阳江铺地毡 Yangjiangpudizhan 43.61 25.65 37.56 42.33 2.57 珍珠豆型 Spanish type

图5

不同花生种质的FAB2蛋白进化树"

图6

不同花生种质FAB2蛋白序列多重比对"

表2

FAB2启动子区间保守元件"

保守元件
Conversed elements		 结合序列
Binding sequence		 功能
Function
ABRE ACGTG Involved in the abscisic acid responsiveness
ARE AAACCA cis-acting regulatory element essential for the anaerobic induction
AT-rich sequence TAAAATACT Element for maximal elicitor-mediated activation
AT-TATA-box TATATA Transcription initiation
AuxRR-core GGTCCAT cis-acting regulatory element involved in auxin responsiveness
Box-4 ATTAAT Part of a conserved DNA module involved in light responsiveness
CAAT-box CAAT Common cis-acting element in promoter and enhancer regions
CGTCA-motif CGTCA cis-acting regulatory element involved in the MeJA-responsiveness
G-Box CACGTT cis-acting regulatory element involved in light responsiveness
GA-motif ATAGATAA Part of a light responsive element
GATA-motif AAGATAAGATT Part of a light responsive element
GC-motif CCCCCG Enhancer-like element involved in anoxic specific inducibility
GT1-motif GGTTAA Light responsive element
MRE AACCTAA MYB binding site involved in light responsiveness
MSA-like TCAAACGGT cis-acting element involved in cell cycle regulation
MYB TACCT MYB transcription factor binding site
MYC CATTG MYC transcription factor binding site
O2-site GATGATGTGG cis-acting regulatory element involved in zein metabolism regulation
P-box CCTTTTG Gibberellin-responsive element
TC-rich repeats ATTCTCTAAC cis-acting element involved in defense and stress responsiveness
TCCC-motif TCTCCCT Part of a light responsive element
TCT-motif TCTTAC Part of a light responsive element
TGA-element AACGAC Auxin-responsive element
TGACG-motif TGACG cis-acting regulatory element involved in the MeJA-responsiveness
WRE3 CCACT WRE transcription factor binding site
WUN-motif AAATTTCTT Wun transcription factor binding site
chs-CMA1a TTACTTAA Part of a light responsive element
as-1 TGACG As transcription factor binding site

图7

油酸调控FAB2表达的模型 A: FAB2 启动子区域内AT碱基富集区域示意图; B: 油酸反馈调节FAB2表达的假定模型。"

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