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作物学报 ›› 2019, Vol. 45 ›› Issue (10): 1586-1594.doi: 10.3724/SP.J.1006.2019.84177

• 研究简报 • 上一篇    下一篇

花生FAD2基因家族表达分析及其对低温胁迫的响应

薛晓梦1,李建国1,白冬梅2,晏立英1,万丽云1,康彦平1,淮东欣1,*(),雷永1,廖伯寿1,*()   

  1. 1中国农业科学院油料作物研究所 / 农业部油料作物生物学与遗传育种重点实验室, 湖北武汉 430062
    2山西省农业科学院经济作物研究所, 山西汾阳 032200
  • 收稿日期:2018-12-27 接受日期:2019-05-12 出版日期:2019-10-12 网络出版日期:2019-09-10
  • 通讯作者: 淮东欣,廖伯寿
  • 基金资助:
    本研究由湖北省自然科学基金项目(2017CFB161);国家自然科学基金项目(31671734);国家自然科学基金项目(31871662);国家重点研发计划项目资助(2018YFD1000901)

Expression profiles of FAD2 genes and their responses to cold stress in peanut

XUE Xiao-Meng1,LI Jian-Guo1,BAI Dong-Mei2,YAN Li-Ying1,WAN Li-Yun1,KANG Yan-Ping1,HUAI Dong-Xin1,*(),LEI Yong1,LIAO Bo-Shou1,*()   

  1. 1Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture / Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, Hubei, China
    2Industrial Crops Research Institute, Shanxi Academy of Agricultural Sciences, Fenyang 032200, Shanxi, China
  • Received:2018-12-27 Accepted:2019-05-12 Published:2019-10-12 Published online:2019-09-10
  • Contact: Dong-Xin HUAI,Bo-Shou LIAO
  • Supported by:
    This study was supported by the Hubei Provincial Natural Science Foundation of China(2017CFB161);the National Natural Science Foundation of China(31671734);the National Natural Science Foundation of China(31871662);the National Key R&D Program of China.(2018YFD1000901)

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摘要：

为了探究FAD2在花生低温响应中的作用, 本研究从普通油酸花生中花16 (ZH16)和高油酸花生中花413 (ZH413)中克隆得到花生AhFAD2家族的全部基因, 共7个。通过分析这些基因的表达模式发现, 在ZH16和ZH413中各FAD2基因表达模式相似, AhFAD2-1A/B主要在花和发育的种子中表达, AhFAD2-3A/B主要在营养组织中表达, AhFAD2-4A/B主要在根和花中表达, 表明AhFAD2基因在花生不同发育阶段和不同组织中发挥各自的生物学功能。在15℃下发芽6 d发现, ZH413的发芽率未显著下降, 而ZH16的发芽率显著下降。种子萌发过程中, AhFAD2-1A/B和AhFAD2-4A/B均受低温诱导表达。在ZH16中AhFAD2-1A/B在低温诱导第6天开始显著上调表达, 而在ZH413中第1天显著上调表达; 在ZH16中AhFAD2-4A/B在低温诱导第3天出现显著上调表达, 之后表达量下降, 但在ZH413中第1天就显著上调表达, 且始终维持在高水平表达。基于以上研究结果推测, 高油酸花生在受到低温胁迫时, AhFAD2-1A/B编码蛋白失活, 但AhFAD2-4A/B的高量表达在一定程度上弥补了这部分功能。同时也说明AhFAD2-1A/B功能的缺失并不是决定花生耐寒性的主要因素。本研究的开展为培育抗寒的高油酸花生品种奠定了理论基础, 为高油酸花生在高纬度、高海拔地区推广提供了理论支持。

关键词: 脂肪酸脱氢酶(FAD2), 基因克隆, 低温胁迫, 基因表达

Abstract:

To explore the roles of FAD2s in response to cold stress in peanut, we cloned seven AhFAD2 genes from normal oleate peanut ZH16 and high oleate peanut ZH413, respectively. The results of qRT-PCR showed that the expression patterns of FAD2 genes were similar in ZH16 and ZH413. AhFAD2-1A/B was highly expressed in flower and developing seed, AhFAD2-3A/B was mainly expressed in leaf and stem, and AhFAD2-4A/B was expressed specifically in root and flower, indicating that AhFAD2 genes played their respective roles in different developmental stages and tissues of peanut. At 6 days after inducing under 15℃, the germination rate of ZH16 was significantly decreased while that of ZH413 was not significantly affected. The expression of both AhFAD2-1A/B and AhFAD2-4A/B were induced by cold stress. The expression of AhFAD2-1A/B was significantly up-regulated at 6 DAI in ZH16, while at 1 DAI in ZH413, suggesting that AhFAD2-1A/B was induced by cold more quickly in high oleate peanut. Furthermore, the expression of AhFAD2-4A/B was significantly up-regulated at 3 DAI in ZH16 and then decreased, while it was increased immediately and maintained at high level for six days in ZH413. Based on these results, we speculate that up-regulation of AhFAD2-4A/B may compensate the function of AhFAD2-1A/B that is deactivated under cold stress in high oleate peanut, and the deactivation of AhFAD2-1A/B is not the most important factors affecting peanut cold tolerance. This study provides a theoretical basis in breeding of high oleate peanut with high tolerance to cold stress, and the theoretical support for extension of high oleate peanut in both high latitude and high altitude regions.

Key words: fatty acid desaturase 2 (FAD2), gene cloning, cold stress, expression profile

表1

克隆AhFAD2基因使用的引物及其序列"

基因
Gene		 基因注册号
Accession No.		 上游引物
Forward primer (5°-3°)		 下游引物
Reverse primer (5°-3°)
AhFAD2-1A Aradu.G1YNF GATTATTGACTTGCTTTGTAGTAGTGC ACACAAACGTTTTCAACTCTGAC
AhFAD2-1B Araip.S3GXY CAGAACCATTAGCTTTGTAGTAGTGC ACACAAACGTTTCCAACTCTGAC
AhFAD2-2 Araip.65EGG CAGAACCATTAGCTTTGTAGTAGTGC ACACAAACGTTTCCAACTCTGAC
AhFAD2-3A Aradu.10027244 AGCACAACCGATAATACTCTCCCAA TTATTCTGAGCACAACCGATAATA
AhFAD2-3B Araip.D6HPL CTCTGTTCCTCCTTCAAACAGGACG TAATCAACCATCCTAATTGGCAGAA
AhFAD2-4A Aradu.7W39T ATGGGGTCTGGGGGTCATTCTTCCG TTAAAGCTTATTATACCAAAGAACACCTTT
AhFAD2-4B Araip.WI51C ATGGGGTCTGGTGGTCATTCTG TTAAAGCTTATTATACCAAAGAACACCT

表2

Real time PCR引物及其碱基序列"

基因
Gene		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
AhFAD2-1A and AhFAD2-1B RTAhFAD2-1-F TTTATGTCTCAGATTCATCTGT
RTAhFAD2-1-R CCTCTTAACCAGTCCCATTCG
AhFAD2-2 RTAhFAD2-2-F TTCTAATTTATGTCTCAGGACTCAG
RTAhFAD2-2-R CCATAATCTCTGTCCACTGTTGCCA
AhFAD2-3A and AhFAD2-3B RTAhFAD2-3-F GGTCTTATCCGTCTTGTCATGG
RTAhFAD2-3-R AGATGAATCGTAATGTGGCAATG
AhFAD2-4A and AhFAD2-4B RTAhFAD2-4-F TGGCCTTTGTACTGGTTCG
RTAhFAD2-4-R GGACGAGAAGGAAAGAATGGAG
AhActin Actin-F TAAGAACAATGTTGCCATACAGA
Actin-R GTTGCCTTGGATTATGAGC

表3

花生FAD2家族基因信息"

基因编号
Gene		 基因注册号
Accession No.		 染色体
Chr.		 翻译阅读框
ORF (bp)		 氨基酸残基
Len (aa)		 相对分子质量
Molecular weight (kDa)		 等电点
pI
AhFAD2-1A Aradu.G1YNF A09 1140 379 43.65 8.90
AhFAD2-1B Araip.S3GXY B09 1140 379 43.65 8.90
AhFAD2-2 Araip.65EGG B09 453 150 17.20 8.30
AhFAD2-3A Aradu.10027244 A06 1050 349 39.70 8.85
AhFAD2-3B Araip.D6HPL B06 1152 383 43.80 8.80
AhFAD2-4A Aradu.7W39T A09 1164 387 45.00 8.96
AhFAD2-4B Araip.Wi51C B09 1164 387 45.00 9.04

图1

AhFAD2基因在15个花生组织中的表达水平 A: 本研究中涉及到的15个花生组织。I: 播种6 d后的胚根; II: 播种6 d后的胚轴; III: 播种6 d后的子叶; IV: 播种6 d后的真叶; V: 播种60 d后的根; VI: 播种60 d后的果针; VII: 播种60 d后的茎; VIII: 播种60 d后的幼嫩叶片; IX: 播种60 d后的成熟叶片; X: 播种60 d后的花; XI: 白色、扁平的发育中的种子; XII: 白色、水滴形的发育中的种子; XIII: 白色、鱼雷形的发育中的种子; XIV: 浅粉色、圆形的发育中的种子; XV: 深粉色、成熟的种子。B: AhFAD2基因在15个花生组织中的表达水平。白色表示普通油酸材料ZH16; 灰色表示高油酸材料ZH413。经单因素方差分析差异达到显著水平。* P < 0.05; ** P < 0.01; *** P < 0.001。C: AhFAD2基因在花生发育种子中的表达分析。标有不同小写字母的柱值表示经单因素方差分析和最小显著差异法(LSD)检验差异达到显著水平(P < 0.05)。"

表4

不同温度处理条件下普通油酸花生ZH16和高油酸花生ZH413的种子发芽率"

温度
Temperature
(℃)		 ZH16种子发芽率
Germination rate of ZH16 (%)		 ZH413种子发芽率
Germination rate of
ZH413 (%)
25 91.33±5.25 bc 94.66±3.77 b
15 45.00±7.34 c 98.67±0.94 a

图2

不同温度处理下普通油酸花生ZH16和高油酸花生ZH413的种子发芽情况"

图3

在不同温度处理下各AhFAD2基因在ZH16和ZH413中的表达分析 a: 在不同温度处理下, AhFAD2-1A/B在ZH16中的表达模式; b: 在不同温度处理下, AhFAD2-3A/B在ZH16中的表达模式; c: 在不同温度处理下, AhFAD2-4A/B在ZH16中的表达模式; d: 在不同温度处理下, AhFAD2-1A/B在ZH413中的表达模式; e: 在不同温度处理下, AhFAD2-3A/B在ZH413中的表达模式; f: 在不同温度处理下, AhFAD2-4A/B在ZH413中的表达模式。经单因素方差分析差异达到显著水平, * P < 0.05; ** P < 0.01; *** P < 0.001。"

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