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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (10): 1586-1594.doi: 10.3724/SP.J.1006.2019.84177

• RESEARCH NOTES • Previous Articles     Next Articles

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 Online:2019-10-12 Published:2019-09-10
  • Contact: Dong-Xin HUAI,Bo-Shou LIAO E-mail:dxhuai@caas.cn;lboshou@hotmail.com
  • 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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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

Table 1

Sequences of primers for cloning of AhFAD2"

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

Table 2

Sequences of primers for real-time PCR"

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

Table 3

Information of FAD2 family genes in peanut"

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

Fig. 1

Expression levels of AhFAD2 genes in 15 peanut tissues A: 15 peanut tissues involved in this study. I: radicle after 6 days of sowing; II: hypocotyl after 6 days of sowing; III: cotyledon after 6 days of sowing; IV: true leaves after 6 days of sowing; V: root after 60 days of sowing; VI: peg after 60 days of sowing; VII: stem after 60 days of sowing; VIII: yong leaves after 60 days of sowing; IX: mature leaves after 60 days of sowing; X: flowers after 60 days of sowing; XI: white and flat developing seeds; XII: white and drop-shaped developing seeds; XIII: white and torpedo-shaped developing seeds; XIV: light pink and round developing seeds; XV: dark pink and mature seeds. B: expression analysis of AhFAD2 genes in 15 tissues of peanut. The white column indicates normal oleate peanut ZH16, the gray column indicates high oleate peanut ZH413. Differences reached a significant level by one-way analysis of variance. * P < 0.05, ** P < 0.01, *** P < 0.001. C: expression analysis of AhFAD2 genes in developing seeds. Bars superscripted by different lowercase letters are significantly different at P < 0.05 by one-way ANOVA and least significant difference (LSD) test."

Table 4

Seed germination rates of normal oleate ZH16 and high oleate ZH413 under different temperatures"

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

Fig. 2

Seed germination test of normal oleate ZH16 and high oleate ZH413 under different temperatures"

Fig. 3

Expression analysis of AhFAD2 genes in ZH16 and ZH413 under different temperature a: expression pattern of AhFAD2-1A/B in ZH16 under different temperature treatment conditions; b: expression pattern of AhFAD2-3A/B in ZH16 under different temperature treatment conditions; c: expression pattern of AhFAD2-4A/B in ZH16 under different temperature treatment conditions; d: expression pattern of AhFAD2-1A/B in ZH413 under different temperature treatment conditions; e: expression pattern of AhFAD2-3A/B in ZH413 under different temperature treatment conditions; f: expression pattern of AhFAD2-4A/B in ZH413 under different temperature treatment conditions. Significant differences are determined by one-way analysis of variance. * P < 0.05, ** P < 0.01, *** P < 0.001."

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