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作物学报 ›› 2023, Vol. 49 ›› Issue (1): 73-85.doi: 10.3724/SP.J.1006.2023.14217

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

向日葵DGATs基因家族的鉴定及表达分析

张程1(), 张展2, 杨佳宝1, 孟晚秋1, 曾令露1, 孙黎1,*()   

  1. 1新疆石河子大学生命科学学院, 新疆石河子 832003
    2兵团兴新职业技术学院, 新疆巴州841007
  • 收稿日期:2021-11-23 接受日期:2022-05-05 出版日期:2023-01-12 网络出版日期:2022-05-24
  • 通讯作者: 孙黎
  • 作者简介:E-mail: 2020002371@qq.com
  • 基金资助:
    国家自然科学基金项目(31760064);国家自然科学基金项目(31360052)

Genome-wide identification and relative expression analysis of DGATs gene family in sunflower

ZHANG Cheng1(), ZHANG Zhan2, YANG Jia-Bao1, MENG Wan-Qiu1, ZENG Ling-Lu1, SUN Li1,*()   

  1. 1College of Life Science, Shihezi University, Shihezi 832003, Xinjiang, China
    2Bingtuan Xingxin Vocational and Technical College, Bazhou 841007, Xinjiang, China
  • Received:2021-11-23 Accepted:2022-05-05 Published:2023-01-12 Published online:2022-05-24
  • Contact: SUN Li
  • Supported by:
    National Natural Science Foundation of China(31760064);National Natural Science Foundation of China(31360052)

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摘要：

二酰基甘油酰基转移酶(Diacylglycerol acyltransferase, DGAT)在植物油脂代谢和抗逆过程中发挥重要作用。为探究DGAT基因在向日葵(Helianthus annuus L.)中的进化及在油脂积累和非生物胁迫应答中的功能, 本研究以拟南芥AtDGATs基因为基础序列, 通过同源比对从向日葵基因组中获得18个DGAT同源基因序列, 并对其染色体分布、基因结构、蛋白保守结构域、系统进化关系、组织特异性表达以及非生物胁迫下的表达模式进行系统分析。结果表明, 向日葵DGATs基因可分为4个亚族(DGAT1、DGAT2、DGAT3和WSD), 且同一亚族成员具有相似的基因结构和蛋白保守基序; HaDGATs基因的启动子区富含逆境和植物激素响应相关元件; 片段复制是导致该基因家族扩张的主要因素; qRT-PCR结果表明, HaDGAT1、HaDGAT2和HaDGAT3主要在种子发育的早、中期表达, 与种子油脂快速积累密切相关, 而HaWSDs亚家族基因主要在茎、叶和花中表达。盐、低温、干旱和ABA处理后, 多数HaWSDs基因在向日葵根、茎和叶中呈现诱导表达, 推测其可能在对逆境胁迫的响应中起重要作用。这些结果为进一步研究向日葵DGATs基因的功能奠定了基础。

关键词: 向日葵, DGAT基因家族, 非生物胁迫, 基因表达

Abstract:

Diacylglycerol acyltransferase (DGAT) plays an important role in plant oil metabolism and stress resistance. To explore the evolution of DGAT gene in sunflower (Helianthus annuus L.) and its function in oil biosynthesis and response to abiotic stresses, 18 homologous gene sequences of DGAT were obtained from the sunflower genome database by alignment with Arabidopsis thaliana AtDGATs genes. Then, chromosome distribution, gene structure, conserved protein motifs, phylogenetic relationship, tissue-specific expression of HaDGATs, and their expressing patterns under abiotic stresses were systematically investigated. The results showed that sunflower DGATs genes were divided into four subfamilies (DGAT1, DGAT2, DGAT3, and WSD), and the members of the same subfamily shared similar gene structures and conserved motifs. The promoter regions of HaDGATs harbored multiple cis-elements related to environmental stresses and plant hormone response. The main factor for HaDGATs amplification was fragment duplication. The qRT-PCR indicated that HaDGAT1, HaDGAT2, and HaDGAT3 were mainly expressed in the early and middle stages of sunflower seed development, which coincided with the rapid accumulation period of oil in seeds, while HaWSD subfamily genes were mainly expressed in stems, leaves, and petals. Most of HaWSD genes could be induced by NaCl, low temperature, drought, and ABA treatments in sunflower roots, stems, and leaves, indicating that they might play essential roles in dealing with various abiotic stresses. The results revealed that HaDGATs had functional differentiation in regulating lipid biosynthesis and abiotic stresses. This study provides an important foundation for further understanding the function of sunflower DGAT genes.

Key words: sunflower, DGAT gene family, abiotic stresses, gene expression

表1

qRT-PCR引物序列"

基因名称
Gene name		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
HaDGAT1-1 TACGTAGGCGTCCGATCACA AATAGGCCTGCATGACTCTGTTT
HaDGAT1-2 GAGGAGCGTGAAGCCTGATG ATGCGCTGGAGAAGAAGCAC
HaDGAT1-3 GCCTAACATCGCCGAATCCA AACGCAACACACTCGTCTGA
HaDGAT2-1 AGTACGGGCGTGGCTATTTT GCCAATGACAGTCCCATCCA
HaDGAT2-2 TCTGGCCGATTGGAGTTGTC TGAACGCCACCTGGTATGAC
HaDGAT3 GTGGGTGTTAAGCAGCGGAA TTGACTTGCTGACCCAACCC
HaWSD1 GAGGATACCGAAACGCCGAT ACCTTCGTCTTTTTGCCCCT
HaWSD2 TGGATGCCGCTAAACACGTC CGGCGATGAAACCGTGAGAT
HaWSD3 TGGAGGATAGGCCAAGACGA TTGTTTCCCCATGTGCCATCT
HaWSD4 GAAAGGAGAGGACCACGACC GCTCCCGTTTTCCTTGTCCA
HaWSD5 GTTGGTCACGGACAAACACG ACGATAGAAGCGAAACGCCA
HaWSD6 GGGGCCAAAGATGACCAAGA CCACCACCGTTGCTTCTAGT
HaWSD7 TTCGTTAGGCGACGGTTTGT GCCCATGCTGCCTTTTAACG
HaWSD8 TGTGTGCACTGGGCTGTTTC ACACCCGCGATTTCACTGTATC
HaWSD9 CACTAAAAGGGTCCGCAGGT GTCGAACGCCTTTGGGAATG
HaWSD10 GCACGACGAACCACTATCCC TCCGCCAATGTTCATGTCCG
HaWSD11 AGCGTGTTTGGCCTCGAAC GTGAGGTCGGCTAGGTATTCG
HaWSD12 TGTGTGGAGGATGTGACCATGT GGTCTTCCCCCTTCACTTCAAC
18S rRNA CTACCACATCCAAGGAAGGCAG CGACAGAAGGGACGAGTAAACC

表2

向日葵HaDGATs基因基本信息"

转录本ID
Transcript ID		 基因名称
Gene name		 氨基酸数量
Amino acid amounts (aa)		 分子量
Molecular
weight (kD)		 等电点
pI		 亲水性系数
Hydrophilicity coefficient		 跨膜结构域数量Transmembrane
domain amounts
OTG30061 HaDGAT1-1 479 55.74 9.24 0.243 8
OTG13343 HaDGAT1-2 517 59.81 8.56 0.166 9
OTG03739 HaDGAT1-3 507 58.52 9.11 0.219 9
OTF98266 HaDGAT2-1 330 37.47 9.12 0.294 2
OTF96040 HaDGAT2-2 336 38.09 9.85 0.191 2
OTG25557 HaDGAT3 329 35.59 8.63 -0.416 0
OTG29248 HaWSD1 666 74.73 6.84 -0.249 0
OTG29986 HaWSD2 455 50.68 8.62 -0.037 0
OTG23138 HaWSD3 476 54.00 8.95 -0.241 0
OTG23434 HaWSD4 486 55.54 9.20 -0.244 0
OTG11715 HaWSD5 474 52.63 8.55 -0.003 0
OTG11716 HaWSD6 461 51.91 8.77 -0.025 0
OTG12880 HaWSD7 491 56.20 9.00 -0.221 1
OTG03949 HaWSD8 508 57.33 9.21 -0.176 1
OTG03950 HaWSD9 509 57.11 9.21 -0.190 1
OTF91362 HaWSD10 458 51.51 8.41 -0.079 0
OTF91364 HaWSD11 456 50.79 8.65 -0.057 0
OTF86778 HaWSD12 475 53.16 7.11 -0.111 0

图1

向日葵和其他植物DGAT家族蛋白系统发育树 Ha: 向日葵; At: 拟南芥; Gm: 大豆; Ah: 花生; Bn: 甘蓝型油菜; Sb: 高粱; Os: 水稻: Eg: 油棕: Aa: 黄花蒿; Ct: 红花; Zm: 玉米。"

图2

向日葵HaDGATs基因结构"

图3

向日葵HaDGATs蛋白的保守基序"

图4

向日葵HaDGATs染色体分布(A)及启动子顺式作用元件(B)分析"

表3

向日葵HaDGATs家族成员基因复制事件和发散时间分析"

重复基因对
Duplicated gene pairs		 非同义替换率
Ka		 同义替换率
Ks		 Ka/Ks 重复类型
Duplicated type		 选择类型
Selective type		 分化时间
Differentiation time (Mya)
HaDGAT1-1/HaDGAT1-2 0.1424 1.1312 0.1259 片段重复Segmental 纯化Purifying 37.70
HaDGAT1-1/HaDGAT1-3 0.1334 1.0506 0.1270 片段重复Segmental 纯化Purifying 35.02
HaDGAT1-2/HaDGAT1-3 0.0906 0.4019 0.2255 片段重复Segmental 纯化Purifying 13.39
HaWSD3/HaWSD4 0.1488 0.4223 0.3523 串联重复Tandem 纯化Purifying 14.07
HaWSD7/HaWSD8 0.1271 0.5899 0.2155 片段重复Segmental 纯化Purifying 19.66
HaWSD7/HaWSD9 0.1349 0.5677 0.2377 片段重复Segmental 纯化Purifying 18.92
HaWSD8/HaWSD9 0.0635 0.3272 0.1942 串联重复Tandem 纯化Purifying 10.90
HaWSD10/HaWSD11 0.1251 0.3299 0.3793 串联重复Tandem 纯化Purifying 10.99

图5

共线性关系分析 A: 向日葵种内HaDGATs共线性分析; B: 向日葵与拟南芥、红花种间DGATs基因共线性分析。"

图6

HaDGATs在向日葵不同组织的表达模式 DAF: 开花后天数。HaDGATs基因的相对表达量以根的表达量作为对照。"

图7

向日葵种子发育不同时期的油脂含量分析 不同字母表示数值有显著差异(P < 0.05)。"

图8

向日葵HaDGATs在盐(A)和低温(B)胁迫下的表达"

图9

向日葵HaDGATs在外源ABA (A)和干旱(B)处理下的表达"

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