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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 426-437.doi: 10.3724/SP.J.1006.2023.14251

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

向日葵HaLACS9基因的克隆与功能分析

杨佳宝1(), 张展2, 周至铭1, 吕新华1, 孙黎1,*()   

  1. 1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, 新疆铁门关 841007
  • 收稿日期:2021-12-29 接受日期:2022-06-07 出版日期:2022-07-07 网络出版日期:2022-07-07
  • 通讯作者: 孙黎
  • 作者简介:E-mail: 2516040371@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)

Cloning and function analysis of a HaLACS9 gene in Helianthus annuus L.

YANG Jia-Bao1(), ZHANG Zhan2, ZHOU Zhi-Ming1, LYU Xin-Hua1, SUN Li1,*()   

  1. 1College of Life Sciences, Shihezi University, Shihezi 832003, Xinjiang, China
    2Bingtuan Xingxin Vocational and Technical College, Tiemenguan 841007, Xinjiang, China
  • Received:2021-12-29 Accepted:2022-06-07 Published:2022-07-07 Published online:2022-07-07
  • 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：

长链酰基CoA合成酶(long-chain acyl-CoA synthetase, LACS)催化游离脂肪酸合成酰基CoA, 在植物脂质代谢中发挥重要作用。本研究从向日葵中筛选并克隆得到1个LACS家族基因HaLACS9, 同源比对表明HaLACS9具有LACS酶的保守结构域, 与拟南芥(Arabidopsis thaliana)、莴苣(Lactuca sativa)和刺菜蓟(Cynara cardunculus) LACS9蛋白具有较高的相似性。亚细胞定位预测HaLACS9定位于叶绿体。HaLACS9启动子区含有多种逆境及激素响应相关元件。qRT-PCR分析表明, HaLACS9在向日葵多个器官中表达, 在花后10 d的种子中优势表达。干旱、盐、外源脱落酸和赤霉素处理均能诱导HaLACS9基因在向日葵根、茎和叶中的表达。分析HaLACS9在向日葵种子发育不同时期的相对表达量发现, HaLACS9在向日葵种仁发育早期高水平表达, 与籽油的快速积累密切相关。随着种子发育和籽油积累速率的减慢, HaLACS9转录水平呈下降趋势。缺陷型酵母互补试验证明HaLACS9具有酰基CoA合成酶活性, 并且油酸是其偏好的催化底物。推测HaLACS9与向日葵种子发育过程中的油脂积累和非生物胁迫响应相关。

关键词: 向日葵, HaLACS9, 表达模式, 酵母功能互补

Abstract:

Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which plays important roles in lipid metabolism in plant. Here, a LACS family gene named HaLACS9 was screened and cloned from sunflower (Helianthus annuus L.). Homologous protein comparison showed that HaLACS9 had conserved LACS domains and had higher homology with Arabidopsis thaliana, Lactuca sativa, and Cynara cardunculus LACS9 proteins. Subcellular localization prediction indicated that HaLACS9 was localized in the chloroplast. The promoter region of HaLACS9 gene contained a large number of hormone and stress responses related elements. The qRT-PCR revealed that HaLACS9 gene was ubiquitously expressed in sunflower organs, and the highest expressed in seeds at 10 days after flowering. The transcription levels of HaLACS9 were induced by drought, salt, ABA, and GA3 treatments in sunflower roots, stems, and leaves. The relative expression pattern of HaLACS9 at seed developmental stages in sunflower was higher at the early seed developmental stages, related to sunflower seed oil rapid accumulation periods. The relative expression of HaLACS9 was gradually decreased at late stages of seed development and the slow accumulation rate of oil. HaLACS9 was proved to have the synthetase activity of acyl-CoA in complementation test of deficient yeast mutant and prefers to activate oleic acid substrate. It was speculated that HaLACS9 participated in the regulation of sunflower seed lipid biosynthesis and abiotic stress responses.

Key words: sunflower, HaLACS9, expression patterns, yeast complementation

表1

本研究的引物及序列"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Function
HaLACS9-F ATGAGTGCATATATTGTTGGAGTTT 全长cDNA克隆
Full-length cDNA cloning
HaLACS9-R TCAAGACTGGTAGAACTTTTCTAGC
18S rRNA-F CTACCACATCCAAGGAAGGCAG 实时荧光定量PCR
qRT-PCR
18S rRNA-R CGACAGAAGGGACGAGTAAACC
HaLACS9-qRT-F ACTCTCGCCCTTCGGAAATC 实时荧光定量PCR
qRT-PCR
HaLACS9- qRT-R TTTCAAATGCCCTCCCGTCT
pHaLACS9-F GGGGTACCATGAGTGCATATATTGTTGG (Kpn I) pYES2-HaLACS9载体构建
Construction of pYES2-HaLACS9 vector
pHaLACS9-R TGCTCTAGATTAAGACTGGTAGAACTTTT (Xba I)

图1

向日葵HaLACS9的克隆(A)及染色体定位分析(B)"

图2

HaLACS9与同源蛋白的序列比对及序列分析 At: 拟南芥; Ha: 向日葵; Cc: 刺菜蓟; Ps: 山杏; Os: 水稻; Ta: 小麦; Ls: 莴苣。"

图3

不同植物LACS9序列的保守基序分布(A)和保守基序分析(B) At: 拟南芥; Ha: 向日葵; Cc: 刺菜蓟; Ps: 山杏; Os: 水稻; Ta: 小麦; Ls: 莴苣。"

图4

HaLACS9的结构分析 A: HaLACS9的三级结构预测; AMP结合结构域用红色方框表示。B: HaLACS9的信号肽预测。"

图5

HaLACS9与同源蛋白的系统进化树 At: 拟南芥; Ha: 向日葵; Ls: 莴苣; Cc: 刺菜蓟; Si: 芝麻; Rc: 蓖麻; Ps: 山杏; Gm: 大豆; Ah: 花生; Eg: 油棕; Bn: 甘蓝型油菜; Zm: 玉米; Os: 水稻; Ta: 小麦。红三角标记为目的蛋白。"

图6

HaLACS9启动子顺式作用元件分析"

图7

HaLACS9的组织表达分析 不同小写字母表示数值有显著差异(P < 0.05)。"

图8

PEG (A)和NaCl (B)胁迫对HaLACS9相对表达量的影响 不同小写字母表示数值有显著差异(P < 0.05)。"

图9

外源ABA (A)和GA3 (B)处理对HaLACS9相对表达量的影响 不同小写字母表示数值有显著差异(P < 0.05)。"

图10

向日葵种仁不同发育时期HaLACS9相对表达水平和含油量 不同小写字母表示数值有显著差异(P < 0.05)。"

图11

酵母生长状况 不同小写字母表示数值有显著差异(P < 0.05)。"

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