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作物学报 ›› 2020, Vol. 46 ›› Issue (8): 1283-1290.doi: 10.3724/SP.J.1006.2020.94192

• 研究简报 • 上一篇    下一篇

紫苏二酰基甘油酰基转移酶2基因克隆与功能研究

鲁庚,唐鑫,陆俊杏,李丹,胡秋芸,胡田,张涛()   

  1. 重庆师范大学生命科学学院, 重庆 401331
  • 收稿日期:2019-12-11 接受日期:2020-03-24 出版日期:2020-08-12 网络出版日期:2020-04-26
  • 通讯作者: 张涛
  • 作者简介:E-mail: 778448973@qq.com
  • 基金资助:
    国家自然科学基金项目(31171588);重庆市技术创新与应用发展项目(cstc2019jscx-msxm0612)

Cloning and function analysis of a type 2 diacylglycerol acyltransferase (DGAT2) from Perilla frutescens

LU Geng,TANG Xin,LU Jun-Xing,LI Dan,HU Qiu-Yun,HU Tian,ZHANG Tao()   

  1. College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
  • Received:2019-12-11 Accepted:2020-03-24 Published:2020-08-12 Published online:2020-04-26
  • Contact: Tao ZHANG
  • Supported by:
    National Natural Science Foundation of China(31171588);Chongqing Technology Innovation and Application Development Project(cstc2019jscx-msxm0612)

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摘要：

二酰基甘油酰基转移酶(diacylglycerol acyltransferase, DGAT)是植物合成三酰甘油(TAG)最后一步的关键酶, 其中DGAT2在某些植物的种子油中能选择性积累更多不饱和脂肪酸。本文成功克隆了紫苏二酰基甘油酰基转移酶2基因(PfDGAT2), 并进行生物信息学分析。PfDGAT2实时荧光定量结果表明, 不同器官中PfDGAT2基因均有表达, 10 d种子的表达量最高, 在根中的表达量次之,在种子发育中后期, PfDGAT2表达量逐渐降低。与野生型拟南芥相比, 过表达PfDGAT2拟南芥种子含油率提高了21.68%~77.89%, 其中种子含油率增加最多的4个株系, 其亚麻酸(C18:3)增加4.57%, 花生一烯酸(C20:1)增加7.44%, 花生二烯酸(C20:2)增加5.4%, 二十二一烯酸(C22:1)增加10.37%, 而棕榈酸(C16:0)、硬脂酸(C18:0)和亚油酸(C18:2)含量分别降低了3.47%、6.64%和4.83%, 油酸(C18:1)和花生酸(C20:0)分别只降低了0.18%和1.91%。本研究结果表明, 紫苏PfDGAT2基因不仅能提高种子含油率, 还能促进亚麻酸、花生一烯酸等不饱和脂肪酸的积累, 这为研究植物不饱和脂肪酸的合成积累提供了参考及理论依据。

关键词: 二酰基甘油酰基转移酶(DGAT2), 种子含油率, 不饱和脂肪酸, 紫苏

Abstract:

Diacylglycerol acyltransferase (DGAT) is a key enzyme in the final step of triacylglycerol (TAG) synthesis in plant. In seed oil of certain plants, DGAT2 can selectively accumulate more unsaturated fatty acids. In this paper, we successful cloned PfDGAT2 from Perilla frutescens and performed bioinformatics analysis. Real-time fluorescence quantitative analysis showed that PfDGAT2 was expressed in different organs, with the highest in seeds at 10 d after anthesis, the medium in roots, and gradual decrement in the middle and late stages of seed. Compared with the wild-type, the oil content of seeds in PfDGAT2 transgenic Arabidopsis was increased by 21.68%-77.89%. The fatty acid components of the four strains with the largest increase in seed oil content were analyzed. Compared with the control, linolenic acid (C18:3), arachidonic acid (C20:1), arachidonic acid (C20:2), docosaenoic acid (C22:1) increased significantly by 4.57%, 7.44%, 5.40%, and 0.37%, respectively. Palmitic acid (C16:0), stearic acid (C18:0), and linoleic acid (C18:2) were obviously reduced by 3.47%, 6.64%, and 4.83%, respectively. Oleic acid (C18:1) only decreased by 0.18% and arachidic acid (C20:0) by 1.91%. In conclusion that PfDGAT2 gene can not only increase the oil content, but promote the accumulation of unsaturated fatty acids such as linolenic acid and arachidonic acid, which provides a reference and theoretical basis for studying the synthesis and accumulation of unsaturated fatty acid in plants.

Key words: diacylglycerol acyltransferase, seed oil content, unsaturated fatty acid, Perilla frutescens

表1

PCR引物"

引物名称
Primer name		 引物序列
Sequences (5'-3')
PfDGAT2 F CTCGCTTACTGCTACTTCAATG
PfDGAT2 R CGACAATTAGAGAATCCTGAGC
PfDGAT2-DL F AGTCCGAGCCCAACGGCGATGTCAG
PfDGAT2-DL R GGATGCCCAACCCTTGCTTTGTGCC
Actin F AGACCTTCAATGTGCCAGCCA
Actin R CACGACCAGCAAGATCCAACC
18S RNAF CGGCGACGCATCATTCAAA
DGAT2-BglII F ACTCTTGACCATGGTAGATCTGGAGTCCGAGCCCAACGGCG
DGAT2-BglII R GGACGTAAACTAGTCAGATCTAGAATCCTGAGCTCTAAGTCG
JD-F TTTCATTTGGAGAGAACACGGGGGA
JD-R CGCTGATCAATTCCACAGTTTTCGC

图1

PfDGAT2基因的克隆与分析 A: PfDGAT2的PCR扩增产物, M: DL2000 DNA marker。B: PfDGAT2 保守结构域的预测结果。"

图2

不同植物DGAT2氨基酸序列的系统进化树 分支上的数字表示Bootstrap验证中基于1000次重复该节点的可信度。"

图3

不同植物DGAT2蛋白序列比对分析 PfDGAT2: 紫苏; SiDGAT2: 芝麻(XP_011098009.1); StDGAT2: 马铃薯(XP_006365015.1); AtDGAT2: 拟南芥(OAP06431.1); GmDGAT2: 大豆(NP_001299586.1); NtDGAT2: 烟草(AGL46984.1); CsDGAT2: 亚麻荠 Camelina sativa(XP_010426724)。黑色方框内依次为DGAT2蛋白的YFP、EPHS、GGVQE、RXGFX(K/R)XAXXXGXX(L/V)VPXXXFG(E/Q)和VVGRPI的保守结构域。"

图4

PfDGAT2基因不同组织和种子不同时期的相对表达量 柱值标以不同字母表示在P < 0.05水平差异显著性。数据点为平均值±标准误(n = 3)。"

图5

转PfDGAT2拟南芥种子含油率 柱值标以不同字母表示在P < 0.05水平差异显著性。数据点为平均值±标准误(n = 3)。"

图6

转 PfDGAT2 拟南芥种子脂肪酸相对含量分析 图中*为PD2-1、PD2-3、PD2-5、PD2-5与野生型Col-0之间各组分显著性分析。*代表在P < 0.05时显著性的差异, **代表在P < 0.01时显著性的差异, ***代表在P < 0.001时显著性的差异。数据点为平均值±标准误(n = 3)。"

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