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作物学报 ›› 2018, Vol. 44 ›› Issue (02): 218-226.doi: 10.3724/SP.J.1006.2018.00218

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棉花半乳糖基转移酶基因GhGalT1启动子的克隆及表达分析

秦丽霞, 李静, 张换样, 李盛, 竹梦婕, 焦改丽, 吴慎杰*   

  1. 山西省农业科学院棉花研究所, 山西运城 044000;
  • 收稿日期:2017-04-11 接受日期:2017-09-10 出版日期:2018-02-12 网络出版日期:2017-10-27
  • 通讯作者: 吴慎杰
  • 作者简介:

    qinlixia11@163.com, Tel: 0359-2161515

  • 基金资助:
    本研究由国家自然科学基金项目(31601350), 山西省基础研究项目(2015021152)和国家转基因生物新品种培育重大专项(2016ZX08005)资助

Cloning and Expression Analysis of Galactosyltransferase Gene GhGalT1 Promoter in Cotton

Li-Xia QIN, Jing LI, Huan-Yang ZHANG, Sheng LI, Meng-Jie ZHU, Gai-Li JIAO, Shen-Jie WU*   

  1. Institute of Cotton, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, Shanxi, China;
  • Received:2017-04-11 Accepted:2017-09-10 Published:2018-02-12 Published online:2017-10-27
  • Contact: Shen-Jie WU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31601350), the Shanxi Province Fundamental Research Foundation (2015021152), and the National Major Project for Developing New GM Crops (2016ZX08005).

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摘要：

糖基转移酶(glycosytransferase, GT)是催化活化的供体糖基转移到特异受体生成糖苷键的酶类, 在应答多种生物和非生物胁迫中起重要作用。本研究利用PCR技术从陆地棉品种Coker 312基因组中分离克隆了GhGalT1基因的启动子, 序列长度为539 bp, 命名为pGhGalT1。启动子分析软件PlantCARE分析表明pGhGalT1含有CAAT-box、TATA-box核心元件, 以及响应干旱、热、脱水、防御与胁迫应答的顺式作用元件MBS、HSE、MYCCONSE、TC-rich repeats和CGTCA-motif等。因此将pGhGalT1构建到启动子检测载体pBI101-GUS上, 形成pBI101-pGhGalT1-GUS融合表达载体, 以检测其启动子活性。通过农杆菌介导的浸花法转化拟南芥, 经卡那霉素抗性筛选及PCR检测成功获得阳性转基因植株。对T3代转基因拟南芥进行组织化学染色分析显示该启动子主要在生长5~15 d的幼苗主根及侧根根尖表达, 在子叶及莲座叶边缘也有微弱表达。非生物胁迫和激素处理后的组织化学染色、GUS酶活性及GUS基因定量分析结果显示GhGalT1基因的启动子受盐、渗透胁迫和激素(6-BA、MeJA、BL)的诱导, 该结果为合理选用启动子改良作物提供理论依据。

关键词: 棉花, 糖基转移酶, 启动子, 顺式作用元件, GUS组织化学染色, GUS定量分析

Abstract:

Glycosytransferases (GTs) transfer an activated sugar donor to a specific acceptor to form glucosidic bond, which are regulated by various abiotic and biotic stresses, and may play a role in plant responses to changes in living conditions. In this study, a 539 bp fragment of GhGalT1 5′-flanking sequence was isolated from upland cotton Coker 312 by PCR, designated pGhGalT1. Analysis of pGhGalT1 sequence by PlantCARE revealed it contained not only putative CAAT box, TATA box sequence, but also MBS, HSE, TC-rich repeats, MYCCONSE and CGTCA-motif cis-acting element which involved in drought, heat, dehydration, defense and stress responsiveness. Thus, we constructed it into pBI101-GUS vector and formed pGhGalT1::GUS fusion expression vector (pBI101-pGhGalT1-GUS), then transferred the vector into Arabidopsis by the Agrobacterium-mediated floral dip method, and successfully obtained positive transgenic plants by screening test of resistance to kanamycin and PCR detection. Histochemical assay of T3 generation of transgenic Arabidopsis revealed that GUS activities were mainly accumulated in root tips of primary and lateral roots in 5- to 15-day-old seedlings, and less strongly in cotyledons and rosette leaves. The histochemical staining results and the assay of quantitative GUS activity and GUS gene expression under abiotic stresses and hormone treatments revealed that the GhGalT1 promoter was salt-/osmotic-/6-BA-/MeJA-/BL-inducible. These findings contribute to the selection of a suitable promoter for crop molecular improvement.

Key words: cotton (Gossypium hirsutum L.), glycosytransferase, promoter, cis-acting element, GUS histochemical staining, quantitative GUS assay

表1

本试验中所用引物序列"

引物名称
Primer name		 上游引物序列
Forward primer sequence (5′-3′)		 下游引物序列
Reverse primer sequence (5′-3′)
pGhGalT1 GGGGTCGACTAACAACGGTGAAGAAAGAA GGTGGATCCAACTGTGAATTTGAAGAGAAG
GUS ATGTTACGTCCTGTAGAAACC TCATTGTTTGCCTCCCTGCTGC
NPTII ATGATTGAACAAGATGGATTGC TCAGAAGAACTCGTCAAGAAGGC
AtActin2 GAAATCACAGCACTTGCACC AAGCCTTTGATCTTGAGAGC
GUS-RT ATGTTCTGCGACGCTCAC CCCGGCTAACGTATCTGT

图1

pBI101-pGhGalT1-GUS表达载体构建示意图"

图2

棉花GhGalT1基因启动子的克隆 M: DNA marker DL2000; 1: 阴性对照; 2: pGhGalT1目的片段。"

图3

GhGalT1启动子序列及预测的顺式作用元件"

表2

GhGalT1启动子主要顺式作用元件及预测的功能"

顺式作用元件
cis-acting element		 核心序列
Core sequence		 位置
Location (bp)		 功能
Function
3-AF binding site CACTATCTAAC -472 to -462 光响应元件 Light responsive element
ATCT-motif AATCTAATCT -401 to -392 光响应元件
Part of a conserved DNA module involved in light responsiveness
Box 4 ATTAAT -505 to -499 光响应元件
Part of a conserved DNA module involved in light responsiveness
Box 1 TTTCAAA -387 to -381 光响应元件Light responsive element
GAG-motif AGAGAGT -246 to -240 光响应元件 Part of a light responsive element
MYCCONSE CACCTG -298 to -293 脱水响应 Dehydration responsiveness
CGTCA-motif CGTCA -175 to -171 茉莉酸甲酯响应元件 MeJA responsive element
MBS CAACTG -141 to -136 干旱诱导的MYB 结合位点
MYB binding site involved in drought-inducibility
TC-rich repeats ATTTTCTTCA -460 to -451 防御及胁迫响应元件
Cis-acting element involved in defense and stress responsiveness
HSE AAAAAATTTC -249 to -259 热胁迫响应元件 Heat stress responsiveness
Sp 1 CC(G/A)CCC -152 to -147 光响应元件Light responsive element
Skn-1 motif GTCAT -520 to -524, -325 to -321,
-206 to -202		 胚乳特异表达
Endosperm specific expression
TCCC-motif TCTCCCT -156 to -148 光响应元件 Light responsive element
Circadian CAANNNNATC -364 to -355 调控生物节律
Cis-acting regulatory element involved in circadian control

图4

GhGalT1启动子载体的构建 M: DNA marker DL2000; A: pBI101-pGhGalT1-GUS质粒的PCR检测; 1和2: 阴性对照; 3: 阳性对照; 4~5: pBI101-pGhGalT1-GUS质粒; B: 重组质粒pBI101-pGhGalT1-GUS经Sal I和BamH I双酶切电泳."

图5

T1代转基因拟南芥阳性植株的卡那霉素抗性筛选箭头所指部位为具有卡那霉素抗性的绿色小苗。"

图6

转基因拟南芥阳性植株的PCR检测 A~C: T1代转基因拟南芥阳性植株的PCR检测; D: T3代转基因拟南芥阳性植株的PCR检测; A: 用抗性标记基因NPTII引物扩增的产物; B和D: 用GhGalT1启动子pGhGalT1引物扩增的产物; C: 用GUS基因引物扩增的产物; 1: 野生型对照; 2: 阳性质粒对照; 3~11: 不同株系T1和T3代转基因植株。"

图7

GhGalT1启动子转基因拟南芥的GUS活性检测 A~B: 5 d幼苗, B为A的局部放大; C~D: 10 d幼苗, D为C的局部放大; E~F: 15 d小苗, F为E的局部放大; G~H: 成熟叶片, H为G的局部放大。"

图8

盐、干旱和激素处理GhGalT1启动子转基因拟南芥的GUS活性检测生长10 d幼苗在MS液体培养基(A~C)和分别添加150 mmol L-1 NaCl (D~E)、300 mmol L-1甘露醇(F~G)、100 μmol L-1 6-BA (H~J)、50 μmol L-1 MeJA (K~L)和500 nmol L-1 BL (M~N)的MS液体培养基处理后的GUS染色结果; 其中B和C为A的局部放大、E为D的局部放大、G为F的局部放大、I和J为H的局部放大、L为K的局部放大、N为M的局部放大。"

图9

胁迫条件下转基因拟南芥GUS定量分析 mannitol: 甘露醇; NaCl: 氯化钠; MeJA: 茉莉酸甲酯; BL: 油菜素内酯; 6-BA: 6-苄氨基嘌呤。误差棒表示±标准差, n > 50。柱子上大、小写字母表示胁迫处理与对照在0.01和0.05概率水平的差异显著(t检验)。WT: 野生型; L2和L6: pGhGalT1:GUS转基因拟南芥株系2和株系6。"

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