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作物学报 ›› 2020, Vol. 46 ›› Issue (4): 503-512.doi: 10.3724/SP.J.1006.2020.94082

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

费尔干猪毛菜病程相关蛋白SfPR1a基因的异源表达增强了烟草对干旱、盐及叶斑病的抗性

衡友强,游西龙,王艳()   

  1. 新疆大学生命科学与技术学院 / 新疆生物资源与基因工程重点实验室, 新疆乌鲁木齐 830046
  • 收稿日期:2019-06-04 接受日期:2019-12-26 出版日期:2020-04-12 网络出版日期:2020-01-15
  • 通讯作者: 王艳
  • 作者简介:E-mail: hengyouqiang706567@163.com
  • 基金资助:
    本研究由新疆生物资源基因工程重点实验室开放课题(2017D04026);国家自然科学基金项目资助(31860061)

Pathogenesis-related protein gene SfPR1a from Salsola ferganica enhances the resistances to drought, salt and leaf spot disease in transgenic tobacco

HENG You-Qiang,YOU Xi-Long,WANG Yan()   

  1. Xinjiang Key Laboratory of Biological resources and Genetic Engineering / College of Life Science and Technology, Xinjiang University, Urumqi 830046, Xinjiang, China
  • Received:2019-06-04 Accepted:2019-12-26 Published:2020-04-12 Published online:2020-01-15
  • Contact: Yan WANG
  • Supported by:
    This study was supported by the Xinjiang Key Laboratory of Biological Resources Genetic Engineering(2017D04026);the National Natural Science Foundation of China(31860061)

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摘要：

为明确一年生草本盐生植物费尔干猪毛菜(Salsola ferganica Drob.)病程相关蛋白基因SfPR1a (GenBank登录号为JQ670917)是否参与了植物对逆境胁迫的响应, 采用qRT-PCR检测了该基因在不同组织部位和脱落酸(ABA)、茉莉酸(JAs)、乙烯合成直接前体(ACC)等相关激素胁迫及NaCl处理下的表达规律, 同时对转基因烟草在盐、旱及丁香假单胞菌等胁迫下的抗性进行了鉴定。结果显示, SfPR1a基因在费尔干猪毛菜根中的表达量显著高于茎叶中, 且受到ABA、JAs、ACC、NaCl的积极诱导; 干旱胁迫下, 转基因烟草的丙二醛(MDA)含量显著低于野生型烟草, 显示出较强的抗旱表型; 盐胁迫下, 异源表达SfPR1a的转基因烟草幼苗生长显著优于野生型烟草; 丁香假单胞菌攻毒后的转基因烟草叶片呈现严重的坏死反应, 但植株的整体抗性表型显著优于野生型烟草; 亚细胞定位结果显示该蛋白定位于植物细胞质外体空间。以上结果表明, 费尔干猪毛菜病程相关蛋白SfPR1a基因参与了植物对非生物及生物胁迫的抗性。

关键词: 病程相关蛋白基因SfPR1a, 表达规律, 转基因烟草, 抗性功能, 亚细胞定位

Abstract:

In order to investigate whether SfPR1a, a pathogenesis-related protein gene from an annual halophytic species Salsola ferganica Drob., was involved in the response to plant defense, qRT-PCR was employed to detect its expression patterns under abscisic acid (ABA), jasmonic acid (JAs), ethylene synthesis direct precursor (ACC), and NaCl treatments. We also identified resistances to salt, drought and Pseudomonas syringae tomato (PstDC3000) of transgenic tobacco were identified. The expression of SfPR1a gene in roots was significantly higher than that in shoots, and positively induced by ABA, JAs, ACC, and NaCl treatments. The malondialdehyde (MDA) content of transgenic tobacco was significantly lower than that of wild-type tobacco, showing a strong resistance to drought. The ectopic expression of SfPR1a gene improved plant growth under salt stress. After infection of P. syringae, transgenic tobacco leaves showed serious necrosis reaction, but the overall resistance phenotype of the plants was significantly better than that of WT. Subcellular localization analysis showed SfPR1a was localized in the plant cell apoplastic space. The above results indicated that the SfPR1a gene is involved in plant resistance to abiotic and biotic stresses.

Key words: pathogenesis-related protein gene SfPR1a, expression pattern, transgenic tobacco, resistance function, subcellular localization

图1

费尔干猪毛菜SfPR1a蛋白与其他PR1蛋白家族成员的比较 不同物种PR1蛋白GenBank登录号: 水稻OsPR1 (AJR16763.1)、大豆GmPR1 (XP003545775.1)、青稞HvPR1 (CAA79703.1)、橡胶树HbPR1 (ALS87256.1)、费尔干猪毛菜SfPR1a (AFR90191.1)。"

图2

SfPR1a基因在费尔干猪毛菜不同组织部位以及不同处理下的表达分析 SfPR1a基因在茎叶和根中(A)、ABA (B)、ACC (C)、JAs (D)以及NaCl (E)处理下的相对表达量。每组实验设3个生物学重复, 不同字母表示存在显著差异(P < 0.05)。"

图3

SfPR1a基因在转基因烟草中的表达分析 标以不同小写字母的柱值差异显著(P < 0.05)。"

图4

转SfPR1a基因烟草在干旱胁迫下的抗性 A: 干旱胁迫处理2周后的烟草生长表型; B: 干旱胁迫处理2周后的烟草MDA含量变化; C: 干旱胁迫处理4周复水1周后的烟草生长表型; D: 干旱胁迫处理4周复水1周后的烟草存活率统计。图中不同小写字母间表示存在显著性差异(P < 0.05)。"

图5

转SfPR1a基因烟草在盐胁迫下的抗性 200 mmol L-1 NaCl胁迫下的烟草萌发生长表型(A)及相对生长量(B); 250 mmol L-1 NaCl胁迫下的烟草成苗抗盐表型(C)。图中标以不同小写字母的柱值差异显著(P < 0.05)。"

图6

转SfPR1a基因烟草对叶斑病的抗性 A: 接菌10 d后烟草叶片菌斑面积; B: 接菌45 d后的烟草叶片; C: 接菌45 d后烟草叶片菌斑面积; D: 接菌60 d后烟草植株的生长表型。图中标以不同小写字母的柱值差异显著(P < 0.05)。"

图7

SfPR1a的亚细胞定位 A: 空载对照mGFP的荧光图; B: 融合蛋白SfPR1a-mGFP的荧光图; C: 明场下的图B; D: 荧光图B与明场图C的叠加效果图。"

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