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作物学报 ›› 2018, Vol. 44 ›› Issue (04): 483-492.doi: 10.3724/SP.J.1006.2018.00483

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

BnA7HSP70分子伴侣结合蛋白超表达能够提高甘蓝型油菜耐旱性

万丽丽1,*(), 王转茸2, 辛强2, 董发明2, 洪登峰2, 杨光圣2   

  1. 1 武汉市农业科学院作物研究所, 湖北武汉 430065
    2 华中农业大学作物遗传改良国家重点实验室, 湖北武汉 430070
  • 收稿日期:2017-09-04 接受日期:2018-01-08 出版日期:2018-01-31 网络出版日期:2018-01-31
  • 通讯作者: 万丽丽
  • 作者简介:

    zhuzhanwang@163.com

  • 基金资助:
    本研究由湖北省武汉市农业科学院特种经济作物种质资源创新与产业开发技术研究项目(CX201710), 国家自然科学基金青年科学基金项目(31401413)和湖北省重大技术创新项目(2016ABA084)资助

Enhanced Accumulation of BnA7HSP70 Molecular Chaperone Binding Protein Improves Tolerance to Drought Stress in Transgenic Brassica napus

Li-Li WAN1,*(), Zhuan-Rong WANG2, Qiang XIN2, Fa-Ming DONG2, Deng-Feng HONG2, Guang-Sheng YANG2   

  1. 1 Institute of Crop, Wuhan Academy of Agricultural Sciences, Wuhan 430065, Hebei, China
    2 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430065, Hebei, China
  • Received:2017-09-04 Accepted:2018-01-08 Published:2018-01-31 Published online:2018-01-31
  • Contact: Li-Li WAN
  • Supported by:
    This study was supported by Germplasm Innovation Foundation of Wuhan Academy of Agricultural Sciences (CX201710), the National Natural Science Foundation of China (31401413), and the High Technology Innovation Foundation of Hubei Province (2016ABA084).

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摘要：

分子伴侣结合蛋白广泛参与植物生长发育过程, 在逆境下能够保护植物细胞免受胁迫。在甘蓝型油菜中超表达油菜含有HSP70热激蛋白结构域的分子伴侣基因BnA7HSP70, 所得到的转基因植株在缺水条件下延缓萎蔫。通过生理生化实验证明, 干旱条件下转基因植株有着更高的相对含水量、更强的渗透调节能力和较低的脂质膜过氧化性。另外, 转基因植株的幼苗在萌发期表现出对糖基化酶抑制剂衣霉素处理的耐受性。Evans blue染色实验证明, 转基因植株逆境下叶片死亡细胞数目比非转基因植株减少, 叶片衰老相关标记基因BnCNX1在转基因植株中下调表达证明, 超表达BnA7HSP70基因所介导的途径能够减轻逆境胁迫下的植株衰老, 保持叶片持绿性。在转基因植株中内质网和渗透胁迫产生的细胞死亡标记基因N-Rich蛋白BnNRP延迟表达证明, 在油菜中增强BnA7HSP70基因的表达能够缓解未折叠蛋白途径(unfold protein response, UPR)和NRP (N-rich pathway)途径介导的叶片黄萎, 并降低油菜叶片失绿的标记基因BnLSC222和BnLSC54的表达。研究结果表明, 在油菜中超表达BnA7HSP70基因能够提高植株在干旱条件下内质网胁迫的耐受性。

关键词: BnA7HSP70, 耐旱性, 甘蓝型油菜, 叶片衰老, 抗氧化酶

Abstract:

The molecular chaperone binding protein gene participates in the constitutive function of plant growth and protects plant cells against stresses. In this study, we found that BnA7HSP70 overexpressed transgenic lines did not wilt and showed only a small decrease in water potential. However, the wild type lines showed a large decrease in leaf water potential. The transgenic plants had higher relative water content, better osmotic adjustment and less lipid membrane peroxidation. In addition, the leaves from the elevated levels of BnA7HSP70 in transgenic lines conferred tolerance to the glycosylation inhibitor tunicamycin during germination. BnA7HSP70 overexpression-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell and the reduced induction of the senescence-associated marker gene BnCNX1. These phenotypes were accompanied by a delay in the induction of the cell death marker genes BnNRP, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP (N-rich protein)-mediated signaling pathway. Enhanced expression of BnA7HSP70 delayed unfold protein response and NRP pathway mediated chlorosis and the appearance of senescence-associated markers BnLSC222 and BnLSC54 in Brassica napus. These results suggest that overexpression of BnA7HSP70 in Brassica napus alleviate ER stress and osmotic stress-integrating cell death response confronted with water stress.

Key words: BnA7HSP70, drought tolerance, Brassica napus, leaf senescence, antioxidant enzymes

图1

BnA7HSP70-GFP融合蛋白在拟南芥原生质体中的定位(A) BnA7HSP70和GFP融合后蛋白的亚细胞定位, 在488 nm下原生质体中细胞质显示绿色荧光; (B)在580 nm下同样原生质体中叶肉细胞显示红色荧光信号; (C)自然光照视野下细胞图; (D)自然光和荧光下的融合图片。"

图2

BnA7HSP70基因超表达植株(OE)和非转基因对照(WT)在干旱和20% PEG渗透胁迫处理后表现出耐旱性(A)对生长4~5周油菜苗进行断水10 d处理, 可见转基因植株表现出更好的耐受性; (B)对生长4~5周油菜苗进行断水15 d处理, 可见转基因植株表现出更好的耐受性; (C)干旱10 d后复水3 d, 大多数非转基因植株不能恢复正常生长, 而转基因植株能够恢复正常生长。(D)在营养液中生长40 d的非转基因和转基因植株; (E)将生长在营养液中40 d的非转基因和转基因植株转移到20% (w/v) PEG-6000的培养液中48 h。通过处理过程中每天更换新鲜的PEG溶液来保证处理时的浓度保持不变; (F)转基因和非转基因植株在干旱10 d、15 d和复水3 d后叶片的相对含水量测定; (G)转基因和非转基因植株从营养液中正常生长转移到20% PEG-6000渗透胁迫处理48 h后叶片的相对含水量。"

图3

20% PEG处理下转基因植株和非转基因植株中的H2O2和MDA含量(A) 20% PEG处理后第3天植株H2O2含量; (B) 20% PEG处理后第3天植株中MDA含量。每次实验重复3次。"

图4

20% PEG胁迫条件下转基因和非转基因植株抗氧化酶活性(A) SOD酶活性; (B) POD酶活性。每次实验重复3次。"

图5

超表达BnA7HSP70转基因油菜能够在断水10 d后具有较高持水能力(A)转基因植株(OE2、OE3、OE7和OE8)与非转基因植株在干旱条件下的相对含水量; (B)对转基因和非转基因材料每个家系取样6~8片叶子, 测定叶绿素含量。Control: 正常生长条件下的野生型和超表达植株; Stress: 生长到4周的转基因和非转基因植株进行连续7 d干旱处理。"

图6

BnA7HSP70超表达能够延迟逆境下转基因植株的叶片衰老相关基因表达野生型(WT)和超表达(OE)转基因家系(OE2、OE3、OE7和OE8)在生长4周后断水10 d, 取样检测油菜叶片衰老相关基因BnLSC45和BnLSC222的表达。Control: 正常灌溉条件下的植株。Stress: 受到干旱胁迫后的植株。每次实验3次重复。干旱条件下诱导衰老相关基因BnLSC45 和BnLSC222基因的表达。从正常灌溉和断水胁迫处理下的非转基因和转基因植株成熟叶片中提取RNA, 用qRT-PCR实验检测基因的表达量。"

图7

BnA7HSP70超表达植株表现出对衣霉素诱导细胞死亡的耐受性(A)转基因和非转基因植株种子和幼苗对2.5 µg mL-1或者5.0 µg mL-1衣霉素的敏感性实验。(B)~(C) Evans Blue染色方法测定转基因和非转基因植株在衣霉素处理后7 d、14 d的细胞死亡程度。Abs (600 nm)测定值反映死细胞含量, 每个实验重复3次。"

图8

衣霉素处理后转基因和非转基因植株内质网胁迫的标签基因的表达分析取衣霉素处理0、24、48和72 h的非转基因和转基因植株的叶片, 分析内源BnA7HSP70、BnCNX1、BnNRP和BnBiP3基因的表达量。DMSO处理的植株为对照。BnCNX1、BnBiP3和BnNRP基因是内质网胁迫的标志基因。"

图9

BnA7HSP70调控bZIP 28基因介导的未折叠蛋白途径的模式(A)在逆境胁迫下, BnA7HSP70蛋白与未折叠蛋白结合后从bZIP 28中释放下来, 定位于高尔基体的S1P和S2P将bZIP 28蛋白水解后释放得到bZIP 28n, 转录因子bZIP 28n定位到细胞核中上调BnBiP3和BnCNX1基因的表达; (B)当BnA7HSP70超表达后, 得到足够的BnA7HSP70蛋白质与bZIP28和胁迫下产生的未折叠蛋白结合, 使得bZIP28保留在内质网上, 从而不会上调细胞核中基因的表达。"

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