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作物学报 ›› 2018, Vol. 44 ›› Issue (12): 1829-1836.doi: 10.3724/SP.J.1006.2018.01829

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转cry1C*及cry2A*基因早粳稻Bt蛋白的时空表达和抗螟虫性

李荣田1,王新宇1,田崇兵1,周青1,刘长华1,2,*()   

  1. 1黑龙江大学 / 分子生物学黑龙江省高校重点实验室, 黑龙江哈尔滨150080
    2黑龙江大学农业资源与环境学院, 黑龙江哈尔滨150080
  • 收稿日期:2018-01-23 接受日期:2018-08-20 出版日期:2018-12-12 网络出版日期:2018-09-29
  • 通讯作者: 刘长华
  • 基金资助:
    本研究由国家转基因生物新品种培育重大专项(2016ZX08001001-001-007);国家高技术研究发展计划(863 计划)项目(2014AA10A600);分子生物学黑龙江省高校重点实验室开放课题(1206)资助

Spatio-temporal Expression of Bt Protein and Stem Borer Resistance of Transgenic Early Japonica Rice with cry1C* or cry2A* Gene

Rong-Tian LI1,Xin-Yu WANG1,Chong-Bing TIAN1,Qing ZHOU1,Chang-Hua LIU1,2,*()   

  1. 1 Key Laboratory of Molecular Biology, College of Heilongjiang Province / Heilongjiang University, Harbin 150080, Heilongjiang, China
    2 College of Agricultural Resources and Environment, Heilongjiang University, Harbin 150080, Heilongjiang, China
  • Received:2018-01-23 Accepted:2018-08-20 Published:2018-12-12 Published online:2018-09-29
  • Contact: Chang-Hua LIU
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops(2016ZX08001001-001-007);the National Major Project for Developing New GM Crops(2014AA10A600);the Open Project of Key Laboratory of Molecular Biology, College of Heilongjiang Province

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摘要：

早粳稻空育131为受体, 以根瘤农杆菌介导遗传转化法创制了转ubi启动子调控下的cry1C*及cry2A*基因早粳稻空育131 (cry1C*)和空育131(cry2A*)。为了研究转基因水稻Bt蛋白的时空表达特性及抗螟虫性, 将不同转化事件形成的转基因早粳稻品系种植于田间, 利用酶联免疫吸附测定(ELISA)法检测转基因水稻不同生长发育阶段不同器官的、以及成熟期糙米的Bt蛋白量, 采用室内离体茎秆法接虫鉴定转基因水稻的抗螟虫性。结果显示, 转基因早粳稻不同抗虫基因Bt蛋白量不同, cry1C*基因总是低于cry2A*基因的蛋白质表达量; 不同生长发育时期Bt蛋白量不同, 叶片和茎鞘等器官的Bt蛋白量为分蘖期<抽穗期<灌浆期, 幼穗或糙米等器官的Bt蛋白量为抽穗期幼穗>灌浆期幼穗>成熟期糙米; 不同器官Bt蛋白量不同, 高低次序在分蘖期为叶片、茎鞘, 抽穗期为叶片、幼穗和茎鞘, 灌浆及成熟期为叶片、茎鞘、幼穗和糙米; 同一抗虫基因不同转基因品系间抽穗期叶片、茎鞘和幼穗等器官Bt蛋白量及抗螟虫性、糙米Bt蛋白量等性状存在差异, 抽穗期各器官Bt蛋白量与抗螟虫性及成熟期糙米Bt蛋白量之间相关不显著, 不论Bt蛋白量高或低的品系均表现为高抗螟虫。转基因早粳稻营养器官生长发育前期Bt蛋白量较低、后期较高, 繁殖器官生长发育早期Bt蛋白量较高、晚期较低, 营养器官通常比繁殖器官的Bt蛋白量高。在本研究范围内, 不同基因及不同转化事件培育的转基因水稻Bt蛋白表达量高低不同, 但所有的转基因早粳稻品系均表现高抗螟虫。

关键词: cry1C*基因, cry2A*基因, 转基因早粳稻, Bt蛋白, 时空表达, 抗螟虫性

Abstract:

The early japonica rice Kongyu 131 (Oryza sativa) was transformed with cry1C* or cry2A* gene driven by ubi promoter using Agrobacterium tumefaciens mediated method to create the transgenic early japonica rice Kongyu 131 (cry1C*) and Kongyu 131 (cry2A*). The experiments were conducted by transplanting the different lines of Kongyu 131 (cry1C*) and Kongyu 131 (cry2A*) from independent transformation events in the field to detect Bt protein content (BPC) of organs of the transgenic rice at different growth stages with enzyme-linked immune sorbent assay (ELISA) and resistance to rice striped stem borer as evidenced by insect feeding bioassays. The BPC of cry1C* gene was always lower than that of cry2A* in early japonica rice. There was a difference in BPC at different growth stages of transgenic rice, displaying tillering stage < heading stage < filling stage in leaf blade or sheath-stem and heading stage > filling stage > maturity in young panicle or brown rice. BPC of organs of transgenic rice was different at every stage, the order of BPC from high to low was leaf blade and sheath-stem at tillering stage, leaf blade, young panicle and sheath-stem at heading stage, leaf blade, sheath-stem, young panicle and brown rice at filling stage and maturity. There were some obvious differences in BPC of leaf blade, sheath-stem and young panicle at heading stage or brown rice at maturity and in borer-resistant property among transgenic lines with cry1C* or cry2A*. Correlations between BPC of each organ at heading stage and borer-resistant property or BPC of brown rice were not found in transgenic rice lines with cry1C* or cry2A*. The BPC increased in vegetative organs while descended in reproductive organs in the process of growth and development, and BPC of vegetative organs was usually higher than BPC of reproductive organs in both of Kongyu 131 (cry1C*) and Kongyu 131 (cry2A*). All transgenic early japonica rice showed effective resistance to stem borer in the experiment, though there was significant difference in BPC either between cry1C* and cry2A* genes or among transgenic rice lines.

Key words: cry1C* gene, cry2A* gene, transgenic early japonica rice, Bt protein content, spatio-temporal expression, stem borer resistance

图1

Ti质粒pBar13-cry1C*(A)和pBar13-cry2A*(B)的T-DNA区 RB: 右边界; ubi: ubi启动子; cry1C*: cry1C*基因; cry2A*: cry2A*基因; 35S: CaMV35S启动子; bar: 膦丝菌素乙酰转移酶基因; LB: 左边界。"

图2

转基因早粳稻HD1和HD2的Bt蛋白量 A: 叶片; B: 茎鞘; C: 幼穗或糙米; BPC: Bt蛋白量。*和**: 差异达0.05和0.01概率显著水平。"

图3

转基因早粳稻不同生长发育阶段的Bt蛋白量 A: 叶片; B: 茎鞘; C: 幼穗或糙米; BPC: Bt蛋白量。*和**: 差异达0.05和0.01概率显著水平。"

图4

转基因早粳稻器官的Bt蛋白量 A: 分蘖期; B: 抽穗期; C: 灌浆期及成熟期; BPC: Bt蛋白量。**差异达0.01概率显著水平。"

表1

转基因早粳稻不同品系Bt蛋白量及抗虫性"

品系
Line		 叶片Bt蛋白量
BPC of leaf blade (μg g-1)		 茎鞘Bt蛋白量
BPC of sheath-culm
(μg g-1)		 幼穗Bt蛋白量
BPC of young panicle (μg g-1)		 糙米Bt蛋白量
BPC of brown rice (μg g-1)		 校正幼虫死亡率
Adjusted larval mortality rate (%)
HD1-1 5.33±0.04** 0.50±0.03** 0.52±0.02** 0.09±0.01** 96.46±1.42**
HD1-2 2.74±0.03** 0.49±0.02** 0.51±0.03** 0.15±0.02** 91.34±1.02**
HD1-3 1.16±0.02** 0.44±0.03** 0.43±0.03** 0.06±0.02** 92.08±2.70**
HD1-4 1.16±0.02** 0.31±0.03** 0.36±0.02** 0.10±0.02** 96.20±1.06**
HD2-1 21.81±0.02** 2.87±0.04** 3.95±0.02** 0.33±0.03** 94.21±1.05**
HD2-2 29.65±0.02** 5.69±0.02** 4.73±0.02** 0.24±0.02** 93.73±3.55**
HD2-3 21.63±0.04** 3.63±0.02** 4.02±0.05** 0.55±0.03** 91.56±4.10**
HD2-4 49.63±0.06** 4.64±0.02** 5.43±0.04** 0.13±0.03** 98.06±0.88**

表2

转基因早粳稻不同品系抽穗期Bt蛋白量和糙米Bt蛋白量及螟虫抗性关系"

转基因早粳稻
Transgenic early japonica rice		 抽穗期性状
Characteristic at heading stage		 成熟期糙米Bt蛋白量
BPC of brown rice at maturity		 校正幼虫死亡率
Adjusted larval mortality rate
HD1 叶片Bt蛋白量 BPC of leaf blade 0.233 0.375
茎鞘Bt蛋白量 BPC of sheath-culm 0.254 -0.389
幼穗Bt蛋白量 BPC of young panicle 0.381 -0.267
HD2 叶片Bt蛋白量 BPC of leaf blade -0.796 0.910*
茎鞘Bt蛋白量 BPC of sheath-culm -0.533 0.250
幼穗Bt蛋白量 BPC of young panicle -0.827 0.838
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