一个水稻铜锌SOD酶基因在应答亚砷酸盐胁迫中的作用

doi:10.16819/j.1001-7216.2018.7154

摘要/Abstract

摘要：

【目的】水稻Os08g44770.1基因编码一个铜锌SOD酶,但其在响应亚砷酸盐[As(Ⅲ)]胁迫中的生物学功能未知。本研究旨在深入揭示由该基因调控水稻砷耐性改变的分子机理并为水稻抗逆育种提供理论参考。【方法】以野生型日本晴(WT)和2个Os08g44770.1过表达转基因株系为试材,通过胁迫处理、生理指标测定和基因表达分析等,系统探究了转基因植株对As(Ⅲ)的耐受性表现,并初步揭示了其调控水稻砷耐性的生理和分子机理。【结果】与WT相比,过表达转基因株系对As(Ⅲ)更加敏感;转基因植株在砷胁迫下的根系相对伸长量、生物量(干质量)、叶绿素含量、根系细胞质膜完整性、叶片抗氧化程度等均显著低于WT;Os08g44770.1在砷处理后的WT和转基因植株叶片中的表达模式略有不同,但均表现为处理24 h时被显著诱导表达。【结论】过度表达Os08g44770.1基因可导致水稻的砷耐受性极显著下降。

关键词: 水稻, 铜锌SOD, 亚砷酸盐, 转基因, 生理机理, 基因表达

Abstract:

【Objective】Os08g44770.1 encodes a Cu/Zn-SOD protein in rice, but its biological function in response to arsenite [As(Ⅲ)] remains unclear. The results presented here would be intriguing for further investigating the molecular mechanisms of Os08g44770.1-mediated arsenic tolerance in rice, and also providing useful new idea for rice stress resistance breeding. 【Methods】Using wild type(WT) Nipponbare and two transgenic rice lines overexpressing Os08g44770.1 as materials, we investigated the phenotypes of WT and transgenic plants after exposure to As(Ⅲ), and further uncovered its physiological and molecular mechanisms in regulation of As(Ⅲ) tolerance in rice through stress treatment, physiological index measurement, and gene expression analysis, etc. 【Result】The results showed that, compared with WT, transgenic rice plants were more sensitive to As(Ⅲ) stress; Under As(Ⅲ) exposure, transgenic plants showed shorter relative root elongation, lower biomass (dry weight) and chlorophyll content, as well as lower root cell membrane integrity and antioxidant abilities in leaves. Moreover, Os08g44770.1 exhibited slightly different expression patterns but both showing expression peak at 24 h after As(Ⅲ) treatment in leaves of WT and transgenic plants. 【Conclusion】Strong overexpression of Os08g44770.1 in rice would result in its hypersensitivity to arsenite stress.

Key words: rice, Cu/Zn SOD, arsenite, transgenic, physiological mechanism, gene expression

中图分类号:

窦玲玲, 胡海超, 马龙, 柯笑楠, 刘明月, 练旺民, 金珂, 谢玲娟, 刘庆坡. 一个水稻铜锌SOD酶基因在应答亚砷酸盐胁迫中的作用[J]. 中国水稻科学, 2018, 32(5): 437-444.

Lingling DOU, Haichao HU, Long MA, Xiaonan KE, Mingyue LIU, Wangmin LIAN, Ke JIN, Lingjuan XIE, Qingpo LIU. Functional Analysis of a Copper/Zinc SOD Encoding Gene in Response to Arsenite Stress in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(5): 437-444.

图/表 10

Table 1 The real-time RT-PCR primer sequences.

基因名称 Gene symbol	正向引物 Forward primer（5′-3′）	反向引物 Reverse primer（5′-3′）
Os08g44770.1	GCTTCCACCTCCACGAGTTT	GCCACCCTTCCCCAAATCAT
β-actin	TGTGGATTGCCAAGGCTGAG	ACGGCGATAACAGCTCCTC

图1 部分转基因水稻的PCR检测 M–DNA标记 2000; 1–阳性对照;2, 3, 5~7, 9~12, 14, 15, 19, 21, 23, 24为转基因阳性植株;4, 8, 13, 16~18, 20, 22为转基因阴性植株。

Fig. 1. PCR analysis of transgenic rice plants carrying overexpressed Os08g44770.1. M–DNA marker 2000; 1, Positive control; 2, 3, 5-7, 9-12, 14, 15, 19, 21, 23, 24 are transformed plants; The others are untransformed plants.

图2 WT和各转基因株系叶片中Os08g44770.1的表达量

Fig. 2. Expression level of Os08g44770.1 gene in the leaves of wild type(WT) and transgenic rice lines.

图3 亚砷酸盐处理3 d和6 d后野生型(WT)和两个转基因株系的表型

Fig. 3. Comparison of phenotypes of WT and transgenic rice plants after As(Ⅲ) treatment for 3 and 6 days.

图4 亚砷酸盐处理6 d后WT和各转基因株系的生物量相同小写字母表示不同材料不同处理间差异未达0.05显著水平。

Fig. 4. Comparison of biomass of WT and transgenic rice plants after 6-day As(Ⅲ) treatment(n=8). Common lowercase letters above the bars indicate no significant difference among treatments or materials (P≤0.05).

图5 亚砷酸盐处理6 d后WT和各转基因株系根系相对伸长量的比较(n=8) 相同小写字母表示不同材料不同处理间差异未达0.05显著水平。

Fig. 5. Comparison of relative root elongation of WT and transgenic rice plants under As(Ⅲ) stress for 6 days (n=8). Common lowercase letters above the bars indicate no significant difference among treatments or materials (P≤0.05).

Table 2 Effects of As(Ⅲ) stress on chlorophyll contents of wild type(WT) and transgenic rice plants (n=3).

处理 Treatment	供试材料 Tested material	叶绿素a含量 Chlorophyll a content / (mg∙g^-1)	叶绿素b含量 Chlorophyll b content / (mg∙g^-1)	总叶绿素含量 Chlorophyll a+b content / (mg∙g^-1)	类胡萝卜素含量 Carotenoid content / (mg∙g^-1)	叶绿素a /叶绿素b Chlorophyll a/ Chlorophyll b	类胡萝卜素 /总叶绿素 Carotenoid/ Chlorophyll a+b
对照CK	WT	24.25±1.16 a	8.14±1.16 a	32.39±1.79 a	4.69±0.30 abc	2.98±0.35 a	0.14±0.02 b
	OE-44770-4	22.90±1.30 b	6.20±1.30 ab	29.21±2.68 ab	4.94±0.25 ab	3.67±0.50 a	0.17±0.01 ab
	OE-44770-13	23.90±1.34 a	7.27±1.34 ab	31.20±2.44 a	5.20±0.07 a	3.30±0.49 a	0.17±0.01 ab
亚砷酸处理As(Ⅲ) stress	WT	23.83±0.44 a	7.03±0.20 ab	30.86±0.59 a	4.23±0.07 cd	3.38±0.07 a	0.14±0.00 b
亚砷酸处理As(Ⅲ) stress	OE-44770-4	22.05±0.89 b	5.41±0.07 b	25.13±0.77 b	3.78±0.07 d	3.64±0.16 a	0.15±0.00 ab
	OE-44770-13	23.00±0.30 b	5.33±0.16 b	24.81±0.48 b	4.50±0.29 bc	3.65±0.17 a	0.18±0.00 a

图6 亚砷酸盐处理后WT和各转基因株系根尖细胞质膜透性比较

Fig. 6. Comparison of plasma membrane integrity of WT and transgenic rice plants after As(Ⅲ) treatment.

图7 亚砷酸盐处理后WT和各转基因株系叶片活性氧平衡变化的比较

Fig. 7. Comparison of reactive oxygen balance in the leaves of WT and transgenic rice plants under As(Ⅲ) stress.

图8 亚砷酸盐处理后Os08g44770.1在WT和转基因植株叶片中的表达变化

Fig. 8. Temporal expression profiles of Os08g44770.1 in the leaves of As(Ⅲ)-treated WT and transgenic rice plants.

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