OsDIS1通过抗氧化途径负调控水稻耐旱性

doi:10.13560/j.cnki.biotech.bull.1985.2022-0538

摘要/Abstract

摘要：

干旱作为一种非生物胁迫因子，严重影响水稻产量和品质。挖掘抗旱相关功能基因有助于培育抗旱新品种。本研究通过根癌农杆菌介导转化方法构建了日本晴OsDIS1-RNAi转基因水稻，并对其和野生型进行一系列生理生化实验和农艺性状分析。结果显示，干旱处理后，与野生型相比，OsDIS1-RNAi转基因水稻中抗氧化酶的活性显著增强，丙二醛含量显著降低，活性氧清除基因表达量显著上升。而经过ABA处理后，OsDIS1-RNAi转基因植株没有表现出显著的形态变化，这表明OsDIS1介导的抗旱性可能独立于ABA途径。同时OsDIS1-RNAi水稻植株的株高、穗长、产量等农艺性状没有变化。以上结果表明，OsDIS1通过激活抗氧化途径增加细胞内活性氧的清除能力，从而负调节水稻抗旱性，对于水稻耐旱育种具有潜在应用价值。

关键词: 水稻, OsDIS1, 抗氧化酶, 耐旱性, 育种

Abstract:

Drought, as an abiotic stress factor, seriously affects the yield and quality of rice. Exploring drought-related functional genes will help to breed new drought-resistance varieties. In this study, the Nipponbare OsDIS1-RNAi transgenic rice was constructed by Agrobacterium-mediated transformation method, and a series of physiological and biochemical experiments and agronomic traits analysis were conducted on it and wild type. The results showed that the activities of antioxidant enzymes after drought treatment significantly enhanced, the content of malondialdehyde significantly reduced and the expression of reactive oxygen species clearance gene significantly increased in OsDIS1-RNAi transgenic rice compared with the wild type. After ABA treatment, OsDIS1-RNAi transgenic plants did not show significantly morphological changes; these results indicated that OsDIS1-mediated drought tolerance may be independent of the ABA pathway. Meanwhile, agronomic traits such as plant height, spike length and yield of OsDIS1-RNAi rice plants did not change. The above results indicate that OsDIS1 negatively regulates drought tolerance in rice by activating the antioxidant pathways to increase the intracellular scavenging capacity of reactive oxygen species, and it may have potential applications for drought tolerance breeding in rice.

Key words: rice, OsDIS1, antioxidant enzyme, drought tolerance, breeding

杨茂, 林宇丰, 戴阳朔, 潘素君, 彭伟业, 严明雄, 李魏, 王冰, 戴良英. OsDIS1通过抗氧化途径负调控水稻耐旱性[J]. 生物技术通报, 2023, 39(2): 88-95.

YANG Mao, LIN Yu-feng, DAI Yang-shuo, PAN Su-jun, PENG Wei-ye, YAN Ming-xiong, LI Wei, WANG Bing, DAI Liang-ying. OsDIS1 Negatively Regulates Rice Drought Tolerance Through Antioxidant Pathways[J]. Biotechnology Bulletin, 2023, 39(2): 88-95.

图/表 8

表1 实时荧光定量PCR引物

Table 1 Primers for RT-qPCR

引物Primer	序列Sequence（5'-3'）
Ubq-F	AACCAGCTGAGGCCCAAGA
Ubq-R	ACGATTGATTTAACCAGTCCATGA
OsDIS1-F	GTATCATCACAGTGCGAGAAT
OsDIS15-R DSM1-F DSM1-R	CATCTCATGAATTGCCATTAG GTGGTCATGGTCCATTATTGCC CCCAAACCCTCAACTGGCTTA
OsSIK1-F	TCTGCTAGTCTGCCCGAGGAA
OsSIK1-R	TATGTACTGGTTGCAATCAG
OsSKIPa-F	TACAGATGCGATCCAAGGTG
OsSKIPa-R	AGTGCCCTTAGCTCTTGCTC
OsDSM2-F	GCTTCGCCTGGCAAATGG
OsDSM2-R	ACCGTCCAAATGAGCTTCCA
OsBZIP23-F OsBZIP23-R	GGAGCAGCAAAAGAATGAGG GGTCTTCAGCTTCACCATCC

图1 脱落酸处理下OsDIS1-RNAi转基因植株的表型 A: OsDIS1-RNAi转基因植株在含有不同浓度ABA的1/2 MS培养基上萌发表型；B: 不同浓度ABA处理下野生型和OsDIS1-RNAi植株的种子发芽率；C、D: OsDIS1-RNAi转基因植株的根长和株高统计；以RNAi-21为代表结果作图，下同；数据为3个生物学重复 ± 标准差

Fig. 1 Phenotypes of OsDIS1-RNAi transgenic plants treated with abscisic acid (ABA) A: OsDIS1-RNAi transgenic plants were germinated on 1/2 MS medium containing different concentrations of ABA. B: Seed germination rates of wild-type and OsDIS1-RNAi plants under different concentrations of ABA. C, D: OsDIS1-RNAi transgenic root length and plant height statistics of plants; RNAi-21 as the representative result of the graph, the same below. The error bar represents ± SD of triplicate experiments

图2 RT-qPCR检测ABA途径基因在OsDIS1-RNAi植株中的表达量

Fig. 2 Expressions of ABA pathway genes in OsDIS1-RNAi plants detected by RT-qPCR

图3 野生型和RNAi的活性氧含量检测

Fig. 3 Detection of reactive oxygen content of wild-type and RNAi

图4 OsDIS1-RNAi转基因植株中抗氧化酶活性和MDA含量检测 A-C: 干旱胁迫下OsDIS1-RNAi幼苗叶片CAT、SOD、POD活性和含量；D：MDA含量；数据为3个生物学重复 ± 标准差

Fig. 4 Detection of antioxidant enzyme activity and MDA content in OsDIS1-RNAi transgenic plants A-C: CAT, SOD, POD activity and content in the leaves of OsDIS1-RNAi seedlings under drought stress. D: MDA content. The error bar represents ± SD of triplicate experiments

图5 RT-qPCR检测OsDIS1-RNAi植物中相关活性氧（ROS）清除基因的表达量

Fig. 5 Expressions of related reactive oxygen species （ROS）scavenging genes in OsDIS1-RNAi plants by RT-qPCR

图6 OsDIS1-RNAi转基因水稻的生长表型

Fig. 6 Growth phenotypes of OsDIS1-RNAi transgenic rice

表2 转基因水稻OsDIS1-RNAi的农艺性状

Table 2 Agronomic characteristics of transgenic rice OsDIS1-RNAi

品种名称 Variety	株高 Plant height /cm	穗长 Panicle length /cm	单株有效穗数 Effective panicle per plant	每穗实粒数 Filled grains per panicle	每穗总粒数 Total grains per panicle	结实率 Seed setting /%	千粒重 1 000 grain weight/g	单株产量 Yield per plant /g
RNAi-8	94.4±5.2	19.4±1.6	26.8± 2.7	94.9±10.6	106.9±17.7	88.8±6.7	24.7±0.7	75.1±7.7
RNAi-21	96.7±6.2	20.4±1.4	28.6± 2.5	96.9±11.7	111.2±14.8	87.1±4.0	25.8±0.6	77.3±8.7
RNAi-22	95.7 ±4.1	19.6±0.9	30.4± 2.9	89.7±9.0	106.3±19.4	84.3±5.6	25.4±0.4	72.9±7.2
NPB	96.2±5.2	19.1±1.4	26.2±2.6	95.6±13.3	108.6±19.3	88.0±7.5	24.9±0.5	73.1±6.8

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