AtERF49在拟南芥应答盐碱胁迫中的作用

doi:10.13560/j.cnki.biotech.bull.1985.2022-0479

摘要/Abstract

摘要：

盐碱胁迫是造成作物减产的主要逆境因素之一。植物AP2/ERF（APELATA2/ethylene response factors）转录因子在植物生长发育及其响应非生物逆境胁迫过程中发挥重要作用。探究AtERF49在拟南芥中对盐碱胁迫的应答，为深入解析AtERF49参与植物对盐碱胁迫的分子机理奠定基础。选取拟南芥野生型Col-0、过表达AtERF49转基因拟南芥和CRISPR/Cas9突变体erf49为试验材料，用150 mmol/L混合盐碱（摩尔比NaHCO₃∶Na₂CO₃=9∶1）溶液进行处理，使用荧光定量PCR技术对该基因的基本特性、盐碱胁迫及光合响应基因表达模式等进行分析。结果表明，盐碱胁迫处理后，突变体erf49叶片萎蔫并发生白化，而过表达AtERF49植株叶片稍有变黄。此外，在盐碱胁迫条件下，过量表达AtERF49上调盐碱胁迫响应基因（RD29A和RAB18）以及光合响应基因rbcL的表达。拟南芥叶片叶绿素荧光参数测定结果表明，过表达AtERF49植株的光系统Ⅱ实际量子产能Y（Ⅱ）、光化学淬灭系数（qP）显著高于Col-0，光损伤程度（NO）和非光化学淬灭系数（qN）显著低于Col-0，而突变体erf49与之相反。因此，AtERF49通过调控下游盐碱胁迫响应基因的表达以及植物的光合作用效率，改变参与植物对盐碱胁迫的应答。

关键词: AtERF49, 盐碱胁迫, 光合作用, 拟南芥

Abstract:

Saline-alkali stress is one of the main environmental constraints that cause crop failures. AP2/ERF（APELATA2/ethylene response factors）transcription factors play important roles in plant growth, development and response to abiotic stress. Investigating the responses of AtERF49 to Saline-alkali stress would lay a foundation for explaining the molecular mechanism of plant AtERF49 involving in Saline-alkali stress. Arabidopsis wild-type, namely, Col-0, AtERF49-overexpressed lines of Arabidopsis and CRISPR/Cas9 mutant erf49 of Arabidopsis were treated with 150 mmol/L Saline-alkali solution（NaHCO₃ and Na₂CO₃ molar ratio 9∶1 mixed）. The RT-qPCR assays were used to analyze the basic characteristics and expression pattern of this gene under saline-alkali stress and photosynthetic response. Results showed that the leaves of erf49 mutant were wilt and bleach under Saline-alkali stress, and the leaves of AtERF49-overexpressed lines turned to be yellow slightly. Moreover, the overexpression of AtERF49 up-regulated the expression of Saline-alkali stress inducible genes（RD29A and RAB18）and photosynthetic gene rbcL under Saline-alkali stress. Chlorophyll fluorescence parameters assay of Arabidopsis leaves revealed that actual photochemical efficiency（Y(II)）and non-photochemical quenching coefficient（qP）in AtERF49-overexpressed plants were significantly higher than that on Col-0, and the photodamage（NO）and photochemical quenching coefficient（qN）were significantly lower compared to Col-0. However, the opposite trend was apparent in erf49 mutant. These results suggested that AtERF49 might participate in response to Saline-alkali stress through regulating its downstream Saline-alkali stress-responsive genes or affecting photosynthesis efficiency.

Key words: AtERF49, salt-alkali stress, photosynthesis, Arabidopsis

阮航, 多浩源, 范文艳, 吕清晗, 姜述君, 朱生伟. AtERF49在拟南芥应答盐碱胁迫中的作用[J]. 生物技术通报, 2023, 39(1): 150-156.

RUAN Hang, DUO Hao-yuan, FAN Wen-yan, LV Qing-han, JIANG Shu-jun, ZHU Sheng-wei. Role of the AtERF49 in the Responses to Salt-alkali Stress in Arabidopsis[J]. Biotechnology Bulletin, 2023, 39(1): 150-156.

图/表 6

表1 荧光定量PCR AtERF49所用引物

Table 1 Primers used for fluorescence quantitative PCR

引物名称Primer name	引物序列Primer sequence（5'-3'）
RAB18-F	TCCAGCTCTAGCTCGGAGGATGA
RAB18-R	GGATCCCATGCCGCCCATCG
RD29A-F	GCCGGAATCTGACGGCGGTT
RD29A-R	CCCGTCGGCACATCCTTGTCG
AtUBC30-F	TCACTTCCCACCAGATTACCC
AtUBC30-R	TCGACAGAAGAACCTTGGATACG
AtERF49-F	AGACTACACCAAGCAGCAACACC
AtERF49-R	TTGGATGAACACGGCGACTCAG
rbcl-F	TTGGCAGCATTCCGAGTAACTCCT
rbcl-R	CTGGTAAGTCCATCGGTCC
psaA-F	CTACTTTGCCACCCACTGC
psaA-R	TGAGTGCTTTAGGGCGTCC

图1 盐碱胁迫下AtERF49表达量分析 *：P<0.05；***：P<0.001。下同

Fig. 1 Analysis of AtERF49 expression under Saline-alkali stress *: P<0.05; ***: P<0.001. The same below

图2 过表达AtERF49增强拟南芥对盐碱胁迫的抗性 A：盐碱胁迫处理前后AtERF49转基因拟南芥表型，标尺为2 cm；B：转基因株系中AtERF49的表达分析

Fig. 2 Overexpression of AtERF49 gene increasing Arabidopsis resistance to Saline-alkali stress A: Phenotypic analysis of AtERF49 transgenic Arabidopsis phenotype with Saline-alkali stress, the scale is 2 cm. B: Relative expression of AtERF49 in wild type and transgenic lines

图3 转基因AtERF49植株中RAB18（A）和RD29A（B）的表达分析

Fig. 3 Expression analysis of RAB18（A）and RD29A（B）in AtERF49 transgenic lines

图4 盐碱胁迫对转基因AtERF49拟南芥叶片叶绿素荧光参数的影响 A：能量耗散量子产能；B：光系统Ⅱ实际量子产量；C：非光化学淬灭系数；D：光化学淬灭系数

Fig. 4 Effects of Saline-alkali stress on the chlorophyll fluorescence parameters of leaves of AtERF49 transgenic Arabidopsis A: Energy dissipation quantum yield. B: Actual quantum yield of photosystem II. C: Coefficient of non-photochemical quenching. D: Coefficient of photochemical quenching

图5 AtERF49转基因株系中rbcL和psaA的表达

Fig. 5 Relative expressions of rbcL and psaA in AtERF49 transgenic lines

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