天山雪莲SiRVE8基因对小球藻抗寒性的影响

Effect of the SiRVE8 gene from Saussurea involucrata on the cold resistanceof Chlorella

摘要

RVE8基因作为响应冷胁迫的关键调控因子,在植物低温适应中发挥重要作用。本研究以天山雪莲(Saussurea involucrata Kar. et Kir.)SiRVE8基因为研究对象,旨在通过功能验证评估其在单细胞真核生物中的抗寒潜力。首先,克隆了SiRVE8基因,生物信息学分析表明其编码包含一个典型MYB结构域的蛋白。进而,将该基因构建至由CaMV 35S启动子驱动的pET-2300表达载体,并通过农杆菌介导法转化至沙漠小球藻(Chlorella sp. TLD6B)中。RT-PCR证实SiRVE8基因在藻株中稳定整合与转录表达。经-20 ℃低温胁迫处理后,转基因藻株恢复培养的比生长速率较野生型提高21.6%,生物量积累增加10.4%;生理指标显示,其可溶性糖含量升高19.7%,丙二醛(MDA)含量降低21.5%,电解质渗透率减少14.8%,超氧化物歧化酶(SOD)活性提升11.5%。研究表明,SiRVE8基因通过促进渗透调节物质积累、维持细胞膜完整性和增强抗氧化能力协同提升小球藻耐寒性。本研究为抗冻微藻种质创制及作物抗逆遗传改良提供了候选基因资源。

Abstract

The RVE8 gene, as a key regulatory factor in response to cold stress, plays an important role in plant low-temperature adaptation. In this study, the SiRVE8 gene from Saussurea involucrata Kar. et Kir. was taken as the research object, aiming to evaluate its potential for cold resistance in unicellular eukaryotes through functional validation. Firstly, the SiRVE8 gene was cloned, and bioinformatics analysis revealed that it encodes a protein containing a typical MYB domain. Subsequently, the gene was inserted into the pET-2300 expression vector driven by the CaMV 35S promoter and transformed into Chlorella sp. TLD6B via Agrobacterium-mediated transformation. RT-PCR confirmed the stable integration and transcriptional expression of the SiRVE8 gene in the transgenic algal strains. Under -20 ℃cold stress, the transgenic algae exhibited significantly enhanced cold resistance compared with the wild type (WT). After recovery culture, the specific growth rate and biomass accumulation of transgenic lines increased by 21.6% and 10.4%, respectively. Physiological analyses revealed that soluble sugar content was elevated by 19.7%, while malondialdehyde (MDA) content decreased by 21.5%, electrolyte leakage reduced by 14.8%, and superoxide dismutase (SOD) activity increased by 11.5%. These results demonstrate that SiRVE8 synergistically enhances the cold resistance of Chlorella by promoting the accumulation of osmoregulatory substances, maintaining cell membrane integrity, and enhancing antioxidant capacity. This study provides a candidate gene resource for the breeding of freeze-tolerant microalgae strains and for genetic improvement of crop stress resistance.

关键词

天山雪莲 / SiRVE8基因 / 小球藻 / 低温胁迫 / 膜透性

Key words

Saussurea involucrata Kar. et Kir. / SiRVE8 gene / Chlorella sp. 6B / low-temperature stress / membrane permeability

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基金资助

国家自然科学基金项目(32160061)