高耐盐紫球藻资源筛选及其miR398-CSD盐胁迫应答模式研究

doi:10.11924/j.issn.1000-6850.casb2023-0263

摘要/Abstract

摘要：

紫球藻是一类极具开发潜力的特色微藻，本研究旨在筛选、鉴定一批高耐盐紫球藻株，并对其miR398-CSD盐胁迫应答模式进行研究，以期为特色紫球藻的工业化高效应用及其耐盐机理机制的研究奠定基础。以国内外不同品系的紫球藻为试材，采用形态学、生理学、系统进化的方法分析其基本特征，从中筛选高耐盐品系；用高通量测序及生物信息学技术，鉴定紫球藻miR398及其靶基因CSD序列，分子生物学技术对其抑制类型进行验证；用stem-loop qRT-PCR和qRT-PCR技术，对紫球藻高耐盐品系不同盐度胁迫下的miR398及CSD表达模式进行分析。7株藻株均属于紫球藻属，系统进化上可划分为3簇，其细胞形态均符合典型的紫球藻特征，P1和P4属于高耐盐品系；miR398以切割抑制的方式对其靶基因CSD进行抑制，紫球藻miR398的表达随盐度的升高而下调，CSD则随盐度的升高而上调。研究结果为高耐盐紫球藻资源的开发利用及miRNAs分子定向改良藻株品质奠定基础。

关键词: 紫球藻, miR398, 铜/锌超氧化物歧化酶, 盐胁迫响应

Abstract:

Porphyridium is a kind of characteristic microalgae with tremendous development potential. The purpose of this study is to explore and identify some high-salt tolerant Porphyridium strains, and to research their miR398-Cu, Zn-superoxide dismutase gene (CSD) salt stress response pattern, which will lay a foundation for the industrialized application of the special Porphyridium strains and exploring their salt tolerance mechanism. In this study, seven different Porphyridium strains were collected from home and abroad, and their morphological, physiological, phylogenetic evolution, and salt tolerance characteristics were analyzed, respectively. The high-salt tolerance strains among them were screened. Furthermore, the sequence of miR398 and its targeted gene CSD in Porphyridium were identified through the high-throughput sequencing and bioinformatics analyzing, respectively. Moreover, the inhibition type of miR398-CSD of the microalga was validated via molecular biology analysis. Lastly, based on the stem-loop qRT-PCR and qRT-PCR techniques, the expression patterns of miR398 and CSD in the high-salt tolerant Porphyridium strains under different salinity stresses were clarified. The results showed that, altogether, seven microalgae strains belonged to the Porphyridiumgenus, and were divided into three clusters via the phylogenetic analysis. Their cells morphology was consistent with the typical characteristics of Porphyridium. P1 and P4 were the high-salt tolerant strains. MiR398 inhibited its targeted gene CSD expression in the microalgae via slicing the gene targeted mRNA. The expression of miR398 in Porphyridium was down-regulated with the salinity increasing, while the CSD up-regulated. Our researches would lay a foundation for the development and utilization of highly-salt tolerant Porphyridium resources and molecular improvement of the novel algal strains based on the specific miRNAs.

Key words: Porphyridium, miR398, Cu/Zn-superoxide dismutase (CSD), salt-stress response

高帆. 高耐盐紫球藻资源筛选及其miR398-CSD盐胁迫应答模式研究[J]. 中国农学通报, 2024, 40(3): 103-111.

GAO Fan. High-Salt Tolerance Porphyridium: Resources Screening and Its miR398-CSD Salt Stress Response Pattern[J]. Chinese Agricultural Science Bulletin, 2024, 40(3): 103-111.

图/表 13

编号	名称	大小	形状	颜色	蛋白核	载色体	来源
P1	P. marinum	H：12~13 μm；W：12~13 μm	圆形	紫色	有	有	中科院水生生物研究所
P2	P. aerugineum	H：10~11 μm；W：9~11 μm	圆形	紫红色	有	有	中科院水生生物研究所
P3	P. cruentum	H：13~15 μm；W：13~15 μm	圆形	紫红色	有	有	中科院水生生物研究所
P4	P. griseum	H：12~14 μm；W：12~13 μm	圆形	紫色	有	有	山西大学藻类实验室
P5	P. purpureum	H：12~14 μm；W：10~12 μm	椭圆形	紫色	有	有	山西大学藻类实验室
P6	P. sordidum	H：13~16 μm；W：13~15 μm	圆形	紫色	有	有	光语生物科技有限公司
P7	P. violaceum	H：14~17 μm；W：15~16 μm	圆形	紫红色	有	有	光语生物科技有限公司

表1. 7株紫球藻形态特征

图1. 紫球藻细胞显微形态

图2. 基于ITS基因的紫球藻ML系统进化树

图3. 紫球藻生长曲线图

图4. 不同盐度胁迫下的紫球藻细胞渗透势差

图5. 不同盐度胁迫下的紫球藻细胞数量

图6. 紫球藻miR398前体二级结构

图7. 紫球藻miR398靶基因CSD氨基酸保守区功能域分析

图8. 紫球藻miR398-靶标的RLM-RACE扩增示意图

图9. 紫球藻靶标所在转录本CL640核心区扩增 P1/P2为第一轮扩增产物，P3/P4为第二轮扩增产物，M为DNA marker，NC为阴性对照

图10. 紫球藻miR398切割CSD靶标序列示意图（红色箭头指向切割位点）

图11. 高耐盐紫球藻miR398盐胁迫下的相对表达

图12. 高耐盐紫球藻CSD基因盐胁迫下的相对表达

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