巨藻中谷胱甘肽S转移酶基因对细长聚球藻PCC7942耐镉性的影响

Effects of the glutathione S-transferase gene extracted from giant kelp (Macrocystis pyrifera) on the cadmium tolerance of Synechococcus elongatus PCC7942

中文关键词: 谷胱甘肽S转移酶基因  转基因  镉离子胁迫  巨藻  细长聚球藻PCC7942

英文关键词: Glutathione S-transferase genes  Transgenesis  Cadmium ion stress  Macrocystis pyrifera  Synechococcus elongatus PCC7942

中文摘要:

      谷胱甘肽S转移酶(glutathione S-transferase, GST)是一个较大的基因家族，在生物体生长发育和对环境变化响应中发挥重要的调控作用。本研究从巨藻(Macrocystis pyrifera)配子体中克隆了6个完整的GST基因(mpgst1、mpgst2、mpgst3、mpgst4、mpgst5和mpgst6)。随后将6个巨藻GST基因分别转化至细长聚球藻(Synechococcus elongatus PCC7942)中，提取细长聚球藻转化株基因组DNA作为模板进行PCR验证及测定转化株GST酶活进行基因功能验证，结果显示，6个mpgst基因都分别成功整合到细长聚球藻的基因组中，但只有含mpgst1、mpgst4和mpgst6基因的细长聚球藻转化株(MG1、MG4和MG6)具有耐镉性。在镉离子胁迫下，细长聚球藻转化株MG1、MG4和MG6的生长、光合色素含量和叶绿素荧光参数Fv/Fm值均显著高于野生株(P<0.05)。本研究结果为进一步研究巨藻GST基因的抗重金属胁迫功能奠定了基础。

英文摘要:

      Giant kelp (Macrocystis pyrifera, Laminariales), has long been considered one of the most promising macroalgal species for biomass production because of its large size, rapid growth rate, and dynamic life history strategies. Brown seaweeds are economically important and commonly used for agricultural and industrial purposes. Intertidal and subtidal habitats, which most brown algae inhabit, are shaped by fluctuating levels of salinity-, temperature-, and light-related stresses. The responses of brown algae to abiotic stress have been comparatively well studied. With the rapid development of modern industry and agriculture as well as the exploitation of mineral resources, the pollution of ecological environments, particularly heavy-metal contamination of water, is becoming increasingly serious. Natural quantities of heavy metals in seawater do not adversely affect marine life, and some heavy metals even serve as trace nutrients essential for the normal growth and metabolism of algae. However, at excess concentrations, heavy metals act as pollutants and harm algae, and the magnitude of their impact varies depending on the degree of pollution. High metal concentrations negatively affect diatoms by inhibiting growth, triggering oxidative damage, modifying gene expression, damaging photosynthetic cells and mitochondria, and disrupting various cellular processes. Among the various metals, cadmium is particularly toxic and can easily accumulate in many marine organisms. Usually, cadmium concentrations in the sediment and open seawater are low, although these values may increase in some offshore and estuarine areas due to leakage or anthropogenic emissions. Glutathione S-transferase (GST) is a phase Ⅱ enzyme in cells that catalyzes the formation of chelates between reduced glutathione and metal ions as well as the binding of sulfur atoms of reduced glutathione to phase Ⅰ electrophilic groups, thereby reducing the levels of intracellular toxic substances, such as reactive oxygen species, and accelerating their exogenous release. GST belongs to a large gene family that plays important regulatory roles in growth, development, and responses to environmental fluctuations. Owing to the lack of a stable genetic operating system in M. pyrifera, the functions of some genes and proteins remain unclear. To date, there has been no successful genetic transformation of M. pyrifera. Synechococcus elongatus PCC7942 is easy to culture, has a small genome size, and can easily be genetically manipulated through natural transformation or conjugation with Escherichia coli, making it a good protein expression system for studying prokaryotic circadian rhythms, nutrient regulation, environmental responses, and lipid metabolism. In the present study, S. elongatus PCC7942 was selected to verify the functions of the GST gene in M. pyrifera under Cd stress. Total RNA was extracted from M. pyrifera gametophytes frozen in liquid nitrogen and reverse-transcribed to cDNA. Gene-specific primers containing enzyme restriction sites at both ends were designed to construct an expression vector based on the transcriptome sequence of M. pyrifera (accession number CNP0001061 in China National GenBank). Six complete GST genes (mpgst1, mpgst2, mpgst3, mpgst4, mpgst5, and mpgst6) were cloned using RT-PCR. Subsequently, the six MPGST genes were transformed into S. elongatus PCC7942, and the transformed strains containing mpgst1–mpgst6 were labelled MG1–MG6, respectively. Transformation was verified by genomic DNA extraction and GST enzyme activity assays. In this study, 0.2 mg/L was determined as the Cd2+ concentration that was lethal to the wild strain but enabled normal growth of some transformed strains. Some of the transformed strains did not exhibit resistance, which may be due to differences in the GST gene sequences of M. pyrifera or because they may belong to different GST gene families, serving different functions. The selected resistant transformed strains MG1, MG4, and MG6 were tested for growth, photosynthetic pigment content, and photosynthetic parameters at 0.2 mg/L cadmium ion stress to verify their functions. The transformed strains showed an upward trend of light absorbance, but most of the wild type strains died. Furthermore, the transformed strains presented values for photosynthetic pigment content and photosynthetic parameters even under stress, but the wild strain died, which was consistent with the growth curve. In particular, the carotenoid content of MG6 slightly increased following Cd2+ stress, indicating elevated antioxidant activity. However, differences in the physiological indices of different genes before and after stress may be related to their specific mechanisms of action, which warrants further study. Our findings laid a foundation for further research into the stress resistance function of GST genes in M. pyrifera and for the future breeding of pollution-tolerant algal strains.