坛紫菜转录因子NhbZIP1克隆和功能验证

cDNA cloning and functional verification of transcription factor NhbZIP1 from Neoporphyra haitanensis

中文关键词: 坛紫菜  高温胁迫  bZIP转录因子  转基因  莱茵衣藻

英文关键词: Neoporphyra haitanensis  Heat stress  bZIP transcription factor  Transgenic  Chlamydomonas Reinhardtii

中文摘要:

      高温是制约坛紫菜(Neoporphyra haitanensis)产业发展的主要因素之一，阐明坛紫菜高温胁迫应答机理对耐高温品种选育至关重要。人们已分离多个坛紫菜抗逆相关基因，但尚不清楚这些基因的表达调控机制。本研究通过分子生物学和生物信息学技术分离了坛紫菜转录因子NhbZIP1基因。该基因开放阅读框长825 bp，编码274个氨基酸。从开放阅读框推导的氨基酸序列有5个低复杂度区域和1个BRLZ结构。其中，BRLZ是bZIP家族的保守结构域，含有一个α卷曲螺旋结构(121~171 aa)。实时荧光定量PCR (qRT-PCR)检测发现，NhbZIP1受高温胁迫显著诱导。为进一步阐明NhbZIP1的功能，将其转入莱茵衣藻(Chlamydomonas reinhardtii)中。结果显示，高温胁迫下转基因藻株生物量始终高于野生型，且随处理时间增加差异越来越显著。转基因藻株中热激蛋白家族和抗氧化系统相关基因的表达量显著高于野生型。研究表明，NhbZIP1激活下游抗逆基因表达，在坛紫菜应答高温胁迫中发挥重要作用。研究结果有助于阐明bZIP调控坛紫菜响应高温胁迫的分子机制，为耐高温新品种选育提供了基础信息。

英文摘要:

      Neoporphyra haitanensis is a macroalgae available in the south coast of China, and it is one of the most widely cultivated seaweeds in China. In recent years, due to global warming, the continuously high temperatures following the White Dew solar term, has led to the decomposition of seedlings of N. haitanensis in Fujian, Zhejiang and other provinces. This has had a huge impact on the coastal N. haitanensis cultivation industry in terms of production and development. Therefore, investigation of the molecular mechanism of high temperature stress response of N. haitanensis and the high-temperature resistance related genes is essential, and the results can also lay a foundation for the breeding of high-temperature resistant varieties of N. haitanensis. A previous study revealed that the basic region Leucine Zipper (bZIP) family transcription factors are one of the largest and most conserved transcription factor families in plants. The family plays an important role in plant response to abiotic stresses, such as high temperature, drought, and osmosis. bZIP transcription factors regulate plant response to abiotic stress by binding to functional genes or regulatory gene promoter cis-elements to activate and induce downstream gene expression. So far, 127, 89, and 216 bZIP transcription factors have been found in Arabidopsis, rice, and maize, respectively. However, previous studies on bZIP have mainly focused on model plants and only some field crops, and the functions of bZIP in macroalgae have not been reported. To this end, NhbZIP1 was screened based on the whole genome and transcriptomic data of N. haitanensis, and the NhbZIP1 gene was cloned and functionally analyzed by molecular biology and bioinformatics techniques. Its structure and expression pattern were also analyzed. Finally, the NhbZIP1 gene was transformed into Chlamydomonas reinhardtii by the “glass bead transformation” method for gene function verification. In this study, a gene product with a length of approximately 1000 bp was obtained by PCR amplification. After sequencing and BLAST analysis, the gene was identified as the bZIP gene of N. haitanensis and named NhbZIP1. Studies have shown that the open reading frame of NhbZIP1 gene is 825 bp in length and encodes 274 amino acids. There are five low-complexity domains and one BRLZ (115~179 aa) structure. BRLZ is a conserved domain of bZIP family and contains a α-coiled helix structure (121~171 aa). The molecular formula of NhbZIP1 is C1193H1935N339O375S7, and the predicted molecular weight of NhbZIP1 is 27 251.95 Da; its theoretical isoelectric point is 5.03, and it contains 32 negative charge residues and 26 positive charge residues in total. Ala (A) content of the protein was 27%, and Arg (R) content was 4.7%. The total average hydrophilic coefficient was 0.089, which indicates that the protein is hydrophilic, and the instability coefficient was 42.68, which indicates that the protein is unstable. There were 15 potential phosphorylation sites and 12 potential O-linkage glycosylation sites in this protein, which had no signal peptide or transmembrane structure. The protein was located in the nucleus and its characteristics were consistent with those of the genes encoding transcription factors, indicating that NhbZIP1 was a bZIP family transcription factor. Phylogenetic analysis showed that NhbZIP1 gene was isolated from Porphyra umbilicalis and was different from that higher plants, indicating that NhbZIP1 gene was relatively conserved in Porphyra and was genetically distant from higher plants. It is speculated that the NhbZIP1 gene of N. haitanensis has a different evolutionary mode from that of other species. Real-time fluorescence quantitative PCR (qRT-PCR) showed that NhbZIP1 gene was significantly induced by high temperature stress, and the expression level of NhbZIP1 gene was about 3.4 times that of the initial level after 6 h of stress. Under long-term high temperature stress, the heat tolerance of N. haitanensis could be enhanced by enhancing the expression of the resistance gene NhbZIP1. To further clarify the molecular function of NhbZIP1 gene, we transformed it into C. reinhardtii for functional verification. The results showed that the expression level of NhbZIP1 gene was relatively stable before 30 min of high temperature treatment, while the gene expression level was significantly increased after 60 min of the treatment, which was 1.8 times that of the initial level. Under subsequent high temperature stress, the gene expression level remained high, which was about 2.6 times that of the initial level after 180 min of high temperature treatment. The biomass of transgenic lines under heat stress was always higher than that of the wild type, and the difference became more significant with the increase of treatment time. The expression levels of heat shock protein family and genes related to antioxidant system in transgenic lines were significantly higher than that of the wild type. The results showed that NhbZIP1 gene plays an important role in activating the expression of downstream stress-resistant genes in the response to heat stress in N. haitanensis, suggesting that NhbZIP1 gene may enhance the heat tolerance of algae by regulating the expression of HSPs and activating the expression of genes encoding antioxidant enzymes. This study helps to clarify the molecular mechanism of bZIP transcription factor in regulating the response of N. haitanensis to high temperature stress and provides theoretical basis for guiding the breeding of new varieties with high temperature tolerance.