Structure and Expression Analysis of vv-SPK Gene in VEGF Signaling Pathway of Volvariella volvacea
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摘要: VEGF通路是一条调控细胞增殖、分化、迁移、应激反应以及生存的信号通路。本研究从草菇基因组中获得1个在VEGF通路中编码SPK蛋白的基因，将其命名为vv-SPK，并对该基因进行结构分析，结果显示：基因vv-SPK全长1 996 bp，包含11个内含子；ORF长为1 401 bp，编码466个氨基酸。通过BLASTP比对显示，vv-SPK基因与纹缘盔孢伞、双色蜡蘑以及紫蜡蘑的相似度最高；经过荧光定量PCR验证后发现，SPK蛋白的基因表达量与菌柄的伸长有关。根据SPK在VEGF通路中参与的细胞增殖途径以及SPK蛋白本身的激酶特性，推测其对草菇菌柄生长过程的细胞分裂和伸长有显著的促进作用，且草菇中可能存在SPK激酶介导的促进细胞增殖、伸长的信号通路。Abstract: VEGF signaling pathway regulates cell proliferation, differentiation, migration, stress response, and survival of fungi. A gene, vv-SPK, in the pathway coding SPK protein from the mushroom, Volvariella volvacea, was obtained. A structure analysis showed that the gene was 1 996 bp, including 11 introns, and the open reading frame (ORF) of 1 401 bp encoding 466 amino acids. BLASTP comparison showed vv-SPK sharing a high similarity with the genes from Galerina patagonica, Laccaria bicolor and Laccaria amethystine. RT-qPCR verified the gene expression of SPK protein being associated with the stipe elongation. Since SPK, with its protein kinase characteristics, involves in the cell proliferation pathway in VEGF, the protein was postulated to play a significant role in enhancing the cell division during the stipe development of V. volvacea. There might also be a SPK-mediated signaling pathway that promoted the cell proliferation in V. volvacea.
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Key words:
- VEGF pathway /
- Volvariella volvacea /
- stipe elongation /
- promote proliferation
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图 4 vv-SPK基因在草菇生长不同时期菌柄中表达量变化
注：不同小写字母表示差异显著 (P < 0.05)。
Figure 4. Differential expression of vv-SPK in stipe at different growing stages
表 1 荧光定量PCR所使用的引物
Table 1. Primers used for RT-qPCR
引物名称 序列 (5′-3′) GAPDH-F 5′-ATTGGCGTGGTGGTCGTAG-3′ GAPDH-R 5′-ACGGAAACATCAAGGGTAGGG-3′ vv-SPK-F 5′-GTTACGGTTGCTGGAGACGG-3′ vv-SPK-R 5′-CGGAGTTTACTGCCGCCAC-3′ 
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表 2 SPK蛋白的基本理化性质
Table 2. Properties of SPK
名称 鞘氨醇激酶 氨基酸残基/个 549 分子量/Da 60461.2 等电点 8.53 分子式 C2700H4264N752O787S19 稳定性 是 亲水性 -0.157 (Arg+Lys) 数量/个 63 (Asp+Glu) 数量/个 59
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