发菜G6PDH基因(GND)克隆及其干旱胁迫下的差异表达分析
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国家自然科学基金(31660066,31360054,31360025);


Clone of G6PDH Gene (GND) from Nostoc flagelliforme and Differential Expression under Drought Stress
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    摘要:

    GND编码的葡萄糖6磷酸脱氢酶(G6PDH)是戊糖磷酸途径的关键酶。为了解发菜GND分子信息以及对干旱胁迫的响应机制,该研究设计了特异性引物克隆发菜GND全长,进行序列分析、原核表达和MALDITOFTOF/MS鉴定,并对干旱胁迫下发菜GND的差异表达水平和G6PDH活性进行分析。结果表明:(1)发菜GND全长1 431 bp(GenBank登录号为KX553955),与点形念珠藻(73102)的GND核苷酸序列相似性为96%,G6PDH氨基酸相似性为98%;氨基酸序列中第26和27位的Ile疏水性最强,在第302位的Arg亲水性最强,Thr有19个磷酸化位点,Ser有18个磷酸化位点,Tys有5个磷酸化位点,二级结构和三级结构主要由α螺旋、β折叠、β转角和随机卷曲构成。(2)将GND基因进行原核表达,获得47.23 kD的外源表达蛋白G6PDH。(3)随干旱胁迫程度加剧,GND在转录水平的表达量和G6PDH活性均逐渐增加。研究结果为进一步探讨干旱胁迫条件下发菜GND表达调控奠定了基础。

    Abstract:

    GND coded glucose 6phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway. In order to understand molecular information of GND and mechanism in response to drought stress, we cloned GND by designed specific primers based on sequences of homologous cyanobacteria species. The sequence was analysed by bioinformatics methods, and the differential expression of GND and G6PDH activities of N. flagelliforme under drought stress were also discussed. The results indicated that: (1) the full length of GND is 1 431 bp(GenBank access number: KX553955). Nucleotide sequence and deduced amino acid sequences of GND from N. flagelliforme were similar to that of N. punctiforme PCC 73102 with similarities above 96% and 98%, respectively. Ile is the most hydrophobic in site 26 and 27, but Arg is the most hydrophilic in site 302. Thr, Ser and Tys have 19, 18 and 5 phosphorylation sites, respectively. Secondary and tertiary predicted structures of G6PDH were made up of α helix, β sheet, β turn and random coil. (2) GND was expressed in E. coil then a 47.23 kD heterologous protein was observed and tested by MALDITOFTOF/MS. (3) GND expression at the transcription level and G6PDH activity were all gradually increased under drought stress. The results provided a foundation for further research in GND expression and regulation of N. flagelliforme in response to drought stress.

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刘 阳,严奉坤,丁苗苗,等.发菜G6PDH基因(GND)克隆及其干旱胁迫下的差异表达分析[J].西北植物学报,2017,37(7):1263-1270

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  • 在线发布日期: 2017-08-09
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