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野生大豆肌醇磷酸水解酶Gs5PTase8的原核表达纯化及活性鉴定
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作者 陈媛 范寒雨 +3 位作者 刘雨杭 梁康迳 林文雄 贾琪 《生物工程学报》 CAS CSCD 北大核心 2024年第10期3588-3602,共15页
多磷酸肌醇-5-磷酸酶(inositol polyphosphate 5-phosphatase,5PTase)是肌醇信号传导途径中的关键酶,能水解肌醇磷酸(inositol phosphate,IP)或磷脂酰肌醇磷酸(phosphatidylinositol phosphates,PIP)的肌醇环5-磷酸。然而,大豆中该类基... 多磷酸肌醇-5-磷酸酶(inositol polyphosphate 5-phosphatase,5PTase)是肌醇信号传导途径中的关键酶,能水解肌醇磷酸(inositol phosphate,IP)或磷脂酰肌醇磷酸(phosphatidylinositol phosphates,PIP)的肌醇环5-磷酸。然而,大豆中该类基因研究较少。本研究从野生大豆(Glycine soja S.&Z.)中克隆出耐盐基因Gs5PTase8并对其底物进行探究。Gs5PTase8编码493个氨基酸。经序列比对与系统进化树分析,发现其在植物中具有保守性。利用实时荧光定量PCR对不同大豆组织分析该基因的表达,发现Gs5PTase8主要在大豆根中表达。为了探究其水解底物,分别构建了大肠杆菌表达载体pET28a-Gs5PTase8和pGEX4T1-Gs5PTase8,但只成功诱导表达了GST-Gs5PTase8重组蛋白。使用不同浓度异丙基β-D-硫代半乳糖苷(isopropyl beta-D-thiogalactopyranoside,IPTG),分别在16、30、37℃条件下进行诱导表达条件优化,发现重组蛋白在16℃、0.2 mmol/LIPTG过夜诱导时表达量最高。SDS-PAGE检测重组蛋白的相对分子量约为75 kDa,经纯化后条带单一,纯度达95%以上,二辛可酸法(bicinchoninic acid assay,BCA)检测出重组蛋白的得率为4.9 mg/L。体外底物酶活检测表明,Gs5PTase8蛋白可以水解IP3[inositol(1,4,5)trisphosphate]、IP_(4)[inositol(1,3,4,5)tetrakisphosphate]、PI(4,5)P_(2)[phosphatidylinositol(4,5)bisphosphate]和PI(3,4,5)P3[phosphatidylinositol(3,4,5)trisphosphate],本研究为进一步探索Gs5PTase8参与耐盐的分子作用机制提供了科学依据。 展开更多
关键词 大豆 Gs5PTase8 原核表达 蛋白纯化
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Fine mapping of cisc(t),a gene for cold-induced seedling chlorosis,and identification of its candidate in rice 被引量:5
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作者 LAN Tao WANG Bin +6 位作者 LING QiuPing XU ChunHua TONG ZhiJun liang kangjing DUAN YuanLin JIN Jing WU WeiRen 《Chinese Science Bulletin》 SCIE EI CAS 2010年第27期3149-3153,共5页
The seedlings of indica rice cultivar Dular are susceptible to chlorosis under low temperature conditions.Our previous studies indicated that low temperature-induced seedling chlorosis is controlled by a recessive gen... The seedlings of indica rice cultivar Dular are susceptible to chlorosis under low temperature conditions.Our previous studies indicated that low temperature-induced seedling chlorosis is controlled by a recessive gene,located between SSR markers RM257 and RM242,on the long arm of chromosome 9.We temporarily named the gene cisc(t).Using a large F2 population derived from a cross between Dular and the japonica cultivar Lemont,which displays a normal green color at low temperatures,cisc(t)was fine mapped to within a 12-kb interval.There is only one annotated gene in this interval,which encodes a pentatricopeptide repeat(PPR)protein.Sequence analysis indicated that 8 bases were deleted at the 60th base in the Dular allele,resulting in a frame-shift mutation and loss of function of the gene.This is consistent with the chlorosis mutant phenotype of Dular.In addition,previous studies have shown that many chlorosis mutants of seedlings are related to PPR proteins.Hence,we presume that the PPR gene is the candidate for cisc(t). 展开更多
关键词 黄化现象 隐性基因 CISC 精细定位 幼苗 鉴定 水稻 低温条件
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