CCCH型锌指蛋白基因以基因家族的形式存在，在信号转导调控、形态发生和逆境应答等诸多生物学过程中起着重要作用。为给该基因的克隆及功能研究提供参考信息，本研究针对二穗短柄草的基因组序列，运用多种生物信息学方法在全基因组范围...CCCH型锌指蛋白基因以基因家族的形式存在，在信号转导调控、形态发生和逆境应答等诸多生物学过程中起着重要作用。为给该基因的克隆及功能研究提供参考信息，本研究针对二穗短柄草的基因组序列，运用多种生物信息学方法在全基因组范围对CCCH型锌指蛋白基因进行了鉴定分析。结果表明，二穗短柄草共有61个含CCCH基序的蛋白质，它们的基因 DNA序列全长825~13 288 bp，内含子数量0~13个，编码区全长498~4 944 bp，编码165~1 007个氨基酸；共检测到145个CCCH基序，以C X8 C X5 C X3 H和C X7 C X5 C X3 H最为常见；基因不均等地分布在5条染色体上，其中2号染色体分布最多，有21个，5号染色体分布最少，仅4个；系统发育分析表明，61个成员可分为8组。展开更多
RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和...RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.展开更多
The CCCH proteins play important roles in plant growth and development,hormone response,pathogen defense and abiotic stress tolerance.However,the knowledge of their roles in thermotolerance are scarce.Here,we identifi...The CCCH proteins play important roles in plant growth and development,hormone response,pathogen defense and abiotic stress tolerance.However,the knowledge of their roles in thermotolerance are scarce.Here,we identified a heat-inducible CCCH gene LlC3H18 from lily.LIC3H18 was localized in the cytoplasm and nucleus under normal conditions,while it translocated in the cytoplasmic foci and co-located with the markers of two messenger ribonucleoprotein(mRNP)granules,processing bodies(PBs)and stress granules(SGs)under heat stress conditions,and it also exhibited RNA-binding ability.In addition,LIC3H18 exhibited transactivation activity in both yeast and plant cells.In lily and Arabidopsis,overexpression of LIC3H18 damaged their thermotolerances,and silencing of LIC3H18 in lily also impaired its thermotolerance.Similarly,Arabidopsis atc3h18 mutant also showed decreased thermotolerance.These results indicated that the appropriate expression of C3H18 was crucial for establishing thermotolerance.Further analysis found that LIC3H18 directly bound to the promoter of LIWRKY33 and activated its expression.Besides,it was found that LIMYB305 acted as an upstream factor of LlC3H18 and activated its expression.In conclusion,we demonstrated that there may be a LIMYB305-LlC3H18-LIWRKY33 regulatory module in lily that is involved in the establishment of thermotolerance and finely regulates heat stress response.展开更多
文摘CCCH型锌指蛋白基因以基因家族的形式存在，在信号转导调控、形态发生和逆境应答等诸多生物学过程中起着重要作用。为给该基因的克隆及功能研究提供参考信息，本研究针对二穗短柄草的基因组序列，运用多种生物信息学方法在全基因组范围对CCCH型锌指蛋白基因进行了鉴定分析。结果表明，二穗短柄草共有61个含CCCH基序的蛋白质，它们的基因 DNA序列全长825~13 288 bp，内含子数量0~13个，编码区全长498~4 944 bp，编码165~1 007个氨基酸；共检测到145个CCCH基序，以C X8 C X5 C X3 H和C X7 C X5 C X3 H最为常见；基因不均等地分布在5条染色体上，其中2号染色体分布最多，有21个，5号染色体分布最少，仅4个；系统发育分析表明，61个成员可分为8组。
文摘RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.
基金Open access funding provided by Shanghai Jiao Tong Universitysupported by the National Natural Science Foundation of China(32272761,31902055)+1 种基金the Fundamental Research Funds for the Central Universities(KYZZ2022004)the Project for Crop Germplasm Resources Conservation of Jiangsu(2021-SJ-011).
文摘The CCCH proteins play important roles in plant growth and development,hormone response,pathogen defense and abiotic stress tolerance.However,the knowledge of their roles in thermotolerance are scarce.Here,we identified a heat-inducible CCCH gene LlC3H18 from lily.LIC3H18 was localized in the cytoplasm and nucleus under normal conditions,while it translocated in the cytoplasmic foci and co-located with the markers of two messenger ribonucleoprotein(mRNP)granules,processing bodies(PBs)and stress granules(SGs)under heat stress conditions,and it also exhibited RNA-binding ability.In addition,LIC3H18 exhibited transactivation activity in both yeast and plant cells.In lily and Arabidopsis,overexpression of LIC3H18 damaged their thermotolerances,and silencing of LIC3H18 in lily also impaired its thermotolerance.Similarly,Arabidopsis atc3h18 mutant also showed decreased thermotolerance.These results indicated that the appropriate expression of C3H18 was crucial for establishing thermotolerance.Further analysis found that LIC3H18 directly bound to the promoter of LIWRKY33 and activated its expression.Besides,it was found that LIMYB305 acted as an upstream factor of LlC3H18 and activated its expression.In conclusion,we demonstrated that there may be a LIMYB305-LlC3H18-LIWRKY33 regulatory module in lily that is involved in the establishment of thermotolerance and finely regulates heat stress response.
