背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
【目的】研究旨在对努比亚山羊大脑富集Ras同源物(Ras homolog enriched in brain,Rheb)基因进行克隆和分析,并构建其真核表达载体,为进一步揭示Rheb对努比亚山羊骨骼肌调控的分子机理奠定基础。【方法】试验采用RT-PCR方法从努比亚山...【目的】研究旨在对努比亚山羊大脑富集Ras同源物(Ras homolog enriched in brain,Rheb)基因进行克隆和分析,并构建其真核表达载体,为进一步揭示Rheb对努比亚山羊骨骼肌调控的分子机理奠定基础。【方法】试验采用RT-PCR方法从努比亚山羊背最长肌组织中扩增Rheb基因,经琼脂糖凝胶电泳及测序验证正确后进行生物信息学分析,同时构建该基因真核表达载体,转染细胞后进行实时荧光定量PCR检测来验证所构建载体的正确性。【结果】努比亚山羊Rheb基因编码区序列长度为555 bp,编码184个氨基酸,Rheb蛋白分子式为C 910 H 1456 N 236 O 279 S 6,分子质量为20359.34 u,原子总数为2887,理论等电点为5.93。Rheb蛋白属于稳定的亲水性蛋白,不包含跨膜结构域。蛋白二级结构预测结果显示,努比亚山羊Rheb蛋白中α-螺旋、β-转角、延伸链和无规则卷曲分别占40.76%、7.07%、22.83%和29.35%。构建的真核表达载体pcDNA3.1-Rheb转染山羊骨骼肌细胞后,与空载体组相比,Rheb基因表达量极显著升高(P<0.01)。【结论】本试验成功克隆努比亚山羊Rheb基因编码区序列,并构建pcDNA3.1-Rheb真核表达载体,这为深入理解Rheb基因在努比亚山羊肌肉中的作用提供了理论支持。展开更多
萜类化合物是植物中最大的代谢物群组之一,在植物应对生物和非生物胁迫过程中发挥着重要作用。萜烯合成酶(Terpene synthases, TPSs)是植物萜类化合物合成的关键酶,然而目前关于油菜萜烯合成酶研究很少报道。为探究TPSs在油菜中的作用,...萜类化合物是植物中最大的代谢物群组之一,在植物应对生物和非生物胁迫过程中发挥着重要作用。萜烯合成酶(Terpene synthases, TPSs)是植物萜类化合物合成的关键酶,然而目前关于油菜萜烯合成酶研究很少报道。为探究TPSs在油菜中的作用,本文对甘蓝型油菜萜类合酶基因BnaTPSA08-1 (BnaA08T0101600Z)进行了功能研究。在克隆得到甘蓝型油菜BnaTPSA08-1基因CDS序列基础上,成功构建了BnaTPSA08-1基因的过表达双元载体pFGC5941-35s::BnaTPSA08-1。利用农杆菌介导的甘蓝型油菜下胚轴遗传转化方法成功获得5株稳定遗传的BnaTPSA08-1基因过表达转基因植株。通过收集过表达转基因植株释放的有机挥发性气体,并用GC-MS测定气体组分,结果显示:与对照植株的有机挥发性组分比较,过表达植株产生25种不同类型的挥发性气体,其中十一烷及其衍生物占比最多,这说明BnaTPSA08-1基因产物的生化功能是促进十一烷及其衍生物的合成,这为进一步研究BnaTPSA08-1基因在甘蓝型油菜中的生物学功能打下了良好基础。Terpenoids are one of the largest groups of metabolites in plants and play an important role in plant response to biotic and abiotic stresses. Terpene synthases (TPSs) are key enzymes in the synthesis of terpene compounds in plants. However, there are few reports on terpene synthases in rapeseed. In order to explore the role of TPSs in rapeseed, the function of the terpene synthase gene BnaTPSA08-1 (BnaA08T0101600Z) in Brassica napus was studied. Based on the cloned CDS sequence of BnaTPSA08-1 gene in Brassica napus, the over-expression vector pFGC5941-35s::BnaTPSA08-1 was successfully constructed. Five transgenic plants with stable BnaTPSA08-1 gene over-expression were successfully obtained by Agrobacteria-mediated hypocotyl genetic transformation of Brassica napus. The organic volatile gases released by overexpressed transgenic plants were collected and the gas components were determined by GC-MS. The results showed that: Compared with the organic volatile components of control plants, over-expressed plants produced 25 different types of volatile gases, among them, undecane and undecane derivatives account for the most, indicating that the biochemical function of BnaTPSA08-1 gene product is to promote the synthesis of undecane and undecane derivatives. This provides a good basis for further study on the biological function of BnaTPSA08-1 gene in Brassica napus.展开更多
目的构建含有小鼠血清和糖皮质激素诱导蛋白激酶3(serum and glucocorticoid-induced protein kinase 3,SGK3)基因的真核表达载体pcDNA3.1-MYC-SGK3-mCherry,并观察和验证其在转染细胞HEK293中的表达。方法通过聚合酶链式反应将实验室...目的构建含有小鼠血清和糖皮质激素诱导蛋白激酶3(serum and glucocorticoid-induced protein kinase 3,SGK3)基因的真核表达载体pcDNA3.1-MYC-SGK3-mCherry,并观察和验证其在转染细胞HEK293中的表达。方法通过聚合酶链式反应将实验室保存的真核表达质粒pcDNA3.1-MYC-SGK3中目的基因SGK3与mCherry融合并扩增出来,然后定向克隆至pcDNA3.1-MYC质粒中,经限制性内切酶消化和测序证实后,通过脂质体法转染HEK293细胞,Western blotting法检测目的基因的蛋白表达情况。结果测序结果与之前预期结果相符,证实pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。Western blotting结果显示,转染pcDNA3.1-MYC-SGK3-mCherry的HEK293细胞出现清晰的阳性反应条带,说明目的片段成功表达。结论pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。展开更多
文摘【目的】研究旨在对努比亚山羊大脑富集Ras同源物(Ras homolog enriched in brain,Rheb)基因进行克隆和分析,并构建其真核表达载体,为进一步揭示Rheb对努比亚山羊骨骼肌调控的分子机理奠定基础。【方法】试验采用RT-PCR方法从努比亚山羊背最长肌组织中扩增Rheb基因,经琼脂糖凝胶电泳及测序验证正确后进行生物信息学分析,同时构建该基因真核表达载体,转染细胞后进行实时荧光定量PCR检测来验证所构建载体的正确性。【结果】努比亚山羊Rheb基因编码区序列长度为555 bp,编码184个氨基酸,Rheb蛋白分子式为C 910 H 1456 N 236 O 279 S 6,分子质量为20359.34 u,原子总数为2887,理论等电点为5.93。Rheb蛋白属于稳定的亲水性蛋白,不包含跨膜结构域。蛋白二级结构预测结果显示,努比亚山羊Rheb蛋白中α-螺旋、β-转角、延伸链和无规则卷曲分别占40.76%、7.07%、22.83%和29.35%。构建的真核表达载体pcDNA3.1-Rheb转染山羊骨骼肌细胞后,与空载体组相比,Rheb基因表达量极显著升高(P<0.01)。【结论】本试验成功克隆努比亚山羊Rheb基因编码区序列,并构建pcDNA3.1-Rheb真核表达载体,这为深入理解Rheb基因在努比亚山羊肌肉中的作用提供了理论支持。
文摘萜类化合物是植物中最大的代谢物群组之一,在植物应对生物和非生物胁迫过程中发挥着重要作用。萜烯合成酶(Terpene synthases, TPSs)是植物萜类化合物合成的关键酶,然而目前关于油菜萜烯合成酶研究很少报道。为探究TPSs在油菜中的作用,本文对甘蓝型油菜萜类合酶基因BnaTPSA08-1 (BnaA08T0101600Z)进行了功能研究。在克隆得到甘蓝型油菜BnaTPSA08-1基因CDS序列基础上,成功构建了BnaTPSA08-1基因的过表达双元载体pFGC5941-35s::BnaTPSA08-1。利用农杆菌介导的甘蓝型油菜下胚轴遗传转化方法成功获得5株稳定遗传的BnaTPSA08-1基因过表达转基因植株。通过收集过表达转基因植株释放的有机挥发性气体,并用GC-MS测定气体组分,结果显示:与对照植株的有机挥发性组分比较,过表达植株产生25种不同类型的挥发性气体,其中十一烷及其衍生物占比最多,这说明BnaTPSA08-1基因产物的生化功能是促进十一烷及其衍生物的合成,这为进一步研究BnaTPSA08-1基因在甘蓝型油菜中的生物学功能打下了良好基础。Terpenoids are one of the largest groups of metabolites in plants and play an important role in plant response to biotic and abiotic stresses. Terpene synthases (TPSs) are key enzymes in the synthesis of terpene compounds in plants. However, there are few reports on terpene synthases in rapeseed. In order to explore the role of TPSs in rapeseed, the function of the terpene synthase gene BnaTPSA08-1 (BnaA08T0101600Z) in Brassica napus was studied. Based on the cloned CDS sequence of BnaTPSA08-1 gene in Brassica napus, the over-expression vector pFGC5941-35s::BnaTPSA08-1 was successfully constructed. Five transgenic plants with stable BnaTPSA08-1 gene over-expression were successfully obtained by Agrobacteria-mediated hypocotyl genetic transformation of Brassica napus. The organic volatile gases released by overexpressed transgenic plants were collected and the gas components were determined by GC-MS. The results showed that: Compared with the organic volatile components of control plants, over-expressed plants produced 25 different types of volatile gases, among them, undecane and undecane derivatives account for the most, indicating that the biochemical function of BnaTPSA08-1 gene product is to promote the synthesis of undecane and undecane derivatives. This provides a good basis for further study on the biological function of BnaTPSA08-1 gene in Brassica napus.
文摘目的构建含有小鼠血清和糖皮质激素诱导蛋白激酶3(serum and glucocorticoid-induced protein kinase 3,SGK3)基因的真核表达载体pcDNA3.1-MYC-SGK3-mCherry,并观察和验证其在转染细胞HEK293中的表达。方法通过聚合酶链式反应将实验室保存的真核表达质粒pcDNA3.1-MYC-SGK3中目的基因SGK3与mCherry融合并扩增出来,然后定向克隆至pcDNA3.1-MYC质粒中,经限制性内切酶消化和测序证实后,通过脂质体法转染HEK293细胞,Western blotting法检测目的基因的蛋白表达情况。结果测序结果与之前预期结果相符,证实pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。Western blotting结果显示,转染pcDNA3.1-MYC-SGK3-mCherry的HEK293细胞出现清晰的阳性反应条带,说明目的片段成功表达。结论pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。
