应用基因敲除快速构建大肠杆菌突变体改造脂肪酸代谢途径

【目的】基因敲除技术是研究基因功能的重要手段。我们试图建立一种快速、高效的大肠杆菌基因敲除方法。【方法】利用大肠杆菌(Escherichia coli)BW25113单基因缺失体Keio文库,将经典的Red同源重组技术与P1噬菌体转导技术相结合,对E.coli MG1655脂肪酸代谢基因进行快速敲除。【结果】获得了大肠杆菌β-氧化途径的缺失菌株△fadD、△fadE和△fadD-△fadE;脂肪酸合成途径缺失菌株△fabH、△fabF和△fabH-△fabF。敲除fadD和fadE对生长情况没有影响;敲除fabH后,生长速度明显减慢;敲除fabF对生长几乎没有影响。FadD、FadE及双敲缺失体的脂肪酸含量18.2 mg/L、20.0mg/L和19.2 mg/L,略高于野生型17.5 mg/L;FabH、FabF及双敲缺失体的含量分别为12.6 mg/L、15.2 mg/L和11.2 mg/L,明显低于野生型。【结论】在单基因突变体文库基础上,利用P1噬菌体转导、Red同源重组和抗性基因消除进行基因敲除,简化了构建大肠杆菌单基因和多重突变体的方法。     

Non‐dormant Axillary Bud 1 regulates axillary bud outgrowth in sorghum

Tillering contributes to grain yield and plant architecture and therefore is an agronomically important trait in sorghum (Sorghum bicolor). Here, we identified and functionally characterized a mutant of the Non‐dormant Axillary Bud 1 (NAB1) gene from an ethyl methanesulfonate‐mutagenized sorghum population. The nab1 mutants have increased tillering and reduced plant height. Map‐based cloning revealed that NAB1 encodes a carotenoid‐cleavage dioxygenase 7 (CCD7) orthologous to rice (Oryza sativa) HIGH‐TILLERING DWARF1/DWARF17 and Arabidopsis thaliana MORE AXILLARY BRANCHING 3. NAB1 is primarily expressed in axillary nodes and tiller bases and NAB1 localizes to chloroplasts. The nab1 mutation causes outgrowth of basal axillary buds; removing these non‐dormant basal axillary buds restored the wild‐type phenotype. The tillering of nab1 plants was completely suppressed by exogenous application of the synthetic strigolactone analog GR24. Moreover, the nab1 plants had no detectable strigolactones and displayed stronger polar auxin transport than wild‐type plants. Finally, RNA‐seq showed that the expression of genes involved in multiple processes, including auxin‐related genes, was significantly altered in nab1. These results suggest that NAB1 functions in strigolactone biosynthesis and the regulation of shoot branching via an interaction with auxin transport.     

皖南尖吻蝮蛇毒出血毒素Ⅳ的纯化与初步鉴定

从皖南尖吻蝮蛇（Ａｇｋｉｓｔｒｏｄｏｎａｃｕｔｕｓ）毒液中经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ和ＳｅｐｈａｃｒｙｌＳ－２００两步凝胶柱层析首次纯化出一种中分子量出血毒素（简称ＡａＨⅣ）．经ＳＤＳ－ＰＡＧＥ和等电聚焦凝胶电泳测定其分子量为４４ｋＤ,等电点为ｐＨ５．０．从５００ｍｇ粗毒中可获得２０ｍｇＡａＨⅣ纯品．ＡａＨⅣ有较强的出血活性,最小出血剂量（ＭＨＤ）为０．４μｇ．     

用DNA杂交技术检测绿脓杆菌氨基糖甙类钝化酶基因的研究

目前已知的氨基糖甙类抗菌素钝化酶基因有许多种,2″—0—腺苷转移酶[ANT(2″)]基因为其中之一。本文从一株含ANT(2″)基因的重组质粒pFCT3103的基因克隆株,分离出310bp的ANT(2″)基因片段,将它制成基因探针,用该探针检查了30株绿脓杆菌是否含有氨基糖甙类钝化酶基因的存在情况,结果表明ANT(2″)基因的检出率为43.3%。     

寡肽转运蛋白PepT2原核表达质粒的构建及其表达初探

寡肤转运蛋白(PepT2,peptide transporter,SLC15A2)是哺乳动物体内能够转运二肤、三肽的蛋白.研究表明,一些类肽的小分子药物也是PepT2的底物,但PepT2的结构与生物学功能尚待研究.建立稳定表达PepT2的表达体系是研究PepT2的重要环节.根据GenBank中人PepT2基因序列,借助Primer5.0设计了1对寡核苷酸引物,经PCR合成长达2 190bp的目的序列,通过重组构建pET30a(+)/PepT2表达质柱,测序分析确认目的基因中的3个碱基发生突变.初步研究了pET30a(+)/PepT2在大肠杆菌BL21(DE3)pLysS中的表达,为PepT2原核表达的进一步科研和实际应用奠定了基础.     

籼粳亚种间杂交稻米脂肪含量的遗传分析

用包括基因型×环境互作效应的种子性状遗传模型,研究了籼粳亚种间杂交稻米脂肪含量的遗传特性,结果表明：在籼粳杂种中,脂肪含量的遗传表达主要受控于种子直接加性效应和母体加性效应,以前者为主．基因型X环境互作主要表现为显性（包括直接显性和母体显性）X环境以及细胞质X环境工作．直接近传率和母体遗传率都极显著．此外,根据遗传效应预测值对供试条本的利用价值作了评价．