烟草突变体库及其在功能基因组研究中的应用

烟草突变体是烟草功能基因组学研究的重要材料。本文综述了烟草突变体在功能基因研究中的作用,构建烟草突变体库的必要性以及烟草突变体库的构建方法,并展望了烟草突变体库在烟草功能基因组学中的应用。     

基于变异测试的错误定位研究进展

随着软件规模和复杂度的不断提高，软件的质量问题成为了关注的焦点，如何高效地找出软件中的错误成为一个亟需解决的问题。错误定位是软件质量保证的重要途径之一，近年来已经成为软件工程中一个非常重要的研究课题。基于变异测试的错误定位通过比较原程序和对应变异体的差异来计算每条语句的怀疑度，再由怀疑度大小进行排序，程序员根据排序逐个检查找出错误语句。汇总近7年（2012-2018）国内外的基于变异测试的错误定位技术的研究成果，介绍了错误定位的基本方法，介绍基于变异测试的错误定位思想，从变异算子、变异体及等价变异体3个方面对已有的研究工作进行分类归纳和总结，探讨了基于变异测试的错误定位未来可能的研究方向、机遇和挑战。     

Effects of exogenously applied ferulic acid,a potential allelopathic compound,on leaf growth,water utilization,and endogenous abscisic acid levels of tomato,cucumber, and bean

To determine the relative sensitivities of tomato, cucumber, and bean to exogenously applied concentrations of ferulic acid (FA) and to determine whether FA-induced stress responses increase endogenous levels of abscisic acid (ABA), wild-type andFlacca (ABA-deficient mutant) tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig), cucumber, (Cucumis sativus L. cv. Early Green Cluster), and bean (Phaseolus vulgaris L. cv. Oregon 91) were treated with FA (0.0, 0.2, 0.4, 0.8 mM) in nutrient solution every other day for a total of two or three treatments. FA inhibited leaf growth and water utilization of wild-type tomato,Flacca tomato, and cucumber, but not of bean. Acclimation to FA was observed following the first FA treatment and increased endogenous ABA levels were found in wild-type tomato,Flacca tomato, and cucumber following multiple FA treatments. Induction of ABA biosynthesis occurred in wild-type tomato within 8 hr of FA treatment and maximum ABA levels were observed 24 hr after treatment. At that time, ABA levels of tomato treated with 0.4 and 0.8 mM FA were 13.7 times and 2.6 times higher than control levels, respectively. A second FA (0.4 or 0.8 mM) treatment, 48 hr after the first, did not appear to affect ABA levels. Ninety-six hours after the first treatment, ABA levels of tomato treated with 0.4 mM FA approached control levels; ABA levels of plants treated with 0.8 mM FA were 1.9 times higher than control levels. Control ABA levels increased gradually with time. The data showed that plant sensitivity and ability of subsequent acclimation to phenolic acids, such as FA, were taxa dependent.The use of trade names in this publication does not imply endorsement by North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.     

Analysis of the Quaternary Structure of Hemoglobin Beckman Variant and Molecular Interpretation of Its Functional Abnormality: A Mass‐Spectrometry‐Based Approach

Electrostatic attraction between α and β globin chains holds the subunits together in a tetrameric human hemoglobin molecule (α₂β₂). Compared to normal globin chains, the affinity of a mutant chain to its partner globin might be different in genetic variants of hemoglobin. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous samples, even though the rates of synthesis of both the normal and variant chains are the same. The aforementioned affinities across various globin chains might be assessed by quantification of the different forms of the tetramers present in a variant hemoglobin sample. In the present study, by exploiting mass differences between globin chains, differently populated hemoglobin tetramers present in hemoglobin (Hb) Beckman, a β variant (βA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α₂β₂, α₂ββ^V, and α₂β^V₂) indicated that both β and β^V have different affinities towards the α globin chain. Conformational dynamics analyzed from hydrogen/deuterium exchange kinetics of the three peptide fragments of Hb Beckman in its oxy state displayed molecular insight into its functional abnormality. However, in comparison to normal hemoglobin (α₂β₂), the point mutation did not show any change in the collision cross‐sections of the functionally active conformers of the variant hemoglobin molecules (α₂ββ^V and α₂β^V₂).     

Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability

There have been two major problems preventing applications of termite cellulases; one was difficulty for their hetelologous overexpression, and another is their low thermostability. We previously achieved adaptation of termite cellulase genes to an overexpression system of Escherichia coli by family shuffling of four orthologous cDNAs (Biosci. Biotechnol. Biochem., 2005; 69: 1711-1720). Using the adapted mutant cDNAs as parental genes combined with native-form cDNAs, we performed further family shuffling and obtained mutant cDNAs, which gave enzymes with improved thermostability. The best-evolved clone (PA68) was improved by 10 degrees C in maximum stability (retaining 90% original activity for 30 min incubation) from the parental enzymes, and kept 54% of its original activity for 150 min at 50 degrees C, whereas the most thermostable enzyme amongst the parents (A18) retained 30% of its original activity. PA68 showed 889 (micromoles of reducing sugars/min/mg of protein) in V(max) and 560 (micromoles of reducing sugars/min/mg of protein) in the specific activity against carboxymethylcellulose, which corresponds to 9.8 and 13.1 times of those of one of the ancestral enzymes rRsEG. In summary, we improved thermostability of the termite cellulase and increased the V(max) value and specific activity by combining only cDNAs encoding enzymes adapted for normal temperatures.     

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.     

The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.     

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.     

重离子束诱变呼吸缺陷型酵母菌株的筛选与快速鉴定

利用单核能为5.19 MeV/u的22Ne5 辐照啤酒酵母菌株,采用TTC筛选培养基筛选出呼吸缺陷型酵母菌株。通过一种新型而简便的限制性酶切分析手段,有效地对所筛选出的8株呼吸缺陷型酵母菌株进行遗传学鉴定。     

保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株的原生质体制备及融合

为获得具有抗噬菌体功能且发酵性能优良的乳酸菌融合子,采用单亲灭活及正交分析方法,研究了保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株的原生质体制备及融合条件。结果表明：保加利亚乳杆菌原生质体制备的最适条件是以磷酸盐缓冲液和甘露醇制作的高渗溶液为原生质体稳定剂,1.0 mg/mL的溶菌酶36 ℃处理30 min,原生质体的形成率为（89.02±2.31）%,再生率为（4.62±0.22）%。嗜热链球菌抗噬菌体菌株原生质体制备的最适条件是以Tris-HCl和蔗糖制作的高渗溶液为原生质体稳定剂,0.1 mg/mL的溶菌酶42 ℃处理30 min,原生质体的形成率为（99.15±0.23）%,再生率为（5.79±0.17）%。单亲灭活保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株原生质体融合的最适条件为聚乙二醇6000（质量浓度为400 g/L,添加0.01 mol/L CaCl2、0.02 mol/L MgCl2）40 ℃促融2 min,融合率可达（1.85±0.12）×10－6。所得融合子各项性能优良,适合于酸奶生产。