Functional analyses of the Dof domain, a zinc finger DNA-binding domain, in a pumpkin DNA-binding protein AOBP

AOBP, a DNA-binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA-binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA-binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA-binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.     

Functional analysis of poly(ADP-ribose) polymerase in Drosophila melanogaster

Poly(ADP-ribose) polymerase (PARP) is conserved in eukaryotes. To analyze the function of PARP, we isolated and characterized the gene for PARP in Drosophila melanogaster. The PARP gene consisted of six translatable exons and spanned more than 50 kb. The DNA binding domain is encoded by exons 1-4. Although the consensus cleavage site of CED-3 like protease during apoptosis is conserved from human to Xenopus laevis PARPs, it is neither conserved in the corresponding region of Drosophila nor Sarcophaga peregrina. There are two cDNAs species in Drosophila. One cDNA could encode the full length PARP protein (PARP I), while the other is a truncated cDNA which could encode a partial-length PARP protein (PARP II), which lacks the automodification domain and is possibly produced by alternative splicing. The expression of these two forms of PARP in E. coli demonstrated that while PARP II has the catalytic NAD-binding domain and DNA-binding domain it is enzymatically inactive. On the other hand PARP I is active. A deletion mutant of PARP gene could grow to the end of embryogenesis but did not grow to the adult fly. These results suggest that the PARP gene plays an important function during the development of Drosophila.     

葡萄NCED基因家族进化及表达分析

9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物体内ABA生物合成的关键限速酶, 参与植物对干旱、外源ABA和高盐的响应过程, 降低环境胁迫对植株的危害。基于全基因组鉴定分析葡萄(Vitis vinifera) NCED基因家族成员, 探讨各成员的物种进化关系及各个基因成员在不同组织中的时空表达模式及对干旱、ABA和高盐(NaCl)胁迫的响应, 为进一步揭示该基因家族成员的生物学功能奠定基础。在葡萄基因组中共发现12个NCED基因。其推测的编码蛋白质长度在510 (VvNCED2)-625 aa (VvNCED10)之间。VvNCED蛋白的分子量最大值是70.53 kDa (VvNCED10), 最小值是57.85 kDa (VvNCED2)。在从祖先基因分化之后, 葡萄NCED基因发生了5次复制事件, 同时有2次丢失事件。NCED1/2、NCED3/4、NCED6/7和NCED9/10基因对被认为是通过片段复制产生。上述4对复制基因复制时间分布在3.08-120.0百万年前, 晚于单双子叶植物分化的时间。与对照相比, VvNCED1在ABA处理48小时后显著上调(72.1%), 而VvNCED2显著下调(84.0%)。VvNCED6只在干旱处理14、21和28天的根系中表达量高于对照, 分别为对照的2.49、1.05和1.09倍。VvNCED7只在干旱处理14天的根系中表达量高于对照, 为对照的1.07倍。在ABA处理72小时后, VvNCED3表达量较对照显著下调(59.5%), 而VvNCED4较对照显著上调(169.9%)。VvNCED3/VvNCED4分别在NaCl处理24和48小时出现显著性峰值, 较对照分别上调219.2%和114.4%。保守结构域不同组成和不同胁迫处理下差异表达模式是NCED蛋白发生功能分化的基础。推测NCED在进化过程中发生的功能分化有利于复制事件的发生。     

葡萄座腔菌Mcm基因家族全基因组鉴定及其对硫化物的响应特性

由葡萄座腔菌Botryosphaeria dothidea引起的轮纹病是严重威胁我国苹果产业健康持续发展的重要病害之一。前期研究表明,韭菜及其主要成分硫化物能显著致死病原菌并能高效防控该病的发生。为了明确微小染色体维持蛋白（Mcm）基因家族在葡萄座腔菌中的基本功能,在全基因组水平对葡萄座腔菌Mcm基因家族进行了鉴定,并分析了葡萄座腔菌Mcm基因家族对硫化物的响应特性。结果显示：葡萄座腔菌Mcm基因家族共有6个成员（BdMcm2-BdMcm7）,分别位于5条重叠群（contig）上,含有3-14个CDS区。葡萄座腔菌Mcm基因家族编码产物主要定位于细胞核,每个成员均具有Mcm-box结构域及其Walker A、Walker B及R-finger结构特征,长度为718-1 618aa,等电点为5.04-8.33,分子量为79.78-180.30kDa。RNA-seq数据和qRT-PCR分析表明硫化物三硫醚能高效抑制葡萄座腔菌Mcm基因家族的表达,与对照相比,6个葡萄座腔菌Mcm基因表达量分别下调20.56%-78.10%和10.74%-73.83%,两者呈现高度一致性（R²=0.8958）。细胞流式仪检测表明硫化物也显著阻滞了葡萄座腔菌细胞周期。该研究为进一步揭示韭菜及其主要成分防控苹果轮纹病机理提供了理论依据。     

葡萄NCED基因家族进化及表达分析

9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物体内ABA生物合成的关键限速酶, 参与植物对干旱、外源ABA和高盐的响应过程, 降低环境胁迫对植株的危害。基于全基因组鉴定分析葡萄(Vitis vinifera) NCED基因家族成员, 探讨各成员的物种进化关系及各个基因成员在不同组织中的时空表达模式及对干旱、ABA和高盐(NaCl)胁迫的响应, 为进一步揭示该基因家族成员的生物学功能奠定基础。在葡萄基因组中共发现12个NCED基因。其推测的编码蛋白质长度在510 (VvNCED2)-625 aa (VvNCED10)之间。VvNCED蛋白的分子量最大值是70.53 kDa (VvNCED10), 最小值是57.85 kDa (VvNCED2)。在从祖先基因分化之后, 葡萄NCED基因发生了5次复制事件, 同时有2次丢失事件。NCED1/2、NCED3/4、NCED6/7和NCED9/10基因对被认为是通过片段复制产生。上述4对复制基因复制时间分布在3.08-120.0百万年前, 晚于单双子叶植物分化的时间。与对照相比, VvNCED1在ABA处理48小时后显著上调(72.1%), 而VvNCED2显著下调(84.0%)。VvNCED6只在干旱处理14、21和28天的根系中表达量高于对照, 分别为对照的2.49、1.05和1.09倍。VvNCED7只在干旱处理14天的根系中表达量高于对照, 为对照的1.07倍。在ABA处理72小时后, VvNCED3表达量较对照显著下调(59.5%), 而VvNCED4较对照显著上调(169.9%)。VvNCED3/VvNCED4分别在NaCl处理24和48小时出现显著性峰值, 较对照分别上调219.2%和114.4%。保守结构域不同组成和不同胁迫处理下差异表达模式是NCED蛋白发生功能分化的基础。推测NCED在进化过程中发生的功能分化有利于复制事件的发生。     

金针菇CA基因家族分析及其对CO2的响应

金针菇栽培通过提高栽培室CO₂浓度来抑制菌盖生长,提高商品质量。碳酸酐酶能调节CO₂/HCO₃ ^-在细胞内的平衡,它可能是响应CO₂胁迫的关键酶。本研究利用我们已完成的金针菇Flammulina filiformis的基因组测序数据,以双色蜡蘑碳酸酐酶家族基因为参照,通过本地BLAST,得到7个金针菇碳酸酐酶家族的基因,分别命名为CA-1、CA-2、CA-3、CA-4、CA-5、CA-6、CA-7。基因结构、保守结构域和系统进化分析结果均支持这7个序列是碳酸酐酶家族的编码基因。高浓度CO₂胁迫处理金针菇子实体12h,CA-1、CA-2表达量与CO₂浓度正相关,CA-5负相关,CA-3、CA-4和CA-6没有响应,CA-7无明显规律。据此推测,CA-1、CA-2和CA-5可能是金针菇响应CO₂胁迫的关键基因之一,参与调控菌盖生长发育。     

尖孢镰刀菌FoHeli转座元件的结构特征及功能验证

Helitron是一种广泛存在于真核生物中的可移动遗传元件。与其他转座子不同,自主Helitron元件可编码具有复制引发（Rep）和解旋酶（Hel）结构域的转座酶,并通过滚环复制的方式在基因组中进行扩张。本研究对9种尖孢镰刀菌中的自主Helitron元件进行系统分析,结果表明尖孢镰刀菌中存在两类自主Helitron元件FoHeli1与FoHeli2。其中FoHeli1成员间序列高度相似,并具有明晰的边界特征：3’端为保守的“TATTTT”序列,其上游可形成稳定的发夹结构,且发夹上游可与5’端形成12bp的反向互补结构。基于上述分析结果,从尖孢镰刀菌Fo4287菌株中克隆获得完整的FoHeli1元件,并通过构建双元转座系统及PEG介导的原生质体转化,证明尖孢镰刀菌中的FoHeli元件可在禾谷镰刀菌PH-1菌株的基因组中发生跳转。     

极性蛋白Crumbs胞内域功能保守性研究

跨膜蛋白Crumbs(Crb)是细胞顶部的决定因子,对上皮细胞顶-底极性的建立和维持起着关键的作用。其胞内域虽然仅有37个氨基酸,但对Crb的功能必不可少。在果蝇(Drosophila melanogaster)中,如果胞内域发生突变,将造成胚胎发育异常、上皮细胞顶底极性丧失等严重后果。Crb胞内域从果蝇到小鼠(Mus musculus)和人类(Homo sapiens)具有很高的同源性,但线虫(Caenorhabditis elegans)两个Crb蛋白的胞内域与果蝇和哺乳动物却较为不同。为验证线虫Crb蛋白胞内域是否功能保守,本文利用基因组工程法(Genomic engineering),将果蝇基因组中Crb基因编码胞内域的部分替换为一致性和相似性较远的线虫Crb2基因的相应区段。与其他Crb胞内域突变果蝇不同,替换突变体胚胎发育正常,Crb及其他极性蛋白的表达和定位正常,胚胎上皮细胞顶底极性能够正确的建立和维持。这些结果证实虽然线虫和果蝇Crb蛋白胞内域之间存在大量序列变异,但重要的氨基酸位点和功能模块则完全保守。     

Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells

Four new members of the ERF (ethylene-response factor) family of plant-specific DNA-binding (GCC box) factors were isolated from tomato fruit (LeERF1–4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding affinities differed between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding affinity of the LeERFs was affected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain.     

Domain libraries: Synthetic diversity for de novo design of antibody V-regions

A completely synthetic gene library encoding the variable light (VL) immunoglobulin domains has been constructed in vitro. The library was constructed by assembling a set of six oligodeoxyribonucleotides (oligos) using the polymerase chain reaction (PCR). Three out of the six overlapping oligonucleotides were synthesized with randomized complementarity determining regions (CDR) with the codon pattern, (NNS)_n, where N is any of the four nucleotides (nt) and n is the number of codons with variation in the CDR. The framework regions, taken from the D1.3 anti-lysozyme antibody (Ab), were kept intact. Overlapping regions of approx. 20 nt, together with two additional flanking primers carrying the desired restriction sites, allowed the construction of a library in one single PCR reaction. The VL library was cloned into the phage display vector pEXmide3, and ten randomly picked clones were sequenced. These sequences exhibited complete diversity in all the three CDR and the codons for five canonical amino acid (aa) residues were kept intact and identified. Seven clones contained the full-length gene for the VL domain while deletions were observed in three clones. The restricted use of nt at the third position successfully avoided the stop codons TGA and TAA, whereas the stop codon TAG is read as Gln in an amber suppressor strain. We call this synthetic Ab diversity Domain Library, and it represents an example of syntheticlibraries with extensive, multiple randomized sequences. The use of Domain Libraries opens up the possibility for design in Ab engineering, e.g., additional CDR regions can be added or their length varied. Furthermore, the use of synthetic gene libraries, constructed with the Domain Library strategy, is not limited to the construction of synthetic Ab fragments, but can be used in the design of other types of proteins.     

白菜型冬油菜β-1,3-葡聚糖酶基因在低温胁迫下的表达

为探明β-1,3-葡聚糖酶基因(β-1,3-glucanase)对油菜(Brassica campestris)抵御低温胁迫能力的作用,通过蛋白质谱分析得到β-1,3-葡聚糖酶蛋白,采用RT-PCR技术克隆白菜型冬油菜(B.rapa)陇油6号和天油4号β-1,3-葡聚糖酶的c DNA序列;并对该序列进行生物信息学分析;进而采用实时荧光定量PCR及半定量PCR检测β-1,3-葡聚糖酶基因在低温胁迫下的表达模式。结果获得长度为1 032 bp的陇油6号β-1,3-葡聚糖酶基因开放阅读框,编码343个氨基酸,相对分子量为38.102k Da,理论等电点为6.63,其与菜心(B.rapa subsp.chinensis)和甘蓝型油菜(B.napus)的蛋白质氨基酸序列同源性高达93.94%。该基因编码的酶是一个主要由α-螺旋组成的亲水性稳定蛋白,含有1个信号肽,存在2个跨膜结构域。该基因在进化上高度保守,其保守序列属于植物的糖基水解酶家族17特有的保守结构域。β-1,3-葡聚糖酶基因表达模式分析显示,4°C时该基因上调表达,继续低温(–4°C)胁迫处理,该基因上调表达至峰值,至–8°C时其表达下调。研究表明从白菜型冬油菜中克隆的β-1,3-glucanase在冬油菜品种陇油6号抗寒过程中发挥作用。     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.