香蕉NPR1基因家族的鉴定及在枯萎病菌胁迫下表达分析

以拟南芥NPR1基因家族成员蛋白序列为查询序列,在香蕉基因组数据库中鉴定香蕉NPR1基因家族成员,并对其进行生物信息学分析及在枯萎病菌侵染下的表达分析。共鉴定得到15个成员,将其命名为MaNPR1～MaNPR15。理化性质、保守功能结构域、重要的氨基酸残基及motif分析结果与其他物种所报道的有较高的一致度。香蕉NPR1基因家族成员种内进化树、基因结构及结构域的分类情况呈现出高度一致,表明了香蕉NPR1基因家族成员间有着明确的分工。种间进化树显示香蕉的NPR1基因分为3个分组,每个分组都含有拟南芥NPR1成员。主栽品种巴西蕉(Musa acuminata Colla. AAA group′Brazilian′)易感香蕉枯萎病,从巴西蕉CK及巴西蕉受枯萎病侵染2 d的根系转录组数据中综合表达量及表达趋势选定8个成员,并在巴西蕉与抗(耐)Foc TR4品种GCTCV-119中验证其表达模式。基因MaNPR4及MaNPR11在抗感品种中表现出明显的差异表达,在抗病品种GCTCV-119中随着Foc TR4侵染时间延长表达量不断增加,而在感病品种巴西蕉中表达量呈下降趋势。表明MaNPR4及MaNPR11参与香蕉抗枯萎病过程。本研究结果为进一步利用香蕉NPR1基因进行香蕉抗枯萎病遗传改良奠定基础。     

番茄WOX转录因子家族的鉴定及其进化、表达分析

WUSCHEL相关的同源异型盒(WUSCHEL-related homeobox,WOX)是一类植物特异的转录因子家族,具有调控植物干细胞分裂分化动态平衡等重要功能。本研究利用番茄(Solanum lycopersicum)基因组数据,通过建立隐马尔科夫模型并进行检索,鉴定了番茄10个WOX转录因子家族成员。多序列比对发现,番茄WOX转录因子家族成员具有高度保守的同源异型结构域;以拟南芥WOX转录因子家族成员序列为参照,通过邻接法、极大似然法、贝叶斯法重建了系统发育树,三者呈现出类似的拓扑结构,番茄和拟南芥WOX转录因子家族共25个成员被分为3个进化支(Clade)和9个亚家族(Subgroup);利用MEME和GSDS对WOX转录因子家族成员的蛋白保守结构域和基因结构进行了分析,同一亚家族内的WOX转录因子家族成员的保守结构域的种类、组织形式以及基因结构具有高度的一致性;利用Perl和Orthomcl对家族成员的染色体定位和同源性关系进行分析,结果表明串联重复的SlWOX3a和SlWOX3b可能来源于一次复制事件;利用番茄转录组数据和qRT-PCR进行表达分析,结果显示家族成员在不同组织中的表达存在差异,暗示了WOX家族的不同成员在功能上可能具有多样性。本研究对番茄WOX转录因子家族成员进行GO(Gene Ontology)注释和比较分析,结果表明该家族成员作为转录因子,可能在组织器官发育、细胞间通讯等过程中发挥作用。     

茶树BES1转录因子全基因组鉴定与分析

植物特异性转录因子(BRI1-EMS-SUPPRESSOR1,BES1)家族参与调节油菜素内酯(brassinosteroids,BR)信号通路,在植物生长和应对非生物胁迫的反应中发挥重要作用。该研究利用生物信息学方法对茶树BES1转录因子家族进行鉴定,分析茶树CsBES1基因在8个茶树组织中的表达模式,并对CsBES1转录因子家族在低温、干旱及ABA激素处理下的表达进行分析,以揭示茶树CsBES1转录因子家族的功能及其对环境胁迫的响应。结果显示:(1)从茶树全基因组中鉴定获得10个茶树BES1转录因子家族成员,均具有完整的BES1_N结构域;根据系统进化关系将10个CsBES1转录因子家族分成3组,分别包含2个、3个和5个成员;该家族成员每个基因含有2～11个外显子。(2)组织表达模式分析显示,茶树BES1转录因子家族在生长活跃的茶树嫩梢和成熟叶中表达量较高,不同成员之间表达存在差异性。(3)上游启动子区域分析发现大量与植物激素和非生物胁迫响应相关的顺式作用元件。(4)荧光定量检测发现,BES1基因在茶树不同胁迫处理下的表达模式不同,在低温和脱落酸处理下,CsBES1-1、CsBES1-7、CsBES1-8和CsBES1-9基因的表达量显著提高,而在干旱处理下,CsBES1-2、CsBES1-4、CsBES1-5、CsBES1-6、CsBES1-7、CsBES1-8和CsBES1-9基因的表达量显著提高。     

花生bZIP基因家族全基因组鉴定及抗旱表达分析

本研究利用野生二倍体花生基因组信息鉴定出112个bZIP转录因子家族成员,其中AA基因组中55个家族成员,BB基因组中57个家族成员,分别被命名为AradubZIP1～AradubZIP55与AraipbZIP1～AraipbZIP57。通过生物信息学方法,分析了其基因结构、保守基序、理化性质,研究了其与拟南芥bZIP家族成员和部分四倍体花生bZIP的系统进化关系,预测了其在植物细胞中的位置,并利用转录组测序技术探究了部分家族成员在栽培种花生L422生长后期叶片响应干旱胁迫的基因表达规律。结果表明:野生二倍体中bZIP成员分布于AA和BB基因组的10条染色体上,均为不稳定蛋白,大多数定位在细胞核内(AA基因组36个、BB基因组39个),少数定位在叶绿体(14个与15个)和线粒体(4个与3个);具有相似基因结构与基序bZIP成员各25个,然而,不同成员间存在外显子数目差异,最多为15个外显子(AraipbZIP5),最少为1个(AraipbZIP1与AradubZIP10等12个成员);通过与拟南芥bZIPs氨基酸序列进化分析发现,花生bZIP家族成员划分为A～M、S等14个亚组,其中四倍体bZIP成员在第I亚组中分布最多(7个);32个四倍体bZIP成员在干旱胁迫下的表达模式基本分为4类,第Ⅰ类前、后期高,中期低,第Ⅱ类前期低,中、后期高,第Ⅲ类前、中期高,后期低,第Ⅳ类前、后期低,中期高。上述结果为研究花生bZIP基因家族在生长后期耐旱生长过程的调控作用提供参考依据。     

苹果TIPs亚家族成员的鉴定与干旱胁迫响应分析

【目的】鉴定并分析苹果液泡膜内在蛋白 (Tonoplast intrinsic proteins, TIP) 亚家族成员,探究该亚家族成员在苹果干旱胁迫下的表达模式。【方法】通过生物信息学方法对苹果MdTIPs全基因组进行鉴定,分析其家族成员的理化性质、基因结构、保守基序、顺式作用元件、系统进化树等,并结合实时荧光定量PCR (qRT-PCR) 对其在干旱胁迫下不同器官中的表达模式进行分析。【结果】在苹果基因组中,共检索得到了13个MdTIP基因,大部分成员亚细胞定位预测在质膜上,分布于10条染色体上,且每条染色体定位有1~3个成员;该基因家族成员的启动子区域包含多种响应激素和逆境胁迫的应答元件;qRT-PCR显示,MdTIPs亚家族成员在根中除MdTIP1;1外其余均都受上调表达,且MdTIP1;3和MdTIP1;4与对照相比,分别上调表达了5.27倍和5.69倍,推测是参与调控干旱胁迫的关键基因。【结论】初步鉴定并提供了MdTIPs亚家族成员信息,10个MdTIPs亚家族成员在根、茎、叶中差异表达,12个亚家族成员在根中高表达。结果为进一步研究苹果抗旱性基因资源的挖掘和利用提供了有价值的信息。     

血管内皮生长因子家族及其受体与肿瘤血管生成研究进展

血管内皮生长因子(vascular endothelial growth factor,VEGF),又名血管通透性因子(vascular permeability factor,VPF)是重要的血管生成正性调节因子,是目前抗癌治疗的研究靶点之一。现已发现的VEGF家族成员包括VEGF—A、VEGF—B、VEGF—C、VEGF—D、VEGF—E和胎盘生长因子(placenta growth factor,PLGF)。VEGF的受体有VEGFR—1(fit—1)、VEGFR-2(flk-1／KDR)、VEGFR-3(fit-4)、neuropilin(NPR1／NPR2)。该家族的成员可以选择性地增强血管和／或淋巴管内皮细胞的有丝分裂,刺激内皮细胞增殖并促进血管生成,提高血管特别是微小血管的通透性,使血浆大分子外渗沉积在血管外的基质中,促进新生毛细血管网的建立,为肿瘤细胞的生长提供营养等。作者对VEGF家族成员及其受体的理化特征、VEGF与肿瘤的关系、VEGF抑制剂的研制作一综述。     

KLFs转录因子在山羊成肌细胞分化过程中的表达模式

本研究旨在研究KLFs家族6个成员(KLF3,KLF5,KLF7,KLF8,KLF9和KLF12)在山羊成肌细胞诱导分化过程中的表达模式及各个成员的相关性。利用qPCR检测KLFs在成肌细胞不同分化阶段中的表达丰度,并分析该家族成员间的相关性。结果显示,KLF3、KLF5、KLF7、KLF8、KLF9和KLF12在成肌细胞诱导分化过程中的表达水平逐渐升高,在诱导分化96 h的表达水平均极显著高于未诱导细胞中的表达水平(p0.01);相关性分析结果显示6个成员mRNA表达水平存在极显著相关(p0.01)。本研究明确了KLF3、KLF5、KLF7、KLF8、KLF9和KLF12在山羊成肌细胞诱导分化过程中的表达模式,为阐明该家族在山羊肌肉生长发育中的调控作用提供重要的基础数据。     

人14-3-3蛋白家族的生物信息学分析

目的:分析人14-3-3蛋白家族的同源性及分子进化关系。方法:利用已公布的人基因组数据库,采用BLASTN程序检索人14-3-3蛋白家族各成员的编码基因和假基因,并利用DNAMAN软件进行序列联配,绘制其分子进化树。结果:该家族半数成员具有多个假基因序列,为返转座类型假基因。进化分析表明该家族有共同的祖先,可归为3个亚类。结论:人14-3-3蛋白家族每个成员长期进化所形成的多样性提示其功能具有独特性。     

普通烟草ATⅢ氨基转移酶家族序列鉴定与表达分析

氨基转移酶是5'-磷酸吡哆醛依赖酶,在植物的生长发育和非生物胁迫的反应中起重要作用。ATⅢ氨基转移酶家族(classⅢ aminotransferase family)是转氨酶家族中一个非常重要的亚家族。本研究利用普通烟草(Nicotiana tabacum)基因组序列信息,鉴定出26个ATⅢ家族成员,对烟草ATⅢ家族进行理化性质分析表明,普通烟草ATⅢ家族成员之间的理化性质差异较大;系统进化和结构域分析显示,烟草ATⅢ家族成员可形成4个分支,同一分支内ATⅢ家族成员的保守结构域的种类和组织形式高度一致;将19个家族成员定位在12条染色体上;分析普通烟草转录组数据,结果显示大多数家族成员在不同组织中都有表达,主要集中在叶脉、打顶后茎和叶、离体叶片等组织。对NtATⅢ1和NtATⅢ2基因的qRT-PCR分析显示,这两个基因主要在植物地上组织中表达。本研究为普通烟草ATⅢ基因的功能研究提供依据。     

苹果SRS基因家族的鉴定与功能分析北大核心CSCD

SHI-related sequence(SRS)基因家族通过介导激素变化以调控植物成花及生长发育,并且在适应环境胁迫中起重要调控作用。该研究基于苹果(Malus domestica Borkh.)基因组数据,通过生物信息学手段鉴定苹果SRS基因家族成员,并分析SRS基因家族特点与功能及表达情况。结果表明:(1)苹果MdSRS基因家族共包含11个成员,分别命名为MdSRS1-MdSRS11,不均匀地分布在苹果的9条染色体上。(2)MdSRS蛋白包含229~414个不等的氨基酸残基,等电点分布在6.38~9.36之间;亚细胞定位结果表明,MdSRS蛋白大多分布于细胞膜,在细胞核、叶绿体中也有分布。(3)通过引入拟南芥、水稻、番茄及杨树的SRS基因进行系统发育分析表明,将11个MdSRSs分成5个亚族(A-A),在A4中分布最多。(4)顺式作用元件分析表明,11个MdSRSs启动子上游2 000 bp序列分布有激素、环境适应性和逆境诱导等响应元件。(5)荧光定量PCR结果显示,苹果MdSRS基因家族在盐胁迫和干旱胁迫下总体呈下调表达,在ABA胁迫后大多呈上调表达,是具有很大潜力的抗性候选基因,说明SRS家族对ABA调节等非生物胁迫具有调控作用。研究认为,SRS家族的11个成员均参与了调控干旱、盐及ABA胁迫多种逆境的响应,推测在实际苹果生产中对抵御不良环境具有重要作用。     

Salicylic acid promotes autophagy via NPR3 and NPR4 in <Emphasis Type="Italic">Arabidopsis</Emphasis> senescence and innate immune response

In Arabidopsis thaliana, the non-expresser pathogenesis-related (NPR) multigene family members NPR1, NPR3, and NPR4 are necessary for salicylic acid (SA) perception. NPR3 and NPR4 are the CUL3 E3-ligase substrate adaptors allowing for the ubiquitination and turnover of NPR1 by the 26s proteasome. Concurrently, roots treated with the SA agonist benzothiadiazole accumulate autophagic bodies via NPR1-dependent signal pathway. However, the mechanisms by which NPR3 and NPR4 regulate autophagy remain unclear. In the present study, using single, double, and triple npr1-, npr3-, and npr4-null mutants and wild-type plants, the following results were obtained: (1) leaf senescence progressed faster in npr3/npr4 mutants than in wild type, suggesting that NPR3 and NPR4 negatively regulated leaf senescence. Moreover, npr3/npr4 promoted the expression of pathogenesis-related 1 (PR1) gene and enhanced resistance in response to avirulent pathogen infections suppressing cell death. Still, all mutants had similar SA levels, suggesting that NPR3 and NPR4 positive regulation of cell death and disease resistance was not associated with SA levels; (2) the number of autophagosomes, ATG7, and ATG8a-phosphatidylethanolamine and the concentration of free green-fluorescence protein were lower in npr3/npr4 mutants than in wild-type plants, indicating that NPR3 and NPR4 affected the two ubiquitination-like conjugation systems during the autophagosome formation and degradation of autophagic bodies.     

Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs

NPR1 is required for systemic acquired resistance, and there are five NPR1 paralogs in Arabidopsis. Here we report knockout analysis of two of these, NPR3 and NPR4. npr3 single mutants have elevated basal PR-1 expression and the npr3 npr4 double mutant shows even higher expression. The double mutant plants also display enhanced resistance against virulent bacterial and oomycete pathogens. This enhanced disease resistance is partially dependent on NPR1, can be in part complemented by either wild-type NPR3 or NPR4, and is not associated with an elevated level of salicylic acid. NPR3 and NPR4 interact with TGA2, TGA3, TGA5 and TGA6 in yeast two-hybrid assays. Using bimolecular fluorescence complementation analysis, we show that NPR3 interacts with TGA2 in the nucleus of onion epidermal cells and Arabidopsis mesophyll protoplasts. Combined with our previous finding that basal PR-1 levels are also elevated in the tga2 tga5 tga6 triple mutant, we propose that NPR3 and NPR4 negatively regulate PR gene expression and pathogen resistance through their association with TGA2 and its paralogs.     

A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway.     

Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.     

New species from the Fusarium solani species complex derived from perithecia and soil in the old World tropics

A large collection of strains belonging to the Fusarium solani species complex (FSSC) was isolated from soil and perithecia in primary forests in Sri Lanka (from fallen tree bark) and tropical Australia (Queensland, from fallen tree fruits and nuts). Portions of the translation elongation factor 1-alpha (tef1) gene, the nuclear large subunit (NLSU) and internal transcribed spacer regions (ITS) of the nuclear ribosomal RNA genes were sequenced in 52 isolates from soil and perithecia. The FSSC was divided previously into three clades with some biogeographic structure, termed Clades 1, 2 and 3. All Sri Lankan and Australian soil isolates were found to be members of Clade 3, most grouping with the cosmopolitan soil-associated species F. falciforme. All but two Sri Lankan perithecial isolates were associated with a set of five divergent phylogenetic lineages that were associated with Clade 2. Australian perithecial isolates resided in a subclade of Clade 3 where most of the previously defined mating populations of the FSSC reside. Isolates from perithecia and those cultured from soil were always members of different species lineages, even when derived from proximal locations. The previous biogeographic assignment of Clade 2 to South America is now expanded to the worldwide tropics. Sri Lanka appears to be an important center of diversity for the FSSC. Nectria haematococca is epitypified with a collection from the type locality in Sri Lanka; its anamorph is described as a new species, Fusarium haematococcum. Neocosmospora E.F. Smith is adopted as the correct genus for Nectria haematococca. These new species are described: F. kurunegalense/Neo. kurunegalensis, F. rectiphorus/Neo. rectiphora/, F. mahasenii/Neo. mahasenii/, F. kelerajum/Neo. keleraja.