甘蓝型油菜扩展蛋白家族的全基因组鉴定及其对缺硼胁迫响应的差异分析

以甘蓝型油菜(Brassica napus L.)硼高效品种‘青油10号’和硼低效品种‘Westar 10’为研究对象,采用生物信息学分析、转录组测序和实时荧光定量PCR技术,鉴定其基因组中扩展蛋白的家族成员,并对该基因家族响应缺硼胁迫的表达差异进行分析。结果显示,甘蓝型油菜基因组中包含109个扩展蛋白,可分为4个亚家族,包括:79个扩展蛋白A(BnaEXPAs)、21个扩展蛋白B(BnaEXPBs)、5个类扩展蛋白A(BnaEXLAs)和4个类扩展蛋白B(BnaEXLBs)。同一亚家族中的扩展蛋白具有相对保守的基因结构和蛋白质基序组成。这些扩展蛋白基因分布在19条染色体上,其中10个位于硼高效QTL区间内。转录组测序分析结果表明,缺硼胁迫时‘青油10号’的根、幼叶和老叶中分别有40、18和30个扩展蛋白基因显著上调或下调表达;而‘Westar10’中分别有27、24和41个扩展蛋白基因显著上调或下调表达。其中‘青油10号’根中的BnaC04.EXPA6a,幼叶中的BnaA09.EXPA5以及老叶中的BnaA09.EXPA16、BnaC04.EXPA3、BnaCnn.EXPA5b和BnaA03.EXPA8基因的表达水平均显著高于‘Westar10’。研究结果说明甘蓝型油菜基因组中扩展蛋白基因家族数量庞大,其中高、低效品种间和不同硼水平中差异表达的扩展蛋白可能在甘蓝型油菜低硼适应性中发挥重要作用。     

甘蓝型油菜扩展蛋白家族的全基因组鉴定及其对缺硼胁迫响应的差异分析（英文）

以甘蓝型油菜(Brassica napus L.)硼高效品种‘青油10号’和硼低效品种‘Westar 10’为研究对象,采用生物信息学分析、转录组测序和实时荧光定量PCR技术,鉴定其基因组中扩展蛋白的家族成员,并对该基因家族响应缺硼胁迫的表达差异进行分析。结果显示,甘蓝型油菜基因组中包含109个扩展蛋白,可分为4个亚家族,包括:79个扩展蛋白A(Bna EXPAs)、21个扩展蛋白B(Bna EXPBs)、5个类扩展蛋白A(Bna EXLAs)和4个类扩展蛋白B(Bna EXLBs)。同一亚家族中的扩展蛋白具有相对保守的基因结构和蛋白质基序组成。这些扩展蛋白基因分布在19条染色体上,其中10个位于硼高效QTL区间内。转录组测序分析结果表明,缺硼胁迫时‘青油10号’的根、幼叶和老叶中分别有40、18和30个扩展蛋白基因显著上调或下调表达;而‘Westar10’中分别有27、24和41个扩展蛋白基因显著上调或下调表达。其中‘青油10号’根中的Bna C04.EXPA6a,幼叶中的Bna A09.EXPA5以及老叶中的Bna A09.EXPA16、Bna C04.EXPA3、Bna Cnn.EXPA5b和Bna A03.EXPA8基因的表达水平均显著高于‘Westar10’。研究结果说明甘蓝型油菜基因组中扩展蛋白基因家族数量庞大,其中高、低效品种间和不同硼水平中差异表达的扩展蛋白可能在甘蓝型油菜低硼适应性中发挥重要作用。     

干旱高温复合胁迫下sHSPs基因在不同耐旱性玉米中的表达差异及其对ABA和H2O2的响应

以4个玉米(Zea mays L.)品种——‘郑单958’(耐干旱)、‘浚单20’(耐高温)、‘隆玉602’(耐干旱高温复合胁迫)和‘驻玉309’(对3种胁迫均敏感)为实验材料,分别采用脱落酸(ABA)及其抑制剂氟定酮(F)、H2O2及其清除剂碘化钾(I)和丙酮酸钠(P)预处理,探讨干旱高温复合胁迫下小热休克蛋白(sHSPs)在不同耐旱性玉米品种中的表达以及ABA和H2O2对其表达的影响。结果显示:(1)高温、干旱高温复合胁迫诱导的sHSP16.9、sHSP17.2、sHSP17.4、sHSP17.5、sHSP22和sHSP26等6个sHSPs基因表达增加量明显高于干旱和对照。(2)在6个sHSPs中,sHSP17.2基因仅在‘郑单958’中表达;sHSP16.9、sHSP17.4和sHSP26基因在‘隆玉602’中表达增加量最高,在‘驻玉309’中表达增量最低;sHSP17.5和sHSP22基因在‘郑单958’中表达增加量最高,在‘驻玉309’中最低。(3)ABA、F、I和P预处理后,对干旱高温复合胁迫诱导的6个sHSPs基因表达增加量仅有略微影响,但显著影响了sHSP26的蛋白表达。研究表明,sHSPs在不同耐旱性玉米品种中的表达存在显著差异,ABA和H2O2仅稍微提高了sHSPs基因表达,但显著提高了sHSP26蛋白表达,这些结果为进一步研究植物在多胁迫条件下耐逆机理奠定了基础。     

小麦TaHDA19基因的克隆及胁迫表达分析

利用基因克隆技术从小麦(Triticum aestivum L.)品种‘科农199’中扩增得到一个RPD3/HDA1型组蛋白去乙酰化酶基因Ta HDA19,采用生物信息学方法对该基因序列的结构特征进行分析,并对植株不同组织及不同胁迫条件下该基因的表达量进行检测。结果显示:Ta HDA19的开放阅读框长1560 bp,共编码519个氨基酸;该基因的氨基酸序列存在组蛋白去乙酰化酶(HDAC)家族典型的结构域Hist-deacety1。该基因上游启动子区含有多种响应元件,如:光响应元件I-box和G-box,脱落酸响应元件ABRE和低温响应元件LTR等。‘科农199’Ta HDA19基因的表达结果表明:该基因在根、茎、叶片和幼穗中均有表达,其中在叶片中表达量最高;采用脱落酸、氯化钠和PEG分别处理植株0、1、3、6、12、24 h后,基因的表达水平出现差异;在对植株的12个生育时期进行35℃和42℃热胁迫处理1 h后,该基因表达量均出现上调。研究结果表明Ta HDA19可能在小麦响应非生物胁迫过程中发挥重要作用。     

小麦TaDRLea3 2基因的克隆及胁迫诱导表达分析

胚胎发育后期丰富蛋白(late embryogenesis abundant protein,LEA蛋白) 是植物体中广泛存在的一类与渗透调节相关的家族蛋白,植物受非生物胁迫会大量表达。该研究采用同源克隆技术,从干旱诱导的小麦品种‘郑引1号’ (Triticum aestivum L.)中获得1个新的LEA3家族基因（ TaDRLea3 2）,该基因全长668 bp,编码区为570 bp,编码189个氨基酸。生物信息学分析表明该蛋白为亲水性蛋白,二级结构以α 螺旋为主,含有9个由11个氨基酸组成的保守结构域,为典型的LEA3蛋白,存在3个磷酸化位点,无信号肽结构域及跨膜结构域,可能定位于细胞质中;实时定量PCR结果表明, TaDRLea3 2基因受干旱、高盐、低温诱导表达,同时也受外源ABA诱导,推测 TaDRLea3 2为ABA依赖型 LEA3基因,以不同机制参与小麦对非生物胁迫的应答过程,为深入分析小麦LEA3家族蛋白的抗逆机制奠定了基础。     

盐及干旱胁迫对油菜抗氧化系统和RbohC、RbohF基因表达的影响

以白菜型油菜‘陇油6号’和‘天油2号’为试验材料,经MAPK抑制剂U0126、H₂O₂清除剂DMTU、NADPH氧化酶抑制剂DPI和IMD预处理后再分别进行盐胁迫、PEG-6000模拟干旱胁迫,研究其对两种油菜幼苗活性氧、抗氧化酶活性和RbohC、RbohF基因表达的影响.结果表明: 盐胁迫和PEG-6000模拟干旱胁迫下,两种白菜型油菜中H₂O₂积累量上升,O₂^-·积累量下降,抗氧化酶(超氧化物歧化酶SOD、过氧化氢酶CAT、抗坏血酸过氧化物酶APX和谷胱甘肽还原酶GR)活性和RbohC、RbohF基因表达均升高.与单独胁迫处理相比,两种油菜O₂^-·积累、抗氧化酶活性和RbohC、RbohF基因的表达量均明显降低,经DMTU、DPI和IMD预处理后再分别进行盐和干旱胁迫,H₂O₂积累量下降,但U0126预处理后再进行胁迫处理,H₂O₂积累量上升.说明NADPH氧化酶、MAP激酶级联途径、H₂O₂参与了盐、干旱胁迫下活性氧产生、抗氧化酶活性变化和RbohC、RbohF基因表达的调控.     

甘蓝型油菜PGK基因的克隆与表达分析

为研究甘蓝型油菜磷酸甘油酸激酶(PGK)基因表达特性,在对拟南芥PGK基因家族生物信息学分析的基础上,通过电子克隆方法获得3个甘蓝型油菜PGK基因(BnPGK1、BnPGK2、BnPGK3)。分别设计特异引物,以甘蓝型油菜雄性不育系09A和保持系09B的cDNA为模板克隆BnPGK基因全长序列。根据获得的cDNA序列设计实时荧光定量特异引物,采用实时荧光定量PCR技术,研究油菜雄性不育系与保持系PGK基因表达差异。结果显示:BnPGK基因在甘蓝型油菜雄性不育系09A和保持系09B的根、茎、叶、花蕾中均有表达,属组成性表达。除茎中的BnPGK3外,BnPGK其它基因在根、茎、叶中的表达均表现为09A高于09B,而在花蕾中均为09B高于09A,BnPGK1和BnPGK3在09B中的表达量是09A中的2倍以上。     

白菜型冬油菜HDACs基因家族的鉴定及表达分析北大核心CSCD

为了研究HDACs(组蛋白去乙酰化酶)在白菜型冬油菜响应低温胁迫中的影响,该研究采用强抗寒品种‘陇油7号’、耐寒品种‘天油4号’和弱抗寒品种Lenox等白菜型冬油菜,分析HDACs抑制剂曲古抑菌素A(TSA)处理后白菜型冬油菜种子的萌发,及低温胁迫下抗氧化酶活性和渗透调节物质含量变化,并全基因组鉴定HDACs的基因家族,分析其在不同抗寒性白菜型冬油菜中的表达特性。结果表明:(1)1μmol/L和3μmol/L的TSA对3个品种的根长均具有抑制作用,不同温度下外源喷施TSA均可增加抗氧化酶活性和渗透调节物质含量。(2)在白菜型冬油菜中共鉴定出21个HDACs;系统发育分析显示,HDACs可分为3个亚家族,所有基因不均等分布在8条染色体上,但7号和8号染色体上没有基因分布;HDACs启动子序列中包含胁迫响应、光响应、激素应答等相关的作用元件。(3)组织表达结果显示,大部分基因在生长锥中表达量明显高于其他组织部位,BrapaHDA19、BrapaHDA6-1、BrapaHDA8和BrapaHDA7-2在白菜型冬油菜花中表达量较高,且BrapaHDA5-2在‘天油4号’的茎生叶、角果、新基叶和老基叶中的表达量较高,而BrapaHDA7-2在Lenox的花中表达量较高。(4)低温胁迫表达结果显示,BrapaHDA8、BrapaHDA14、BrapaHDA2、BrapaHDT2-2和BrapaHDA15-1在3个抗寒性不同的材料中呈现出差异表达。研究推测,所鉴定的这些基因可能在白菜型冬油菜生长发育及抗寒性方面发挥重要功能。     

甘蓝型油菜BnTCP7基因的克隆和表达分析

该研究克隆获得了甘蓝型油菜预测转录因子TCP7-like同源编码基因,命名为BnTCP7,与甘蓝型油菜TCP7-like NC_027757基因的核苷酸序列相似性为95.68%。BnTCP7基因开放阅读框全长为648bp,编码215个氨基酸。BnTCP7氨基酸序列与十字花科(Brassicaceae)中其他19条TCP7或者TCP7-like氨基酸序列比对发现,BnTCP7与芸薹属和萝卜属等TCP7同源蛋白具有很强的相似性和保守性,尤其在靠近N端的TCP保守结构域,具有典型的螺旋-环-螺旋结构,且BnTCP7属于TCP家族Ⅰ类,为亲水性不稳定蛋白。通过BnTCP7和拟南芥(Arabidopsis thaliana)AtTCP7(NC_003076)蛋白的二级和三级结构在线预测比对分析,进一步证实BnTCP7具有TCP家族典型结构。进化树分析表明,BnTCP7蛋白与甘蓝型油菜TCP7-like NC_027757蛋白聚在同一分支,两者亲缘关系最近。利用转录组数据对不同时期不同器官BnTCP7基因表达模式分析发现,其表达量呈现一定差异性,其中营养器官中的表达量高于生殖器官。非生物胁迫及激素处理对甘蓝型油菜幼苗植株中BnTCP7基因的表达影响分析发现,BnTCP7基因不仅响应了冷、热、损伤等非生物胁迫,而且参与了ABA和GA3激素的信号转导,表明BnTCP7基因在植物维持正常生长发育和逆境胁迫中可能发挥重要作用。     

白菜型油菜RbohC和RbohF基因克隆与表达分析

该研究以白菜型油菜（Brassica rapa L.）‘陇油6号’为实验材料,采用RT PCR方法克隆油菜RbohC和RbohF基因,并采用实时荧光定量PCR技术对RbohC、RbohF基因在不同组织及非生物胁迫下的表达进行分析,为深入研究油菜RbohC、RbohF基因的生物学功能提供依据。结果显示：(1) 成功克隆得到2个全长分别为3 050 bp和2 995 bp的油菜RbohC (GenBank登录号：XM_009134386) 和RbohF (GenBank登录号：XM_009114548) 基因序列。(2) 生物信息学分析显示,油菜RbohC和RbohF基因开放阅读框（ORF）分别为2 733 bp和2 847 bp,编码910和948个氨基酸,推测二者的蛋白质分子量分别为103 kDa和108 kDa,理论等电点分别为9.47和9.21; 油菜RbohC、RbohF编码的氨基酸序列与萝卜等多种植物相应蛋白氨基酸序列具有较高的同源性,且这些序列高度保守并含有NADPH氧化酶的典型保守结构域,包括2个可以与Ca²⁺结合的EF手性模体结构、6个跨膜结构域、黄素腺嘌呤二核苷酸结合结构域、NAD焦磷酸结合结构域和C末端区域中的NADP核糖保守结合位点。(3) 油菜RbohC和RbohF基因在根、茎、叶和下胚轴中均表达,无组织特异性,但RbohC基因在根中表达量最高, RbohF基因在下胚轴中表达量最高。(4) 低温、干旱、盐、ABA、H₂O₂处理都能够诱导油菜RbohC和RbohF基因的表达,但抗寒性强的 ‘陇油6号’的RbohC和RbohF基因对胁迫的响应更敏感,且RbohC基因的表达量均高于RbohF基因。(5) 用H₂O₂清除剂DMTU、NADPH氧化酶抑制剂DPI和IMD、MAPKK抑制剂U0126处理后,油菜RbohC和RbohF基因的表达均较对照下降,说明U0126和DMTU对油菜RbohC和RbohF基因的表达有抑制作用。研究认为,油菜RbohC和RbohF基因在油菜适应逆境胁迫中具有重要作用,两基因的表达均受MAPK激酶信号途径的调节,并受到H₂O₂的反馈调节,而且抗寒性强的‘陇油6号’品种中RbohC和RbohF基因对H2O2和MAPK激酶信号途径的响应更敏感。     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

Evolution of Tom, 297, 17.6 and rover retrotransposons in Drosophilidae species

LTR retrotransposons are the most abundant transposable elements in Drosophila and are believed to have contributed significantly to genome evolution. Different reports have shown that many LTR retrotransposon families in Drosophila melanogaster emerged from recent evolutionary episodes of transpositional activity. To contribute to the knowledge of the evolutionary history of Drosophila LTR retrotransposons and the mechanisms that control their abundance, distribution and diversity, we conducted analyses of four related families of LTR retrotransposons, 297, 17.6, rover and Tom. Our results show that these elements seem to be restricted to species from the D. melanogaster group, except for 17.6, which is also present in D. virilis and D. mojavensis. Genetic divergences and phylogenetic analyses of a 1-kb fragment region of the pol gene illustrate that the evolutionary dynamics of Tom, 297, 17.6 and rover retrotransposons are similar in several aspects, such as low codon bias, the action of purifying selection and phylogenies that are incongruent with those of the host species. We found an extremely complex association among the retrotransposon sequences, indicating that different processes shaped the evolutionary history of these elements, and we detected a very high number of possible horizontal transfer events, corroborating the importance of lateral transmission in the evolution and maintenance of LTR retrotransposons.     

Chromosomal Mapping ofTmp(Emp1),Xmp(Emp2), andYmp(Emp3), Genes Encoding Membrane Proteins Related toPmp22

We have recently characterized a novel mammalian gene family, encoding membrane glycoproteins with four trans-membrane domains. This gene family includes the previously studiedPMP22,which is involved in the Charcot–Marie–Tooth neuropathy, and three novel genes:TMP, XMP,andYMP(HGMW-approved symbolsEMP1, EMP2andEMP3,respectively). TheTmp(tumor-associated membrane protein) gene was isolated from a c-mycinduced mouse brain tumor and is expressed in several highly proliferative cell types. We have now isolated cDNAs of the mouseXmpandYmpgenes and determined the chromosomal localization of mouseTmp, Xmp,andYmp. Tmpwas mapped to mouse chromosome 6,Xmpwas mapped to chromosome 16, andYmpwas mapped to chromosome 7.TmpandYmpmap to paralogous chromosomal regions, whereasXmpmaps to a chromosomal region that is putatively paralogous to a region on chromosome 11, to whichPmp22was previously mapped. These data suggest that this family of membrane glycoproteins evolved as a result of chromosomal duplications.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

麻疯树种子发育过程中JcOle14.3和JcOle16.6基因的表达模式研究

油质蛋白基因对种子中油体的形成至关重要,该研究通过实时荧光定量PCR,对麻疯树的两个油质蛋白基因JcOle14.3和JcOle16.6在种子不同发育时期的表达模式进行了分析。结果表明:两个基因在种子发育初期(10~30 d)表达量逐渐升高,但表达水平均较低;40 d时表达量急剧增加并达到最高,而种子发育后期(50~55 d)两个基因表达水平均逐渐降低。由此可初步推测,JcOle14.3和JcOle16.6基因的表达量可能与种子油脂积累量存在正相关。该研究结果为麻疯树油体形成机理和油质蛋白的深入研究提供了理论基础。     

Genomic organization of Tropomodulins 2 and 4 and unusual intergenic and intraexonic splicing of YL-1 and Tropomodulin 4

Background  

The tropomodulins (TMODs) are a family of proteins that cap the pointed ends of actin filaments. Four TMODs have been identified in humans, with orthologs in mice. Mutations in actin or actin-binding proteins have been found to cause several human diseases, ranging from hypertrophic cardiomyopathy to immunodefiencies such as Wiskott-Aldrich syndrome. We had previously mapped Tropomodulin 2 (TMOD2) to the genomic region containing the gene for amyotrophic lateral sclerosis 5 (ALS5). We determined the genomic structure of Tmod2 in order to better analyze patient DNA for mutations; we also determined the genomic structure of Tropomodulin 4 (TMOD4).     

Genomic distribution of retrotransposons 297, 1731, copia, mdg1 and roo in the Drosophila melanogaster species subgroup

The intragenomic distribution of five retrotransposon families (297, 1731, copia, mdg1 and roo) in the species of the melanogaster complex was studied by comparing results of the Southern blotting technique in males and females with those of in situ hybridization. The degree of structural polymorphism of each family in the different species was also investigated by restriction enzyme analysis. It was found that genomic distribution is a trait that depends on the family and species. The distribution of roo is mainly euchromatic in the four species and 1731 is heterochromatic, but the distribution of families 297, copia and mdg1 is markedly different in the melanogaster and simulans clades. These families were mainly euchromatic in D. melanogaster but heterochromatic in its sibling species. In the simulans clade most copia and mdg1 elements are located on chromosome Y. Differences in genomic distribution are unrelated with structural conservation. The relation of intragenomic distribution to phylogeny, transpositional activity and the role of the host genome are discussed.     

Characterization of CaGSP1, the Candida albicans RAN/GSP1 homologue

Gsp1p is a small nuclear-located GTP binding protein from the yeast Saccharomyces cerevisiae. It is highly conserved among eucaryotic cells and is involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules. To learn more about the GSP1 structure/function, we have characterized its Candida albicans homologue. CaGsp1p is 214 amino acids long and displays 91% identity to the ScGsp1p. There is functional complementation in S. cerevisiae, and its mRNA is constitutively expressed in the diploid C. albicans grown under various physiological conditions. Disruption of both alleles was not possible, suggesting that it could be an essential gene, but heterozygous mutants exhibited genomic instability.     

TFS1: A suppressor of cdc25 mutations in Saccharomyces cerevisiae

Summary The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.