水稻OsWRKY89启动子紫外线-B反应元件区的鉴定

植物需要利用太阳光能进行光合作用,因而不可避免地受到紫外线-B(UV-B) 辐射的影响．为了鉴定水稻WRKY转 录因子OsWRKY89基因启动子中的UV-B反应相关元件,分析了转启动子不同缺失片段与gus融合基因的水稻幼苗,发现在该启动子中存在UV-B反应元件,位于基因翻译起始位点上游-1 213～-1 188之间的25 bp区域,碱基序列为AAGATCTACCATTGCTCTATAGCTT．结合OsWRKY89和UV-B诱导上调表达基因启动子序列分析发现,该元件区在水稻UV-B反应基因启动子上具有高度的保守性,而且与已知保守的光反应元件位置邻近,表明该区域在水稻UV-B反应的转录调控中可能具有重要功能．     

水稻转录因子WRKY42的转录、表达及其与W-box的结合特征分析

WRKY是植物中最大的转录因子家族之一.本文对水稻WRKY42基因的转录分析发现,该基因在水稻苗期和花药中转录,其蛋白质可在各时期的叶片中检测到.在Xa21介导的抗白叶枯病过程中,接菌后期可检测到明显的诱导表达条带,比较其在抗、感和对照反应中的表达丰度发现,在抗、感反应中的表达相似但均明显大于对照反应,推测WRKY42蛋白质在水稻-白叶枯病菌互作反应中发挥作用.我们克隆并在细菌中表达了WRKY42蛋白质,采用微量热泳动(microscale thermophoresis)技术,调查了WRKY42与病程相关基因PR1a和PR1b启动子区顺式元件W-box的互作,发现它们之间可发生特异结合,其解离常数分别为73.3μmol/L和58.3μmol/L.上述数据提供了WRKY42调控下游基因的直接证据,支持WRKY42在水稻抗病过程中发挥作用.文章提出了WRKY转录因子在水稻与白叶枯病菌互作过程中的作用模式.     

水稻类Tubby蛋白质在叶片生长和白叶枯病抗性反应中的表达

类Tubby蛋白质（Tubby-like protein,TLP）在动植物中广泛存在,暗示其在生命过程中发挥重要的作用。水稻（Oryza sativa）基因组中有14个TLP家族成员,首先制备了这些蛋白质的抗体,用免疫印迹方法检测了它们在水稻叶片不同生长时期的表达情况,揭示其表达模式;然后对Xa21介导的水稻白叶枯病抗性反应不同时间点进行检测,发现OsTLP2、OsTLP7、OsTLP8和OsTLP9等4个蛋白质的表达发生了变化;进一步比较它们在抗病、感病反应和对照处理中的表达情况,发现不同反应间的表达也有区别。该研究结果为阐释水稻TLP在叶片生长过程中的功能,尤其是在水稻-白叶枯病菌互作过程中的作用提供了重要线索。     

芥菜BjuWRKY75基因表达及其与开花整合子BjuFT互作

芥菜(Brassica juncea)是十字花科芸薹属一年或二年生蔬菜,其产品器官的产量和品质会受到开花时间的影响。WRKY家族成员具有响应生物和非生物胁迫、发育调控和信号转导等作用。WRKY75是WRKY家族中能够调节开花的重要成员,但在芥菜中的开花调控机制还未见报道。本研究克隆了芥菜BjuWRKY75基因,发现其编码蛋白具有高度保守的WRKY结构域,属于Ⅱ类WRKY蛋白,与黑芥BniWRKY75同源性最高。BjuWRKY75在花中表达丰度显著高于叶和茎,并且在叶中表达较为稳定。BjuWRKY75定位于细胞核,能够与含有W-box应答元件的开花整合子BjuFT的启动子相互作用,且能转录激活下游基因表达。BjuWRKY75转入拟南芥可显著提早开花。综上说明,BjuWRKY75能够直接靶向BjuFT从而促进开花。这为深入研究BjuWRKY75开花分子调控奠定了基础。     

OsPDR在酵母中异源表达增强了酵母对钴的耐受性

在通过RNA-Seq技术得到的镉响应转录组图谱中,用50 μmol/L Cd处理24 h后,一个镉响应金属离子转运蛋白OsPDR被鉴定出其在水稻(Oryza sativa ssp. japonica cv. Nipponbare)茎中的表达量显著上调．本研究中,从水稻(Oryza sativa cv. Nipponbare)中分离了OsPDR基因,并对其金属离子转移活性进行了分析．金属耐受性实验结果表明,过表达OsPDR能提高酵母对Co的耐受性,但对Zn、Ni和Cd的耐受性不强,并且经电感耦合等离子体质谱法(ICP-MS)测定Co含量后,与空载体转化酵母相比,过表达OsPDR的酵母中Co的积累更高．利用共聚焦显微镜观察发现,EGFP-OsPDR融合蛋白定位于液泡膜上．这些数据表明OsPDR可能在Co稳态中起着重要作用．OsPDR在植物中的作用,还需要进一步的研究．     

小麦TaWRKY47基因的克隆与表达分析

为了探究小麦WRKY基因的功能,该研究采用RT PCR方法,在小麦叶片组织中克隆WRKY基因,并对其进行生物信息学和不同逆境胁迫下的表达分析。结果表明：(1)成功克隆得到1个小麦WRKY基因,命名为TaWRKY47。(2)TaWRKY47基因开放阅读框长度为900 bp,编码299个氨基酸,含有一个WRKY保守结构域和一个C₂HC锌指结构域,属于WRKY基因家族的第Ⅲ类成员。(3)亚细胞定位分析结果显示,TaWRKY47蛋白定位于细胞核。(4)荧光定量PCR结果表明,TaWRKY47基因在小麦根、茎、叶、雄蕊和雌蕊中均有表达,其中在雌蕊中表达量最高,且受低温、干旱、盐、ABA和H₂O₂等胁迫表达增强,推测TaWRKY47基因参与了小麦的逆境胁迫过程。该研究结果为进一步研究TaWRKY47基因功能与抗逆机制奠定了理论基础。     

Cre-LoxP系统在炭疽芽胞杆菌基因敲除中的应用及eag基因的敲除

将Cre-LoxP系统应用于Bacillus anthracis中并成功敲除eag基因．以B.anthracis基因组为模板扩增得到上下游同源臂,联合两端带有LoxP位点的壮观霉素抗性基因片段构建好同源重组载体,转化B．anthracis AP422,通过一系列筛选得到带有抗性标记的重组菌．然后,通过转入Cre重组酶表达质粒,去除抗性标记,得到eag基因缺失的重组菌,并在DNA水平、RNA水平和蛋白质水平进行了系统的鉴定．最终建立了Cre-LoxP系统在B．anthracis中的应用方法,并成功敲除eag基因.     

沉默大豆GmWRKY33B基因导致大豆抗病性降低

WRKY转录因子基因家族是植物特有的转录因子,在防御中起着重要作用。通过生物信息学分析,本研究在古四倍体大豆(Glycine max)基因组中找到一对同源性高达93%的WRKY33同源基因,并将其命名为GmWRKY33B。从GmWRKY33B的两个同源基因保守区域选取一个315 bp片段构建至菜豆豆荚斑驳病毒(bean pod mosaic virus, BPMV)沉默载体(BPMV-VIGS)上,以期同时沉默上述2个GmWRKY33B基因。结果表明,同时沉默2个GmWRKY33B基因并不显著改变沉默植株的表型,但却显著降低了大豆对大豆斑点病菌以及大豆花叶病毒的抗性,说明GmWRKY33B在大豆免疫反应中起正调控作用。激酶分析表明,GmWRKY33B沉默植株中flg22诱导的GmMPK6的磷酸化水平较空载体BPMV-0植株显著降低,说明GmWRKY33B可以通过调控GmMPK6的激酶活性而参与大豆的免疫反应。抗毒素为大豆中主要起防御作用的植保素,而大豆异黄酮类特异性异戊烯基转移酶(prenyltransferase, PT)基因家族是参与大豆抗毒素生物合成的主要基因,许多PT基因启动子区含有与WRKY特异性结合的W-box序列。在丁香假单胞菌pv.甘氨酸(Pseudomonas syringae pv. glycinea, Psg)侵染条件下,4个PT基因的表达水平在沉默株系中显著降低,说明GmWRKY33B参与PT基因的转录激活。综上所述,GmWRKY33B通过调控GmMPK6的激活以及调控大豆抗毒素生物合成途径中关键酶编码基因的表达而参与免疫反应。     

p14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探

ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53, 诱导细胞周期阻断或凋亡.利用高表达p14^ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14^ARF高表达能将细胞周期阻断在G1和G2期, p53, p21^cip1和p27^kip1蛋白水平明显增强, 而p-ERK1/2,CyclinD1和CyclinE蛋白水平下降, 明显抑制细胞生长. 提示p14^ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖.     

水稻BCAT4基因突变株幼苗对PEG模拟干旱胁迫的响应

以水稻野生型‘日本晴’(NIP)及其BCAT4基因突变体BCAT4 1为材料,在苗期进行PEG 6000模拟干旱处理,分析其对幼苗形态、生长和抗逆生理指标的影响,以探究BCAT4基因在水稻响应干旱胁迫中的作用。结果表明：(1)20% PEG处理后野生型NIP幼苗叶片中BCAT4表达量显著高于对照(处理0 d),复水后幼苗存活率显著高于突变体BCAT4 1。(2)20% PEG处理后,两水稻材料幼苗叶片的相对叶绿素含量下降,脯氨酸和可溶性糖含量上升,抗氧化酶活性先上升后下降,且突变体BCAT4 1中上述各指标均显著低于同期NIP。(3)两材料幼苗叶片中丙二醛和过氧化氢含量及相对电导率随胁迫处理天数增加而上升,且BCAT4 1均显著高于同期NIP。(4)在20% PEG处理后,两水稻材料间根系各形态、生长和生理指标的差异均小于相应叶片。研究发现,BCAT4基因突变加剧了干旱胁迫下水稻幼苗叶片叶绿素含量的下降,抑制了地上部渗透调节物质的积累及抗氧化酶活性上升的幅度,促进了丙二醛和过氧化氢积累以及相对电导率增加,从而降低了水稻的耐旱性。     

Exploring the mechanism and efficient use of a durable gene-mediated resistance to bacterial blight disease in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, the most devastating bacterial disease of rice worldwide. The major disease resistance gene Xa3/Xa26 confers a durable resistance to Xoo with a dosage effect. However, the mechanism of Xa3/Xa26-mediated resistance remains to be elucidated. We created near-isogenic lines carrying Xa3/Xa26 with a background of indica and japonica, the two major subspecies of Asian cultivated rice. Analyzing these rice lines showed that the japonica background facilitated resistance to Xoo, which was associated with increased Xa3/Xa26 expression, compared with rice lines with an indica background. This characteristic of Xa3/Xa26 was related to the WRKY45 locus, which had higher expression with the japonica background than with the indica background. However, the two alleles of the WRKY45 locus had different expression levels, with the WRKY45-1 expression level being higher than that of WRKY45-2 for both japonica and indica backgrounds. In addition, the resistance level conferred by Xa3/Xa26 was higher in the presence of WRKY45-1 than in the presence of WRKY45-2 for both japonica and indica backgrounds. Xa3/Xa26-mediated resistance was associated with increased accumulation of jasmonic acid (JA), JA-isoleucine, and terpenoid and flavonoid phytoalexins. Exogenous JA application enhanced Xa3/Xa26-mediated resistance. These results not only provide more knowledge toward understanding the mechanism of Xa3/Xa26-mediated resistance but also offer the best choice for using Xa3/Xa26 for rice resistance improvement, specifically, a japonica background with the WRKY45-1 allele.     

Breeding bacterial blight-resistant hybrid rice with the cloned bacterial blight resistance gene Xa21

The cloned bacterial blight (BB) resistance gene Xa21 was transferred into Minghui63, a widely used restorer line of indica hybrid rice in China, through an Agrobacterium-mediated system. Molecular and resistance analyses revealed that the Xa21 gene was integrated in the genomes of transgenic plants and their progeny inherited resistance stably. For the purpose of hybrid breeding, Xa21 transgenic homozygous restorer lines were selected through `within-lane' dosage comparison of hybridization signal in combination with PCR and resistance analyses. The selected transgenic restorer lines were then crossed with a commonly used sterile line, Zhenshan97A, to produce Xa21 transgenic hybrid rice, Shanyou63-Xa21. The hybrid rice plants with Xa21 displayed high broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo) races and maintained elite agronomic characters of Shanyou63. The propagation of this BB-resistant hybrid variety with Xa21 will benefit rice production.     

Characteristic expression of rice pathogenesis-related proteins in rice leaves during interactions with <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Pathogenesis-related (PR) proteins play an important role in the disease resistance response. To better understand the function of rice PR proteins, we examined the expressions of ten PR proteins in rice leaves at different developmental stages with or without the interaction between rice and Xanthomonas oryzae pv. oryzae (Xoo). The results showed that most of the PR proteins were expressed in rice leaves in normal growth conditions, suggesting that they play a role in rice growth. Six out of ten PR proteins (PR1, PR2, PR3, PR4b, PR8, and PR-pha) showed enhanced expression in Xa21-mediated resistance responses at late stages after inoculation with Xoo. The remaining four PR proteins (PR5, PR6, PR15, and PR16) did not show changes in expression in the resistance response. The expressions of PR proteins in the resistance reaction were further compared with those in the susceptible reaction and a mock treatment. Interestingly, several of the PR proteins were expressed at the highest levels in the susceptible reaction and at the lowest levels in the mock treatment. Among the other four PR proteins, PR5 and PR16 showed changes in the abundance only in the susceptible response, while PR6 and PR15 showed no detectable difference in expression. These data provide fundamental knowledge about the expression of PR proteins in the interaction between rice and Xoo.     

Evaluation of resistance genes in rice against local isolates of Xanthomonas oryzae pv. oryzae in Punjab Province of Pakistan

Absence of resistance/tolerance against bacterial leaf blight (BLB), incited by Xanthomonas oryzae pv. oryzae, in famous basmati varieties is one of the main reason for BLB epidemic in Punjab in 2007–2008. For developing resistance against BLB, the response of 26 IRBB lines of IRRI including 10 near isogenic lines (NILs) and 16 gene pyramids carrying two to five resistance genes (Xa series) was evaluated against 61 indigenous Xoo isolates under artificial inoculation field conditions. None of the NILs or gene pyramid provides complete protection against all the isolates. However, Xa21 and xa13 were found resistant against the majority of Xoo isolates, followed by Xa14 and Xa7. Of the 16 gene pyramids used in this study, IRBB-54 (Xa5 + Xa21), IRBB-55 (Xa13 + Xa21) followed by IRBB-58 (Xa4 + Xa13 + Xa21) were found effective against the majority of the Xoo isolates. These resistance genes (individually and in combinations) can be incorporated for the improvement of basmati rice cultivars cultivated in Punjab province of Pakistan. Effectiveness of gene combination supports the strategy of pyramiding appropriate resistance genes. Newly identified resistant genes may also be evaluated for achieving broad spectrum resistance against more Xoo isolates of the area.     

Small RNAs and Gene Network in a Durable Disease Resistance Gene—Mediated Defense Responses in Rice

Accumulating data have suggested that small RNAs (sRNAs) have important functions in plant responses to pathogen invasion. However, it is largely unknown whether and how sRNAs are involved in the regulation of rice responses to the invasion of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, the most devastating bacterial disease of rice worldwide. We performed simultaneous genome-wide analyses of the expression of sRNAs and genes during early defense responses of rice to Xoo mediated by a major disease resistance gene, Xa3/Xa26, which confers durable and race-specific qualitative resistance. A large number of sRNAs and genes showed differential expression in Xa3/Xa26-mediated resistance. These differentially expressed sRNAs include known microRNAs (miRNAs), unreported miRNAs, and small interfering RNAs. The candidate genes, with expression that was negatively correlated with the expression of sRNAs, were identified, indicating that these genes may be regulated by sRNAs in disease resistance in rice. These results provide a new perspective regarding the putative roles of sRNA candidates and their putative target genes in durable disease resistance in rice.