玉米线立体类质粒的克隆和鉴定

S型细胞质雄性不育玉米的线粒体类质粒S-1为一个长6.4kb的双链线状DNA分子。本文对基本上全长的S-1（只用限制内切酶PstI在两端各切掉128bp）在pUC18和pBR322两种质粒中进行了克隆得到4种杂合的质粒。限制性酶切图谱证明它们分别是S-1以不同方向克隆在两种质粒中的结果,Southern印迹杂交也证明S-1克隆在pBR322中.     

玉米细胞分裂素反应调节因子基因的克隆与进化分析

在植物的生长发育过程中,细胞分裂素在调节细胞分裂和组织发育中起着非常重要的作用.研究表明细胞分裂素的信号转导是一种双组分信号转导途径,在这个系统中,由3种蛋白组成,分别是组氨酸激酶、磷酸转移蛋白和反应调控因子.利用已经克隆的玉米和水稻细胞分裂素反应调节因子基因,进行BLAST分析从玉米全基因组中获得候选ZmRR类型基因.然后设计全长基因引物,通过Trizol法提取玉米叶片总RNA,利用RT-PCR技术克隆出全长候选基因.序列分析表明所扩增序列含有完整的编码框,共编码156个氨基酸残基.序列比对分析其与ZmRR1-10基因具有较高的同源性,并命名为ZmRR11,系统进化树分析证实其属于A类细胞分裂素调控因子,并对所有ZmRR类型基因进行motif分析,共发现37个保守的motif.该基因的克隆和进化分析对阐明玉米双元信号传导体系具有重要的意义.     

玉米Dof转录因子家族基因的全基因组分析

Dof转录因子家族在植物生长发育和基因表达调控过程中具有重要的作用,本文利用公布的玉米基因组草图数据,利用生物信息学方法对玉米全基因组Dof基因的结构、系统进化关系和保守motif进行了分析。结果表明:玉米中共有18个Dof类型基因,命名为ZmDof1-ZmDof18,其蛋白质长度在211aa至618aa之间,通过系统进化树分析后,18个Dof基因可以明显的分为三类,此外玉米Dof基因的数目远远小于水稻和拟南芥,基因复制现象较少是玉米Dof基因数量较少的原因之一,MEME分析证实了Dof基因含有三个保守的motif。对玉米Dof类型基因的系统分析,将有助于玉米Dof类型基因的克隆和功能的进一步研究。     

一个新的玉米NAC类基因(ZmNAC1)的克隆与分析

NAC转录因子是近十年来新发现的具有多种生物功能的植物特异转录因子,在植物生长发育、激素调节和抵抗逆境等方面发挥着重要的作用。本研究利用电子克隆方法,结合RT-PCR分析,从玉米中克隆了一个与NAC类基因同源的全长cDNA序列,命名为ZmNAC1（GenBank登录号EU224278）,该基因全长为1029bp,具有完整的开放阅读框架（ORF,26~907bp）,推测编码蛋白含有293个氨基酸,分子量为32.3KD,等电点为8.65。半定量RT-PCR分析表明,ZmNAC1可以被低温、聚乙二醇（polyethylene glycol, PEG）、高盐和ABA诱导。这些结果表明,ZmNAC1可能在植物逆境反应中起作用。本研究是有关玉米NAC转录因子基因的首次报道。     

A Nicotiana benthamiana receptor-like kinase regulates Phytophthora resistance by coupling with BAK1 to enhance elicitin-triggered immunity

Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs) are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana benthamiana.We used the cell-death phenotype caused by INF1,an oomycete e...     

玉米中一种新的蛋白激酶电子克隆与序列分析

目的：电子克隆玉米中一种新的蛋白激酶基因。方法：以拟南芥中一个蛋白激酶的氨基酸序列为探针,对玉米的EST数据库进行同源性检索和筛选并克隆。结果：序列分析显示该cDNA长1121bp,有一个450bp的开放阅读框,编码149个氨基酸,且具有保守苏氨酸/丝氨酸蛋白激酶结构域和TEY基元,说明所克隆的cDNA序列为玉米的MAPK全长cDNA。结论：所克隆的cDNA序列为玉米的MAPK全长cDNA。     

玉米特异DNA通过有性杂交导入小麦DH后代的分子证据

构建了玉米的随机基因组文库,筛选了近100个玉米基因组特异的重复DNA克隆,用它们作探针分别对2个来自小麦×玉米的小麦DH群体进行RFLP分析.结果发现,玉米的MR64克隆导入到2个小麦群体的各1株后代中,即在普通小麦DH系的18号株和波斯小麦DH系的15号株中检测到强杂交信号,这个结果首次从DNA水平上证明某些玉米特异的DNA序列,可通过受精作用以很低的频率转移到小麦DH后代的基因组中.测序分析证实,克隆MR64的插入片段长度为695Pb,A+T含量为58%,用多种酶切玉米基因组进行RFLP分析表明它是一个带1～3个主串联重复单位的散布重复序列.同时还对它的序列结构、甲基化图谱、拷贝数和在玉米染色体上的分布以及在其它小麦品种和禾本科种基因组中的序列同源性情况进行了详细的研究.     

水稻MYB转录因子OsDUO1的cDNA克隆及其表达模式初步分析

我们利用RT-PCR方法成功从水稻中克隆了R2R3类MYB转录因子OsDUO1（Oryza sativa duo pollen1）的全长为1032bp的cDNA,该基因编码一个343个氨基酸残基的蛋白。RT-PCR分析结果表明OsDUO1只在水稻的花粉发育后期表达,说明OsDUO1可能对水稻花粉发育具有生物学功能。生物信息学分析表明,OsDUO1在短柄草、高粱、玉米、拟南芥、烟草、葡萄、蓖麻、杨毛果、小立碗藓植物中有相近同源序列,暗示该基因在进化中具有保守的生物学功能。     

转Bt-cry1Ah基因玉米花粉对意大利蜜蜂幼虫的影响

新型杀虫蛋白基因crylAh基因是中国农业科学院植物保护研究所从Bt菌株BT8中鉴定克隆的,其编码蛋白对鳞翅目害虫具有强毒力,尤其对亚洲玉米螟Ostrinia furnacalis(Guenée)的毒力强于目前使用的cry1A类基因.转cry1Ah基因抗虫玉米具有很好的应用前景.花粉是蜜蜂重要的食物来源,蜜蜂是转基因植物安全性评价的关键测试生物.因此,开展转crylAh基因玉米对蜜蜂的安全性研究很有必要.给意大利蜜蜂Apis mellifera ligustica Spinola蜂群中4～6日龄幼虫饲喂转基因玉米花粉、常规玉米花粉、杂花粉,哺育蜂饲喂为对照.转基因玉米花粉对意大利蜜蜂封盖率、出房率和发育历期没有显著影响.表明转cry1Ah基因玉米花粉对意大利蜜蜂幼虫的存活和发育没有不良影响.     

抑制差减杂交法分离玉米幼苗淹水诱导表达基因

以淹水处理(submergence-treated, ST)的玉米(Zea mays L.)幼苗根部cDNA为目标群体,未处理(untreated, UT)的玉米幼苗根部cDNA为对照群体,进行抑制差减杂交.用经过UT差减的ST cDNA构建了一个含有大约2 000个独立克隆的差减文库.对随机挑取的408个克隆进行差异筛选,获得了184个在ST中特异表达或表达增强的候选克隆.对其中155个cDNA克隆测序并去除重复克隆后,共得到95个差异表达的cDNA片段.GenBank中BLAST查询结果表明:6个克隆为已知的玉米核苷酸序列;68个克隆与已知基因或EST序列部分区域的同源性为60%～90%;21个克隆在GenBank中无法查到对应的同源序列,可能代表了新基因,或者由于序列位于变异丰富的3′端而无法查到与其他物种基因的同源性.     

Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma

Leucine-rich-repeat receptor-like kinases (LRR-RLKs) play central roles in sensing various signals to regulate plant development and environmental responses. The extracellular domains (ECDs) of plant LRR-RLKs contain LRR motifs, consisting of highly conserved residues and variable residues, and are responsible for ligand perception as a receptor or co-receptor. However, there are few comprehensive studies on the ECDs of LRR-RLKs due to the difficulty in effectively identifying the divergent LRR repeats. In the current study, an efficient LRR motif prediction program, the “Phyto-LRR prediction” program, was developed based on the position-specific scoring matrix algorithm (PSSM) with some optimizations. This program was trained by 16-residue plant-specific LRR-highly conserved segments (HCS) from LRR-RLKs of 17 represented land plant species and a database containing more than 55,000 predicted LRRs based on this program was constructed. Both the prediction tool and database are freely available at http://phytolrr.com/ for website usage and at http://github.com/phytolrr for local usage. The LRR-RLKs were classified into 18 subgroups (SGs) according to the maximum-likelihood phylogenetic analysis of kinase domains (KDs) of the sequences. Based on the database and the SGs, the characteristics of the LRR motifs in the ECDs of the LRR-RLKs were examined, such as the arrangement of the LRRs, the solvent accessibility, the variable residues, and the N-glycosylation sites, revealing a comprehensive profile of the plant LRR-RLK ectodomains. The “Phyto-LRR prediction” program is effective in predicting the LRR segments in plant LRR-RLKs, which, together with the database, will facilitate the exploration of plant LRR-RLKs functions. Based on the database, comprehensive sequential characteristics of the plant LRR-RLK ectodomains were profiled and analyzed.     

Plant receptor kinases bind and phosphorylate 14-3-3 proteins

14-3-3 proteins are pSer/pThr-binding proteins that interact with a wide array of cellular ‘client’ proteins. The plant brassinosteroids (BRs) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is a member of the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that contain cytoplasmic protein kinase domains. At least two LRR-RLKs are involved in BR perception and signal transduction: BRI1 and BRI1-associated receptor kinase 1 (BAK1). We determined that several 14-3-3 proteins bind to BRI1-CD and are phosphorylated by BRI1, BAK1 and At3g21430 receptor kinases in vitro. Moreover, we observed14-3-3 s are phosphorylated on threonine residue(s) with BR-dependent manner. To reveal the function of 14-3-3 proteins interacting with LRR-RLKs, we treated tyrosine phosphatase (PTP1B) to the BRI1-CD recombinant protein, which is autophosphorylated on tyrosine residue(s). Tyrosine autophosphorylation signal was disappeared, suggesting that 14-3-3 proteins cannot protect BRI1 tyrosine phosphorylation from PTP1B phosphatase. Our study suggests that 14-3-3 proteins may be important for plant growth and development through BR signaling.     

Overexpression of rice <Emphasis Type="Italic">LRK1</Emphasis> restricts internode elongation by down-regulating <Emphasis Type="Italic">OsKO2</Emphasis>

Rice (Oryza sativa) has the potential to undergo rapid internodal elongation which determines plant height. Gibberellin is involved in internode elongation. Leucine-rich repeat receptor-like kinases (LRR-RLKs) are the largest subfamily of transmembrane receptor-like kinases in plants. LRR-RLKs play important functions in mediating a variety of cellular processes and regulating responses to environmental signals. LRK1, a PSK receptor homolog, is a member of the LRR-RLK family. In the present study, differences in ectopic expression of LRK1 were consistent with extent of rice internode elongation. Analyses of gene expression demonstrated that LRK1 restricts gibberellin biosynthesis during the internode elongation process by down-regulation of the gibberellin biosynthetic gene coding for ent-kaurene oxidase.     

Evolutionary and expression dynamics of LRR-RLKs and functional establishment of KLAVIER homolog in shoot mediated regulation of AON in chickpea symbiosis

Chickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON.     

SGT1 is not required for plant LRR-RLK-mediated immunity

Plant immune signalling activated by the perception of pathogen-associated molecular patterns (PAMPs) or effector proteins is mediated by pattern-recognition receptors (PRRs) and nucleotide-binding and leucine-rich repeat domain-containing receptors (NLRs), which often share cellular components and downstream responses. Many PRRs are leucine-rich repeat receptor-like kinases (LRR-RLKs), which mostly perceive proteinaceous PAMPs. The suppressor of the G2 allele of skp1 (SGT1) is a core immune regulator required for the activation of NLR-mediated immunity. In this work, we examined the requirement of SGT1 for immune responses mediated by several LRR-RLKs in both Nicotiana benthamiana and Arabidopsis. Using complementary genetic approaches, we found that SGT1 is not limiting for early PRR-dependent responses or antibacterial immunity. We therefore conclude that SGT1 does not play a significant role in bacterial PAMP-triggered immunity.     

Heterozygosity of Knob-Associated Tandem Repeats and Knob Instability in Mitotic Chromosomes of Zea （Zea mays L. and Z. diploperennis Iltis Doebley）

Knobs are blocks of heterochromatin present on chromosomes of maize （Zea mays L.） and its relatives that have effects on the frequency of genetic recombination, as well as on chromosome behavior. Knob heterozygosity and instability in six maize inbred lines and one Z. diploperennis Iltis Doebley line were investigated using the fluorescence in situ hybridization （FISH） technique with knob-associated tandem repeats （180 bp and 350 bp （TR- 1）） as probes. Signals of seven heterozygous knobs containing 180- bp repeats and of one heterozygous knob containing TR- 1 were captured in chromosomes of all materials tested according to the results of FISH, which demonstrates that the 180-bp repeat is the main contributor to knob heterozygosity compared with the TR- 1 element. In addition, one target cell with two TR- 1 signals on one homolog of chromosome 2L, which was different from the normal cells in the maize inbred line GB57, was observed, suggesting knob duplication and an instability phenomenon in the maize genome.     

Autophagy: A critical regulator of cellular metabolism and homeostasis

Plants possess a variety of extracellular leucine-rich repeats receptor-like kinases (LRR-RLKs) to coordinate developmental programs with responses to environmental changes. Out of sixteen families of LRR-RLKs in Arabidopsis, the LRR-RLKII family consists of fourteen individual members, including five Arabidopsis thaliana somatic embryogenesis receptor kinases (AtSERKs). BAK1/AtSERK3 was first identified as a dual co-receptor of BRI1 and FLS2, mediating BR signaling and pathogen-associated molecular pattern (PAMP) triggered immunity (PTI), respectively. Since its identification, many researchers have attempted to elucidate the phosphorylation mechanisms between receptor complexes and identify additional components that interact with receptor complexes to transduce the signaling downstream. Relatively detailed early events in complex formation, phosphorylation sites on the BRI1/BAK1 complex and BAK1-interacting proteins, such as BIK1 and PUB13, have been identified. Small receptor complexes consisting of BAK1 and BIR1 or BAK1 and AtSERK4 regulate cell death during steady state conditions. Moreover, the redundant and distinct functions of AtSERK proteins and other members of the LRR-RLKII family have been revealed. This review focuses on the integration of the information from the most recent studies concerning BAK1 and its homologs.     

农杆菌介导的玉米遗传转化系统研究进展

摘要: 农杆菌介导的遗传转化由于具有独特的优点, 一直受到育种家、分子生物学家和微生物学家的重视, 因此近10年发展很快。玉米是重要的粮食作物, 农杆菌介导的玉米遗传转化也取得了重大成就。文章就近年来玉米遗传转化取得的进展、影响转化的重要因素作一小结, 并就目前转化存在的问题和前景作简单的讨论。     

Responses of transgenic maize hybrids to variant western corn rootworm larval injury

Abstract:  In 2005 and 2006, transgenic insecticidal maize hybrids (YieldGard Rootworm, MON 863, Cry3Bb1, Vector ZMIR 13L) were evaluated for their ability to limit root injury caused by western corn rootworm ( Diabrotica virgifera virgifera LeConte) larval feeding. Hybrids in each year of the experiment were planted in plots that had been devoted to a trap crop (late-planted maize interplanted with pumpkins) the previous growing season. All maize hybrids were provided by Monsanto Company and the genetic backgrounds remain unknown to the investigators. In 2005, the experiment was conducted in Urbana, Illinois. Urbana is located in east central Illinois, an area of the state in which a variant of the western corn rootworm has overcome the pest management benefits of crop rotation. Variation in root injury was noted across the maize hybrids in 2005 and the level of pruning increased from 20 July to 9 August for most hybrids. In 2006, the experiment was conducted in two locations, Monmouth and Urbana, Illinois. Monmouth is located in north-western Illinois and is within an area of the state in which densities of the variant of the western corn rootworm are lower than in east-central Illinois. In 2006, variation in root protection was again observed across the maize hybrids. Root injury differences among the hybrids were more prominent at the Urbana site. Similar to the previous year, root injury increased from the third week in July to the first week of August at both locations with this increase most noticeable at the Urbana location. We hypothesize that the variant western corn rootworm may be able to inflict more root injury to these transgenic insecticidal maize hybrids than the non-variant population of this species.