甘蓝型油菜BnWRKY41的理化性质分析及BnWRKY41-2 在花青素合成中的功能解析

甘蓝型油菜BnWRKY41-1通过负调控花青素合成相关基因的表达而显著抑制拟南芥叶片花青素的积累。为[JP2]深入了解同源拷贝BnWRKY41-2在花青素合成过程中的作用,对BnWRKY41-1和BnWRKY41-2进行生物信息学分析,并从甘蓝型油菜品种‘中双11’中成功克隆BnWRKY41-2 的全长编码序列,分别转化烟草和拟南芥 wrky41-2 突变体,对其亚细胞定位和在花青素合成中的调控作用进行分析。结果表明,BnWRKY41两个拷贝均具有典型WRKY结构域,N端不含跨膜结构和信号肽,二级结构主要由无规则卷曲组成,蛋白质修饰以磷酸化为主。BnWRKY41-2 定位在细胞核中,在拟南芥 wrky41-2 突变体背景下异源表达 BnWRKY41-2 基因使突变体叶片中的花青素含量恢复到野生型水平,而且可显著调控花青素积累相关基因的表达。这些结果表明BnWRKY41-2 与BnWRKY41-1在蛋白质结构及理化性质方面相似,并且在调控拟南芥叶片花青素积累方面发挥类似的生物学功能,起负调控作用。     

Prediction and Expression Analysis of miRNAs Associated with Heat Stress in Oryza sativa

Computational prediction of potential microRNAs （miRNAs） and their target genes was performed to identify the miRNAs and genes associated with temperature response in rice. The data of temperature-responsive miRNAs of Arabidopsis, and miRNAs and the whole genome data of rice were used to predict potential miRNAs in Oryza sativa involved in temperature response. A total of 55 miRNAs were common in both the species, and 27 miRNAs were predicted at the first time in rice. Target genes were searched for these 27 miRNAs in rice genome following stringent criteria. Real time PCR based on expression analysis of nine miRNAs showed that majority of the miRNAs were down regulated under heat stress for rice cultivar Nagina 22. Furthermore, miR169, miR1884 and miR160 showed differential expression in root and shoot tissues of rice. Identification and expression studies of miRNAs during heat stress will advance the understanding of gene regulation under stress in rice.     

不结球白菜BcLBD37基因的克隆及其对硝酸盐吸收的调节

LBD转录因子是高等植物特有的一类转录因子。为研究不结球白菜LBD基因对硝酸盐吸收的影响,以紫色不结球白菜自交系NJZX3-4为材料,采用同源克隆的方法克隆BcLBD37基因;通过构建PRI101过表达载体,异缘转化拟南芥,实现BcLBD37基因的过表达;同时借助病毒介导的基因沉默技术(VIGS),使不结球白菜中的BcLBD37基因沉默;并利用实时荧光定量PCR(qRT-PCR)分析BcLBD37基因过表达以及沉默后对硝酸盐吸收相关基因表达量的影响。结果表明,该基因片段长度为840 bp,编码279个氨基酸。与野生型相比,转基因拟南芥中硝酸盐代谢相关基因NIA1、NIA2、NRT1.1、NRT1.7、NRT2.1、NRT2.5的表达量均显著下调,硝酸还原酶活性和硝态氮含量亦显著降低。而在不结球白菜中沉默该基因后,沉默植株PTY-2/4中硝酸盐代谢相关基因的表达显著上调,硝酸还原酶活性和硝态氮含量亦显著提高。综上可知,BcLBD37基因通过抑制硝酸盐代谢相关基因的表达,调节硝酸还原酶的活性,进而抑制$\text{NO}_{3}^{-}$的吸收。本研究为提高不结球白菜营养品质提供了理论依据。     

Regulation of C/N Interaction in Model Plant Species

Summary

Carbon (C) and nitrogen (N) metabolisms are two major metabolic pathways which have been intensively studied in plants. Both N and C metabolisms are tightly linked in numerous plant biochemical pathways. Therefore the C to N ratio has to be carefully regulated to ensure proper functioning of the huge metabolic network. In order to maintain a viable C/N status under a large range of growth conditions plants have evolved complex mechanisms to regulate the delicate network of these two major assimilatory pathways. C and N metabolisms are both highly regulated. We will present the current knowledge on the regulation of N and C metabolisms by sugars and N metabolites. Players involved in these regulatory processes are just starting to be uncovered and possible signaling molecules involved in these regulations as well as known or potential candidate regulatory genes willbe discussed.     

拟南芥悬浮细胞及其愈伤组织对潮霉素的反应

研究了潮霉索对拟南芥悬浮细胞及其愈伤组织的作用效果和特性．结果表明：潮霉素对拟南芥悬浮细胞生长、愈伤组织诱导及其生长均有极明显的抑制作用。但作用效果和特性均有所不同；拟南芥悬浮细胞继代培养阶段、愈伤组织诱导阶段和愈伤组织增殖阶段的临界致死质量浓度分别为25、2．5、1．5mg／L，液体与固体培养条件下差异极大；潮毒素需要一定处理时间才能充分表现其抑制细胞生长或杀死细胞的效果，一般液体培养条件下3d以后、固体培养条件下5d以后效果趋于稳定，但不同的处理质量浓度有较大差异．还就液体与固体培养条件下作用效果差异的原因进行了讨论，并提出了对潮毒素抗性标记筛选的建议．     

外源施加棉子糖对离体拟南芥叶片失水速率的影响

[目的]研究外源施加棉子糖与植物成熟器官耐逆的相关性。[方法]将拟南芥离体叶片分别浸泡在Hoagland水溶液和0.1%棉子糖(W/V)的Hoagland水溶液中,黑暗中浸泡24 h,重复5次,探讨了含0.1%(W/V)棉子糖的Hoagland溶液对拟南芥离体叶片失水速率的影响。[结果]含0.1%棉子糖的Hoagland水溶液处理叶片的饱和鲜重为对照的104.46%。在正常温度光照条件下,含0.1%棉子糖的Hoagland处理的叶片失水速率与对照的基本保持一致,但同样时间点测量的鲜重较高。含0.1%棉子糖的Hoagland处理的叶片干重较对照的偏高,为对照的104.20%。[结论]外源施加棉子糖增加了饱和鲜重和干重,提高了拟南芥离体叶片的脱水胁迫耐性,同样时间点的生物量下降较少,失水速率基本不变。     

No DNA loss in autotetraploids of Arabidopsis thaliana

To address the issue of genome evolution in autopolyploids and particularly to investigate whether rapid sequence elimination also occurs in autopolyploids as in allopolyploids, amplified fragment length polymorphism (AFLP) fingerprinting was employed to examine a large number of genomic loci in F₁ hybrids between two different autotetraploids of Arabidopsis thaliana accessions, namely Ler and Col. Using this approach, perfect additivity in the F₁ hybrids was found between the newly‐formed autopolyploids when compared with their parental lines. Using flow cytometry, the study was extended in a quantitative manner, in which the nuclear DNA contents in one autotetraploid A. thaliana accession Ler, was determined. The increase in genome size of the autotetraploid line was additive. Taken together, no evidence was found for genome size reduction due to autopolyploidization of A. thaliana. The results indicating that there was no DNA loss in autotetraploid A. thaliana suggest that a different type of genome evolution may occur in autopolyploids during the initial stages of their formation when compared with allopolyploids.     

Constructive Approaches to Intellectual Property Complexity in Today＇s Agricultural Technology World

Essential to progress in plant biotechnology for deliverable applications, patent and patent application data must be transparent in order to determine where there is whitespace for innovation.     

Evaluating the utility of Arabidopsis thaliana as a model for understanding heterosis in hybrid crops

Despite the phenomenon of heterosis having been used to improve crop productivity for almost a century, there is little understanding of the molecular mechanisms involved. Heterosis has been described recently in the widely used plant model Arabidopsis thaliana. To assess the opportunity afforded by this system, we have developed, through pollination to a male sterile female parent (Ler ms1), a range of hybrids of A. thaliana accessions. We tested this method of hybrid production in A. thaliana by comparing seed produced by natural pollination and hand pollination of emasculated plants. Our results demonstrate that using male sterile lines, which more closely represent methods used for hybrid crop production, circumvents the problems associated with the analysis of hybrids produced from emasculated plants. We identified hybrid combinations exhibiting mid-parent heterosis for vegetative fresh weight ranging from −9 to 103% and characterised heterosis in the reciprocal hybrids of one strongly heterotic combination. We found no evidence of a relationship between the extent of genetic relatedness of A. thaliana accessions and the strength of heterosis exhibited by their hybrids. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

拟南芥未知基因At018融合蛋白原核表达条件的优化及纯化

本课题组在前期的研究中利用Al Cl3胁迫筛选碱茅全长c DNAs酵母表达文库获得到一个与铝毒害相关的未知基因(基因编号为018,用put018表示),该基因与拟南芥中未知基因(基因号为At5g57345,本研究中用At018表示)有较高的同源性。为了获得At018的纯化蛋白,本研究将拟南芥At018基因克隆后构建原核表达载体p GEX-6p-3-At018,再将其转入大肠杆菌BL21(DE3)菌株,利用异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达。同时运用传统方法优化诱导条件,以提高重组融合蛋白的表达效率。SDS-PAGE分析结果表明,30℃下0.1 mmol/L IPTG的条件下诱导3 h后,GST-At018融合蛋白的表达量最大,蛋白分子质量与预测值相符,该蛋白主要以可溶性形式存在;接着利用Glutathione Sepharose4 Fast Flow亲核层析树脂纯化最终获得GST-At018融合蛋白。本研究可为进一步进行At018的功能解析提供基础。