病原诱导的小麦ERF转录因子TaERF1b的原核表达及纯化

为了得到纯化的TaERF1b活性蛋白,将TaERF1 b基因含有AP2/ERF结构域的片段插入原核表达载体pGEX-4T-1的多克隆位点中,构建GST-TaERF1b融合蛋白表达载体,并转化到大肠杆菌BL21(DE3)中。0.1mmol/L IPTG即能诱导融合蛋白表达,37℃诱导4h或30℃诱导8h,融合蛋白均以包涵体的形式表达,16℃诱导12h,融合蛋白不表达。包涵体经溶解及稀释复性后,过GST亲和层析柱,获得纯化的融合蛋白,考马斯亮蓝法测得纯化蛋白的浓度约为0.5μg/μl,凝胶阻滞实验表明包涵体复性成功,所得蛋白具有生物活性。

6个玉米ERF基因的克隆、序列及表达分析

利用同源克隆的方法分离了6个玉米ERF(ethylene-responsive element binding factor)基因,分别命名为ZmERF4、ZmERF26、ZmERF28、ZmERF64、ZmERF89和ZmERF202。基因结构分析显示,ZmERF与拟南芥的AtERF可能存在差异。系统发育分析显示,ZmERFs与OsERFs的亲缘关系更加亲近,而与AtERFs的关系则稍远。RT-PCR表达分析表明,在乙烯利(ET)处理之后,这6个ZmERFs的mRNAs水平在叶中都是先积累后下降的,但其中4个基因(ZmERF4、ZmERF28、ZmERF64和ZmERF89)在玉米根中都是呈现负调控的。在脱落酸(ABA)和茉莉酸(JA)处理下,6个ZmERFs的mRNAs水平在叶中都发生了或多或少的变化,但是大部分基因在根中的表达量没有明显变化,特别是在JA处理之后。这些结果暗示ZmERFs可能在玉米根和叶的激素信号通路中发挥不同的作用。

Comparative Proteomics Analysis of the Sieve Tube from Rubber Tree(Hevea brasiliensis) Seedlings under Contrasting Ethylene Treatments

[Objective]To elucidate the role of ethylene(ET),a latex yield stimulant of the rubber tree,on the sieve tube(ST)transport efficiency of materials(especially sucrose)needed for natural rubber biosynthesis.[Method]Rubber tree seedlings were treated with ET solution or water which was used as a control on the bark,and latex samples and ST tissue samples were collected for proteomic analyses and latex sucrose content determination respectively.[Results]After ET treatment,the sucrose content of the latex was found significantly decreased.A total of 66 ethylene-responsive proteins(ERPs)were distinguished by two-dimensional gel electrophoresis(2-DE),and 54 were successfully identified by MALDI-TOF/TOF and database searching.The majority of these ERPs were involved in carbohydrate transport and metabolic processes in the ST.[Conclusion]Our findings suggest that the application of ET may increase the transport efficiency of the ST and that the application of ET promotes the consumption of energy and sucrose in the ST.

Isolation and characterization of an ERF-B3 gene associated with flower abnormalities in non-heading Chinese cabbage

BrcERF-B3 gene, a member of ethylene-responsive factor family, was screened from a mutant plant in non-heading Chinese cabbage（Brassica rapa ssp. chinensis） by cDNA-AFLP technology. We got full length cDNA of two BrcERF-B3 genes by homology-based cloning from two materials and found that their nucleotide sequences were the same by sequencing. The BrcERF-B3 protein, belonging to the B3 subgroup of the ERF subfamily, shared a close relationship with B. rapa. RT-PCR result showed that BrcERF-B3 expressed only in mutant stamen rather than maintainer stamen. qRT-PCR results indicated that BrcERF-B3 expressed highly during reproductive growth development and in the early of mutant buds, suggesting BrcERF-B3 might be involved in the formation of abnormal flower in mutant. What＇s more, the expression of BrcERF-B3 was more significant to ABA, Me JA and cold stresses in mutant than in maintainer and was down-regulated in NaCl treatment in two lines, implying BrcERF-B3 might be different roles in biotic and abiotic stresses.

Identification of Rice Ethylene-Response Mutants and Characterization of MHZ71OsEIN2 in Distinct Ethylene Response and Yield Trait Regulation

Ethylene plays essential roles in adaptive growth of rice plants in water-saturating environment; how- ever, ethylene signaling pathway in rice is largely unclear. In this study, we report identification and characterization of ethylene-response mutants based on the specific ethylene-response phenotypes of etiolated rice seedlings, includ- ing ethylene-inhibited root growth and ethylene-promoted coleoptile elongation, which is different from the ethylene triple-response phenotype in Arabidopsis. We establish an efficient system for screening and a set of rice mutants have been identified. Genetic analysis reveals that these mutants form eight complementation groups. All the mutants show insensitivity or reduced sensitivity to ethylene in root growth but exhibit differential responses in cole0ptile growth. One mutant group mhz7 has insensitivity to ethylene in both root and coleoptile growth. We identified the corresponding gene by a map-based cloning method. MHZ7 encodes a membrane protein homologous to EIN2, a central component of ethylene signaling in Arabidopsis. Upon ethylene treatment, etiolated MHZ7-overexpressing seedlings exhibit enhanced coleoptiie elongation, increased mesocotyl growth and extremely twisted short roots, featuring enhanced ethylene- response phenotypes in rice. Grain length was promoted in MHZ7-transgenic plants and 1000-grain weight was reduced in mhz7 mutants. Leaf senescent process was also affected by MHZ7 expression. Manipulation of ethylene signaling may improve adaptive growth and yield-related traits in rice.