Functional analysis of a reproductive organ predominant expressing promoter in cotton plants

Transgenic Bt insect-resistant cotton plants have high insect resistance in the early stage of development, but relatively low resistance in the late stage. Substituting a reproductive organ-specific promoter for the CaMV35S promoter presently being used could be an ideal solution. For the first time, the promoter sequence of ADP-ribosylation factor 1 (arf1) gene was isolated from Gossypium hirsutumY18 by means of inverse PCR. The sequencing result discovered the unique structure of the arf1 promoter, including four promoter-specific elements, the initiator, TATA box, CAAT box and GC box, and also an intron in 5′-untranslation region. Four plant expression vectors were constructed for functional analysis of the promoter. Based on the pBI121 plant expression vector, four truncated arf1 promoters took the place of the CaMV35S promoter. These vectors were different only in their promoter regions. They were introduced into cotton plants via pollen tube pathway. Histochemical GUS staining and fluorescence quantitative analyses were performed to examine the expression patterns of the GUS gene driven by the 4 arf1 truncated promoters in transgenic cotton plants respectively. The results showed that the arf1 promoter was a typical reproductive organ-specific promoter. Hopefully, the arf1 promoter can be a regulatory element for designing cotton reproductive organs with desired characteristics.     

Functional Analysis of Nodulin-like Promoter in Transgenic Cotton Plants

For the first time, a nodulin-like gene promoter was isolated from Gossypium hirsutum L. Guo Y18 by means of inverse PCR. Three plant expression vectors were constructed for functional identification of the promoter. These vectors were different only in promoter regions; three truncations of the nodulinlike promoter took the place of the CaMV35S promoter in the pBI121 plant expression vector. Then, the three vectors were introduced into cotton plants via the pollen tube pathway. The expression patterns of the gus gene driven by nodulin-like promoter truncations were investigated in the offspring of transgenic cotton plants. Histochemical GUS staining and fluorescence quantitative analysis were performed to achieve this goal. The results showed that the nodulin-like promoter was a strong, highly reproductive organspecific promoter, which demonstrated a much higher driver activity than the CaMV35S promoter did in cotton reproductive organs, but relatively lower activity in vegetation. Identification of the speciality and strength-determining regions of the nodulin-like promoter was also undertaken.     

棉花蕾花铃生物量、氮累积特征及临界氮浓度稀释模型

在大田栽培条件下,于河南安阳（黄河流域黄淮棉区）和江苏南京（长江流域下游棉区）设置了棉花氮素水平试验,对不同氮素水平条件下棉花蕾花铃的生物量、氮素累积及氮浓度的动态变化进行分析,并依据Justes的临界氮浓度稀释模型确定方法,研究棉花蕾花铃临界氮浓度稀释模型。结果表明：棉花蕾花铃的生物量增长和氮吸收累积均受氮素水平的影响,其动态变化符合S型曲线,氮累积的快速起始时间较生物量早1～5d;氮浓度过高或过低均不利于产量形成,蕾花铃等器官存在氮奢侈消费现象;氮浓度随施氮量的增加而升高、随生育进程的推移而降低,其生物量累积量与氮浓度间符合幂函数关系,两试点蕾花铃氮稀释曲线模型形式相同,但模型参数a不同,不同生态区存在独立的临界氮稀释曲线模型。由于临界稀释模型具有明确的生物学意义,可以作为定量诊断蕾花铃氮营养动态变化的指标之一。     

Accumulation characteristics of biomass and nitrogen and critical nitrogen concentration dilution model of cotton reproductive organ

Several nitrogen (N) field experiments were carried out in Nanjing and Anyang, China, to study the dynamic characteristics of biomass accumulation and N uptake, and to define the dilution curve for critical N concentration in cotton reproductive organ over the growth period. The results show that the total biomass and N accumulation were affected significantly by the rate of N application, exhibiting a sigmoid curve over time. The beginning time of fast N accumulation was 1–5 d earlier than that of biomass accumulation. The cotton lint yield was correlated with N concentration in the reproductive organ and fluctuated with varying N concentration, indicating the existence of luxurious N consumption in the cotton reproductive organ. The N concentration increased with increasing N application rates, and decreased gradually during the growth period. The relationship between biomass and N concentration can be described with a power equation. The patterns of the N concentration dilution model were consistent at both experimental sites, but the model parameter values of a differed. The results presented in this paper indicate that a critical N concentration dilution curve for cotton reproductive organ is independent of ecological region and can be described with a power equation.     

Accumulation characteristics of biomass and nitrogen and critical nitrogen concentration dilution model of cotton reproductive organ

Several nitrogen (N) field experiments were carried out in Nanjing and Anyang, China, to study the dynamic characteristics of biomass accumulation and N uptake, and to define the dilution curve for critical N concentration in cotton reproductive organ over the growth period. The results show that the total biomass and N accumulation were affected significantly by the rate of N application, exhibiting a sigmoid curve over time. The beginning time of fast N accumulation was 1–5 d earlier than that of biomass accumulation. The cotton lint yield was correlated with N concentration in the reproductive organ and fluctuated with varying N concentration, indicating the existence of luxurious N consumption in the cotton reproductive organ. The N concentration increased with increasing N application rates, and decreased gradually during the growth period. The relationship between biomass and N concentration can be described with a power equation. The patterns of the N concentration dilution model were consistent at both experimental sites, but the model parameter values of a differed. The results presented in this paper indicate that a critical N concentration dilution curve for cotton reproductive organ is independent of ecological region and can be described with a power equation.     

The Chlorella virus adenine methyltransferase gene promoter is a strong promoter in plants

An upstream region isolated from a eukaryotic algal virus adenine methyltransferase gene was tested for promoter function in plants. Fusion of this region to the chloramphenicol acetyltransferase reporter gene resulted in significantly higher expression than fusion with the cauliflower mosaic virus 35S promoter. Strong levels of expression were also found in electroporated monocot plant cells. The promoter activity in transgenic tobacco plants showed tissue-specific expression. Leaves had the highest expression followed by stems and flowers. The promoter activity was not detected in root tissue. Environmental cues, such as light, and the phytohormones auxin and cytokinines had no effect on the promoter's expression. This promoter might be utilized to achieve high levels of expression of introduced genes in higher plants.     

Effect of water-deficit stress on cotton plants expressing the Bacillus thuringiensis toxin

Bacillus thuringiensis (Bt) crops require a high dosage of Bt toxin to delay development of insect resistance, in particular, when the refuge strategy is applied. This strategy is threatened by plant developmental and environmental factors that might reduce Bt toxin concentration and Bt efficacy in Bt crops. Growth of Bt (Cry1Ac) cotton under prolonged, moderate water deficit as a single stress factor was evaluated. Bt cotton plants were analysed for physiological performance, Bt toxin concentration and Bt efficacy. For performance analysis, leaf and total plant dry weight and leaf area were measured. Bt toxin concentration was determined by an immuno‐assay. Effects of Bt toxin on growth and mortality of African cotton bollworm, Helicoverpa armigera, larvae were measured in different plant organs. Leaves from young plants exposed for 30 days to moderate water deficit had both higher Bt toxin concentrations and were more effective against larvae than leaves, flowers or bolls from mature flowering plants exposed to 60 days of moderate water deficit. Although growth of Bt cotton plants under moderate water‐deficit conditions decreased Bt concentrations in leaves, flowers and bolls, this had no effect on efficacy against first‐instar cotton bollworm larvae. No significant evidence was found that moderate water deficit, as a single stress factor, decreases Bt efficacy in Bt cotton.     

Development of insect resistant transgenic cotton lines expressing cry1EC gene from an insect bite and wound inducible promoter

Transgenic cotton lines were developed for high-level expression of a synthetic cry1EC gene from a wound inducible promoter. The tobacco pathogenesis related promoter PR-1a was modified by placing CaMV35S promoter on its upstream in reverse orientation. The resultant chimeric promoter CaMV35S(r)PR-1a expressed constitutively and was further up-regulated at the site of feeding by insects. It was induced more rapidly by treatment with salicylic acid (SA). The CaMV35S(r)PR-1a cry1EC expressing transgenic lines of cotton showed 100% mortality of Spodoptera litura larvae. The tightly regulated low-level expression of PR-1a was modified to a highly expressing constitutive expression by CaMV35S placed in reverse orientation. Salicylic acid treatment and wounding enhanced the expression further by the chimeric promoter. The leaves expressed more δ-endotoxin around the sites of insect bites. The levels of expression and induction varied among different transgenic lines, suggesting position effect. Some of the transgenic lines that expressed Cry1EC from the chimeric promoter at a low level also showed 100% mortality when induced with salicylic acid. A highly expressing insect bite and wound inducible promoter is desirable for developing insect resistant transgenic plants.     

Insertional mutagenesis and promoter trapping in plants for the isolation of genes and the study of development

Insertional mutagenesis, whether by transposable elements or T-DNAs fromAgrobacterium tumefaciens, provides a powerful experimental strategy to investigate the genetic basis of plant growth, metabolism and development. The linkage of an insertion element with a mutant phenotype of interest greatly facilitates the isolation of the wild-type gene. A further refinement of this strategy is the incorporation of promoter traps or enhancer traps into the insertion elements. These can act as functional tags of regulatory elements associated with genes in the host genome, potentially can improve further the efficiency of screening for target mutant phenotypes, and may provide valuable markers of specific cell types for developmental analysis. We discuss the use of these techniques to study the molecular genetics of plant development.     

A comprehensive study of the use of a homologous promoter in antisense cotton lines exhibiting a high seed oleic acid phenotype

As opposed to first-generation biotechnology products, such as pest-resistant crops and herbicide-resistant crops, second-generation products often utilize plant-derived, homologous or heterologous genes and/or promoters. In this study, we evaluated the ability of a promoter from a gene encoding a major storage protein in cottonseed to drive an antisense sequence of the cotton FAD2 gene to down-regulate the activity of Delta-12 desaturase enzyme in cottonseeds. The oleic acid level in the transgenic cottonseeds approximately doubled from the wild-type level of 15%, with a concomitant decrease in the level of linoleic acid. A more extensive study of one line revealed a higher degree of seed-to-seed variability in the transgenic phenotype. A thorough investigation was conducted to determine the impact of the use of a homologous promoter to drive a transgene on the activity of the endogenous promoter. The results showed that the use of the homologous alpha-globulin B promoter for transgenic purposes did not adversely affect the expression of alpha-globulin B storage protein in cottonseed. The results obtained in this investigation on the use of a homologous promoter and antisense technology will be useful in the design of strategies to alter biosynthetic pathways for nutritional quality improvements and for the production of heterologous proteins of commercial value in seeds.     

土壤养分对菊科一年生入侵种和本地种繁殖性状的影响

入侵植物繁殖性状的研究可为揭示植物入侵机制提供重要的科学依据。为研究土壤养分对入侵植物和本地植物繁殖性状的影响, 并进一步研究养分添加是否更能促进入侵植物的繁殖能力, 我们设置了低、高两个养分水平, 通过同质园实验比较了不同土壤养分对假臭草(Praxelis clematidea)、胜红蓟(Ageratum conyzoides)、三叶鬼针草(Bidens pilosa) 3种菊科一年生入侵种和夜香牛(Vernonia cinerea)、一点红(Emilia sonchifolia)、墨旱莲(Eclipta prostrata) 3种本地种繁殖性状的影响。研究结果显示, 养分添加提高了6种菊科植物的开花株高、株高、地上生物量、单粒种子重量、总花序数、每花序种子数、总种子数量、总种子重量, 并使开花时间提前、花期延长。养分添加对入侵种的开花株高和单粒种子重量的提高幅度要比本地种更显著。相对于部分本地植物(夜香牛、墨旱莲), 养分添加更能促进部分入侵植物(假臭草、胜红蓟)的繁殖能力。三叶鬼针草和一点红的总种子数量和总种子重量在两种土壤养分水平下均较小。本地种墨旱莲的总种子数量和总种子重量在低养分条件下高于3个入侵种。这些结果表明, 高土壤养分仅能促进部分入侵植物相对于部分本地植物的繁殖能力。     

儿童孤独症相关基因NRXN1β启动子功能分析

Neurexins是神经特异性突触蛋白,Neurexin1β结构的异常与孤独症密切相关。为分析孤独症相关基因NRXN1β最小启动子和调节基因转录的功能元件,本文构建了含NRXN1β基因上游调控区不同区域的荧光素酶报告基因质粒,转染HEK293细胞后,利用检测双荧光素酶报告基因的转录活性以确定NRXN1β基因最小启动子区,进而筛选出相应的显著增强或抑制报告基因活性的功能区;同时,为鉴定顺式作用元件,利用基因定点突变技术对基因功能区内和临近DNA序列进行连续的碱基突变;最后,采用转录因子预测工具对启动子功能区内的转录调控元件进行分析。结果首次发现NRXN1β最小启动子区位于-88~+156 bp,-88~-73 bp和+156~+149 bp可增强启动子活性,+229~+419 bp可抑制启动子活性,且-84~-63 bp为能够显著性增强启动子活性的顺式作用元件,该区域可能存在DBP(Albumin D-site-binding protein,DBP)和ABF1(Autonomously replicating sequence-binding factor 1,ABF1)两个转录因子结合位点。