大豆microRNA基因GmMIR160A负调控植物叶片衰老进程

叶片衰老是受内外多种因子影响的遗传发育进程。生长素、细胞分裂素和乙烯等多种植物激素是调控叶片衰老的重要内部因子,它们通过长或短距离运输形成叶片组织内特定的区域分布和浓度梯度,从而直接或间接参与植物叶片衰老过程。分子遗传学表明,细胞分裂素和乙烯分别是叶片衰老的抑制子和正调节子,而生长素如何参与叶片衰老的分子机制目前还不清晰。植物体内成熟小分子RNA由小RNA基因转录并通过特定酶加工形成的21~23bp的双链RNA分子。这些小分子通过不完全配对方式抑制其靶基因转录和/或表达,参与植物生长发育多个过程,然而这类小RNA分子如何调控植物叶片衰老发育过程目前则还鲜有报告。大豆是重要的油料作物,具有典型的单次结实性衰老特征。研究大豆叶片衰老具有重要的科学意义和深远的应用价值。该文采用实时荧光定量PCR(qPCR)技术分析大豆(Glycine max)micro RNA基因GmMIR160A的表达模式,发现大豆第一复叶中GmMIR160A表达受外源生长素和黑暗处理的诱导,暗示该基因是生长素快速响应的叶片衰老相关基因。为进一步探究GmMIR160A在大豆叶片发育中的功能,构建了肾上腺皮质激素(Glucocorticoid,GR)类似物地塞米松(Dexamethasone,DEX)诱导表达GmMIR160A双元表达载体并通过农杆菌介导的子叶节方法转化野生型大豆。通过抗性筛选和基因组PCR鉴定并结合表型分析,共获得了4株诱导表达的稳定遗传转基因植株(株系OX-3、OX-5、OX-7和OX-8)。GmMIR160A过表达植株根、茎、叶、花和果实在形态学上与野生型相比无显著差异,但叶片的叶绿素含量增加、最大光量子效率(Fv/Fm)增强。进一步分子分析发现,转基因大豆叶片中GmARFs和衰老标记基因(GmCYSP1)表达明显下降,表明大豆Gma-miR160通过抑制靶基因GmARFs的表达来负调控植物叶片的衰老进程。该文揭示了生长素通过小分子RNA调控叶片发育一条新途径,为研究植物激素调控植物叶片衰老提供了新的思路。     

生长素响应因子GmARF10正调节大豆叶片的衰老进程

采用实时荧光定量PCR(qPCR)分析了GmARF10表达模式。通过挖掘大豆基因组信息并参考拟南芥同源基因序列,分析了大豆生长素响应因子GmARF10和小RNA分子Gm-miR160作用位点,构建了pGmARF10∷mGmARF10(mGmARF10)抗降解表达载体;利用农杆菌介导法转化大豆,并对转基因植株叶片发育表型进行了分析。结果表明:GmARF10在大豆[Glycine max(L.)Merri.]根、茎、叶、花和果荚中均有一定程度的表达,其中花内表达量最高,茎内最低,第一复叶表达量低于子叶和第一对真叶。mGmARF10转基因植株复叶形状和大小与对照组没有明显的差异,但其叶绿素含量和最大光量子效率(Fv/Fm)明显下降,叶片衰老标记基因GmCYSP1表达显著增加。研究认为,大豆生长素响应因子GmARF10参与了叶片衰老进程并可能是叶片衰老信号的重要组份。     

大豆生长素响应因子GmARF16参与调节叶片衰老进程

生长素响应因子(auxin response factors,ARFs)通过调节下游靶基因广泛参与植物生长发育过程,但ARFs如何调控植物叶片衰老的分子机制还不清楚。该文首先利用实时荧光定量PCR(q PCR)技术,分析大豆生长素响应基因Gm ARF16在叶片自然衰老、人工黑暗诱导衰老、外源植物生长素IAA处理条件下的表达模式,结果表明,该基因与叶片衰老调控密切相关,并且属于生长素的原初响应基因。为了进一步验证Gm ARF16基因的功能,采用农杆菌转化方法分别获得基因敲减(Gm ARF16-RNAi)和抗降解表达(m Gm ARF16)的转基因大豆植株。与非转基因对照相比,Gm ARF16-RNAi转基因大豆植株的叶片叶绿素含量和最大光量子效率(Fv/Fm)显著提高,叶片衰老标记基因(Gm CYSP1)的表达受到抑制,而m Gm ARF16转基因大豆植株则呈现出与Gm ARF16-RNAi转基因大豆植株相反的叶片生理表型。结果表明大豆生长素响应因子Gm ARF16正调节叶片的衰老进程。该研究表明,Gm ARF16在植物生长发育进程中发挥着重要作用。     

草莓生长素响应因子FvARF10和FvARF18基因的克隆及表达分析

该研究以森林草莓(Fragaria vesca)为试验材料,利用RT-PCR技术克隆草莓FvARF10、FvARF18基因全长cDNA序列。生物信息学分析表明,FvARF10、FvARF18基因开放阅读框(ORF)分别为2 056bp和2 100bp,编码685和699个氨基酸,预测其分子量和等电点分别为75.73、76.78kD和8.12、6.55。系统进化树分析表明,FvARF10与苎麻BnARF10聚为一个分支,FvARF18与桑树MnARF18聚集在一个分支上,进化关系较密切。亚细胞定位预测分析表明,FvARF10、FvARF18蛋白均定位于细胞核或细胞质中。启动子分析显示,FvARF10、FvARF18基因均具有多样化的激素应答元件。荧光定量PCR结果显示,FvARF10和FvARF18基因在草莓不同组织中均有表达,FvARF10在果实中表达较高,且在半熟果中表达量最高,而FvARF18在茎中相对表达量最高;外源激素IAA处理1h后,FvARF10和FvARF18基因受到强烈诱导均呈上调表达,而后渐恢复正常水平。研究结果表明,FvARF10和FvARF18基因可能参与草莓的营养生殖及果实的生长发育调控,且与生长素信号转导过程相关。     

大豆GeBP转录因子基因家族的生物信息学分析

GeBP转录因子调控植物表皮毛的生长发育,并且参与控制植物叶片的发育。该文利用生物信息学方法,在大豆全基因组范围内搜索GeBP基因家族,并从氨基酸理化性质、基因结构、染色体的物理分布、系统进化、序列比对、功能结构域、组织表达情况等基本特征方面对GmGeBP基因家族进行分析。结果表明:(1)共获得9个GmGeBP转录因子基因家族成员,其中仅2个基因含有内含子,且都只有1个内含子,表明该家族成员基因构造比较简单但稳定。(2)GmGeBP编码的蛋白分子量为39.65~49.24 kD,理论等电点为4.65~9.08;这些成员基本上都是酸性氨基酸,属于亲水性、不稳定蛋白。(3)这9个基因不均匀的分布于7条染色体上,10和20号染色体上分别分布2个GeBP基因,3、5、13、15、19号染色体上各分布1个基因。(4)系统进化分析表明,大豆与拟南芥对应的GeBP成员亲缘关系较近,分别聚类到4个分支,而与水稻的距离较远。(5)结构域分析表明,9个GmGeBP成员都包含DUF573结构域,推测该部分在GeBP转录因子中很可能是与靶标基因顺式作用元件互作的结构域。(6)通过分析大豆GmGeBP转录因子基因家族的组织表达,发现不同基因在大豆不同组织的表达量不同,具有一定的特异性。该文对大豆GeBP转录因子基因家族的分析和鉴定为进一步研究大豆表皮毛发育的分子作用提供了理论基础。     

大豆膨胀素基因GmEXPB5和GmEXPB7生物信息学及表达分析

膨胀素(expansin,EXP)通过调控细胞壁的松弛在植物应对环境胁迫过程中起着重要作用。为研究EXP基因在大豆应对非生物胁迫过程中的作用,该文对大豆中的两个EXP基因(GmEXPB5和GmEXPB7)及其蛋白序列进行生物信息学分析,通过实时荧光定量PCR(qRT-PCR)检测基因表达量。结果表明:(1)GmEXPB5和GmEXPB7分别位于大豆第10号和第12号染色体上,编码的蛋白序列长度分别为272和267个氨基酸。GmEXPB5蛋白分子量为29.07 kD,理论等电点为7.51; GmEXPB7蛋白分子量为29.09 kD,理论等电点为8.66。GmEXPB5和GmEXPB7均为稳定的亲水蛋白且定位于细胞壁中。GmEXPB5和GmEXPB7蛋白均含有一段信号肽序列和一个保守的DPBB_1结构域。(2)GmEXPB5蛋白与鹰嘴豆CaEXPB15蛋白亲缘关系最近,GmEXPB7蛋白与密花豆、赤豆和豇豆的EXPB3蛋白有着较近的亲缘关系。(3)GmEXPB5和GmEXPB7在大豆根、茎和叶中均有表达且它们在根和叶中的表达量均显著高于茎中的表达量。(4)GmEXPB5和GmEXPB7在大豆幼苗中可以响应盐、干旱和低温胁迫。(5)GmEXPB5启动子区域含有2种与逆境相关的顺式作用元件(ABRE和ARE); GmEXPB7启动子区域含有5种与逆境相关的顺式作用元件(ABRE、ARE、CGTCA-motif、TC-rich repeats和MBS)。综上所述,GmEXPB5和GmEXPB7能够参与大豆对非生物胁迫的应答。     

植物Whirly蛋白调控叶片衰老的研究进展

植物衰老是植物细胞生长发育的最后一个阶段,其启动的早晚对植物生物量和品质的形成有很大影响。叶片衰老是植物衰老的主要形式,受到内外环境因素的诱导,并被多种转录因子介导的信号传导途径所调控。对叶片衰老调控机制的研究一直是植物衰老研究中的重点。Whirly蛋白作为一类广泛存于植物中的特异转录因子小家族,能与单链DNA分子结合,双定位于细胞器(线粒体或叶绿体)与细胞核中,在植物细胞核和细胞器中发挥多种功能,参与对植物叶片衰老的调控。本文概述了植物Whirly蛋白的结构和定位,重点阐述了Whirly蛋白的功能与细胞衰老关系及其对叶片衰老调节机理的研究进展等,并对未来的研究方向进行了展望。     

马尾松细胞分裂素羟化酶基因PmCYP735A克隆与表达分析

该文首次在针叶树种马尾松(Pinus massoniana)中采用cDNA末端快速克隆(RACE)技术克隆,并鉴定出1个CYP735A基因。结果表明:马尾松CYP735A基因(PmCYP735A) cDNA全长为1 744 bp,包括1 647 bp的开放阅读框,44 bp的5'端非翻译区和53 bp的3'端非翻译区。该基因编码蛋白由548个氨基酸残基组成,其二级结构含有丰富的α-螺旋和无规则卷曲。该基因编码蛋白不含跨膜区域,且无信号肽酶切位点,在399～406个和475～484个氨基酸残基存在P450超家族保守特征序列ETLRLYP(ExxRxxP)和血红素结合区域(Heme-binding region) FSFGPRKCVG (FxxGxRxCxG)。系统进化树分析表明,马尾松PmCYP735A与水稻、玉米、拟南芥CYP735A蛋白归属于同一小的进化枝,可可、毛果杨、麻风树和橡胶树等的CYP735A同源蛋白相对集中的定位于另一进化分支。实时定量PCR检测发现,PmCYP735A基因在马尾松根和茎中的表达量显著高于叶,该基因响应外源生长素NAA诱导表达,随着诱导时间呈现先上升再下降的表达趋势。以上结果有助于深入探究CYP735A基因家族的生物学功能及其在物种间的表达调控异同,为进一步挖掘马尾松优异基因资源奠定基础。     

珠子参中HMGR基因对皂苷生物合成的影响

该研究以珠子参愈伤组织为材料,通过同源克隆方法获得了珠子参3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因(PjHMGR,登录号:MG712296)的cDNA序列,并对其进行生物信息学分析。基于PjHMGR序列构建了珠子参过表达载体pCAMBIA2300s-PjHMGR,通过根瘤农杆菌转化法将其转化到珠子参细胞中,成功获得7株阳性转PjHMGR基因细胞系;通过实时荧光定量PCR、比色法及皂化法等技术测定阳性细胞系PjHMGR基因的相对表达量、HMGR酶活、皂苷和植物甾醇含量变化。结果显示:(1)与野生型珠子参细胞系相比,转PjHMGR基因珠子参阳性细胞系中,PjHMGR基因的相对表达量、HMGR酶活和皂苷含量均有不同程度的提高;在效果最好的阳性细胞中,PjHMGR基因的相对表达量、HMGR酶活和皂苷含量分别为对照的7.15、6.14和3.50倍。(2)在研究过程中,发现转基因细胞系的植物甾醇含量也有所升高。研究认为,将珠子参关键酶基因PjHMGR过表达载体导入珠子参愈伤组织细胞中,可引起相关关键酶基因相对表达量和PjHMGR酶活性的增加,从而调控珠子参总皂苷的合成,对皂苷合成途径中关键酶基因进行调控将可能实现对皂苷生物合成的调节。     

大豆GmABI4基因克隆及表达分析

该研究基于大豆基因组数据库,根据拟南芥ABI4蛋白的氨基酸序列,经比对分析,获得了大豆中的2个GmABI4基因,分别命名为GmABI4-1(GenBank登录号为XM_014766551.1)和GmABI4-2(GenBank登录号为NM_001249003)。TMHMM软件和系统进化转录分析表明,这2个基因编码的蛋白均不具有信号肽,二级结构主要以无规则卷曲和延伸链为主;进化树分析表明,大豆GmABI4和野生大豆亲缘关系较近。荧光定量PCR分析表明,GmABI4-1与GmABI4-2基因在大豆种子与豆荚中的表达量均高于根、茎、叶、花等其他组织,推测可能与调控大豆种子生命活动相关。     

Characterization of a class of small auxin-inducible soybean polyadenylated RNAs

Four new auxin-responsive RNAs from soybean (Glycine max (L.) Merr., var. Wayne) are described. The RNAs were identified by hybridization to three cDNA probes obtained from a library enriched for sequences which increase in abundance within 60 min after 2,4-D (2,4-dichlorophenoxyacetic acid) treatment. These RNAs appear to define a new class of small (i.e. approximately 550 nucleotides) RNAs that respond extremely rapidly to application of exogenous auxin. In excised elongating hypocotyl sections, an increase in the abundance of these RNAs can be detected 2 to 5 min after treatment with 50 M 2,4-D. This response is half maximal after 10 min and reaches steady state in 60 min. RNA blot analysis shows that these RNAs are expressed differentially in various parts of the seedling. The degree of inducibility by auxin is also organ-specific, with the elongating hypocotyl being the most responsive of the organs tested. The RNAs display identical response specificities with one exception. Accumulation of one RNA, designated 10A, is completely abolished by simultaneous addition of cycloheximide and 2,4-D. This RNA also displays a different 2,4-D dose response than other RNAs examined. These results suggest that more than one mechanism is involved in rapid modulation of gene expression by auxin.     

Characterization of a stress-induced,developmentally regulated gene family from soybean

We describe a family of stress-induced, developmentally regulated soybean genes for which cDNAs have been obtained from two different cultivars (Glycine max cv. Mandarin and Glycine max cv. Williams). The mRNAs corresponding to these cDNAs, called SAM22 and H4, respectively, accumulate predominantly in the roots of soybean seedlings but are present at high levels in the roots and leaves of mature plants. SAM22 accumulation is especially dramatic in senescent leaves. In addition, SAM22 accumulation can be induced in young leaves by wounding or by transpiration-mediated uptake of salicylic acid, methyl viologen, fungal elicitor, hydrogen peroxide or sodium phosphate (pH 6.9). Taken together, these data indicate that the genes corresponding to SAM22 and H4 are induced by various stresses and developmental cues. Southern blot analysis indicates that multiple copies of sequences related to SAM22 exist in the soybean genome. We also show that the nucleotide sequences of the cDNAs corresponding to SAM22 and H4 are 86% identical at the nucleotide level to each other and 70% identical at the amino acid level to the disease resistance response proteins of Pisum sativum.     

Photosynthesis at ambient and elevated humidity over a growing season in soybean

Daytime rates of net photosynthesis of upper canopy leaflets of soybeans were compared on 17 days for leaflets exposed to air at the ambient humidity and at a higher humidity. Leaflets at the higher humidity had higher rates of net photosynthesis on 16 of the 17 days. The daily total of net photosynthesis of leaflets at the higher humidity was on average 1.32 times that for leaflets at ambient humidity. A strong limitation of net photosynthesis by ambient humidity was found throughout the growing season.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups

In the US, soybean genotypes are classified into maturity groups (MG; total of 13) that represent areas of adaptation generally correlated with latitude bands. To determine if one regeneration procedure could regenerate representatives from diverse areas of adaptation, 18 soybean genotypes representing nine MG were compared for organogenic adventitious regeneration and plant formation from hypocotyl explants following the procedure previously tested on representatives from only three MG. Responding explants were those capable of producing shoots on the acropetal end of the explant from either the outer edge plus central region or the central region only. This enabled determination of the contribution of cotyledonary nodal tissue (outer edge) to shoot regeneration and by discounting those explants, it also enabled estimates of true adventitious regeneration. All 18 genotypes were capable of producing meristemoids/shoots solely from the central region with responses ranging from 28.5 to 64.3% after 4 weeks in culture. All genotypes were also capable of producing elongated shoots that could be successfully rooted. No morphological differences were noted among regenerants, or between them and seed-initiated plants. All regenerants produced viable seed which germinated and produced morphologically normal plants. This study confirmed the genotype- and MG-independent nature of this hypocotyl-based organogenic regeneration procedure and provided conservative estimates for responses that were truly/solely adventitious in nature.     

Macronutrient concentrations of soybean infected with soybean cyst nematode

Soybean cultivars (Glycine max(L.) Merr.) infected with soybean cyst nematode (SCN; Heterodera glycinesIchinohe) often show symptoms similar to K deficiency. The objectives of this experiment were to determine if SCN infection affected macronutrient concentrations in soybean seedling vegetative tissues, determine whether increased K fertility can overcome these possible effects, and to determine if these possible effects are localized at the site of infection or expressed systemically throughout the root system. Soybean plants were grown with root systems split into two halves. This allowed differential K (0.2, 2.4 and 6.0 mM K nutrient solutions) and SCN (0 and 15 000 eggs/plant) treatments to be applied to opposite root-halves of the same plant. Thirty days after plants were inoculated with SCN, macronutrient concentrations of shoot and root tissues were determined. Potassium concentration in leaf blades was not affected; but K concentrations in leaf-petiole and stem tissues were increased with SCN infection. Roots infected with SCN contained lower K concentrations than uninfected roots, but only for the 2.4 mM K treatment. Thus, at the medium level of K fertility, SCN reduced K concentration in soybean roots, and increasing K fertility to the high level overcame the effect. Because K concentrations in the shoot tissues were not reduced by SCN infection, above ground portions of the plant may be able to overcome limitations that occur in roots during the first 30 days of infection. Increasing K fertility level in soybean fields may not benefit vegetative growth of soybean infected with SCN.     

Incidence of the introduced parasitoids Cotesia kazak and Microplitis croceipes (Hymenoptera: Braconidae) from Helicoverpa armigera (Lepidoptera: Noctuidae) in tomatoes, sweet corn, and lucerne in New Zealand

Morphological defense traits of plants such as trichomes potentially compromise biological control in agroecosystems because they may hinder predation by natural enemies. To investigate whether plant trichomes hinder red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), as biological control agents in soybean, field and greenhouse experiments were conducted in which we manipulated fire ant density in plots of three soybean isolines varying in trichome density. Resulting treatment effects on the abundance of herbivores, other natural enemies, plant herbivory, and yield were assessed. Trichomes did not inhibit fire ants from foraging on plants in the field or in the greenhouse, and fire ant predation of herbivores in the field was actually greater on pubescent plants relative to glabrous plants. Consequently, fire ants more strongly reduced plant damage by herbivores on pubescent plants. This effect, however, did not translate into greater yield from pubescent plants at high fire ant densities. Intraguild predation by fire ants, in contrast, was weak, inconsistent, and did not vary with trichome density. Rather than hindering fire ant predation, therefore, soybean trichomes instead increased fire ant predation of herbivores resulting in enhanced tritrophic effects of fire ants on pubescent plants. This effect was likely the result of a functional response by fire ants to the greater abundance of caterpillar prey on pubescent plants. Given the ubiquity of lepidopteran herbivores and the functional response to prey shown by many generalist arthropod predators, a positive indirect effect of trichomes on predation by natural enemies might be more far more common than is currently appreciated.