Structural analysis of the unique insecticidal activity of novel mungbean defensin VrD1 reveals possibility of homoplasy evolution between plant defensins and scorpion neurotoxins

A variety of evolutionarily related defensin molecules is found in plants and animals. Plant gamma-thionins and scorpion neurotoxins, for instance, may be categorized in this functional group, although each class recognizes a distinct receptor binding site. Such molecules are also categorized into the superfamily of cysteine-rich proteins. Plant defensins were generally believed to be involved in antimicrobial or antifungal mechanisms and, unlike scorpion toxins, little is known about whether these molecules are also endowed with the function of insect resistance. We have previously reported the isolation of a cDNA encoding a small cysteine-rich protein designated VrD1 (VrCRP) from a bruchid-resistant mungbean, which is apparently the first discovered plant defensin exhibiting in vitro and in vivo both insecticidal and antifungal activities. Our previous data also successfully demonstrated that VrD1 is toxic to E. coli and able to completely arrest the growth of Sf-21 insect cells at low concentration. However, the molecular and structural basis of this unique insecticidal activity of VrD1 is not clear. Therefore, in the present study, we use structural approach and phylogenic analysis to investigate the evolutionary and functional relations for such unique insecticidal activity. From our results, it is suggested that VrD1, in addition to gamma-thionins and several amylase inhibitors, is highly homologous to scorpion toxins, especially the short toxins. Moreover, based on the observation from our homology structures, VrD1 may utilize a newly found cluster of basic residues to achieve its insecticidal function, whereas all the other plant gamma-thionins were known to use a previously identified basic cluster conserved for gamma-thionins. Considering the general feature of short scorpion toxins to act on insect cell membranes with K(+)- or Cl(-)-channels as molecular targets, our analysis of interaction and recognition modes provides reasonable correlations between this newly found basic cluster and the insecticidal activity of VrD1, which is also comprehended as a possible link for "homoplasy evolution" between plant and animal defensin molecules.     

绿豆VrWOX基因家族鉴定及表达分析北大核心CSCD

WOX(WUSCHEL-related homebox)基因家族是植物特有的一类转录因子,是同源盒(homeobox,HB)转录因子超家族中的重要成员。WOX基因在植物干细胞调节及生殖发育过程中具有重要作用,其功能已在多个植物物种中鉴定。然而绿豆(Vigna radiate)VrWOX基因家族信息尚不清楚。本研究通过同源比对和聚类分析,在绿豆基因组中鉴定了42个VrWOX基因。VrWOX基因在绿豆染色体中分布不均,其中7号染色体含有的VrWOX数量最多。VrWOX基因分为古老进化支(19个VrWOX)、中等进化支(12个VrWOX)和年轻进化支(WUSCHEL进化支,11个VrWOX)3个亚类。种内和种间共线性分析发现,VrWOX基因共有12个重复事件,与拟南芥(Arabidopsis thaliana)AtWOX有15个同源基因对,与菜豆(Phaseolus vulgaris)PvWOX有22个同源基因对。VrWOX基因在基因结构、保守基序等方面存在很大差异,因而可能存在功能差异。VrWOX基因启动子区域含有不同种类和不同数量的顺式作用元件,导致VrWOX基因在不同组织中表现出不同的基因表达模式。本研究对VrWOX基因家族信息和表达模式进行了分析,为绿豆VrWOX基因功能和调控网络的解析奠定了一定的理论依据。     

Foraging behaviour of Helicoverpa armigera first instar larvae on crop plants of different developmental stages

Abstract:  Understanding how insect pests forage on their food plants can help optimize management strategies. Helicoverpa armigera (Hübner) (Lep., Noctuidae) is a major polyphagous pest of agricultural crops worldwide. The immature stages feed and forage on crops at all stages of plant development, damaging fruiting and non-fruiting structures, yet very little is known about the influence of host type or stage on the location and behaviour of larvae. Through semi-continuous observation, we evaluated the foraging (movement and feeding) behaviours of H. armigera first instar larvae as well as the proportion of time spent at key locations on mungbean [ Vigna radiata (L.) Wilczek] and pigeon pea [ Cajanus cajan (L.) Millspaugh] of differing developmental stages: seedling- and mature (flowering/pod fill)-stage plants. Both host type and age affected the behaviour of larvae. Larvae spent more time in the upper parts of mature plants than on seedlings and tended to stay at the top of mature plants if they moved there. This difference was greater in pigeon pea than in mungbean. The proportion of time allocated to feeding on different parts of a plant differed with host and age. More feeding occurred in the top of mature pigeon pea plants but did not differ between mature and seedling mungbean plants. The duration of key behaviours did not differ between plant ages in either crop type and was similar between hosts although resting bouts were substantially longer on mungbeans. Thus a polyphagous species such as H. armigera does not forage in equivalent ways on different hosts in the first instar stage.     

Phosphatase activity and phosphorus partitioning in nodules of developing mungbean

The acid and alkaline phosphatase activities were determined in bacteroid free fraction of nodules during development, using different phosphorylated substrates. Both enzymes change their substrate specificities with nodule development. Alkaline phosphatase, 20 days after sowing (DAS), showed negligible activity with ATP while at later stages maximum activity with ATP was observed. Invariably fructose 1,6 bisphosphate was a better substrate compared to fructose-6-phosphate and glucose-6-phosphate. Using Sephadex G-150 column chromatography, only one peak of acid phosphatase around Ve/Vo of 2.2 to 2.3 was observed at 20 and 30 DAS stages while at 40 DAS stage an additional ATP specific peak at around Ve/Vo of 2.9 was also observed. There was only one alkaline phosphatase peak at 20 and 30 DAS. However, at 40 DAS additional ATP specific peaks of phosphatases were observed at Ve/Vo of 1.4 and 2.6. Alkaline phosphatase could not be detected in the bacteroids whereas activity of acid phosphatase was about 5–7 % of that observed in the bacteroid free preparation. A low activity of both acid and alkaline phytases was observed at all stages of nodule development. However, phytic acid could not be detected. Increase in phosphorus content of water soluble organic phosphate at late stage of nodule development appears to be related with low level of phosphatase activity.     

Further characterization of phytase from Phaseolus aureus

Phytase purified to homogeneity from germinated mungbean cotyledons was inhibited by EDTA although it did not show any absolute requirement for divalent cations. Sodium fluoride, sodium citrate, mercaptoethanol and pCMB also inhibit the phytase activity but l-phenylalanine has no effect on activity. The phytase has a low affinity for inositol monophosphate. The relative rate of dephosphorylation of myo-inositol-1 -phosphate and myo-inositol-5 phosphate by phytase is 6 and 18% respectively of that of myo-inositol-hexaphosphate. Mungbean phytase cannot cleave myo-inositol-2-phosphate, 1,2-cyclic inositol phosphate, Na-β-glycerophosphate or p-nitrophenylphosphate. The relative rates of hydrolysis of different isomers of inositol hexaphosphate are in the following order: myo-IP₆, > neo-IP₆ > scyllo-IP₆ = d-chiro-IP₆, > l-chiro-IP₆. This enzyme seems to be most active with myo-inositol hexaphosphate.     

Screening and identification of random amplified polymorphic DNA (RAPD) markers linked to mungbean yellow mosaic virus (MYMV) resistance in mungbean (Vigna radiata (L.) Wilczek)

Bulk segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) techniques were used to analyse the F2 individuals of susceptible VBN (Gg) 2 × resistant KMG 189 to screen and identify the molecular marker linked to mungbean yellow mosaic virus (MYMV) resistant gene in mungbean. Two DNA bulks namely resistant bulks and susceptible bulks were setup by pooling equal amount of DNA from five randomly selected plants of each disease response. A total of 72 random sequence decamer oligonucleotide primers were used for RAPD analysis. Primer OPBB 05 (5′-GGGCCGAACA-3′) generated OPBB 05 260 fragment in resistant parent and their bulks but not in the susceptible parent and their bulks. Co segregation analysis was performed in resistant and susceptible F2 individuals, it confirmed that OPBB 05 260 marker was tightly linked to mungbean yellow mosaic virus resistant gene in mungbean.     

Effect of relative humidity,temperature and fungicide on germination of conidia of Cercospora canescens caused the Cercospora leaf spot disease in mungbean

The aim of the present investigation was to determine the impact of relative humidity (RH) and temperature on conidial germination, nuclear position and effect of important fungicides on growth and conidial germination of Cercospora canescens. Germination of conidia was observed at RH range 92–100% at 5–35°C. Significant interaction between temperature and RH indicated that higher humidity and high temperature promoted quick germination both in the presence and absence of free moisture. Although in absence of free moisture at 92–95% RH higher temperatures 25–35°C promoted quick evaporation of moisture and no conidial germination. Number of germtube was increased significantly at the optimum temperature 25–30°C and higher humidity (98–100%). But higher temperature 25–35°C with lower RH did not support the conidial germination. This finding is very important for disease forecasting using meteorological data. The spray of Carbendazim as contact fungicide may not be useful since it is not effective against the conidia of C. canescens. Triadimefon did not inhibit the conidia germination but completely inhibited mycelium development at 50 μg/ml. Propriconazole inhibited both conidia germination and mycelial development. Therefore, Propiconazole may be taken as protective as well as curative spray. In non-systemic fungicide, Copper oxychloride gave anticipated result by inhibiting both conidial germination and mycelium development. Therefore, copper oxychloride can be used as protectant fungicides for Cercospora leaf spot caused by C. canescens.     

嫁接导致的绿豆可遗传性变异及其在绿豆育种中的应用

植物嫁接可以导致接穗的后代中有可遗传性变异的发生.我们将绿豆(Vigna radiata (L.) Wilczek)的幼苗嫁接在红薯(Ipomoea batatas (L.) Lam.)的茎上, 维护其生长至结实.将收获的绿豆种子连续几代播种于普通环境时, 其后代中出现了明显的遗传变异.这些变异在未经嫁接的绿豆接穗品系中并不出现.为了研究这种嫁接诱导变异现象可能的机理, 我们对变异品系的细胞质和细胞核DNA进行了分析.结果显示,在原绿豆和变异品系之间未发现细胞质DNA的RFLP(限制性酶切片段多态性)差异.而细胞核DNA却发生了高频率的序列重组.同时,本研究没有发现砧木与接穗间基因转移的迹象.根据以上事实,我们推测远缘嫁接变异很有可能是嫁接生长逆境诱导的抗逆变异.     

Estimation of Photorespiration Rate by Simultaneous Measurements of CO2, Gas Exchange Rate,and Chlorophyll Fluorescence Quenching in the C3 Plant Vigna Radiata (L.) Wilczek and the C4 Plant Amaranthus Mongostanus L.

So far the photorespiration rate (R _P) in a leaf has been determined as the difference between the net photosynthetic rates (P _N) measured in 21 % O₂ air (P _N21%) and 3 % O₂ air (P _N3%). In the C₃ plant Vigna radiata and the C₄ plant Amaranthus mongostanus L., P _N and chlorophyll fluorescence quenching in leaves were monitored simultaneously. R _P of leaves in situ was estimated as termed R _PE from the electron transport rates through photosystem 2 (PS2), and compared with R _PO (P _N3% – P _N21%). In V. radiata R _PO was 11.9 µmol(CO₂) m^–2 s^–1 and the ratio of R _PO to P _N21% was 42.2 %, whereas the ratio of R _PE to P _N21% was 25.7 %. This suggests that R _PO may be over-estimated for the real R _P in normal air. In A. mongostanus, P _N was almost not changed with a decrease in O₂ concentration from 21 to 3 %, whereas the quantum yield of PS2 was evidently affected by the change in O₂ concentration. This fact shows the presence of photorespiration in this C₄ species, where R _PE was equivalent to 3.8 % of P _N21%.     

绿豆产量相关农艺性状的QTL定位

绿豆育种的目标性状大多是受多基因控制的数量性状,表现型受环境影响很大。为深入分析绿豆复杂数量性状的遗传特征,本试验以绿豆Berken/ACC41 F10重组近交系群体为作图群体,利用该群体已经构建的包含79个RFLP标记的分子连锁图谱对北京和广西2个种植环境下考察的11个绿豆产量相关农艺性状进行QTL定位。结果表明,2个环境下共检测到产量相关性状QTL 63个（其中北京25个,广西38个）,分布于除第13连锁群以外的12条连锁群。大部分QTL只在单一环境下被检测到,说明产量相关QTLs与环境之间存在明显的互作。2个环境均能检测到的QTL仅有6个,分别为控制荚长、百粒重、生育期的QTLs,这6个在不同生态环境下同时发挥效应的QTLs对于绿豆分子标记辅助育种具有特殊的意义。研究还发现2个QTLs富集区域和若干成束分布的QTLs,它们可能是发掘通用QTL的候选位点。