Bioaccumulation and phyto-translocation of arsenic,chromium and zinc by Jatropha curcas L.: Impact of dairy sludge and biofertilizer

The present study was planned to remediate the metalloid and metal contaminated soil by using non-edible and economic plant species Jatropha curcas L. The experiment was conducted on pots to improve the survival rate, metal tolerance and growth response of the plant on soil; having different concentrations of arsenic, chromium and zinc. The soil was amended with dairy sludge and bacterial inoculum (Azotobacter chroococcum) as biofertilizer. The results of the study showed that the bioaccumulation potential was increased with increase in metalloid and metal concentration in soil system. Application of dairy sludge significantly reduces the DTPA-extractable As, Cr and Zn concentration in soil. The application of organic amendment stabilizes the As, Cr and Zn and reduced their uptake in plant tissues.     

麻疯树鲨烯合酶基因SQS的克隆及生物信息学分析

以麻疯树(Jatropha curcas L.)总RNA为模板,根据已报道的鲨烯合酶基因序列设计简并引物,用RACE方法克隆得到麻疯树鲨烯合酶基因全长cDNA,命名为JcSQS。JcSQS全长1609 bp,包含1个1242 bp的开放阅读框,预测麻疯树鲨烯合酶基因编码的蛋白含有413个氨基酸。JcSQS具有鲨烯合酶类的保守结构域,JcSQS 蛋白与蓖麻、柿、木榄等植物中SQS基因编码的氨基酸序列具有高度同源性。这为研究麻疯树萜烯类物质的生物合成和调控机制奠定了基础。     

Analysis of core genes supports the reclassification of strains Agrobacterium radiobacter K84 and Agrobacterium tumefaciens AKE10 into the species Rhizobium rhizogenes

Some strains of the former genus Agrobacterium have high biotechnological interest and are currently misclassified. Consequently, in this study, the taxonomic status of the non-pathogenic strain Agrobacterium radiobacter K84, used in biological control, and the tumourigenic strain Agrobacterium tumefaciens AKE10, able to regenerate tobacco transgenic plants, was revised. The phylogenetic analysis of the chromosomal genes rrs, atpD and recA showed that they should be reclassified into Rhizobium rhizogenes. The analysis of virulence genes located in the Ti plasmid (pTi) outside T-DNA showed a common phylogenetic origin among strains AKE10, R. rhizogenes 163C and A. tumefaciens (currently R. radiobacter) C58. However, the genes located inside the T-DNA, mainly the 6b gene, of strain AKE10 were phylogenetically close to those of strain 163C but divergent from those of strain C58. Furthermore, the T-DNA of tumourigenic strains from R. rhizogenes conferred on them the ability to regenerate tumour tissue resembling fasciation in tobacco plants. These results showed the existence of a highly mosaic genetic organization in tumourigenic strains of the genus Rhizobium and provided evidence of the involvement of T-DNA from tumourigenic strains of R. rhizogenes in fasciation of Nicotiana leaves. The data further suggested that pathogenic strains of Rhizobium could be good models to analyse bacterial evolution.     

施氮量对麻疯树幼苗生长及叶片光合特性的影响

采用盆栽土培的方法,研究了不同施氮量(对照N₀ 0 kg N/hm²、低氮N_L 96 kg N/hm²、中氮N_M 288 kg N/hm²、高氮N_H 480 kg N/hm²)对麻疯树幼苗生长、叶片气体交换及叶绿素荧光参数的影响。结果表明,麻疯树幼苗叶片氮含量、可溶性蛋白含量、株高、地径、叶片数量、叶面积、根长、各组分生物量、叶片净光合速率(P_n)、气孔导度(G_s)、蒸腾速率(T_r)和水分利用效率(WUE)均随施氮量的增加先升高后降低,N_M处理下麻疯树幼苗长势最好,各气体交换参数值最高;施氮对麻疯树地上部分的促进作用远大于地下部分,施氮后根冠比显著降低;此外,麻疯树叶绿素含量、PSⅡ最大光化学量子产量(Fv/Fm)、PSⅡ有效量子产量(F'v/F'm)、PSⅡ实际光化学效率(Φ_PSⅡ)、电子传递速率(ETR)和光化学淬灭系数(qP)均随施氮量的增加而升高,非光化学淬灭系数(NPQ)随施氮量增加而降低。适量施氮可通过增强叶绿体光化学活性、气孔导度和羧化能力而提高麻疯树幼苗的光合能力,促进生长;过高施氮对麻疯树幼苗光合与生长的促进效应降低。试验条件下,当年生麻疯树幼苗的最适施氮量为288 kg N/hm²。     

In vitro callus induction and plant regeneration from leaf explants of Ruta graveolens L.

A protocol for multiple shoot bud induction and plant regeneration from leaf segment-derived callus of Ruta graveolens has been developed. Maximum organogenic callus induction frequency (70.6 ± 2.33%) was observed on Murashige and Skoog (MS) medium supplemented with 10 µM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus when transferred to shoot induction media (MS nutrients supplemented with 6-benzyladenine (BA), kinetin (Kn), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) in various concentrations and combinations). The highest shoot multiplication (92.3%) was observed on MS medium with 7.5 µM BA and 1.0 µM NAA. Regenerated shoots were rooted in vitro on MS containing 0.5 µM IBA. Plantlets with well developed root and shoot systems were successfully acclimated (90%) and established in earthen pots containing garden soil; they exhibited normal morphology and growth characteristics.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

不同非生物胁迫对麻疯树幼苗光合速率等生理指标的影响

对麻疯树(Jatropha curcas L.)幼苗在不同非生物胁迫下的净光合速率(Pn)和蒸腾速率(Tr)等生理指标的变化进行了研究。结果表明,在缺磷处理中,麻疯树叶片Pn保持在对照的90%左右,气孔导度(Gs)和胞间CO2浓度(Ci)在处理2 d后显著增加,Gs上升20%～40%,Ci升高4%～16%,Tr变化不大;处理17 d时,麻疯树的P含量下降55%～85%,而干重只下降3%。缺氮处理9 d时麻疯树叶片的Pn下降到最低,之后维持在对照的64%左右,Gs在处理2～7 d显著高出对照15%～57%,处理9～16 d恢复到对照水平,Ci从第2天开始上升,高出对照4%～24%,Tr变化不大;处理17 d时组织中N含量显著下降47%～78%,植株干重下降23%。盐胁迫处理5 d后,麻疯树叶片Pn降低到对照的54%,之后维持在对照的48%左右,Gs、Ci和Tr与Pn的变化一致,均呈下降趋势;处理17 d,叶柄和茎中P含量增加37%～54%,组织中K+/Na+下降87%～96%,植株干重下降18%。干旱胁迫处理6 d,叶片Pn快速下降至29%,Gs、Ci和Tr与Pn整体变化趋势一致;处理第7天,叶片细胞膜透性增加67%,停止浇水17 d后植株干重下降55%,同时叶片卷曲下垂,老叶脱落。麻疯树植株Pn在缺磷胁迫过程中最早达到相对稳定状态,其次为盐胁迫和缺氮胁迫。这说明麻疯树植株对缺磷环境具有良好的适应性,而对缺氮环境适应性相对较差;耐盐类型可能属于逃避盐害中的聚盐植物,适应干旱环境的机制属于御旱性类型。     

小桐子糖原合成激酶基因JcGSK的克隆与表达分析

以小桐子(Jatropha curcas L.)cDNA为模版,克隆了JcGSK基因的CDS序列。序列分析表明,JcGSK基因包含1 230bp完全阅读框(ORF),编码409个氨基酸。预测其编码蛋白质的相对分子量为46.33kD,理论等电点为8.58。Blast搜索结果及进化分析结果表明,JcGSK蛋白与巴西橡胶树GSK蛋白的氨基酸序列一致性最高(94%)且亲缘关系最近;JcGSK基因编码的蛋白具有一个蛋白激酶特有的结构域。组织表达结果显示,JcGSK基因在小桐子根、茎、叶、花、果皮和种子中都有表达,且在根中表达量最高。小桐子幼苗在NaCl、ABA、PEG、低温和机械损伤处理后JcGSK基因表达量有不同程度的上调,推测其参与小桐子非生物胁迫响应和信号传导过程。JcGSK基因在种子中也有较高表达,在种子发育过程中表达量的变化与种子生长发育趋势基本一致,推测JcGSK基因也参与调控小桐子种子的生长发育。     

小桐子果壳化感物质的初步分离和鉴定

为明确小桐子(Jatropha curcas L.)果壳的化感物质,对小桐子果壳水提物的水萃取物进行了分离和初步鉴定。结果表明,AO活性部位为10 g L-1时,对萝卜(Raphanus sativus Linn.)、苏丹草[Sorghum sudanense(Piper)Stapf]幼苗生长的抑制率均高于80%。经对AO部位进行HPLC-MS分析,选择鉴定了含量较高的6个化合物,其含量为总量的60.13%。其中,化合物6初步鉴定为楤木皂苷Ⅴ(AraliasaponinⅤ),化合物1、2、3、4、5相似,初步鉴定为肽和蛋白,其相对含量之和为56.13%。利用小桐子果壳可开发植物源除草剂。     

Efficacy of karanjin and phorbol ester fraction against termites (Odontotermes obesus)

The non-edible oil seeds of Jatropha curcas (physic nut) and Pongamia pinnata (karanja) contain some toxic components (phorbol esters in J. curcas and karanjin in P. pinnata), which may be used as biopesticides. In this study, the active components of J. curcas and P. pinnata oil were extracted and their efficacy against the termites Odontotermes obesus (Rambur), was tested. The phorbol ester fraction of J. curcas and karanjin of P. pinnata oil were found to be effective against termites. A mortality rate of 100% was achieved in 6 h with karanjin and in 12 h with phorbol ester fraction. The LC₅₀ levels of karanjin and phorbol esters fractions were 0.038 and 0.071 g ml⁻¹, respectively, after 24 h at a 95% (0.05) confidence limit.     

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of ¹³C-labeled diC8PC ((methyl-¹³C)₃-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-¹³C)₃-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.     

小桐子JcCIPK2基因的克隆与表达分析

钙调磷酸酶B类似蛋白互作蛋白激酶(CBL-interacting protein kinase, CIPK)是一类植物中特有的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,参与多种生物和非生物胁迫响应过程。该实验通过RT-PCR克隆了小桐子(Jatropha curcas L.)JcCIPK2基因cDNA全长序列,采用荧光定量qRT-PCR分析JcCIPK2基因在不同组织及不同处理(12℃和1℃低温、42℃高温、30%PEG、250 mmol/L NaCl、150μmol/L ABA)下的表达模式。结果显示:(1)小桐子JcCIPK2基因开放阅读框全长1 398 bp,编码465个氨基酸,相对分子量为52.95 kD,等电点为8.89。(2)蛋白质结构分析表明,JcCIPK2的N端含有位于第11～265个氨基酸之间的丝氨酸/苏氨酸激酶_蔗糖非发酵-1型相关蛋白激酶3催化域STKc_SnRK3,在激酶结构域内还具有激活环(Activation Loop);C端含有位于第316～430个氨基酸之间的CIPK蛋白激酶调控域CIPK_C,其调控域中含有CIPK家族典型的能与CBL特异性结合的NAF结构域,位于第314～369个氨基酸之间。(3)系统进化分析显示,小桐子JcCIPK2蛋白与同属于大戟科的木薯(Manihot esculenta Crantz.)同源关系最近,序列一致性达87%。(4)qRT-PCR分析表明,JcCIPK2基因在小桐子根、茎、叶中均有表达,经12℃和1℃低温处理后,叶片中JcCIPK2基因的表达都呈现先上调后下调表达的趋势,且都在低温处理24 h的表达量最高,与对照相比分别上调了6.0倍和16.72倍;小桐子JcCIPK2基因在42℃高温、30%PEG、150μmol/L ABA、250 mmol/L NaCl处理下也受到不同程度的诱导表达。研究推测,JcCIPK2基因在小桐子对逆境的响应与适应中起重要作用。     

小桐子JcACBP基因克隆和序列分析

以小桐子(Jatropha curcas)cDNA为模板,克隆了酰基辅酶A结合蛋白(Acyl-CoA-binding Protein)基因(JcACBP)的CDS序列,对其序列进行了生物信息学分析,并采用实时荧光定量PCR方法,研究了JcACBP基因在小桐子不同器官和果实生长发育阶段的表达模式。结果显示:JcACBP基因完全阅读框(coding sequence,CDS)全长279bp,编码92个氨基酸。预测其编码蛋白质的分子量为10.30kD,具有ACBP家族典型的结构域。JcACBP基因推测氨基酸与油桐(Vernicia fordii,AFZ62125)的亲缘关系最近(96%)。JcACBP基因在小桐子根、茎、叶、花、发育中的胚及果实等组织中都有表达,其中在花后40d的种子中表达最高,其次是果皮,而在根中表达较少;在果实发育过程中的表达与果实油脂积累的变化趋势基本一致。     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

Predicting ultimate methane yields of Jatropha curcus and Morus indica from their chemical composition

In this study, all the components of Jatropha curcus and Morus indica were chemically characterized and their biochemical methane potentials (BMP) were determined. From the variables that showed strong influence on the ultimate methane yield (B_o) of J. curcus, a multiple regression Jatropha model was developed. This model comprised of total carbohydrates, protein, lipid, acid-detergent fiber (ADF), cellulose and ash in ADF as independent variables, with r² value of 0.943. The Jatropha model was validated on 7 samples of M. indica parts and wastes from silkworm rearing trays of this study and 13 samples of heterogeneous organic wastes of earlier studies, to judge the prediction quality. It was found that most of the predicted values differed by less than 15% of their experimental B_o.     

利用微嫁接技术促进麻疯树再生不定芽的生长

该研究以麻疯树种子实生苗的小芽和芽条为接穗,以带有胚根的实生苗下胚轴为砧木进行无菌微嫁接,试图建立新的有效微嫁接方法,解决农杆菌介导的麻疯树遗传转化体系中再生的转化不定芽难以顺利发育成完整植株的问题。结果表明:(1)抗生素对嫁接苗的生长有显著的抑制作用。(2)进行微嫁接所用砧木的苗龄以5d为宜。(3)进行微嫁接时适宜采用的砧木类型为带有部分胚根的下胚轴。(4)嫁接苗在0.3mg/L IBA+2mg/L谷氨酰胺+1/2MS培养基上的生长效果最好。(5)嫁接苗的移栽成活率最高可达76.40%。(6)以小芽或芽条为接穗的嫁接苗均可正常生长。该研究建立的麻疯树微嫁接体系,为解决麻疯树转化不定芽或芽条生长发育困难的问题提供了一条新的途径。     

蜂桶寨自然保护区中华姬鼠及社鼠肥满度的年龄和季节变化

肥满度被广泛用于动物生长状况与环境、生存、繁殖等方面的研究。为揭示横断山脉森林环境中中华姬鼠（Apodemus draco）和社鼠（Niviventer confucianus）肥满度的变化规律及影响原因,我们于2008年4~11月对四川省蜂桶寨国家级自然保护区内中华姬鼠和社鼠肥满度在各年龄组及不同季节中的变化进行了研究。结果显示,中华姬鼠肥满度在各年龄组间的差异显著,其变化趋势为老年组>成年组>亚成年组>幼年组;亚成年组及老年组肥满度在各季节间无显著差异,而在成年组则差异明显,以春季中最高。社鼠肥满度在各年龄组间无显著性差异;亚成年组肥满度在各季节间差异显著,在夏秋季中最低,同时成年组及老年组肥满度在季节间无显著差异。中华姬鼠和社鼠亚成年组、成年组及老年组肥满度与海拔无显著的线性关系。分析认为,中华姬鼠老年组个体可能在应对外界环境方面要强于幼年个体而具有最高的肥满度,成年个体肥满度的季节变化可能受食物资源季节差异和繁殖能量需求的影响;社鼠成年及老年个体能通过相应的季节性调节维持肥满度的稳定,而亚成年个体在调节方面较弱,故其肥满度在夏秋季最低。     

外源水杨酸对盐胁迫下小桐子幼苗脯氨酸代谢的影响

以小桐子幼苗为材料,设置盐胁迫(200mmol·L-1 NaCl)和外源水杨酸处理(0~2.0mmol·L-1 SA)水培试验,通过检测幼苗叶片脯氨酸含量、脯氨酸代谢关键酶活性及相关代谢酶基因的表达水平,研究了外源水杨酸对盐胁迫下小桐子幼苗脯氨酸代谢机理的影响。结果显示:(1)外源0.9mmol·L-1 SA处理可显著提高盐胁迫下小桐子幼苗的脯氨酸含量,上调脯氨酸合成关键酶Δ1-吡咯琳-5-羧酸合成酶(P5CS)和鸟氨酸转氨酶(OAT)活性,以及上调JcP5CS和JcOAT基因的表达水平。(2)SA也显著抑制了脯氨酸降解酶ProDH的活性及JcProDH基因的表达水平。(3)SA处理还显著提高了盐胁迫下小桐子幼苗的组织活力,降低了叶片电解质渗漏率和丙二醛(MDA)含量。研究发现,外源SA可通过活化脯氨酸合成的谷氨酸途径和鸟氨酸途径,以及抑制脯氨酸的降解途径来促进盐胁迫下小桐子幼苗脯氨酸的积累;外源SA处理也可提高小桐子幼苗的耐盐性,且这种提高可能与SA诱导脯氨酸的积累密切相关。