白蜡虫ws基因RNAi载体构建及原核表达dsRNA

[目的]构建白蜡虫(Ericerus pela)蜡酯合酶(wax synthase,WS)基因干扰载体并建立其体外dsRNA(doublestranded RNA,dsRNA)原核表达体系,低成本大量制备白蜡虫ws基因的dsRNA。[方法]克隆白蜡虫蜡酯合酶基因ws片段,连入L4440载体,将重组质粒转入大肠杆菌HT115感受态细胞,经IPTG诱导获得与目的片段相对应的dsRNA。[结果]白蜡虫ws基因RNA干扰(RNA interference,RNAi)载体成功构建,重组质粒转入HT115感受态细胞经IPTG诱导后菌体成功表达dsRNA,dsRNA的平均获得量1 705 ng·m L~(-1)。[结论]该研究通过原核表达白蜡虫ws基因的dsRNA,为后续利用RNAi实验研究白蜡虫ws基因功能及作用机理奠定基础。     

High level expression of glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from <Emphasis Type="Italic">Populus suaveolens</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G (pET30a-G6PDH), which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-â-D-thiogalactoside (IPTG) to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol&#8226;L^–1 IPTG treatment for 4 h and that pET-G product was predominately soluble and not extra-cellular secreting.     

High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli

In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL1 was transformed into expression host M15 (pREP4) and induced by isopropyl-α-D-thiogalactoside (IPTG) to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol·L-1 IPTG induction as the ex-pression temperature declined from 37 to 28°C. The 6×His tag facilitates affinity binding to Ni2 -nitrolotriacetic acid (NTA) and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose cou-pled with Ni2 -NTA affinity chromatography. The optimal substrate for Pt4CL1 was 4-coumarate.     

松材线虫Bxpel2基因的克隆及RNA干扰载体构建

[目的]为了构建松材线虫(Bursaphelenchus xylophilus)果胶酶Bxpel2基因干扰载体.[方法]通过Trizol 法提取松材线虫总RNA,反转录合成cDNA,设计带T₇启动子的果胶酶Bxpel2基因引物,以cDNA为模板扩增出果胶酶Bxpel2基因片段,连接到RNA干扰载体,再以干扰载体为模板,PCR扩增出目的片段后进行测序鉴定,合成果胶酶Bxpel2基因双链RNA(dsRNA),采用RT-PCR检测松材线虫Bxpel2基因干扰后的表达情况.[结果]表明:1)提取的松材线虫总RNA完整性好,无降解;2)成功克隆出松材线虫果胶酶Bxpel2基因片段(790 bp)并将其连接至pMD19-T载体;3)以RNA干扰载体为模板合成dsRNA,浓度分别为1.313 mg·mL^-1和1.152 mg·mL^-1;4)RT-PCR结果显示,松材线虫经过dsRNA干扰后,Bxpel2基因表达基本受到抑制.[结论]成功构建松材线虫果胶酶Bxpel2基因干扰载体,为进一步研究Bxpel2基因在松材线虫致病过程中的作用和功能奠定了良好的基础.     

麻疯树BRI1基因的鉴定及其在不同发育时期花蕾中的表达分析

[目的]研究油菜素内酯受体BRI1的同源基因(JcBRI1)在麻疯树花发育过程中的作用。[方法]利用RT-PCR技术克隆JcBRI1基因的CDS,以pET-30a(+)质粒为框架构建原核表达载体转化至大肠杆菌进行诱导表达,随后利用LC-MS/MS对表达产物进行质谱鉴定,并以氨基酸序列为基础分析JcBRI1蛋白质结构。利用荧光定量PCR分析麻疯树JcBRI1基因在雌雄花发育的关键时期的表达水平,初步确定其在麻疯树花发育过程中的表达模式。[结果]克隆获得了JcBRI1基因的CDS,长度为3 591 bp。SDS-PAGE检测结果表明:JcBRI1基因在大肠杆菌BL21(DE3)和Rosetta(DE3)中均能表达,但在低温环境的表达量更高。原核表达产物的LC-MS/MS鉴定结果表明:JcBRI1氨基酸序列与预期的一致,JcBRI1基因的表达产物为麻疯树BRI1蛋白。对JcBRI1在麻疯树花器官不同发育时期的表达水平进行检测分析,结果表明:JcBRI1基因的表达量在雌花发育的第一个时期即大孢子母细胞时期就达到了最高值,在随后几个时期持续下调;然而,其在雄花各个时期的表达量并无明显差异,表明其很有可能参与了麻疯树雌花大孢子母细胞发育过程。[结论]麻疯树JcBRI1基因为LRR-RLKs家族成员BRI1的同源基因,很有可能参与麻疯树雌花大孢子母细胞的发育过程,至于JcBRI1在麻疯树大孢子母细胞发育过程中的具体作用机制还需进一步研究。     

杨树PtRRI基因的组织特异性表达模式分析

[目的]模式植物在木本植物中鉴定的许多重要调控因子家族在木本植物中出现了基因家族成员扩张,但ARRs家族作为细胞分裂素响应调节因子在杨树基因组中成员数量反而减少,其在木本植物中如何行使功能需要进一步研究。[方法]本研究通过生物信息学构建PtRRI启动子与GUS融合表达载体,检测植物激素处理后PtRRI表达量和检测PPtRRI::GUS转基因植株在生根过程中GUS信号等方法,对杨树PtRRI基因的组织特异性表达模式进行分析。[结果]表明:PtRRI在杨树根部、形成层、木质部表达量相对较高,PtRRI转录受6-BA激素诱导,与其在不定根发育过程中激素调控下的表达相一致。[结论]PtRRI基因可能参与杨树的次级生长。     

泡桐丛枝植原体pPaWBNy-2-ORF4编码蛋白的抗体制备和表达分析

以感病泡桐组培苗提取的DNA为模板,采用PCR方法扩增pPaWBNy-2-ORF4的部分片段.将目的片段克隆到原核表达载体pGEX-4T-3,重组质粒pGEX-p2ORF4转化大肠杆菌Rosseta(DE3)菌株.IPTG诱导表达,分子量约为38 kDa的含GST标签的融合蛋白得到表达.切胶回收目的蛋白,免疫大白兔制备抗血清.间接ELISA测定抗血清的效价约为1:4096,免疫印迹实验显示:抗血清能够与原核表达的GST融合蛋白发生特异的免疫反应,与pPaWBNy-1-ORF5的原核表达蛋白无明显的交叉反应.利用制备的抗血清,在感病泡桐饲毒的茶翅蝽中检测到分子量约为18 kDa的蛋白条带,而在无菌茶翅蝽和感病泡桐组培苗中均未检测到,表明pPaWBNy-2-ORF4在饲毒的茶翅蝽中表达,而在感病泡桐组培苗中未表达或表达量低于检测水平.据此推测,该基因参与茶翅蝽传播泡桐丛枝植原体.     

Comparison of gene delivery methods for transformation ofRobinia pseudoacacia L. by Ti-plasmid

The binary vector plasmid pIG121-Hm carrying the chimeric neomycin phosphotransferase (npt II), β-glucuronidase (gusA) and hygromycin phosphotransferase (hpt) genes was delivered intoRobinia pseudoacacia L. callus using theAgrobacterium tumefaciens-mediated transformation method and particle gun transformation method. It was determined that adding 10 mg/L of acetosyringone toAgrobacterium infection medium and callus-Agrobacterium co-culture medium could increaseAgrobacterium-mediated transformation efficiency. In contrast, particle gun transformation could successfully deliver plasmid DNA into the callus, but its transformation efficiency was lower and gene expression was transient in comparison with theAgrobacterium-mediated method, suggesting that substantially stronger promoters might be required.     

Foliar Carbon Isotope Composition (δ 13C) and Water Use Efficiency of Different Populus deltoids Clones Under Water Stress

Foliar carbon isotope composition (δ ¹³C), total dry biomass, and long-term water use efficiency (WUE_L) of 12 Populus deltoids clones were studied under water stress in a greenhouse. Total dry biomass of clones decreased greatly, while δ ¹³C increased. Single-element variance analysis in the same water treatment indicated that WUE_L difference among clones was significant. Clones J₂, J₆, J₇, J₈, and J₉ were excellent with high WUE_L. Extremely significant δ ¹³C differences among water treatments and clones were revealed by two-element variance analysis. Water proved to be the primary factor affecting δ ¹³C under water stress. It showed that there was a good positive correlation between δ ¹³C and WUE_L in the same water treatment, and that a high WUE_L always coincided with a high δ ¹³C. δ ¹³C might be a reliable indirect index to estimate WUE_L among P. deltoids clones. Translated from Scientia Silvae Sinicae, 2005, 41(1) (in Chinese)     

Development of transgenic hybrid sweetgum (<Emphasis Type="Italic">Liquidambar styraciflua</Emphasis> × <Emphasis Type="Italic">L. formosana</Emphasis>) expressing γ-glutamylcysteine synthetase or mercuric reductase for phytoremediation of mercury pollution

Using Agrobacterium-mediated gene transfer, we generated transgenic hybrid sweetgum (Liquidambar styraciflua × L. formosana) overexpressing two types of genes to enhance plant remediation of mercury-contaminated soil and water: bacterial γ-glutamylcysteine synthetase gene (ECS), the first and most important enzyme in phytochelatin synthesis, or various genes encoding a mercuric ion reductase (merA9, merA18, merA77). Hybrid sweetgum proembryogenic masses (PEMs) constitutively overexpressing ECS were able to grow in the presence of 50 μM HgCl₂, which inhibited wild-type PEMs, but plantlets regenerated from the PEMs had abnormal form and did not survive for more than a few weeks following germination. In contrast, mature somatic embryos generated from PEMs constitutively overexpressing merA9 and merA18 converted to normal plantlets on germination medium containing 25 μM HgCl₂, while control embryos were killed on 25 μM Hg(II)-medium. Transgenic merA plantlets displayed enhanced resistance to Hg(II) and released Hg(0) two to three times more efficiently than the wild-type plantlets.     

Organic acid exudation from the roots of Cunninghamia lanceolata and Pinus massoniana seedlings under low phosphorus stress

Organic acid exudation from the roots of Chinese fir (Cunninghamia lanceolata) and Masson pine (Pinus massoniana) seedlings under low phosphorus stress was studied using the solution culture method. The results revealed that organic acid exudation from the roots of Chinese fir and Masson pine seedlings under low phosphorus stress increased. Compared with P₃ (KH₂PO₄, 0.5 mmol/L), the average organic acid exudation from the root of Masson pine seedlings under P₀ (KH₂PO₄, 0 mmol/L), P₁ (KH₂PO₄, 0.03 mmol/L) and P₂ (KH₂PO₄, 0.09 mmol/L) increased by 328.6%, 267.9% and 126.4% respectively. Masson pine from Zhejiang Province in China had the highest organic acid exudation. Under low phosphorus stress, the increase in organic acid exudation from the different provinces of Chinese fir and Masson pine varied. Masson pine from Zhejiang Province mainly increased oxalic acid, tartaric acid, citric acid and malic acid exudation, that from Guangxi Province mainly increased oxalic acid and tartaric acid exudation, and that from Guizhou Province, China mainly increased oxalic acid, tartaric acid and malic acid exudation. Chinese fir mainly increased oxalic acid and tartaric acid exudation. __________ Translated from Journal of Nanjing Forestry University, 2007, 31(2): 9–12 [译自: 南京林业大学学报]     

Effects of light radiation intensity on photosynthetic characteristics and water use efficiency of Platycladus orientalis and Pinus tabulaeformis seedlings

In order to offer a scientific basis for cultivation and management of forests, effects of light radiation intensity on photosynthetic characteristics and water use efficiency of Platycladus orientalis and Pinus tabulaeformis were studied under different soil moisture contents. By adopting artificial control methods to soil moisture, and under simulated photosynthetic radiation (SPR), the net photosynthetic rate (P _N), transpiration rate (T _r), water use efficiency (WUE) and intercellular CO₂ concentration (C _i) of Platycladus orientalis and Pinus tabulaeformis in the semi-arid region of the Loess Plateau, were studied. Results are as follows: within the photon range of 0–2,200 μmol/(m²·s), P _N, T _r and WUE were enhanced with an increase in SPR in both species. P _N and WUE of Platycladus orientalis and Pinus tabulaeformis, however, declined with continued increase in SPR. P _N, T _r, WUE and light saturation point (LSP) of Platycladus orientalis were higher than those of Pinus tabulaeformis, while light compensation point (LCP) of Platycladus orientalis was lower than that of Pinus tabulaeformis at the same soil moisture content. The efficiency of light energy utilization of Platycladus orientalis was higher than that of Pinus tabulaeformis; P _N, T _r and C _i of Platycladus orientalis and Pinus tabulaeformis were enhanced by increasing soil moisture content, whereas WUE declined. At soil moistures of 7.90%, 13.00% and 19.99%, LSP of Platycladus orientalis was 1,275, 1,450 and 1,675 μmol/(m²·s) respectively, and LCP was 42, 25 and 13 μmol/(m²·s) respectively, with corresponding maximal net CO₂ photosynthetic rates (P _max) of 3.04, 4.06 and 5.53 μmol/(m²·s). At soil moistures of 7.83%, 13.04% and 20.15%, the LSP of Pinus tabulaeformis was 1,100, 1,325 and 1,500 μmol/(m²·s) respectively, and LCP was 60, 30 and 23 μmol/(m²·s), with P _max of 1.08, 3.35 and 4.36 μmol/(m²·s) respectively. __________ Translated from Science of Soil and Water Conservation, 2006, 4(3): 108–113 [译自: 中国水土保持科学]     

Investigation of anti-salt stress on tetraploid Robinia pseudoacacia

Tetraploid Robinia pseudoacacia was used as a main test material and diploid R. pseudoacacia was used as the control. The indices of shape, physiology and biochemistry, photosynthesis and anatomic structure of the young plants were investigated under salt stress (NaCl and Na₂SO₄). The treatment time was 30 d with an interval time of 7 d. Before and after treatment, the indices were measured. Results show that: 1) the growth of diploid R. pseudoacacia inhibited an evident symptom of salt damage and the leaf moisture content was lower under salt stress than that of control. But the tetraploid R. pseudoacacia was contrary. 2) The relative electric conductivity and proline (Pro) of tetraploid R. pseudoacacia increased slightly and had no significant difference compared with its control, which was contrary to diploid R. pseudoacacia. At the same time, three protective enzymes including perocidase (POD), superoxide (SOD) and catalase (CAT) kept higher activities at a post stage of salt stress to tetraploid R. pseudoacacia, which enhanced its anti-salt characteristics. Diploid R. pseudoacacia was sensitive to salt and had contrary information. 3) Salt stress had little influence to photosynthesis of tetraploid R. pseudoacacia. The net photosynthetic rate (P _n) and intercellular CO₂ concentration (C_i) had no significant changes, but those of diploid R. pseudoacacia decreased singificantly. 4) After salt stress, the anatomic structure of tetraploid R. pseudoacacia had a positive reaction, including the palisade parenchyma of diachyma, was prolonged and arranged more tightly. The spongy parenchyma was shrunk and was arranged tightly, which was contrary with diploid R. pseudoacacia. These data demonstrate that tetraploid R. pseudoacacia had superior anti-salt performance. __________ Translated from Journal of Plant Ecology, 2008, 32(3): 654–663 [译自: 植物生态学报]     

Tangential and radial shrinkage variation within trees in sugi (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>) cultivars

The transverse shrinkage variation within trees was examined for five sugi cultivars. The within-tree trends of tangential shrinkage (α _T) were different by cultivar, whereas radial shrinkage (α _R) increased from pith to bark in most cultivars. The tangential/radial shrinkage ratio (α _T/α _R) decreased from pith to bark in most cultivars, because the radial variation of α _R was larger than that of α _T. The cultivars showed significant differences among cultivars in α _T, α _R, and α _T/α _R, but the difference among cultivars for α _T/α _R was smaller. The relationships between transverse shrinkage and microfi bril angle (MFA), basic density (BD), tree ring parameters, and modulus of elasticity were examined. The α _T and α _R showed positive relationships with BD, latewood percentage, latewood density, and modulus of elasticity, and negative relationships with MFA and ring width. The relationships with earlywood density were weak. Sugi exhibited variation in transverse shrinkage within stem and among cultivars, with the variation affected by MFA, density, and tree ring parameters. Part of this article was presented at the 56th Annual Meeting of the Japan Wood Research Society, Akita, Japan, August 2006, and the 57th Annual Meeting of the Japan Wood Research Society, Hiroshima, Japan, August 2007     

At5g54160 gene encodes <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 5-hydroxyconiferaldehyde <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The function of an Arabidopsis thaliana gene, At5g54160 annotated as a caffeic acid O-methyltransferase CAOMT gene was characterized. The recombinant enzyme of this gene (AtOMT1) catalyzed the O-methylation of phenylpropanoid and flavonoid substrates. The specificity constants (k _cat/K _m) for 5-hydroxyconiferaldehyde (5-HCAld) and quercetin were both 0.11 μM⁻¹·min⁻¹. On the other hand, lignins of At5g54160-knockout Arabidopsis mutants lacked syringyl units. In addition, we showed that the gene silencing also resulted in significant accumulation of caffeyl alcohol (CaAlc). These results strongly suggested that At5g54160 gene is involved in syringyl lignin synthesis for the methylation of both 5-hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compound(s). Part of this work was presented at the Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry, March 24–27, 2007     

Potential to harness superior nutritional qualities of exotic baobabs if local adaptation can be conferred through grafting

Baobab leaves form an important part of the local diet in Sahel countries and elsewhere in Africa. Existing leaf nutritional data and agroforestry performance information are based solely on Adansonia digitata L., the baobab of continental Africa. The introduction potential of Adansonia species from the center of diversity in Madagascar and from Australia remains untapped. To assess this potential, the mineral contents and B₁ and B₂ vitamin levels of dried baobab leaves were determined for five-year old trees of A. digitata, A. gibbosa (A. Cunn.) Guymer ex D. Baum, A. rubrostipa Jum. & H. Perrier (syn. A. fony Baill.), A. perrieri Capuron and A. za Baill. grown in an introduction trial in Mali. Nutritional data were evaluated against survival and vigor to identify promising germplasm. Leaf vitamin and crude protein contents were highest in the Madagascar species, especially A. rubrostipa (B₁ 88 mg 100 g⁻¹, B₂ 187 mg 100 g⁻¹, protein 20.7% dry weight). However, the local species far outperformed the introductions in survival, tree height, basal diameter and resistance to termites. We suggest grafting as a way of harnessing the vigor of well-adapted local baobab varieties to the superior nutritional profiles of A. rubrostipa and others. Cross-species grafting tests in Adansonia were successful, thus creating new agroforestry possibilities with different scion/rootstock combinations.     

Programmed cell death of Ulmus pumila L. seeds during aging

The programmed cell death (PCD) characteristics of Ulmus pumila L. seeds were investigated. The seeds were treated at a high temperature of 37°C and 100% relative humidity for six days. DAPI (4′6-diamidino-2-phenylindole) staining revealed that the aging treatment induced condensation and margination of chromatin, as well as the formation of apoptotic bodies. DNA electrophoresis results of U. pumila seeds on an agarose gel showed a characteristic “ladder” pattern. Levels of electrolyte leakage of seed cells showed that membranes retained their integral form during almost the entire aging time. There was an immediate increase in the production rate of superoxide anion (O₂ ⁻) and in the amount of hydrogen peroxide (H₂O₂), which remained at a μmol level. All of these common characteristics indicate that seed aging can be classified as PCD. __________ Translated from Journal of Beijing Forestry University, 2007, 29(3): 8–14 [译自: 北京林业大学学报]