根癌农杆菌介导的反义VeFAD2基因对少根根霉的遗传转化及其影响因子

Agrobacteriurn tumefaciens-mediated transformation (AtMT) method was used to study the genetic transformation in Rhizopus arrhizus of antisense expression vector pBI121-fad2 of VeFAD₂ gene. We studied the key factors influencing transformation, such as A.tumefaciens strains,bacterial cell volume initially used, co-cultivation time, effective period and concentration of AS. The results shows that the ideal A.tumefaciens strain is AGL-1, the optimum bacterial cell volume initially used is 50-100 μL and co-culture 24 hours will get the most transformants. At co-culture period, AS induction is indispensable. It is beneficial to improve transformation ratio by adding to 200 μmol·L^-1 AS at preincubate period and increasing AS concentration to 400-600 μmol·L^-1 at co-culture period. It determined that antisense VeFAD₂ gene has been integrated into the genome of R. arrhizus by PCR-Southern detection.     

花吊丝竹组培快繁育苗技术研究

The rapid propagation technology of Dendrocalamus minor var. amoenus was studied by investigating the effects of some factors such as selection of explant, phytohormone, culture method etc. The results show that: The best month for explant collection of D. minor var. amoenus is May and June. The best position for explant collection is middle-upper part knot of semi-lignification branch. The clump shoot could be induced in medium with 3/4MS+BA 4 mg·L^-1+KT 1 mg·L^-1+CW 100 mL·L^-1. The optimal medium for subculture of D. minor var. amoenus is 3/4MS+BA 2 mg·L^-1+KT 1 mg·L^-1+CW 100 mL·L^-1. Liquid medium is beneficial for improving growth condition and proliferation rate of clump shoot. The medium 1/5MS+IBA 8 mg·L^-1+ NAA 4.5 mg·L^-1 + KT 0.1mg·L^-1 is a relative suitable rooting medium for D. minor var. amoenus, with the rooting method of synchronized treatment first and then rooting. The survival rate of seedlings could be higher than 90% in substrate of fine river sand∶peat soil=3∶ 1.     

牡丹‘乌龙捧盛’组培苗生根及生根解剖学研究

The tissue-cultured seedlings of tree peony ‘Wulong Pengsheng’ were used to study the effects of different plant growth regulators, culture methods, and holdfast on rooting. The morphological structure change during rooting was also observed using the method of paraffin section. The result showed that the best combination of plant growth regulators for rooting was IBA 3.0 mg·L^-1 + NAA 0.6 mg·L^-1. The treatment under the temperature of 4℃ for ten days was benefit to rooting, and the rate could reach 75.67%. It was identified that the adventitious root primordia of shoot in vitro originated from the vascular cambium cells, especially, the cross areas of cambium and pith ray and they started to differentiate at the 5th day and lasted to the 12th day. If the shoots were cultured in the root inducing medium for 12 days, it would lead to not only descend of rooting rate, but also showing callus of stem base, and leaf senescent. However, if they were transferred into the medium without hormone in time, the root primordial protruded the epidermis and developed normally after 5 days’ culture.     

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.     

油茶SRAP-PCR反应体系的优化

Camellia oleifera is one of the important oil tree species in south China, and C. oleifera industry is quickly developed with the support of the national policies in recent years. The disorder of C. oleifera varieties is one of the key issues restricting the development of C. oleifera industry. Because of high polymorphism, good repeatability, less use of DNA and so on, SRAP as a new marker was used in identification of cultivars, analysis of genetic resources and genetic diversity in recent years. In this paper, the orthogonal design was used to optimize SRAP-PCR system for C. oleifera by 5 factors (Mg²⁺, dNTPs, primer, Taq polymerase, DNA template) and 4 levels, respectively. The data were analyzed by software SPSS V13.0. A suitable SRAP-PCR system (20 μL) was established as: 75 ng DNA template, 1.5 mmol·L^-1 Mg²⁺, 0.15 mmol·L^-1 dNTPs, 1U Taq DNA polymerase, 0.4 μmol·L^-1 primer, 1×PCR buffer. The result of optimal SRAP-PCR system will provide a foundation for the identification of C. oleifera cultivars.     

马占相思扦插繁殖技术研究

Effects of substrates, type of auxin, treatment concentration and time on rooting ability of Acacia mangium were systematically studied, and the change trend of rooting ability over time were analyzed. The results obtained from the experiment indicated that the substrate of yellow subsoil could improve the rooting rate of the cuttings significantly. IBA was superior to other auxins in increase the number of mean roots and the longest root length. And general rooting effect for different treatments was evaluated based on subordinate function values analysis. The optimized combinations were the cutting treated by IBA 400 mg·L^-1 soaked for 2 hours. Callus were occurred in 5 d after cutting, and higher rooting period occurred in 10—15 d and 25—30 d after cutting.     

基于SSR分子标记的杜仲遗传多样性体系建立

In order to set up the genetic diversity system of Eucommia ulmoides Oliver based on SSR molecular markers, the establishment of SSR-PCR reaction system and screening out SSR marker primer showing high polymorphism were studied. A L₉(3⁴) orthogonal design was performed to optimize the main factors of the SSR-PCR reaction system. The results indicated that the best SSR-PCR reaction system for E.ulmoides was DNA template 1 μL (30~60 ng·μL^-1), 2×Taq PCR Master Mix 10 μL, primer 1 μL with the total volume of 25 μL. The PCR reaction system had high stability and repeatability, the pairs of SSR primers with high polymorphism were gotten. The 8 E.ulmoides samples' DNA sequence was amplified with 13 pairs of SSR primers by SSR-PCR technique, 34 alleles were detected, 2.6 alleles were detected from per site on average. Each allele's effective number was 1.751 5, and the h value was 0.379 8, the average I value was 0.643 3. This study is helpful in using SSR molecular marker to analyze genetic diversity and genetic relationship in E. ulmoides.     

喷施营养液和NaHSO3对'蓝星'高山桧扦插生根的影响

In this study, we investigated the optimal concentration of nutrient-mist and NaHSO₃ for cutting propagation of Juniperus squamata 'Blue Star' , as well as the variation in the contents of chlorophyll in leaves, and solubility carbohydrate,soluble protein and other nutrition in leaves and bases of cuttings during rooting. Cuttings of grade Ⅲ (the lenghth of cutting bases lignified from 0.1 to 0.2 cm) was treated by 1/4MS+NaHSO₃(200 mg·L^-1),and the rooting rate rose 35% and root qualities were improved compared with control experiment, for there were different effects among three grade cuttings. The treatment of grade Ⅲ cuttings also resulted in higher content of soluble sugar and starch of those bases during rooting, especially obviously different for fifteen days, but less effect on the chlorophyll and some nutrition content of cutting leaves.     

鹅掌楸Genomic-SSR反应体系优化

The optimization of the Genomic-SSR reaction system is a basic protocol when the Genomic-SSR is used for genetic analysis in Liriodendron. The concentrations of Mg²⁺, dNTP, Primer and rTaq were tested by L₉(3⁴) orthogonal experiment and single factor gradient experiment to gain the optimal reaction system. The results indicated that the optimal reaction system should contain 75 ng of genomic DNA, 1 μL of 10×buffer, 0.4 μL of 10 mmol·L^-1 dNTP, 0.75 μL of 2.5 mmol·L^-1 MgCl₂, 0.25μL of 10 μmol·L^-1 Primer, 0.05 μL of 5 U rTaq and final volume of 10μL. Repeated trials and two verification tests showed that this optimal reaction system was stable, reliable, efficient and suitable for the applications of Genomic-SSR in Liriodendron population genetics and quantitative genetics research.     

In vitro propagation of a medicinal plant: Tripterygium wilfordii Hook f.

In this study a reliable protocol was developed for the establishment of commercial in vitro cultures of Tripterygium wilfordii Hook f.. Juvenile shoots from one-year-old elite plants were used as the source of explants. New axillary shoots were obtained after 30 days of culture on a MS medium supplemented with BAP (2.0 mg·L^–1) and NAA (0.1 mg·L^–1). The optimal multiplication medium was a modified MS medium supplemented with BAP (1.0 mg·L^–1) and NAA (0.1 mg·L^–1). This yielded a multiplication rate of 2.4 for each subculture. Slightly more than 92% of shoots rooted when cultured on a modified MS medium containing IBA (0.2 mg·L^–1) and acti-vated charcoal (0.5 mg·L^–1). Activated charcoal promoted both a strong and a high rooting rate during the rooting phase. Plantlets were transferred to pots for a short acclimatization stage in a greenhouse where 95% of the plantlets survived. This highly reproduci-ble procedure can be adopted for large-scale propagation of T. wilfordii.     

Photosynthetic characteristics of Fagus sylvatica and Quercus robur established for stand conversion from Picea abies

Efforts in Europe to convert Norway spruce (Picea abies) plantations to broadleaf or mixed broadleaf-conifer forests could be bolstered by an increased understanding of how artificial regeneration acclimates and functions under a range of Norway spruce stand conditions. We studied foliage characteristics and leaf-level photosynthesis on 7-year-old European beech (Fagus sylvatica) and pedunculate oak (Quercus robur) regeneration established in open patches and shelterwoods of a partially harvested Norway spruce plantation in southwestern Sweden. Both species exhibited morphological plasticity at the leaf level by developing leaf blades in patches with an average mass per unit area (LMA) 54% greater than of those in shelterwoods, and at the plant level by maintaining a leaf area ratio (LAR) in shelterwoods that was 78% greater than in patches. However, we observed interspecific differences in photosynthetic capacity relative to spruce canopy openness. Photosynthetic capacity (A₁₆₀₀, net photosynthesis at a photosynthetic photon flux density of 1600 μmol photons m⁻² s⁻¹) of beech in respect to the canopy gradient was best related to leaf mass, and declined substantially with increasing canopy openness primarily because leaf nitrogen (N) in this species decreased about 0.9 mg g⁻¹ with each 10% rise in canopy openness. In contrast, A₁₆₀₀ of oak showed a weak response to mass-based N, and furthermore the percentage of N remained constant in oak leaf tissues across the canopy gradient. Therefore, oak photosynthetic capacity along the canopy gradient was best related to leaf area, and increased as the spruce canopy thinned primarily because LMA rose 8.6 g m⁻² for each 10% increase in canopy openness. These findings support the premise that spruce stand structure regulates photosynthetic capacity of beech through processes that determine N status of this species; leaf N (mass basis) was greatest under relatively closed spruce canopies where leaves apparently acclimate by enhancing light harvesting mechanisms. Spruce stand structure regulates photosynthetic capacity of oak through processes that control LMA; LMA was greatest under open spruce canopies of high light availability where leaves apparently acclimate by enhancing CO₂ fixation mechanisms.     

一种新的白蜡虫寄生蜂体内Wolbachia的wsp基因分子检测及序列分析

采用Wolbachia的通用引物、A大组和B大组的特异性引物对一种新的白蜡虫寄生蜂--长尾啮小蜂(Aprostocetus sp.)体内Wolbachia的wsp基因进行分子检测,所获得的基因片段分别命名为wApr、wAprB和wAprB,长度分别为620、566和463bp;基因序列分析表明:wApA、wAprB与w...     

黑松和马尾松苗受松材线虫侵染后部分化学物质的响应

为研究松材线虫侵染对寄主植物生理生化物质代谢的影响,以黑松和马尾松4 5年生苗为实验材料,测定了接种松材线虫对2种寄主植物的营养物质和次生代谢物质含量的变化及其规律。结果显示:接种松材线虫初期,黑松和马尾松的总糖含量均较高,随接种时间的延长,总糖含量呈不断下降趋势;黑松的可溶性糖含量一直降低并明显低于对照,马尾松的可溶性糖含量在侵染前期与对照相比变化不明显,而15 d后快速降低;黑松和马尾松的可溶性蛋白含量侵染前期均低于对照,后升高,再降低;黑松的单宁含量明显高于对照,马尾松的单宁含量接种第1天略低于对照,第3天后开始升高;黑松和马尾松的总酚含量均在侵染前期高于对照,而后降低。这些物质的变化趋势显示松材线虫侵染不同寄主植物的生理反应。     

三年桐、千年桐感染枯萎病病原菌后的生理反应

枯萎病是油桐毁灭性病害。中国广泛种植的油桐有三年桐和千年桐,三年桐易感枯萎病,千年桐抗枯萎病。为探讨三年桐与千年桐在枯萎病应答过程中的差异,在对油桐枯萎病灾区调研的基础上,进行枯萎病病原菌的分离、鉴定,并进一步探讨三年桐、千年桐接种枯萎病病原菌后超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量的变化。结果表明:油桐枯萎病病原菌为尖孢镰刀菌,接种试验显示千年桐在接种尖孢镰刀菌后SOD、POD、CAT较三年桐均呈现较高活性,在接种后SOD、POD活性先升高后降低,CAT活性升高并维持高活性,MDA含量变化不明显;三年桐SOD活性和MDA含量均先升高后降低,POD活性先降低再升高,CAT活性先降低后升高又降低。千年桐作为抗病种,其抗病性可能与其本身具有较高的SOD、POD、CAT活性有关,并且和病原菌感染后酶活上升有关;三年桐为易感病种,接种后SOD、POD、CAT活性有变化,但仍不能抵御病原菌的危害。     

麻疯树磷酸烯酮式丙酮酸羧化酶em>pepc基因全长cDNA克隆及序列分析

应用RT-PCR和RACE法从麻疯树总RNA中分离出pepc基因全长cDNA,长度为3 142 bp,阅读框2 898 bp,编码965个氨基酸,在GenBank中登录,序列号为EU069413。它的氨基酸序列与蓖麻、陆地棉、橙、大豆、花生、烟草、油菜、拟南芥的氨基酸同源性分别为94.94%、92.46%、90.60%、90.50%、90.50%、88.33%、84.61%、82.44%。该基因编码了pepc基因家族中的pepc1,蛋白属于C₃型PEPC。推测了pepc编码蛋白分子量为110.6 kD,并对其稳定性、二级结构、疏水性等特性进行了分析,最后确定了该蛋白的功能位点和结构域。     

普通油茶无性系花粉离体萌发特性

以6个普通油茶无性系花粉为试验材料,采用正交试验研究了不同温度、硼元素用量、蔗糖浓度、琼脂用量对花粉萌发的影响,以揭示普通油茶花粉在离体条件下的萌发特性。结果表明:4个影响花粉萌发率的试验因素中,温度是极显著影响因素,以25 ℃最优;蔗糖浓度也能显著影响多数无性系,以10%最优;硼元素用量和琼脂用量仅对个别无性系有显著影响,分别以100 mg·kg^-1和0.5%最优。结合多重比较,处理T9为试验优选组合;花粉管的生长从开始到停止略呈"慢—快—慢"的抛物线趋势;稍高的钙离子浓度显著抑制花粉萌发,低浓度锌、钼离子对花粉萌发略有促进,高浓度则效应为负。     

马尾松钙调素基因的克隆及响应松材线虫侵染的表达特征

松材线虫病是由松材线虫(Bursaphelenchus xylophilus)侵染引起的严重病害,引起重大的经济和生态损失。钙调素(CaM)是真核生物中Ca2+主要传导蛋白,本研究采用RT-PCR方法,首次从马尾松中克隆获得CaM,命名为pmCaM,并检测了其响应松材线虫侵染的表达特征。序列分析表明:pmCaM完整的开放阅读框核苷酸序列为450bp,编码149个氨基酸的蛋白质;该蛋白含有4个EF-hand结构域,具有钙调素的典型特征,与其它植物的CaM蛋白均有较高同源性。实时荧光定量PCR分析显示:接种松材线虫后30 180 min时间段,马尾松苗根、茎、叶器官中的pmCaM均下调表达,但不同器官间显著下调表达的时间点存在差异;同时,不同器官pmCaM表达量变化均存在特异的随时间发展的波动特征,其中发现,根茎pmCaM在松材线虫接种45 min时均处于表达量高峰。本研究结果显示pmCaM表达响应了松材线虫侵染,揭示pmCaM可能参与了调控松材线虫-马尾松互作早期的钙信号响应。     

银杏过氧化氢酶基因CAT1的克隆及表达分析

利用RACE技术从银杏中克隆到过氧化氢酶基因(GbCAT1)的cDNA全长。进化树分析结果表明:GbCAT1和其他物种的CAT源自于相同的祖先。Southern杂交显示:GbCAT1属于1个小的多基因家族。实时定量RT-PCR分析表明:GbCAT1在银杏的根、茎、叶和果中都有表达,在叶中的相对表达量最高,其次为果、茎和根。GbCAT1的转录受到ABA、渗透压、紫外、低温和高温胁迫的诱导。水杨酸处理下,GbCAT1相对表达量迅速降低。CAT1基因在逆境条件下的相对表达变化与环境胁迫有关。