农杆菌介导慈竹4CL基因遗传转化梁山慈竹

以梁山慈竹2种类型成熟胚的愈伤组织为材料,采用农杆菌遗传介导的方法,将已构建好的具有降低木质素含量的PBI121-4CL-RNAi表达载体导入愈伤组织,探讨愈伤组织预培养时间、菌液浓度、侵染时间、共培养时间和温度对遗传转化的影响。研究结果表明,淡黄色、颗粒状、疏松易碎的胚性愈伤组织是较好的遗传转化材料。以在愈伤组织培养基上预培养8天的淡黄色、颗粒状、疏松易碎的胚性愈伤组织为转化受体,在菌液浓度为OD600=0.05的EHA105中侵染20min后,在25℃、黑暗条件下共培养2天(共培养基表面加一层无菌滤纸),在含有卡那霉素为55mg.L-1的抗性筛选培养基上筛选30天,抗性愈伤组织率为90%,经PCR检测,慈竹4CL基因已导入梁山慈竹愈伤组织中。抗性愈伤组织在芽诱导培养基上诱导30天,可获得丛生芽,待丛生芽长至3～5cm后,在生根培养基上经过20～30天的诱导,可产生1～8条根,获得再生植株。经PCR检测,慈竹4CL基因已导入梁山慈竹再生植株中,获得了转基因植株,转化效率为9%。RT-PCR检测结果表明,转4CL基因的梁山慈竹愈伤组织和植株的内源4CL基因的表达受到抑制,且表达量比对照明显降低。     

农杆菌介导高羊茅基因转化体系的建立

以高羊茅(FestucaarundinaceaSchrebb)品系宾哥(Bingo)为材料,建立了农杆菌介导的基因转化方法。高羊茅种子在9mgL-12,4-D作用下,获得胚性愈伤组织。愈伤组织经农杆菌侵染,在30和50mgL-1的潮霉素浓度梯度下筛选,得抗性愈伤组织。组织化学染色法证实,uidA基因在抗性愈伤组织中表达:抗性愈伤组织在分化培养基中分化得到转基因植株。PCR、Southern杂交法证实外源基因已导入到高羊茅植株中。图5参37。     

农杆菌介导的杜仲叶片愈伤组织遗传转化体系

【目的】优化杜仲叶片愈伤组织的再生体系,研究愈伤组织对抗生素和抑菌剂的敏感性,探索影响农杆菌介导杜仲愈伤组织遗传转化的最适转化因子水平,构建农杆菌介导的杜仲愈伤组织瞬时转化体系,使杜仲成年植株外植体为受体的遗传转化成为可能,为杜仲基因功能的研究与定向改良奠定基础。【方法】以杜仲成年植株叶片为材料诱导愈伤组织,通过添加不同浓度植物生长调节剂与大量元素的MS培养基进行不定芽的诱导与增殖,确定最适培养基。在此基础上,在培养基中添加不同浓度抗生素及抑菌剂,研究愈伤组织对其敏感性。以获得的叶片愈伤组织受体系统为基础,通过L_(16)(4~5)的正交试验,探索不同转化因子对农杆菌介导叶片愈伤组织遗传转化的瞬时转化效率的影响,建立最适瞬时转化体系。使用获得的瞬时转化体系对愈伤组织进行遗传转化操作,筛选抗性芽,对抗性芽进行GUS组织化学染色与PCR检验。【结果】再生体系优化的结果表明,3/4大量元素浓度的MS培养基能够促进杜仲愈伤组织不定芽的诱导及生长;愈伤组织诱导不定芽的最适培养基为3/4MS+0. 27μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,诱导率为83%±10. 0%;不定芽复壮的最适培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,平均伸长长度为(2. 47±1. 33) cm。抗生素与抑菌剂敏感性试验表明,遗传转化的选择培养基中,抑菌剂头孢霉素的最适浓度为200 mg·L~(-1),筛选用的抗生素卡那霉素最适浓度为70 mg·L~(-1)。转化因子的正交试验表明,最适的农杆菌介导杜仲叶片愈伤组织遗传转化的转化因子组合为:预培养5天、侵染10 min和共培养3天。使用最适瞬时转化体系对约200个愈伤组织进行遗传转化操作,共筛选获得3个抗卡那霉素的抗性芽; GUS组织化学染色显示GUS基因在抗性芽中得到了表达,PCR检测证明这些抗性芽中存在NPTⅡ基因。【结论】杜仲叶片愈伤组织诱导不定芽的最适培养基为3/4MS+0. 27μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,不定芽复壮的最适培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA。农杆菌介导的杜仲叶片愈伤组织瞬时转化体系为:预培养5天、侵染10 min和共培养3天,筛选培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA+200 mg·L~(-1)Cef+70 mg·L~(-1)Km。利用此体系共获得3个抗性芽,PCR分析和GUS组织化学染色都表明T-DNA已整合到抗性芽基因组中。     

微弹介导的火炬松遗传转化

本文建立了一个微弹介导的火炬松遗传转化系统。这个系统解决了火炬松遗传转化过程中存在的许多困难。运载抗虫基因的质粒载体经微弹转化法进入火炬松成熟合子胚,然后在添加了卡那霉素的培养基上从转化的成熟胚上诱导出有器官发生潜力的愈伤组织,再从转化的愈伤组织上产生转基因植株。利用这一系统生产的转基因植株已经被随机扩增技术、Southern杂交技术和虫试验所证实,并且转化的植株已在土壤中成活。图3表2参28。     

农杆菌介导的楸树遗传转化体系

【目的】以楸树胚性愈伤组织为受体,建立有效的楸树遗传转化体系,为今后楸树性状的遗传改良奠定基础。【方法】通过农杆菌EHA105介导以胚性愈伤组织作为外植体进行遗传转化,通过正交试验获得最优的遗传转化条件,进而将外源基因转入到楸树基因组中。【结果】在1/2 MS培养基中添加不同梯度浓度的卡那霉素(Kana)进行选择压力筛选,在添加了60 mg·L~(-1)Kana的1/2 MS培养基中,楸树胚性愈伤组织的分化率为0.00%,存活率仅为5.71%,因此确定60 mg·L~(-1)为遗传转化的选择压。采用正交设计L18(37)进行农杆菌介导的楸树遗传转化试验,通过GUS化学组织染色统计瞬时表达率,正交试验直观分析和单因素方差分析结果表明:在预培养时间为2天,采用农杆菌菌株EHA105、菌液浓度OD600值为0.7、添加乙酰丁香酮(AS)浓度为300μmol·L~(-1)、侵染时间为10 min,共培养时间为5天的条件下,农杆菌介导的转化效率最高,且对转化效率影响最大的2个因素是乙酰丁香酮浓度和预培养时间。对浸染后的胚性愈伤组织进行8个月的筛选培养,共获得32个抗性组织团,对其中15个增殖较多的抗性愈伤组织进行PCR检测,表明86.67%的抗性组织团中有外源基因整合到楸树基因组中。内源激素水平会对植物体细胞胚分化产生影响,细胞分裂素(CTK)和脱落酸(ABA)促进体胚发生,生长素(IAA)和赤霉素(GA)对体胚发生有抑制作用。通过测定内源激素可知,转基因的抗性组织中内源CTK和ABA水平显著低于野生型的楸树胚性愈伤组织,而内源IAA和GA则显著高于野生型胚性愈伤组织,推测内源激素水平可能是转基因抗性组织体胚分化能力比较差的原因。【结论】建立了农杆菌介导的楸树胚性愈伤组织的遗传转化体系,对筛选获得的15个抗性愈伤组织进行PCR检测,其中13个抗性愈伤组织中有外源基因的整合。内源激素水平的变化可能是导致楸树转基因抗性愈伤组织难以分化的原因。     

甜椒抗菌蛋白基因(harp)转化桉树的研究

用不同浓度的2,4-D与IAA对桉树叶盘进行了愈伤组织的诱导及植株再生的试验,建立了新的桉树再生体系.进一步用含甜椒抗菌基因(hrap)的农杆菌侵染桉树叶盘,发现侵染后对愈伤组织诱导率无明显影响,却抑制了桉树的植株再生.对再生苗的PCR和Sou thern杂交检测证实已将hrap基因已转入桉树植株的基因组中.     

柞蚕抗菌肽D基因转化桉树培育抗青枯病株系的研究

通过二次正交旋转组合设计对尾叶桉叶盘愈伤组织分化培养基优化 ,获得尾叶桉 (Eucalyptusuro phylia)叶盘外植体的再生植株。将携带外源目的基因的根癌农杆菌 (Agrobacteriumtumefaciens)与尾叶桉U6无性系叶盘共培养 ,经愈伤组织诱导、卡那霉素筛选和植株再生 ,获得 2 0个小芽 ,再将小芽转入附加卡那霉素(Kam) 4 0 μg·mL- 1 的E3-B培养基中诱根 ,获得 8株存活且可再生的转化子。取转化苗叶片作胭脂碱合成酶活性检测电泳后呈阳性 ;分别以γ 32 p dCTP及地高辛标记柞蚕抗菌肽D基因作探针 ,与转化苗DNA作点杂交及Southern印迹杂交检测 ,结果均呈阳性。以上结果表明 ,柞蚕抗菌肽D基因已整合到尾叶桉基因组中。将从桉树青枯病重病区分离的强毒青枯菌 (Pseudomonasspp .)株 (9910 16 )以 1× 10 9cfu·mL- 1 浓度接种转化试管苗 ,以未经转基因的尾叶桉U6 无性系试管苗为对照 ,结果表明转化苗接种死亡率 5 6 7% ,对照死亡率为 86 7% ,由此可推出导入柞蚕抗菌肽D基因的尾叶桉转化苗明显提高了对青枯菌的抗性     

四倍体刺槐转甜菜碱醛脱氢酶基因的研究

试验以所建立的四倍体刺槐高频再生体系为基础,通过农杆菌介导法转化甜菜碱醛脱氢酶(BADH)基因,以GUS染色组织分析法为依据探讨了影响转化效率的各种因素,建立了优化转化体系,结果如下:20mg·L~(-1)乙酞丁香酮可显著提高转化效率;菌液浓度OD_(600)值0.3～0.7、预培养2d为宜;转化后暗培养对转化效率没有影响。并在以上研究基础上,成功地建立了高效、可重复的遗传转化体系,选择培养基上头孢霉素400mg·L~(-1)可有效地抑制农杆菌;卡那霉素50mg·L~(-1)时愈伤组织的白化死亡率达96.4％。经PCR检测,外源基因已成功的整合到植株的基因组DNA中,获得了15个转基因株系。     

反义磷脂酶Dγ基因与几丁质酶基因转化美洲黑杨G2

建立美洲黑杨G2组培再生体系,确立美洲黑杨G2分化最适宜培养基和生根培养基.然后分别进行卡那霉素和潮霉素敏感性试验,利用叶盘法依次将反义磷脂酶Dγ(Anti-PLDγ)基因和几丁质酶(CH5B)基因转入美洲黑杨G2中.首先转入反义磷脂酶Dγ基因,经诱导不定芽及生根阶段卡那霉素(选择性抗生素)连续筛选,获得了21株卡那霉素抗性植株.抗性植株经PCR及PCR-Southern杂交检测,有13株均呈阳性,证明Anti-PLDγ基因成功整合到美洲黑杨G2基因组中.耐盐性试验表明,4株转基因植株抗NaCl能力比对照都有不同程度提高.选择4号株系继续转入几丁质酶基因,通过潮霉素(选择性抗生素)筛选不定芽及诱导生根获6株潮霉素抗性植株,经PCR及PCR-Southern杂交检测,6株均呈阳性,证明CH5B基因成功整合到美洲黑杨G2基因组中.由于CH5B基因与报告基因GUS处于同一开放阅读框(ORF)内,因此通过GUS基因的组织化学染色分析,证明了6株转化植株几丁质酶基因均获得正常表达.     

银腺杨转Cry I Ac和API双价抗虫基因的研究

在建立以银腺杨(84K)叶片为外植体的再生系统基础上,通过农杆菌介导法把Cry I Ac和API双价抗虫基因导入银腺杨(84K)基因组中.转化杨树叶片,在含有卡那霉素的培养基上诱导不定芽和诱导生根,获得了400株卡那霉素抗性转化再生植株.抗性植株经PCR检测,有70株呈阳性.通过Southern杂交和ELISA检测进一步证明Cry I Ac和API双价抗虫基因已整合到银腺杨(84K)基因组中,拷贝数1～2个,并得到了表达.转化植株用杨扇舟蛾幼虫进行饲虫试验,结果表明昆虫幼虫的死亡率高达60.0%～80.0%.同时,存活幼虫的生长发育也受到了明显抑制.本研究结果为杨树抗虫育种提供了新的种质资源.     

论法治与德治的辩证统一

法治既是一种治国方式和手段,也是人民行使管理国家、管理社会的权利的途径.德治的核心内容是要建立社会主义的道德体系和弘扬优秀的民族精神.法治和德治既有区别,又有联系,是辨证统一的.法治、德治的辩证统一是贯彻"三个代表"重要思想和建设中国特色社会主义的内在要求.     

Inhibition by pulp juice and enhancement by ingestion on germination of bird-dispersedPrunus seeds

To evaluate what prevents seed germination and why ingestion by birds enhances germination, we carried out germination experiments using bird-dispersed seeds of twoPrunus species (Prunus sargentii andP. ssiori) in the laboratory. Germination of seeds with following four treatments was compared: “Ingested seeds” excreted after feeding of fruits to birds; “Extracted seeds” deliberately extracted from the fruit pulp; “Juiced seeds” which were supplemented with the juice of the pulp after seeds had been deliberately extracted from the pulp; and “Intact seeds” in untreated whole fruits. Many ingested (27%, 23% inP. sargentii, 18% inP. ssiori) and extracted seeds (24% inP. sargentii, 17% inP. ssiori) germinated, and difference in germination percentages between ingested and extracted seeds were not significant, for eitherPrunus species. The Juiced seeds rarely germinated (2% inP. sargentii, 6% inP. ssiori), and seeds within intact fruits did not germinate. These results suggest that the fruit pulp and the juice of the pulp inhibit germination and the seeds could germinate without ingestion by birds once they had been manually extracted from pulp. Therefore, it is considered that bird ingestion enahnces germination by removing pulp, especially juice of the pulp, in these twoPrunus species.     

Suppression of debris movement by forests and damage to forests by debris deposition

 Forest buffer zones have recently been introduced in an attempt to suppress debris movement caused by mass wasting. There are, however, many questions left unsolved regarding the role of forests in suppressing debris movement and damage to forests by debris deposition. A number of debris avalanches occurred in southern Fukushima Prefecture and Hiroshima Prefecture in August 1998 and June 1999, respectively. Suppression of debris movement by forests and damage to forests by debris deposition were investigated in this study using aerial photographic interpretation and topographic analysis. Of the debris avalanches delineated in the Fukushima and Hiroshima areas, 282 and 84 sites, respectively, were forested, and 43 and seven sites, respectively, were nonforested. Topographic parameters, land use, and forest type at each site were surveyed by aerial photographic interpretation and topographic analysis. Suppression of debris movement by forest vegetation was confirmed by higher equivalent coefficients of friction and shorter average deposition zone length (about 28% and 55% shorter in the Fukushima and Hiroshima areas, respectively) in forested sites compared with nonforested sites. While previous studies suggested that the width of the forest zone required to prevent sediment outflow by surface erosion increases with increasing slope gradient, no clear relationship was found in this study. While the length of deposition zone of debris material (this study) is strongly affected by the dynamic solid friction coefficient and fluid friction of debris material, the length of deposition zone of the outflow of surface wash (past studies) is mainly affected by the tractive force of water flow. Among the 65 trees that remained at the terminus of deposition zones at five sites, 36 were alive and 29 were dead. Damage to the forest increases with increasing thickness of deposits and decreases with increasing tree diameter. Such results are useful for designing forest buffer zones. Received: June 18, 2001 / Accepted: August 21, 2002 Correspondence to:Y. Ishikawa     

Deviations of kerf by handsaws III: the bend of saw blade by unsymmetrical set

To clarify the inducing mechanism of kerf deviation caused by unsymmetrical set of the handsaw, we hypothesized that the moment on the bottom edge of the saw blade, which is generated by the unsymmetrical set on handsaw teeth, bends the saw blade and this deformation should be one factor of the kerf deviation. To verify this hypothesis, the bend of a saw blade was examined by experiment and by calculation. In the experiments, a model tooth was attached to an actual handsaw. In the calculations, a saw model with a rectangular prism as the model tooth was analyzed by the finite element method. Loads were applied on the model tooth or the rectangular prism. From both the experiments and the calculations, the following results were obtained: (1) the deformation of the handsaw was caused by the unsymmetrical set in the length direction and the height direction of the saw blade; (2) the deformation and the inclination of the blade was larger at the front part of the saw than at the butt part; and (3) comparing the saw handle fixing conditions, the kerf deviation in the sawing line was supposed to be larger in the free handle condition and that kerf deviation in the depth direction was larger in the fixed handle condition. Based on the above results, it is apparent that one of the kerf deviation mechanisms originated from unsymmetrical set on the saw teeth. Part of this report was presented at the 54th Annual Meeting of the Japan Wood Research Society, Sapporo, Japan, August 2004.     

Penetration of hardwoods by liquids

Summary Oven dry highly evacuated sticks of the sapwood of various hardwoods, 40 cm long by 1.0 to 1.2 cm in the radial and tangential directions, took up water to the extent of 44 to 82% of saturation by capillary flow in one minute. Heartwood sticks took up 15 to 38% of saturation in one minute. The initial rapid penetration was followed by a penetration in which the take up increase directly with the square root of elapsed time. In this range, the slope of toluene take up plots was about twice that of the water take up. This is close to the theoratical value on the basis of viscous flow control. The linear portion of the plots is followed by a curvilinear decrease in rate of take up to a final equilibrium value. Water take ups approached 100% of the theoretical values.Times to 0.99 of theoretical filling with water varied from 9 minutes for tupelo gum sapwood to 14.6 to 17.3 days for white oak heartwood. Complete filling with toluene was never attained. Diffuse porous hardwoods filled to the extent of 93.5 to 96.5% of saturation. Ring porous oaks filled to the extent of 88 to 92%, the lower values being for heartwood. Edge coating of the sapwood of yellow poplar and yellow birch with epoxy glue reduced the rate of penetration only slightly. End coating reduced the rate to a higher degree. Moisture gradients in the fiber direction for tyloses-free hardwoods were negligible at various stages of penetration, indicating that filling of the vessels was practically instantaneous. White oak heartwood gave an initial longitudinal gradient followed by a negligible gradient, indicating that longitudinal penetration is primarily through the lumen of the fibers rather than continously through the vessels. Longitudinal penetration was calculated to be 31.5 times as deep as transverse penetration in 100 minutes and 33.3 times as deep in 25 minutes for white oak heartwood. The ratios are of the same order of magnitude as for Loblolly pine. In both cases the ratios are smaller than values calculated from the fiber dimensions alone as ray cell penetration and cross grain effects tend to lower the ratios.Paper No. 3947 of the Journal Series of North Carolina State University Agricultural Experiment Station, Raleigh, N. C.     

Indentation of wood by wedges

Summary The deformation processes involved in wedge indentation of wood are described. The behaviour of wood is compared to that of an elastic-plastic solid and that of a cellular body. It approximates towards the former when its density is high and towards the latter when its density is low. The hardness of wood is examined as a function of density and of wedge angle.We wish to thank the University Grants Committee and the New Zealand Forest Service for supporting this work     

Quantification by allometric equations of carbon sequestered by Tectona grandis in different agroforestry systems

Non destructive methods for quantification of carbon seques-tration in tropical trees are inadequately developed. We described a stan-dardized method for estimating carbon stock in teak (Tectona grandi...     

Saccharification of cellulose by dry pyrolysis

Pyrolysis of cellulose was studied for the purpose of practical production of 1,6-anhydro-β-D-glucopyranoside (levoglucosan, LG). To minimize secondary degradation of levoglucosan, two methods were examined: (1) conductive heating by glass bottle, and (2) radiation heating from the surface by CO₂ laser beam, both under vacuum and in a nitrogen atmosphere. Glass-bottle pyrolysis under vacuum gave levoglucosan yield of 50%–55% in the optimum temperature range of 350°–410°C, where placing the cold trap in the vicinity of heated area was effective in improving the yield. In contrast, glass-bottle pyrolysis under nitrogen gave low yields of 17%–20%, probably due to slower diffusion of pyrolysis product from hot region. The CO₂ laser pyrolysis under vacuum gave the product as aerosol (white smoke), causing difficulty in recovery of the product, and the maximum yield was 5%–17%. In this case the treatment under nitrogen flow was effective for recovery of aerosol, and the maximum yield reached approximately 25%.