Genetic analyses of ancient tea trees provide insights into the breeding history and dissemination of Chinese Assam tea(Camellia sinensis var.assamica)

Chinese Assam tea(Camellia sinensis var.assamica)is an important tea crop with a long history of cultivation in Yunnan,China.Despite its potential value as a genetic resource,its genetic diversity and domestication/breeding history remain unclear.To address this issue,we genotyped 469 ancient tea plant trees representing 26 C.sinensis var.assamica populations,plus two of its wild relatives(six and three populations of C.taliensis and C.crassicolumna,respectively)using 16 nuclear microsatellite loci.Results showed that Chinese Assam tea has a relatively high,but comparatively lower gene diversity(H_(S)=0.638)than the wild relative C.crassicolumna(H_S=0.658).Clustering in STRUCTURE indicated that Chinese Assam tea and its two wild relatives formed distinct genetic groups,with considerable interspecific introgression.The Chinese Assam tea accessions clustered into three gene pools,corresponding well with their geographic distribution.However,New Hybrids analysis indicated that 68.48%of ancient Chinese Assam tea plants from Xishuangbanna were genetic intermediates between the Puer and Lincang gene pools.In addition,10%of the ancient Chinese Assam tea individuals were found to be hybrids between Chinese Assam tea and C.taliensis.Our results suggest that Chinese Assam tea was domesticated separately in three gene pools(Puer,Lincang and Xishuangbanna)in the Mekong River valley and that the hybrids were subsequently selected during the domestication process.Although the domestication history of Chinese Assam tea in southwestern Yunnan remains complex,our results will help to identify valuable genetic resources that may be useful in future tea breeding programs.

Isolation of the Endophytic Fungi of Paris polyphylla var. yunnanensis and Their Effects on the Embryo Development of P. polyphylla var. yunnanensis Seeds

[Objective] This study aimed to isolate the endophytic fungi of Paris polyphylla var. yunnanensis and investigate their effects on the embryo development of P. polyphylla var. yunnanensis seeds. [Method] The endophytic fungi of P. polyphylla were isolated and identified morphologically, and their effects on the embryo development of P. polyphylla var. yunnanensis seeds were studied by using paraffin sectioning and microphotography. [Result] Nine endophytic fungi, i.e. P. polyphylla var. yunnanensis endophytic fungi PPYEF-1, PPYEF-2, PPYEF-3, PPYEF-4, PPYEF-5, PPYEF-6, PPYEF-7, PPYEF-8 and PPYEF-9 belonging to seven genera in five families, three orders were isolated from the rhizomes. Except PPYEF-4 (Cladosporium sp.), other fungi could promote the embryo development of the P. polyphylla var. yunnanensis seeds, mostly reaching the extremely significant or significant level. PPYEF-9 (Trichoderma sp.) resulted in the highest embryo length and embryo-emerging ratio. [Conclusion] This paper could provide a reference for the application of the endophytic fungi of P. polyphylla var. yunnanensis in the dormancy-breaking of P. polyphylla var. yunnanensis seeds.

三峡库区河岸植物野古草(Arundinella anomala var. depauperata Keng)茎通气组织发生对水淹的响应

野古草(Arundinella anomala var. depauperata Keng)在三峡库区长江及其支流江(河)岸有广泛分布,对水淹有很好的耐受能力。有研究表明许多植物在水淹时通气组织发生增强,通气组织的产生改善了植株通气状况,提高了植物对水淹的抵御能力。为了研究水淹是否会影响野古草的通气组织发生以及野古草通气组织发生对水淹的反应,考察了不同水淹深度、不同水淹时间和不同水淹方式处理时野古草茎中通气组织的发生情况。实验中共设置3个水淹深度:不进行水淹(对照)、植株地下部分淹没、植株完全淹没于水下2m深处;5个淹没时间:植株被淹没的时间长度分别为5、10、20、30d和60d;2种水淹方式:连续水淹和间歇水淹。实验结果表明:(1)在无水淹情况下野古草茎中可以产生通气组织,通气组织产生随植株的生长而增强;水淹加快了野古草通气组织发生的进程,促进了野古草通气组织的提前发生。(2)野古草茎中通气组织并不会因为水淹的时间越长而产生越多,植株通气组织的大小达到一定程度后不再因水淹时间的增长而继续增大。(3)淹没深度对通气组织发生有一定影响,总的看来,地下部分淹没野古草植株的通气组织发生要强于完全淹没植株。(4)不同水淹方式对野古草通气组织发生的影响因水淹深度不同而有差异。在完全淹没情况下,连续水淹植株的通气组织比间歇水淹植株的通气组织发达;在地下部分淹没情况下,除水淹初期外,随水淹时间的延长,连续水淹植株通气组织发生与间歇水淹植株没有差异。

Comparison of stand structure and growth between artificial and natural forests of Pinus sylvestiris var. mongolica on sandy land

Mongolian pine (Pinus sylvestiris Linnaeus var. mongolica Litvinov) as a valuable conifer tree species has been broadly introduced to the sandy land areas in 揟hree North?regions (North, northwest and northeast of China), but many problems occurred in the earliest Mongolian pine plantations in Zhanggutai, Zhangwu County, Liaoning Province (ZZL). In order to clarify the reason, comprehensive investigations were carried out on differences in structure characteristics, growth processes and ecological factors between artificial stands (the first plantation established in ZZL in 1950s) and natural stands (the origin forests of the tree species in Honghuaerji, Inner Mongolia) on sandy land. The results showed that variation of diameter-class distributions in artificial stands and natural stands could be described by Weibull and Normal distribution models, respectively. Chapman-Richards growth model was employed to reconstruct the growth process of Mongolian pine based on the data from field investigation and stem analysis. The ages of maximum of relative growth rate and average growth rate of DBH, height, and volume of planted trees were 11, 22 years, 8, 15 years and 35, 59 years earlier than those of natural stand trees, respectively. In respect of the incremental acceleration of volume, the artificial and natural stands reached their maximum values at 14 years and 33 years respectively. The quantitative maturity ages of artificial stands and natural stands were 43 years and 102 years respectively. It was concluded that the life span of the Mongolian pine trees in natural stands was about 60 years longer than those in artificial stands. The differences mentioned above between artificial and natural Mongolian pine forests on sandy land were partially attributed to the drastic variations of ecological conditions such as latitude, temperature, precipitation, evaporation and height above sea level. Human beings' disturbances and higher density in plantation forest may be ascribed as additional reasons. Those results may be potentially useful for the management and afforestation of Mongolian pine plantations on sandy land in arid and semi-arid areas.

花椰菜(Brassica oleracea var.botrytis)黑腐病抗性基因同源序列分离及克隆的研究

通过从 NBS 保守序列设计简并引物 PCR 的方法,以花椰菜(Brassica oleracea vat.botrytis)抗、感黑腐病的近等基因系为材料,分离得到 NBS-LRR 型抗性基因同源序列,并获得1个克隆,命名为 RGA330-7.Southern 杂交表明,该片段在近等基因系中存在明显的多态性,且该片段在抗黑腐病基因位点至少存在3个以上类似 RGA330-7的同源拷贝.序列分析结果认为该克隆与 NBS-LRR 型抗性基因的部分 CDSs 有很高的同源性,说明该片段属于 NBS-LRR 型.系统进化分析该序列与甘蓝型油菜的2个抗病同源序列归为一类,很可能这3个不同来源的抗性基因同源序列同属于一种抗性基因家族.因此推测该序列与花椰菜抗黑腐病基因紧密相关,为进一步克隆花椰菜抗黑腐病基因提供了可靠的候选基因,对分子标记辅助抗性育种具有重要意义。

Genetic diversity and genetic differentiation of natural populations of Pinus kesiya var. langbinanensis

Genetic diversity and genetic differentiation of natural populations of Pinus kesiya var. langbinanensis were examined by means of electrophoresis technique. Analysis of 9 enzyme systems including 16 loci showed that all the three natural popu-lations of the pine were high in genetic diversity but low in inter-population genetic differentiation. The proportion of polymorphic loci is 0.667, with each locus holding 2.13 alleles, averagely. The average expected and observed heterozygosity was 0.288 and 0.197, respectively. The gene differentiation among populations was 0.052, but the mean genetic distance was only 0.015.

来源于链霉菌Streptomyces fradiae var.k11的抗蛋白酶甘露聚糖酶的基因克隆与鉴定

通过设计简并引物和TAIL-PCR的方法从Streptomyces fradiaevar.k11中克隆得到一个β-l,4-甘露聚糖酶编码基因manS221,其全长1 359 bp,编码452个氨基酸,前端28个氨基酸为预测信号肽。构建表达载体pET-28a-manS221并转化大肠杆菌BL2l(DE3),特异性表达manS221基因,重组的甘露聚糖酶通过Ni-NTA亲和层析纯化。酶学性质分析表明,重组甘露聚糖酶最适温度为52℃,最适pH值为6.0,在pH5.0～9.0的范围内有良好的稳定性,并且对中性蛋白酶具有高抗性。这些优良的性质使得ManS221在食品、饲料和纺织工业生产中具有良好的应用前景。

Effects of peat and weathered coal on the growth of Pinus sylvestrisvar. mongolica seedlings on aeolian sandy soil

The experiment was conducted at the Ganqika Sandy Land Ecological Station in Ke抏rqinzuoyihouqi County, Inner Mongolia, in a growing season from April 28 to October 28, 2001. Peat and weathered coal were added to the aeolian sandy soil in different ratios. Two-year-old Pinus sylvestris var. mongolica seedlings and plastic pots were used in the experiment. The experimental results indicated that: 1) the peat and weathered coal could significantly improve the physical and chemical prop-erties of aeolian sandy soil, and thus promoted the growth of seedlings; 2) the effect of peat on seedling growth, including height, base diameter, root length and biomass, presented an order of 8%>10%>5%>2%>0 in terms of peat contents, and the effect of weathered coal on seedling growth presented an order of 5%>8%>10%>2%>0 in terms of weathered coal contents for height and basal diameter, 5%>8%>2% >10%>0 for root length, and 5%>2%>8% >10%>0 for biomass; 3) the effects of peat were generally greater than that of weathered coal. Meanwhile, 8% peat was the best treatment to promote the growth of P. sylvestris var. mongolica seedlings.

响应面法优化Rhizopus microsporus var.chinensis产生淀粉酶的条件

对Rhizopus microsporus var.chinensis固态发酵产生淀粉酶的条件进行了优化。首先采用单因子实验确定最适固态发酵基质为小麦麸皮,最适碳源和氮源分别为可溶性淀粉和硫酸铵,通过控制培养基初始pH为3.0、初始湿度为70%时,使其生淀粉酶产量提高3.5倍。在此基础上,利用响应面中心组合设计对显著因素进行优化,得出每500mL三角摇瓶中含小麦麸皮13.7g、可溶性淀粉0.063g、硫酸铵0.052g时,生淀粉酶产量达到48.50U/mL,比初始产量提高了8倍。

Flavonoids from Millettia nitita var. hirsutissima

Aim To Methods The constituents tures were identified investigate the chemical constituents of MiUettia nitita var. hirsutissima. were isolated and purified by chromatographic techniques, and the strucby spectral evidences. Results Four flavonoids were isolated from the plant, including liquiritigenin （ 1 ）, naringenin （2）, maackiain （3）, and 3R-vestitol （4）. Conclusion Compounds 1 and 2 were obtained from the genus Millettia for the first time ; 3 and 4 were obtained from the plant for the first time.

Ultrastructure of Oogenesis in Osmunda cinnamomea var. asiatica

The ultrastructure of oogenesis in the fern Osmunda cinnamomea L. var. asiatica Fernald has been studied by electron microscopy. During oogenesis, numerous vesicles not only moved towards the periphery, but also were arranged in line along the inside of plasmalemma, and in addition aggregated outside the plasmalemma by exocytosis. They released or excreted osmiophilic material. It was observed that a few vesicles containing lamellar osmiophilic material situated closely along the plasmalemma, seemed to break open. Simultaneously, a separation cavity between egg cell and archegonium wall formed. Its width was broader than the other advanced ferns reported previously, and an extra egg membrane occurred outside around the plasmalemma of the egg, its thickness being greater than in Pteridium and Dryopteris. Amyloplasts around the nucleus were filled with large triangular semicircular or subelliptical starch grains, but as the egg matured they progressively decreased. Nucleus was large and flattened, and paired membrane in two to three couples laid within the nucleus, close and parallel to the nuclear membrane. No nuclear evagination was observed. Mitochondria seemed to have been undeveloped, but finally recovered normally.

Ultrastructure of the Blepharoplast and the Multilayered Structure in Spermatogenesis in Osmunda cinnamomea var. asiatica

The ultrastructure of the blepharoplast and the multilayered structure (MLS) in the fern Osmunda cinnamomea var. asiatica Fernald have been studied by electron microscopy with respect to spermatogenesis. The blepharoplast appears in the young spermatid. The differentiating blepharoplast is approximately a spherical body, which is composed of densely stained granular material in the center and some cylinders outside of it. The differentiated blepharoplast is also a sphere, but without the densely stained material in the center, consisting of scattered or radially arranged cylinders. The MLS seen in the spermatid lies between the basal bodies and the giant mitochondrion. In the early developmental stage, the MLS only consists of lamellar layers, each of which runs parallel to one another and forms a strip. In the mid stage, the MLS is composed of the microtubular ribbon (MTr), the lamellar layers and a layer of plaque. In the late stage, the MLS forms accessory band, osmiophilic crest and a layer of osmiophilic material. The MTr grows out from the MLS and extends along the surface of the nucleus to unite with the nuclear envelope in a complex. The basal body coming from the cylinder produces the axoneme of the flagella in the distal end and the wedge-shaped structure in the proximal end, respectively. In the present study, the ultrastructural features of blepharoplast and the MLS of the protoleptosporangiopsida fern, O. cinnamomea var. asiatica, have been described and compared with those of other kinds of pteridophytes in detail. The lamellar layers appearing before the formation of the MTr was found and reported for the first time.

Effects of Bacillus amyloliquefaciens Biofertilizer on Brassica juncea var. multiceps Growth and N_2O Emission from Soil

In this study, Bacil us amyloliquefaciens A3 was continual y incubated in shake fIasks contalning wastewater from sweet potato starch production as an ef-fective biofertiIizer for cuItivation of Brassica juncea var. multiceps（XueIihong）. Based on pot experiments in the greenhouse, the effects of chemical fertiIizers （CN）, biofertiIizer （BF）, inactivated broth （BI）, starch wastewater （SW） and the combination of biofertiIizer and chemical fertiIizer （BC） on the yield, NO3- content and NO2- con-tent of XueIihong, soiI physicochemical properties and N2O emission were investi-gated. The resuIts showed that the yield of XueIihong in BC and CN treatments was improved by five times compared with CK; BF and SW treatments had insignifi-cant impact on the yield of XueIihong. Compared with CN treatment, BCL treatment exhibited simiIar improving effects on the yield of XueIihong, in which NO3- content of XueIihong and soiI was reduced by 16.4%-73.6% and 22%-29%, which reduced the risk of nitrogen eIuviations in soiI; average N2O fIux （FPV30） in BCL treatment was reduced by 58.3%-73.1% compared with CN treatment. In concIusion, B. amy-loliquefaciens is a feasibIe Iow-cost biofertiIizer for sustalnabIe vegetabIe farming with a great potential for starch wastewater utiIization.

嗜热子囊菌光孢变种Thermoascus aurantiacus var.levisporusβ-葡萄糖苷酶的分离纯化及特性研究

采用室内培养、测定方法,对嗜热子囊菌光孢变种Thermoascus aurantiacus var.levisporus产生的β-葡萄糖苷酶进行了分离纯化及特性研究。粗酶液经硫酸铵沉淀、DEAE-SepharoseFastFlow阴离子层析、Phenyl-Sepharose疏水层析等步骤获得了凝胶电泳均一的β-葡萄糖苷酶。结果表明,经12%SDS-PAGE测得酶的单亚基分子量约为118kDa,凝胶过滤层析测得酶的分子量约为350kDa。该酶反应的最适温度和最适pH分别为80℃和4.5￣5.0,在pH5.0条件下,该酶在70℃条件下基本稳定,80℃保温30min,剩余酶活为15%。金属离子对β-葡萄糖苷酶活性影响较大,其中Ca2+、Ba2+对酶有激活作用;Ag+、Fe3+、Cu2+对酶有显著的抑制作用。该酶对水杨苷具有很强的底物特异性。

Influence of CO_2 Doubling on Water Transport Process at Root/Soil Interface of Pinus sylvestris var. sylvestriformis Seedlings

Water transport at the root/soil interface of 1 year old Pinus sylvestris Linn. var. sylvestriformis (Takenouchi) Cheng et C. D. Chu seedlings under CO 2 doubling was studied by measuring soil electric conductance to survey soil water profiles and comparing it with root distribution surveyed by soil coring and root harvesting in Changbai Mountain in 1999. The results were: (1) The profiles of soil water content were adjusted by root activity. The water content of the soil layer with abundant roots was higher. (2) When CO 2 concentration was doubled, water transport was more active at the root/soil interface and the roots were distributed into deeper layer. It was shown in this work that the method of measuring electric conductance is an inexpensive, non_destructive and relatively sensitive way for underground water transport process.

Anatomy, Histochemistry and Phytochemistry of Leaves in Aloe vera var. chinensis

A multidisciplinary approach-anatomy, histochemistry and phytochemistry-was used to investigate the leaf structure, the content and the storage location of barbaloin in the leaves of Aloe vera L. var. chinensis (Haw.) Berg. Xeromorphic characteristics including secondary thickened epidermal cell walls, thicker cuticle, ambiguous differentiation of spongy and palisade tissues in the chlorenchyma, and well-developed aquiferous tissue could be seen in the leaves. Several large parenchymatous cells were observed at the phloem pole of the first ring of vascular bundles. The secondary ring of vascular bundles in the leaf base and the stomata, which are surrounded by five cells, have some classification significance in this species. The density of vascular bundles, the content of barbaloin and the intensity of histochemical reaction differed among leaf numbers Ll (annual leaf), L2 (biennial leaf), L3 (triennial leaf) and L4 (quadrennial leaf), and in different parts of the leaf. These three factors were highest in the youngest leaf, Ll, and top parts of all the leaves and lowest in the basal parts and the oldest leaf, L4. The density of vascular bundles had a positive correlation to the content of barbaloin. The histochemical results revealed that the small sheath cells that surrounded the bundles might be the location of barbaloin synthesis and the large parenchymatous cells beneath the sheath might be the storage places of this metabolite.

Optimum conditions for pure culture of major ectomycorrhizal fungi obtained from Pinus sylvestris var. mongolica plantations in southeastern Keerqin sandy lands, China

The effects of medium, pH, water potential and temperature on the culture for three pure strains （Lactarius deliciosus, Boletus edulis and Lactarius insulsus） of ectomycorrhizal fungi from plantation forests of Mongolian pine （Pinus sylvestris var. mongolica） on sandy lands were observed to obtain the optimum conditions for the growth of ectomycorrhizal fungi. The results indicated that the three ectomycorrhizal fungi could grow well in the mediums containing natural components, such as vitamin, pine juice and yeast powder, pH had a slight effect on the growth of the three ectomycorrhizal fungi, and the optimum pH values were 6.0 for L. deliciosus, 5.0 for B. edulis, respectively. However, L. insulsus had a wide pH range, and it grew better than the other two strains in neutral and light alkalescent mediums. Water potential （produced by Polyethylene Glycol, PEG） had significant effects on the ecological adaptability for the tested three fungi strains. All of the three stains grow better at lower PEG concentration （100 g PEG.kg^-1 H2O）. The best water potential was 10% PEG concentration for all of the three stains. Temperatures, especially high temperatures induced the fungi death. The optimum temperature for the growth of ectomycorrhizal fungi was 25-28℃ for all of the three stains.

利用Native制备电泳分离来源于Rhizopus microsporus var. chinensis的葡萄糖淀粉酶同工酶

将Native制备电泳应用于Rhizopus microsporus var. chinensis中2个葡萄糖淀粉酶同工酶的分离,考察了电泳缓冲系统、凝胶浓度和凝胶长度对分离效果的影响。结果表明:先使用Ornstein-Davis系统分离同工酶,再使用0.02mol/L,pH6.2的NaAc.CH3COOH缓冲液洗脱,在长度为6cm的7%的Native电泳胶上,2个性质相近仅电泳迁移率略有差别的同工酶能有效分离。分离回收的蛋白质可保持其活力,便于后续研究。

Research Progress in Cultivation and Pharmaceutical Chemicals of Toona ciliata and Toona ciliata var. pubescens

Toona ciliata and Toona ciliata var. pubescens are rare timber species. In China, Toona ciliata and Toona ciliata var. pubescens distribute sporadical y with smal popuIation size but have huge deveIopment potential. The paper reviewed the research progress of their seedIing breeding, nutrient characteristics, fertiIization technoIogy, afforestation design, timber utiIization, chemistry and pharmacoIogy, and then Iooked into the future research and utiIization of Toona ciliata and Toona ciliata var. pubescens.

Effects of Weak Light on the Ultrastructural Variations of Phloem Tissues in Source Leaves of Three-Year-Old Nectarine Trees(Prunus persica L.var. nectarina Ait.)

Leaves from three_year_old solar greenhouse nectarine trees ( Prunus persica L. var. nectarina Ait. “Zao Hong Yan”) were used as materials in this study. It was the first time that the ultrastructural characteristics of phloem tissues of source leaves were observed and compared in normal and weak light intensities using the transmission electron microscopy. Results showed that the average diameters of companion cells (CC) and sieve elements (SE) of all kinds of veins were bigger in normal than that in weak light intensity, indicating that light could influence the cell development and growth. Dense cytoplasm with abundant mitochondria, endoplasmic reticulums, multivesicular bodies, vesicles and plastids were observed in normal light intensity. On the contrary, CC with small vacuolar structures and few mitochondrias, endoplasmic reticulums were shown in weak light. Misalignment of grana thylakoid margins of nectarine leaves also was seen in weak light. The sieve pores of SEs were obstructed in weak light. Chloroplasts with numerous starch grains and few mitochondrias were noticed in the mesophyll cell (MES) surrounding the bundle sheath in weak light. The storage of starch grains appeared to result from an unbalance between photosynthate production and export of photosynthates. This observation provided a strong support to the point that most leaves export the most of assimilates in the light time. Plasmodesmal densities between SE/CC, CC/PP (phloem parenchyma cell), PP/PP and PP/BSC (bundle_sheath cell) decreased in weak light. Plasmodesmata were observed between CC/SE (NS) (nacreous_walled sieve element), PP/BSC in branch veins in normal light intensity, but not in weak light. Thus apoplasmic pathway may be the main mode of transport of assimilates in weak light, however symplasmic pathway may be the main mode of transport of assimilates in normal light intensity. These results demonstrated that the solar greenhouse nectarine trees could be adapted to the weak light via the ultrastructure variation of phloem tissues of the source leaves.