Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens

In binary vectors, the antibiotic resistance gene used for selection of transformed plant cells is also usually expressed in the transforming Agrobacterium cells. This expression gives the bacterium antibiotic resistance, an unnecessary advantage on selective medium containing the antibiotic. Insertion of a castor bean catalase-1 (CAT-1) gene intron or a Parasponia andersonii haemoglobin gene intron into the coding region of the selectable marker gene, hph, completely abolished the expression of the gene in Agrobacterium, rendering it susceptible to hygromycin B. Use of these modified binary vectors minimized the overgrowth of Agrobacterium during plant transformation. Both of the introns were correctly spliced in plant cells and significantly enhanced hph gene expression in transformed rice tissue. The presence of these introns in the hph coding sequence not only maintained the selection efficiency of the hph gene, but with the CAT-1 intron also substantially increased the frequency of rice transformation. Transgenic lines with an intron-hph gene generally contained fewer gene copies and produced substantially more mRNA of the predicted size. Our results also indicate that transgenic plants with many copies of the transgene were more likely to show gene silencing than plants with 1-3 copies.

植物血红蛋白基因及植物凝集素基因转化水稻的研究

植物血红蛋白在共生固氮根瘤中主要是对固氮酶进行嫌氧保护，确保固氮酶活性，在植物根中协助氧的运输或作为感觉氧压的信号分子．豆科植物凝集素功能之一是对相应专一的根瘤菌有识别作用，并有利于根瘤菌的聚集和侵染。本论文将非豆科结瘤植物Parasponia andersonii血红蛋白基因及豌豆凝集索基因转入水稻，目的是此二基因表达后，豌豆凝集素可聚集、识别豌豆根瘤菌，并有助于它们的侵染，血红蛋白则可对根瘤菌固氮酶进行嫌氧保护，保障其发挥固氮作用，从而为实现水稻结瘤和固氮打下初步基础。 本论文将带有Parasponia血红蛋白基因的pL305质粒，带有潮霉素磷酸转移酶基因及Parasponia血红蛋白基因的农杆菌双元载体质粒pLX412-Hb用花粉管通道法转化水稻l用pLX412-Hb和带有Bar基因、豌豆凝集素基因、Parasponia血红蛋白基因的质粒pLHB用基因枪法转化水稻幼胚及愈伤组织，带有pLX412-Hb的Agrobactmum tumefaciens LBA4404转化烟草，得到如下结果： 1．用pL305质粒花粉管通道法转化水稻，运作1512朵花，得到707粒种子．将230粒种子萌发，发芽220粒;白化苗4株，成苗206株;计发芽率96%，白化苗率1.7%，成苗率90%。取120株提取DNA以Parasponia血红蛋白基因为探针进行点杂交，有6株为阳性结果，阳性率5%．再取4株DNA点杂交阳性植株DNA，以Parasponia血红蛋白基因为探针进行Southem杂交，有3株有阳性结果，阳性率为75%，照此推算转化植株约占总植株的4%．目前，这些转基因植株已开花结实。 2．用pLX412-Hb质粒花粉管通道法转化水稻，运作743朵花，得到340粒种子。将120粒种子萌发，发芽117粒，白化苗3株，成苗111株;计发芽率97. 5%，白化苗率2. 5%，成苗率92. 5%．取30株提取DNA以Parasponia血红蛋白基因为探针进行点杂交，有4株为阳性结果，阳性率13%。再取4株DNA点杂交呈阳性的植株DNA，以Parasponia血红蛋白基因为探针进行Southern杂交，有2株有阳性结果，阳性率为50%，照此推算转化植株约占总植株的7%。目前部分转基因植株巳开花结实． 3.用pLX412 - Hb质粒基因枪法转化水稻幼胚及愈伤组织，抗性筛选得到15株再生植株，以Parasponia血红蛋白基因为探针进行点杂交，有14株为阳性结果，阳性率93%．取7株DNA点杂交呈阳性的植株DNA，以Parasponia血红蛋白基因为探针进行Southern杂交，都为阳性结果，阳性率为100%，照此推算转化植株约占总植株的93%．目前，部分转基因植株已开花结实． 4．用pLX412 -Hb质粒通过Agrobacterrum tumeFaciens LBA4404转化烟草，抗性筛选得到的再生植株以Parasponia血红蛋白基因为探针进行Southern杂交，结果为阳性． 5．用pLHB基因枪法转化的愈伤组织，抗性筛选得到已分化出绿芽点的愈伤组织，以Parasponia血红蛋白基因为探针或以豌豆凝集素基因为探针进行Southern杂交，杂交结果均为阳性． 6．最近国外巳发现大麦有血红蛋白基因．本论文以大麦血红蛋白cDNA为探针对未转基因的水稻进行Southern杂交，结果有阳性带。说明水稻有与大麦血红蛋白基因高度同源序列。以Parasponia血红蛋白基因为探针时杂交结果为阴性，说明水稻血红蛋白基因与Parasponiaa血红蛋白基因核苷酸序列相差较大． 7．提取Southern杂交证明有Parasponia血红蛋白基因整合的水稻根及叶总RNA，以Parasponia血红蛋白基因cDNA为探针进行RNA点杂交，结果根总RNA为阳性，叶总RNA为阴性。初步证明Parasponia血红蛋白基因在水稻根中表达。 8．提取未转基因的水稻根及叶RNA，以大麦血红蛋白基因cDNA为探针进行RNA点杂交，结果：根总RNA为阳性结果，叶总RNA为阴性结果．初步证明水稻血红蛋白基因在水稻根中表达。 总之，本论文证明已将Parasponia血红蛋白基因整合进水稻植株染色体，将豌豆凝集素基因、Parasponia血红蛋白基因整合进水稻愈伤组织染色体，初步证明水稻有血红蛋白基因，内外源血红蛋白基因都有组织特异性表达，从而为本研究的战略设想奠定初步基础。

两个固氮相关的植物基因对烟草和水稻的转化及其表达

豆科植物凝集素基因和血红蛋白基因在对根瘤菌的识别作用和类菌体在低氧分压下的共生固氮中起重要作用。本文的目的是试图将这两个基因转移到非豆料植物烟草和水稻，使其能识别根瘤菌，探讨非豆科植物的共生和联合固氮的可能性。 构建了含有豌豆凝集素（P－Lec）基因、Parasponia andersonii血红蛋白基因、gus基因及植物选择标记潮霉素磷酸转移酶基因（hpt）的两个植物表达载体pCBHL和pCBHUL;同时，还构建了含有P－Lec基因、gus基因及植物选择标记PPT乙酰转移酶基因（bar）的植物表达载体pBBUL。在pCBHL中，CaMV35S启动子调控P－Lec基因的表达，而在pCBHUL和pBBUL中，该基因由玉米Ubiquitin 1启动子调控。 用农杆菌法将pCBHL导入烟草，得到53株再生植株，PCR检测表明转化频率为88％。用基因枪法分别将pCBHUL和pBBUL导入水稻幼胚或幼胚诱导的愈伤组织。转pCBHUL的材料共得到40株再生植株，经分子检测有18株分转基因植株，转化频率为0.9%。转pBBUL的幼胚愈伤组织经PPT筛选，只得到能分化出小芽的抗性愈伤组织。 PCR检测、Southern杂交表明P-Lec基因和Paraspoina血红蛋白基因都已经整合到转基因烟草及水稻的基因组中，转基因水稻植株中两个外源目的基因的拷贝数较高。Western杂交分析转基因植物中P-Lec基因的表达情况，结果表明该基因在转基因的烟草和水稻叶片中得到正确地表达。同时，GUS组织化学染色表明转基因烟草的嫩茎和幼根，转基因水稻的嫩叶和幼根中都有gus基因的表达。转基因烟草中外源基因的表达频率高于转基因水稻。T1代转基因烟草幼根的蛋白原位免疫杂交显示P-Lec正确定位于正在生长的幼根根毛的顶端，与对照豌豆中的P-Lec基因表达部位相一致。关于Parasponia血红蛋白基因，以前本实验室对转基因烟草和水稻的研究表明有转录水平的表达，国外实验室证实转基因烟草中有转译表达。 上述结果有可能为进一步研究转基因非豆科植物与根瘤菌的相互作用奠定一定的基础。

Hybridization between Tilapia zillii (Gervais) and Tilapia andersonii (Casteinau) at the Freshwater Fisheries Institute, at Nyegezi, Tanzania

As a part of an overall project on fishculture development techniques in Tanzania, hybridization between Tilapia zillii and Tilapia andersonii was carried out at the Freshwater Fisheries Institute, Nyegezi, Tanzania. T. andersonii, a plankton feeder, is not indigenous to Tanzania but was introduced in 1968 from Zambia for certain specific purpose. T. zillii, a macrovegetation feeder, is present locally and is common. In the present studies T. zillii (245.0 mm/260.0 g) female was hybridized with T. andersonii (288.0 mm/350.0 g) male. Under cement cistern conditions it was only after about four months of acclimatization that hybridization between the two occurred. About 1,637 interspecific hybrid fry were produced in a single brood. Eggs were adhesive and parental care shown by the female, the male being driven away. Growth under cistern conditions was slow, attaining a size of 134.8 mm/44.3 g in 10 months. But this growth rate need not be taken as ideal. In body shape, colouration and other morphometric characters the hybrids had inherited from both parents. The number of gill rakers among the hybrids was eighteen which was intermediate between T. zillii (12) and T. andersonii (27). Among one hundred and seventy two specimens (106.0 mm - 168.0 mm) cut and examined the sex ration was hundred per cent males and all of them were between II and IV stages of maturity. This is the first report of fish hybridization from Tanzania and possibly the first report on hybridization between T. zillii and T. andersonii. The full significanoe of the findings and its role in African fishculture is discussed.

臭蛙群三种蛙类核型及染色体带型的比较(英文)

本文比较了3种臭蛙,即云南臭蛙（R. andersonii）、无指盘臭蛙（R.grahami）和滇南臭蛙（R.tiennanensis）的核型、C-带和银染核仁组织者区（Ag-NORs）。结果表明,3种臭蛙的核型均为2n=26（NF=52）,由5对大型染色体和8对小型染色体组成,与蛙科大多数种类的核型相似,但是染色体形态和次缢痕的位置和数目有差异。其中,云南臭蛙和无指盘臭蛙的核型相似程度比较大,与其他已报道的臭蛙类的核型相差不大,而滇南臭蛙的核型比较特殊,与其他臭蛙类的核型存在显著差异。 C-带技术显示的结果表明,3种臭蛙所有染色体的着丝点都有比较显著的C-带,而居间区C-带和端点C-带却存在明显的差异。3种中,滇南臭蛙的C-带带型比较特殊。有趣的是在研究中还发现,云南臭蛙的早中期细胞（染色体很长）染色体上发现了很多结构异染色质区,除着丝点C-带外,几乎所有的染色体上都有居间区或端点C-带,而在晚中期细胞中所观察到的C-带数目却较少,除着丝点外,只在少数染色体上发现了C-带。 云南臭蛙和无指盘臭蛙的Ag-NORs位于第10对染色体的长臂上,与该染色体上的次缢痕位置相对应,而滇南臭蛙的Ag-NORs位于第6...

Palinoestratigrafia na passagem do grupo Itararé ao Guatá (carbonífero-permiano) no sul do Estado do Paraná e norte do Estado de Santa Catarina, borda leste da Bacia do Paraná

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

List of plant species found on Victoria Island

While engaged in geoecological field work on Victoria Island, 277 new plants could be recorded for the vicinities of Holman, Cambridge Bay, Wellington Bay, Mt. Pelly, Richardson Islands, Hadley Bay, and Minto lnlet; 8 of them were new for Victoria Island, 6 for the western Canadian arctic archipelago.

A new hemoglobin gene from soybean: a role for hemoglobin in all plants.

We have isolated a new hemoglobin gene from soybean. It is expressed in cotyledons, stems of seedlings, roots, young leaves, and in some cells in the nodules that are associated with the nitrogen-fixing Bradyrhizobium symbiont. This contrasts with the expression of the leghemoglobins, which are active only in the infected cells of the nodules. The deduced protein sequence of the new gene shows only 58% similarity to one of the soybean leghemoglobins, but 85-87% similarity to hemoglobins from the nonlegumes Parasponia, Casuarina, and barley. The pattern of expression and the gene sequence indicate that this new gene is a nonsymbiotic legume hemoglobin. The finding of this gene in legumes and similar genes in other species strengthens our previous suggestion that genomes of all plants contain hemoglobin genes. The specialized leghemoglobin gene family may have arisen from a preexisting nonsymbiotic hemoglobin by gene duplication.

Plant species, biomass and environmental characteristics of relevés along the North America Arctic bioclimate gradient

Question: How do interactions between the physical environment and biotic properties of vegetation influence the formation of small patterned-ground features along the Arctic bioclimate gradient? Location: At 68° to 78°N: six locations along the Dalton Highway in arctic Alaska and three in Canada (Banks Island, Prince Patrick Island and Ellef Ringnes Island). Methods: We analysed floristic and structural vegetation, biomass and abiotic data (soil chemical and physical parameters, the n-factor [a soil thermal index] and spectral information [NDVI, LAI]) on 147 microhabitat releves of zonalpatterned-ground features. Using mapping, table analysis (JUICE) and ordination techniques (NMDS). Results: Table analysis using JUICE and the phi-coefficient to identify diagnostic species revealed clear groups of diagnostic plant taxa in four of the five zonal vegetation complexes. Plant communities and zonal complexes were generally well separated in the NMDS ordination. The Alaska and Canada communities were spatially separated in the ordination because of different glacial histories and location in separate floristic provinces, but there was no single controlling environmental gradient. Vegetation structure, particularly that of bryophytes and total biomass, strongly affected thermal properties of the soils. Patterned-ground complexes with the largest thermal differential between the patterned-ground features and the surrounding vegetation exhibited the clearest patterned-ground morphologies.

Towards an assessment of on-farm niches for improved forages in Sud-Kivu, DR Congo

Inadequate quantity and quality of livestock feed is a persistent constraint to productivity for mixed crop-livestock farming in eastern Democratic Republic of Congo. To assess on-farm niches of improved forages, demonstration trials and participatory on-farm research were conducted in four different sites. Forage legumes included Canavalia brasiliensis (CIAT 17009), Stylosanthes guianensis (CIAT 11995) and Desmodium uncinatum (cv. Silverleaf), while grasses were Guatemala grass (Tripsacum andersonii), Napier grass (Pennisetum purpureum) French Cameroon, and a local Napier line. Within the first six months, forage legumes adapted differently to the four sites with little differences among varieties, while forage grasses displayed higher variability in biomass production among varieties than among sites. Farmers’ ranking largely corresponded to herbage yield from the first cut, preferring Canavalia, Silverleaf desmodium and Napier French Cameroon. Choice of forages and integration into farming systems depended on land availability, soil erosion prevalence and livestock husbandry system. In erosion prone sites, 55–60% of farmers planted grasses on field edges and 16–30% as hedgerows for erosion control. 43% of farmers grew forages as intercrop with food crops such as maize and cassava, pointing to land scarcity. Only in the site with lower land pressure, 71% of farmers grew legumes as pure stand. When land tenure was not secured and livestock freely roaming, 75% of farmers preferred to grow annual forage legumes instead of perennial grasses. Future research should develop robust decision support for spatial and temporal integration of forage technologies into diverse smallholder cropping systems and agro-ecologies.