Marker gene excision in transgenic Brassica napus via Agrobacterium-mediated Cre/lox transient expression system

Oilseed rape (Brassica napus L.) is one of the most important oil crops in the world. However, study on marker-free transgene of B. napus for bio-safety purpose is limited in this allotetraploid crop. In order to advance marker gene excision research, a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B. napus. The system consists of 2 sets of independent vectors including pC35Spro::T7RP carrying T7 RNA polymerase and pCT7pro::Cre carrying T7 promoter respectively. After hybridization of 2 according types of transgenic B. napus, marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression. Totally 52 and 46 positive T₀ transgenic lines of these 2 vectors were obtained after identification by PCR and test trip. T₁ plants from 3 T₀ positive pC35Spro::T7RP lines and 2 T₀ positive pC35Spro::T7RP lines were identified as single copy according to segregation ratio and were chosen for crossing. However, expression of CP4 EPSPS (glyphosate resistance gene) and OXY (bromoxynil resistance gene) were not found in F₁ progeny, which proved that the excision was not complete. The possible reasons for our limited success were investigated and detailed analyses were performed. Although this system is not applicable for generating transgenic B. napus free from selectable marker gene, it provided valuable experience and clue for further improvement of this technique. Many other advantages and further improvement will be progressed in future work.     

Stereochemistry and biosynthesis of 8-O-4′ neolignans in Eucommia ulmoides: diastereoselective formation of guaiacylglycerol-8-O-4′-(sinapyl alcohol) ether

Stereochemistry and biosynthesis of guaiacylglycerol-8-O-4′-(sinapyl alcohol) ether (GGSE), an 8-O-4′ neolignan, which consists of coniferyl and sinapyl alcohol moieties, in Eucommia ulmoides were investigated. Four 8-O-4′ neolignans, GGSE, syringylglycerol-8-O-4′-(coniferyl alcohol) ether (SGCE), guaiacylglycerol-8-O-4′-(coniferyl alcohol) ether (GGCE), and syringylglycerol-8-O-4′-(sinapyl alcohol) ether (SGSE), were synthesized. Their erythro and threo diastereomers were separated through acetonide derivatives, intermediates of the synthesis, and identified by means of nuclear magnetic resonance (NMR) spectroscopy. All of the erythro-acetonide derivatives have larger coupling constants (ca 9 Hz) for the C₇-H resonances than those of the threo ones (1.5–2 Hz). In the case of the four 8-O-4′ neolignans, the C₇-H coupling constants of the threo-isomers are not smaller than those of the erythro ones. GGSE isolated previously from this plant was identified as the erythro isomer by comparison of the ¹³C-NMR data with synthetic erythro-GGSE and threo-GGSE and the other 8-O-4′ neolignans mentioned as above. Administration of a mixture of [8-¹⁴C]coniferyl alcohol and [8-¹⁴C]sinapyl alcohol to excised shoots of E. ulmoides was carried out and the incorporation of ¹⁴C into erythro-[¹⁴C]GGSE was found to be higher than that in threo-[¹⁴C]GGSE. The occurrence of diastereoselective formation of erythro-GGSE by cross coupling of coniferyl and sinapyl alcohols is suggested.Part of this paper was presented at the 47th Lignin Symposium, Fukuoka, October 2002 and the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, April 2003     

Selection,mechanism, cross resistance and stability of spinosad resistance in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Spodoptera litura is one of the most destructive pests in Pakistan and in many other regions of the world. A field collected population of S. litura was selected with spinosad for eleven generations under controlled laboratory conditions to study the cross resistance, mechanism and stability of spinosad resistance in S. litura. The resistance to spinosad in S. litura increased 3921-fold (after eleven generations of selection with spinosad) as compared to a susceptible population of S. litura. No cross resistance between spinosad and emamectin benzoate, methoxyfenozide, fipronil, indoxacarb, profenofos, lufenuron or deltamethrin was found in the spinosad-selected population of S. litura. To find the possible mechanism of spinosad resistance in S. litura two synergists, Piperonyl butoxide (PBO), S, S, S-tributyl phosphorotrithioate (DEF) were tested on the susceptible and resistant strains and on the un-selected field population. The values of the synergism ratios of PBO and DEF were 2.33 and 1.06 for the spinosad-selected strain, 1.36 and 1.06 for the un-selected field population and 1.14 and 1.00 for the susceptible strain, respectively. As high PBO ratio indicates the role of microsomal O-demethylase in causing spinosad resistance in S. litura. The spinosad-resistant and field populations of S. litura were reared without any selection pressure from the 12th to the 16th generation (G₁₂–G₁₆). The spinosad resistance decreased from 3921 to 678-fold in the spinosad-resistant population and from 31.1 to 15.1-fold in the un-selected population of S. litura as compared to the susceptible strain. Spinosad resistance in S. litura has a high reversion rate (−0.15) which indicates that spinosad resistance in S. litura is unstable and can be easily managed by switching off the selection pressure for a few generations or alternating with insecticides having different modes of action.     

欧洲肉鸽与卡奴鸽不同杂交组合生产性能的比较

[目的]比较欧洲肉鸽与卡奴鸽不同杂交组合的生产性能,以寻求最佳杂交配套。[方法]对欧洲肉鸡3个品系与白卡奴鸽正反交,组成6个杂交组合,对6个杂交组合的生产性能进行测定,比较6个杂交组合的总产蛋数、产蛋周期、蛋重、受精率、受精蛋孵化率、初生重、28日龄体重、28日龄成活率等指标。[结果]总产蛋数以E2BC组合最高(22.32个),显著大于BCE1、BCE2、BCE3、E1BC组合(P0.05);BCE2组合总产蛋数最少(19.70个),显著小于其他5个组合(P0.05)。E2BC组合产蛋周期最短(34.37 d),显著小于BCE2组合(38.91 d)(P0.05)。蛋重基本在23 g左右。受精率E2BC组合最高(79.85%),显著高于BCE3组合(71.57%)(P0.05)。BCE1组合受精蛋孵化率最高78.79%,显著高于E3BC组合(74.72%)(P0.05)。各杂交组合的初生重基本都在16 g左右。28日龄上市体重以E2BC组合最高(599.94 g),显著大于E3BC组合(556.13 g)(P0.05)。BCE2组合28日龄成活率为84.77%,显著低于其他5个组合(P0.05)。[结论]E2BC组合的年产蛋量、受精率和28日龄体重均最高,其繁殖性能和生长性能都比较优良,可在生产中加以利用。E3品系在杂交利用上不太理想。     

番木瓜环斑病毒弱毒株的构建及分析

弱毒株是利用交叉保护防治植物病毒病害的关键限制因子。通过对马铃薯Y病毒属病毒(强、弱毒株)的全长及部分氨基酸序列比对分析,筛选出在亲本强毒株PRSV-LM的P1和HC-Pro基因上的8个可能与致病性相关的氨基酸突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))。采用定点突变和Gibson拼接法,分别成功构建了4种含有2个突变位点(L_(754)和N_(787))、4个突变位点(I_(309)、K_(481)、F_(753)和D_(944))、6个突变位点(I_(309)、K_(481)、F_(753)、L_(754)、N_(787)和D_(944))和8个突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))的PRSV-LM全长c DNA侵染性克隆突变体:p Gprsvm~2、p Gprsvm4、p Gprsvm6和p Gprsvm8。经人工接种、病症观察和RT-PCR检测,4种突变体克隆均能系统性侵染非转基因番木瓜植株,除克隆p Gprsvm~2的病症表现与亲本强毒株PRSV-LM一致外,其他多位点突变的克隆p Gprsvm4、p Gprsvm6和p Gprsvm8在番木瓜植株上的病症严重程度出现依次减弱,而克隆p Gprsvm8的病症最弱,且延迟2周发病。本实验初步验证了6个氨基酸突变位点(I_(309)→S、K_(481)→Q、K_(598)→E、F_(753)→L、R_(728)→I和D_(944)→N)与PRSV-LM的病症表现及致病力相关,其中K_(598)→E和R_(728)→I可能是影响致病性的关键位点。     

Pre-breeding for resistance to Verticillium wilt in olive: Fishing in the wild relative gene pool

This study aimed to identify new sources of resistance to Verticillium wilt in olive. We evaluated various types of genotypes: wild olive trees (Olea europaea subsp. europaea var. sylvestris), genotypes belonging to related subspecie (Olea europaea subsp. guanchica) and genotypes coming from crosses between Picual cultivar and wild olive trees. Fifty-six genotypes were inoculated by dipping roots and then screened under controlled conditions to test their resistance to a highly virulent Defoliating isolate of Verticillium dahliae. Picual (susceptible) and Frantoio (resistant) were control cultivars. Wide variability and significant differences were obtained in the evaluated disease parameters. The Relative Susceptibility Index (RSI), summarizing the disease parameters, was used for final classification of genotypes. Thirteen genotypes were categorized as resistant: eight wild olives from different locations, two genotypes belonging to subsp. guanchica populations from Canary Islands and three genotypes obtained from one of the crosses Picual x wild. The identification of high levels of resistance to V. dahliae among wild olive genotypes may be helpful for the management of this disease. The resistant genotypes could be used as rootstocks for susceptible olive cultivars or parents in future breeding programs.     

板栗杂交新品种南垂5号的选育及栽培技术

以垂枝为母本、南沟1号为父本进行杂交,育成板栗新品种南垂5号。该品种树姿开张,枝条开张角度大;坚果单粒质量12.51 g,可溶性糖含量19.52%,淀粉含量50.12%,蛋白质含量5.56%,果肉口感细糯,风味香甜;结果早,丰产性强,适宜中国北方板栗栽培区缓坡丘陵及沙地种植。     

"苏州青"青菜的几个杂交组合的选育及栽培技术

[目的]推广＂苏州青＂青菜几个杂交组合的种植。[方法]对苏州青杂一号、苏州青杂二号、苏州青杂三号的选育过程、产量表现、特征特性、栽培技术等要点进行阐述。[结果]苏州青杂一号、苏州青杂二号、苏州青杂三号植株直立,株形紧凑,束腰性好,商品性好,纤维少、口感好,抗病性强,生长迅速,产量高于常规＂苏州青＂品种,已于2009年通过苏州市科技局主持的成果鉴定。[结论]＂苏州青＂青菜3个杂交组合适合江苏省及类似气候地区分别作早中秋、晚秋冬栽培。其中,苏州青杂一号、苏州青杂二号适合晚秋冬栽培,苏州青杂三号适合早中秋栽培。     

23个黄瓜杂交组合田间初选试验

以抗病、早熟、丰产为主要育种目标,选用16个优良黄瓜自交系进行杂交组合配制,获得杂交组合23个,分为绿色和黄绿色2组,2011年春季进行了露地田间初选试验。结果表明：绿色组中P26（组合A31×A16,A31为母本）综合评价最高,第1雌花节位低（3.5节）,早熟,定植至采收需36 d,前期产量高达7.97万kg/hm2,全生育期总产量达13.43万kg/hm2,瓜把短至2.80 cm,果实横径小（3.40 cm）,果形指数大（9.15）,油亮光泽,口感清甜,高抗枯萎病、霜霉病和白粉病;黄绿色组中P10（组合A40×A15,A40为母本）综合评价最高,果形美观,第1雌花节位低（4.5节）,中早熟,定植至采收需40 d,前期产量可达7.54万kg/hm2,全生育期总产量为11.49万kg/hm2,瓜把长度仅0.80 cm,果实横径大（6.1 cm）,果形指数3.38,有光泽,口感香甜,高抗枯萎病和霜霉病,抗白粉病。