番茄Lehsp23.8启动子的热诱导表达调控序列鉴定

番茄线粒体小分子热激蛋白(Lehsp23.8)启动子是典型的热诱导启动子。为了研究热激条件下该启动子的调控序列,本研究将不同长度的Lehsp23.8启动子序列与gus基因融合,构建5′缺失植物表达载体。然后用农杆菌介导法转化烟草,PCR及Southern blotting结果表明融合基因已经整合到烟草基因组中。GUS组织化学染色结果表明:不同长度Lehsp23.8启动子转基因植株热激处理后,在幼苗根、茎、叶以及花和果实中均表现出GUS活性,只是染色强弱有差异。叶片中GUS荧光活性测定结果表明:在热激处理条件下,565bp的Lehsp23.8启动子介导的GUS表达最强;而255bp的Lehsp23.8启动子介导的GUS表达最弱。说明Lehsp23.8启动子中255bp的序列即能满足该启动子的热激表达,-565bp～-255bp之间存在明显的增强子元件,而-871bp～-565bp之间的片段具有一定的抑制作用。     

白桦BpSPL2基因启动子的克隆及表达分析

SPL(SQUAMOSA promoter-binding protein-like)是植物特有的转录因子,研究表明其在参与发育阶段转变、花和果实发育等方面起着重要作用。利用PCR技术从白桦基因组DNA中扩增获得BpSPL2基因上游1 960 bp启动子序列,使用PLACE和Plant CARE在线软件分析序列,发现BpSPL2基因启动子序列中含有与开花、非生物胁迫及激素响应等相关的顺式作用元件,暗示其在植物的生长发育和胁迫应答中起重要作用。进而构建了BpSPL2基因启动子驱动GUS报告基因的植物表达载体,并利用农杆菌介导将其瞬时转化至白桦和拟南芥,通过GUS组织化学染色检测BpSPL2基因启动子的组织表达特性,结果表明BpSPL2基因启动子具有启动子活性,能够驱动GUS基因在白桦和拟南芥中表达;而其表达活性在白桦的叶片、芽及根部中较强,在拟南芥的花药、雌蕊和叶片较强,为进一步研究白桦BpSPL2基因的表达调控及其功能分析提供参考。     

水稻胁迫相关基因OsPM1启动子的克隆与分析

依据NCBI数据库OsPM1的序列信息,采用PCR技术扩增获取OsPM1的2 100bp的启动子序列。利用PLACE预测启动子的顺式作用元件分析表明,启动子内含有大量与胁迫相关的顺式作用元件,主要有ABA响应相关元件、脱水响应元件、低温响应元件、热激响应元件和转录因子结合元件。构建OsPM1的启动子和GUS基因融合表达载体,转入拟南芥。组织化学染色分析结果显示,非生物胁迫处理前,幼苗中GUS基因表达水平很低;干旱、低温、高盐等胁迫处理后,GUS基因表达量显著升高。研究表明,OsPM1的启动子能够显著提高在干旱、高盐和低温处理后下游基因的表达水平。     

西瓜wml1 5'端启动子区域对番茄的遗传转化及其转录调控功能研究

根据wml1 5’端启动子区域内部的限制性酶切位点,分离得到长度分别为1573bp、1197bp、896bp、795bp的片段,并与GUS基因融合构成转录融合体。用农杆菌介导法将这些片段转入番茄中,对转基因植株进行GUS活性分析,发现1573bp、1197bp、896bp的片段都能诱导GUS在授粉后15天、30天、45天的番茄果实中表达,且表达强度随果实发育而增强,而在叶片、茎、根中未检测到GUS基因表达。而795bp的片段转化的植株中则未检测到GUS基因表达。推定857bp至957bp之间的序列中包含了启动子行使正常功能必需的元件。     

高羊茅FaChit1基因启动子的功能分析

用含有不同长度FaChitl基因启动子区域与GUS基因融合构建植物表达载体pFaChitlP—I、pFaChitlP-Ⅱ以及pFaChitlP-Ⅲ并分别对烟草进行转化,经真菌激发子、干旱、机械损伤以及乙烯等多种胁迫处理后测定GUS活性。启动子缺失分析实验结果显示,真菌激发子对FaChitl基因启动子所介导的GUS诱导表达效果最强,而机械损伤只能微弱地诱导GL靥基因表达;FaChitl基因启动子-651bp以内的序列均能介导GUS基因的诱导表达,同时-935bp与-233bp之间的区域是该启动子响应真菌激发子、乙烯以及机械损伤胁迫所必需的。表明FaChitl启动子是一个多胁迫诱导型启动子。     

西瓜wml1 5＇端启动子区域对番茄的遗传转化及其转录调控功能研究

根据wml1 5‘端启动子区域内部的限制性酶切位点,分离得到长度分别为1573bp、1197bp、896bp、795bp的片段,并与GUS基因融合构成转录融合体。用农杆菌介导法将这些片段转入番茄中,对转基因植株进行GUS活性分析,发现1573bp、1197bp、896bp的片段都能诱导GUS在授粉后15天、30天、45天的番茄果实中表达,且表达强度随果实发育而增强,而在叶片、茎、根中未检测到GUS基因表达。而795bp的片段转化的植株中则未检测到GUS基因表达。推定857bp至957bp之间的序列中包含了启动子行使正常功能必需的元件。     

百子莲脱水素基因ApY2SK2逆境应答模式及启动子调控功能研究

在百子莲胚性细胞中筛选到对超低温保存复合逆境具有积极响应的保护类蛋白脱水素(ApY_2SK_2),为探明ApY_2SK_2基因在复合逆境中的应答模式,该研究采用染色体步移技术克隆并分析了ApY_2SK_2编码基因上游1 200 bp的启动子序列。结果表明:(1)序列分析显示,该启动子含有多个与逆境和激素诱导相关的顺式调控元件;实时荧光定量PCR结果表明,ApY_2SK_2基因的表达具有组织特异性,在百子莲的叶和果中表达量较高,且在多种胁迫处理与ABA激素诱导下,其表达量显著升高。(2)成功构建了5个ApY_2SK_2启动子不同缺失片段驱动GUS基因的融合表达载体,经农杆菌转化、抗性筛选和PCR检测鉴定,获得T_3代纯和转基因拟南芥株系。(3) GUS组织化学染色结果显示,GUS基因在拟南芥幼苗全株、成年苗的叶、花和成熟果实中表达活性较强,但在未成熟果实中无明显表达;烟草瞬时表达结果显示,与对照组相比,在脱水胁迫和ABA处理下的ApY_2SK_2启动子不同缺失片段驱动GUS基因表达具有显著差异。(4)转基因拟南芥GUS活性测定结果显示,ApY_2SK_2启动子MBS元件和ABRE元件可响应干旱与渗透胁迫信号;ApY_2SK_2启动子LTR元件参与低温响应;ApY_2SK_2启动子-1 199～-262 bp区域包含多个串联的ABRE顺式调控元件(-373～-211 bp)对响应ABA信号具有主要调控作用。该研究结果揭示了ApY_2SK_2启动子的组织特异性,且启动子上的关键顺式调控元件对不同的胁迫和激素信号响应具有决定性调控作用。     

水曲柳节律基因LHY启动子的克隆和功能分析

以水曲柳基因组DNA为模板,用Site Finding-PCR法扩增得到节律基因LHY（late elongated hypocotyl）启动子序列,长度为1 360 bp。PLACE启动子预测工具分析表明,序列中含有转录必备的TATA box、CAAT box以及一些非生物胁迫和激素响应元件等。构建植物GFP瞬时表达载体p PXGFP-P-LHY,农杆菌介导转化烟草叶片和白桦悬浮细胞,GFP检测结果表明,LHY启动子能够启动GFP基因在烟草和白桦细胞中表达,且对非生物胁迫（低温、高温、盐）产生响应;构建植物GUS报告基因整合表达载体p PCXGUS-P-LHY,农杆菌介导法瞬时转化烟草,GUS染色结果表明,LHY启动子的活性具有不同程度的时空特性。     

拟南芥AtNCED2基因启动子区域序列克隆及其活性分析

目的:克隆拟南芥AtNCED2基因启动子区域序列,并分析其组织器官特异性及对外界刺激的响应.方法:通过PCR从拟南芥基因组中克隆AtNCED2基因5'侧翼2295bp启动子区域序列(AtNCED2p),并进行生物信息学分析.构建AtNCED2p驱动GUS的植物双元表达载体pAtNCED2p::GUS,通过根癌农杆菌介导法将其转化野生型拟南芥,检测转基因植侏中GUS表达的组织器官特异性.结果:该启动子序列中存在TATA-box、CAAT-box、根器官特异性元件、ABA响应元件、低温响应元件、昼夜节律响应元件等顺式作用元件.GUS活性主要集中在转基因拟南芥根尖及侧根发生部位.外源ABA处理的转基因植株根中GUS活性为174.8nmol 4-MU min-1 mg-1蛋白,明显高于对照值91.7nmol 4-MU min-1mg-1蛋白.结论:AtNCED2基因可能在根的生长和发育中起作用,且外源ABA处理增强其在根中的表达.     

番茄SlWRKY31基因启动子的克隆与逆境应答模式分析

该研究以野生型番茄（Solanum lycopersicum）为材料,采用PCR技术克隆得到了番茄 SlWRKY31 基因起始密码子 ATG 上游启动子序列,并利用该启动子驱动 GUS基因在野生型番茄中表达,对获得的转基因番茄采用不同胁迫处理后进行GUS 染色和定量分析。结果表明：（1）序列分析显示,该启动子全长1 849 bp,含有多个与非生物胁迫和激素响应相关的顺式作用元件,主要包括热胁迫响应元件 HSE、干旱诱导响应元件 MBS、防卫和胁迫响应元件 TC rich repeats、创伤诱导响应元件 WUN motif、脱落酸（ABA）响应元件 ABRE 和水杨酸（SA）响应元件 TCA element。（2）实时荧光定量 PCR 结果显示,SlWRKY31 基因呈组成型表达模式,且在叶和果实中表达量较高,茎中较低;在NaCl、甘露醇、SA、ABA 和42 ℃ 高温的胁迫处理下,其表达量显著升高。（3）构建 SlWRKY31 启动子和 GUS 基因融合的植物表达载体,并通过农杆菌介导法将其转化野生型番茄,对获得的转基因番茄进行 GUS 组织化学染色分析结果显示,SlWRKY31 基因在番茄的各个组织（根、茎、叶、花、果实和种子）中均有表达,表明 SlWRKY31 启动子是组成型表达启动子。（4）对转基因番茄在不同胁迫处理后的 GUS 染色和定量分析显示,SlWRKY31 启动子显著受到NaCl、甘露醇、SA、ABA 和42 ℃ 高温的诱导表达,说明该启动子是一个可以响应多种逆境胁迫的诱导型启动子。     

两个大豆类受体蛋白激酶基因的克隆及其结构和功能的初步分析

利用高等植物类受体蛋白激酶基因的保守域设计简并引物的通过RT-PCR方法,从大豆叶片中克隆到两个新的,可能的类受体蛋白激酶基因的部分cDNA片段。对其基因结构的分析表明：在RLPK2的激酶保守域Vib与Ⅸ之间有一个407bp长的内含子。利用RT-PCR方法对它们的表达特性进行初步研究。发现这两个基因可能参与了对大豆叶片衰老和/或细胞分裂素延缓衰老过程的调节机制。     

Genetic and physiological characterization of tomato cv. Micro-Tom

Based on its compact habit, Micro-Tom, a dwarf cultivar of tomato (Solanum lycopersicum L.), has been proposed as a preferred variety to carry out molecular research in tomato. This cultivar, however, is poorly characterized. It is shown here that Micro-Tom has mutations in the SELF-PRUNING (SP) and DWARF (D) genes. In addition to this, it is also shown that Micro-Tom harbours at least two independently segregating resistance loci to the plant pathogen Cladosporium fulvum. The presence of the self-pruning mutation in Micro-Tom, that generates a determinate phenotype, was confirmed by crossing and sequence analysis. It was also found that Micro-Tom has a mutation in the DWARF gene (d) that leads to mis-splicing and production of at least two shorter mRNAs. The d mutation is predicted to generate truncated DWARF protein. The d sequence defect co-segregates with dark-green and rugose leaves, characteristics of brassinosteroid biosynthesis mutants. Micro-Tom also carries at least another mutation producing internode length reduction that affects plant height but not active gibberellin (GA) levels, which were similar in dwarf and tall Micro-TomxSeverianin segregants. GAs and brassinosteroids act synergistically in Micro-Tom, and the response to GA depends on brassinosteroids because the elongation of internodes was at least six times higher when GA(3) was applied simultaneously with brassinolide. A novel variety, Micro-0 that is fully susceptible to C. fulvum and almost as dwarf as Micro-Tom, has been generated from the cross of Cf0xMicro-Tom. This line represents a valuable resource for future analysis of Cf resistance genes through breeding or transformation.     

Aluminum lightens the adverse effects of excessive Mn on growth of soybean (Glycine max)]

The influence of aluminum (Al) on physiological and biological characteristics of soybean under manganese (Mn) stress was investigated. The results showed that Al suppressed the transport of Mn to shoots (Fig.2B, C), and subsequently alleviated the inhibition of shoot growth (Fig.1), decreased the chlorophyll content (Fig.4). Addition of Al diminished the increase in O(-*)(2) producing rate, the hydrogen peroxide (H(2)O(2)) content and malondialdehyde (MDA) content, and activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) in soybean leaves caused by excessive Mn (Fig.5), and prevented the dropping of CAT activity to a low level under excessive Mn stress (Fig.6). Results of fractional analysis indicated that high levels of Al supply deduced mainly accumulation of Mn both in cell walls and organelles, but had no effect on it in soluble fractions (Fig.3).     

壳聚糖涂膜对菜用大豆荚采后衰老和品质的影响

以"南农二号"菜用大豆(Glycine max)荚为试材,研究不同浓度的壳聚糖涂膜对菜用大豆荚采后衰老、品质和腐烂的影响.结果表明,1.0%和1.5%壳聚糖处理可显著降低豆荚的呼吸强度、蒸腾失水、多酚氧化酶(PPO)及过氧化物酶(POD)活性,保持较低的膜透性、丙二醛(MDA)含量和较高的叶绿素、Vc及还原糖含量,提高豆荚的苯丙氨酸解氨酶(PAL)活性和促进木质素合成,减少豆荚腐烂发生,从而延长贮藏期.     

A new model system for tomato genetics

The purpose of this study was to develop a model system for studying tomato genetics. Agronomic, genetic, and molecular data are presented which show that the miniature Lycopersicon esculentum cultivar, Micro-Tom (Micro tomato), fulfills the requirements for such a model. It grows at high density (up to 1357 plants/m⁻²); it has a short life cycle (70–90 days from sowing to fruit ripening); and it can be transformed at frequencies of up to 80% through Agrobacterium -mediated transformation of cotyledons. Moreover, it differs from standard tomato cultivars by only two major genes. Therefore, any mutation or transgene can be conveniently studied in Micro-Tom's background and, when needed, transferred into a standard background. We took advantage of Micro-Tom's features to improve the infrastructure for mutagenesis in tomato. A screening of 9000 M1 and 20 000 M2 EMS mutagenized plants is described. Mutants with altered pigmentation or modified shape of leaves, flowers and fruits were found. In addition, an enhancer trapping and a gene trapping system, based on the Ac/Ds maize transposable elements, were transformed into Micro-Tom and found to be active. In summary, Micro-Tom opens new prospects to achieve saturated mutagenesis in tomato, and facilitates the application of transposon-based technologies such as gene tagging, trapping and knockout.     

Mehler反应在大豆叶片耗散过剩光能中的作用

从初花期大豆植株茎基部引入不同浓度碘乙酰胺(iodoacetamide,IA),叶片光合速率(Pn)受到不同程度的抑制,2h强光照射后抑制效应更加明显,O     

小麦和大豆叶片的气孔不均匀关闭现象

用14CO2放射自显影的方法研究了田间小麦和大豆叶片在水分胁迫下的气孔关闭状况。正常浇水的小麦和大豆叶片呈现出对14CO2的均匀吸收。在小麦与大豆"片水势分别降至-1．75和-1．32MPa的土壤干旱条件下,两种作物叶片都发生气孔不均匀关闭。离休叶片在空气中快速脱水易引起气孔不均匀关闭。正常供水小麦叶片在晴天中午明显的光合午休时,无CO2的不均匀吸收。某些晴天中午,在大豆光合午休低谷时段观察到较明显的气孔不均匀关闭,用气体交换资料计算出的细胞间隙CO2浓度并不随气孔年度的降低而下降,反而略有回升。     

信号传导拮抗物对大豆细胞植保素和异黄酮积累的影响

磷酸酶的抑制剂花萼海绵诱癌素A、芫菁素和冈田酸都能诱导大豆植保素(大豆素)的积累。相反,激酶的抑制剂K252a几乎完全阻止它们诱导大豆素的合成。与磷酸酶抑制剂相比较,酵母细胞壁激发子(YE)有利于诱导黄苷元、染料木苷等异黄酮中间体的积累,而磷酸酶的抑制剂有利于大豆素的合成。YE和芫菁素还呈现出协同诱导大豆素积累的效果。通过磷脂酶A2的抑制剂与阻止线粒体ATP合酶活性的化合物的分别处理,发现茉莉酸信号传导途径是参与调控大豆植保素合成的重要途径之一,而植保素的合成需要线粒体提供能量。     

Use of Micro-Tom cultivar in a <Emphasis Type="Italic">Bemisia tabaci</Emphasis> biotype B interaction study

Tomatoes of the Micro-Tom cultivar, Solanum lycopersicum L. (Solanaceae), are small, have a short life cycle, high-density growth, high-efficiency protocols for genetic transformation, and hormonal and morphological mutants. These characteristics make this cultivar a good candidate as a helpful tool in resistance studies against the whitefly, Bemisia tabaci (Gennadius 1889) (Hemiptera: Aleyrodidae). The insect behavior in the Micro-Tom cultivar was observed through free-choice and no-choice oviposition preference tests and life cycle in lab conditions, having as reference the Santa Clara cultivar. In these tests, behavioral and biological insect parameters were obtained and the purpose was used to assess the trichome absence effect on oviposition with the hairless mutant. In the studies for oviposition preference, no difference was observed among the three material obtained. A nymphal stage prolongation and a low nymph viability with an adult longevity reduction were observed in relation to the Santa Clara in the Micro-Tom cultivar and hairless mutant. The Micro-Tom cultivar and hairless mutant do not present antixenotic effects to the oviposition. Mutation present in the hairless mutant does not alter the results observed in the ‘Micro-Tom.’ In general, the absence of the trichome did not reduce the Micro-Tom susceptibility to the oviposition. Antibiosis was observed in the Micro-Tom and it was discussed considering its association with salicylic and jasmonic acids, and brassinosteroid levels. These results show that this cultivar is a pest host and suitable for greenhouse and lab tests, in addition to being able to be used as a susceptibility standard for antixenosis.     

午间强光胁迫下SOD对大豆叶片光合机构的保护作用

晴天田间大豆叶片Ｐｎ与Ｐｒ均表现出明显的日变化,Ｐｎ日变化曲线里双峰型,中午前后降低。Ｐｒ随日照强度的增加而增加,至上午１１时左右达最大值,然后缓慢下降。普通空气及低氧空气中的ＡＱＹ均在中午前后降低。ＳＯＤ活性也有明显的日变化,最高值出现在下午１６时左右。强光下喷施ＳＯＤ抑制剂ＤＤＴＣ明显降低Ｐｎ及低氧空气中的ＡＱＹ;而在低于叶片光合作用饱和光强下喷施同样浓度ＤＤＴＣ则对Ｐｎ及低氧空气中的ＡＱＹ无明显影响。中午前后ＳＯＤ活性及Ｐｒ的增加对于保护光合机构免受强光的破坏具有重要意义。