Construction of Lactuca sativa Plastid Transformation Vector and High-level Expression of gfp Gene in Escherichia coli

Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.

Physiological and genome-wide gene expression analyses of cold-induced leaf rolling at the seedling stage in rice(Oryza sativa L.)

Leaf rolling and discoloration are two chilling-injury symptoms that are widely used as indicators for the evaluation of cold tolerance at the seedling stage in rice. However, the difference in cold-response mechanisms underlying these two traits remains unknown. In the present study, a cold-tolerant rice cultivar, Lijiangxintuanheigu, and a cold-sensitive cultivar, Sanhuangzhan-2, were subjected to low-temperature treatments and physiolog-ical and genome-wide gene expression analyses were conducted. Leaf rolling occurred at temperatures lower than 11℃, whereas discoloration appeared at moderately low temperatures such as 13℃. Chlorophyll contents in both cultivars were significantly decreased at 13℃, but not altered at 11℃. In contrast, the relative water content and relative electrolyte leakage of both cultivars decreased significantly at 11℃, but did not change at 13℃. Expression of genes associated with calcium signaling and abscisic acid (ABA) degradation was significantly altered at 11℃ in comparison with 25℃ and 13℃. Numerous genes in the DREB, MYB, bZIP, NAC, Zinc finger, bHLH, and WRKY gene families were differentially expressed. Many aquaporin genes and the key genes in trehalose and starch synthesis were down regulated at 11℃ in comparison with 25℃ and 13℃. These results suggest that the two chilling injury symptoms are temperature-specific and are controlled by different mechanisms. Cold-induced leaf rolling is associated with calcium and ABA signaling pathways and is regulated by multiple transcriptional regulators. The suppression of aquaporin genes and reduced accumulation of soluble sugars under cold stress results in a reduction in cellular water potential and consequently leaf rolling.

Expression Vector Construction and Genetic Transformation of <i>Rosa rugosa β</i>-l,3-Glucanase Gene (<i>RrGlu</i>)

In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.

Variation in Expression of <i>Sub</i>1 Gene and Association with Submergence Stress Related Traits in Advance Rice (<i>Oryza sativa</i>L.) Genotypes as a Tool of Climate Change Adaptation

Floods have now become most detrimental natural catastrophe worldwide due to radical climatic fluxes. Therefore, there is a dire necessity to develop a high yielding rice lines to deal with this scenario. For this purpose, a large scale experiment was conducted including one hundred and fifteen (115) rice genotypes having SUB1 gene imported from International Rice Research Institute (IRRI) Philippines, six local cultivars/approved varieties and three high yielding rice varieties i.e. Sabitri, IR6 and NSICRC222 being used as potential varieties in different countries of Asia as susceptible check and IR64-SUB1 as tolerant check. The genotypic screening was performed using two PCR-based DNA markers i.e. ART5 and SC3. Phenotypic screening was conducted in a natural pond to assess the interaction of SUB1 gene in natural stagnant flood water as well as the suitability of introgression of SUB1 gene into approved varieties and elite rice lines. The genotypes were assessed in terms of plant survival percentage, submergence tolerance index, physical condition, stem elongation, number of grains per panicle, thousand grain weight, grain yields and deviations in these traits after submergence stress. The PCR results suggested that both the primers ART5 and SC3 may be used as potential PCR-based markers for molecular screening of rice genotypes for SUB1 QTL. Furthermore, it confirmed the presence of SUB1 gene in all the lines imported from IRRI, while it was absent in all the local cultivars studied. All the genotypes with submergence tolerant gene (SUB1) showed significantly greater tolerance level in submergence stress of 14 days, as compared to other local cultivars/varieties, authenticating the effectiveness of SUB1QTL in conferring submergence tolerance. Significantly different performances of all the SUB1 genotypes in terms of all the studied traits indicate high Genotypic and Genotypic Environment Interaction (GEI) of SUB1QTL. Employment of SUB1 lines such as R105479:149-18, IR64-SUB1 and Rl05469:81-22-3 in breeding programs for developing flood tolerant rice varieties might further upsurge rice yields in flash flood areas. Correlation analysis revealed that plant survival percentage after submergence, reduced stem elongation during submergence and submergence tolerance index are very important traits for developing submergence tolerant lines.

Improvement of foreign gene expression in transgenic rice(Oryza sativa L.)by matrix attachment regions(MARs)

Matrix attachment regions or matrix associated regions (MARs) were special DNA sequences in chromatin of eukaryotic cells that tightly associated with the nuclear matrix or scaffold in vitro after a combination of nuclease digestion and extraction. They were also called scaffold attachment regions(SARs) . It was found that MARs could improve the expression level and the stability of foreign genes in transgenic plants. The reason might be that a transgene flanked by MARs was transmitted into the plant cells, the MARs would attach the nuclear

Genetic expression and effects of the rolled leaf gene Rl(t) in hybrid rice (Oryza sativa L.)

We constructed a nearisogenic line of rolled leaf gene Rl(t), which ex-pressed incompletely dondnance for the character of rolled leaf(RL), with ge-netic background of Zhenshan 97B. Using RL Zhenshan 97B and the originalZhenshan 97B as the female parents, and Minghui 63 and Yanhui 559 as themale parents, crosses of RL Shanyu 63 (RS63) and Shanyu 63(S63), RLShanyou 559 (RS559) and Shanyou 559 (S559) were made. Inheritance andeffects of Rl(t) in hybrid rice were studied at the flowering and at the 20 d afterflowering, respectively. Results were as follow:

Expression of Cold Resistant Gene CAS19 of Gongnong No.2 Medicago sativa L. in Tobacco

[Objective] The aim was to study the expression of cold resistant gene CAS19 of Gongnong No.2 Medicago sativa L. in tobacco. [Method] A pair of primers was designed according to nucleotide sequences of cold resistant gene CAS19 of M. sativa,and then RT-PCR was used to amplify the protein gene of CAS19,which was then cloned into pMD18-T vector and subcloned into expression vector PBI121. The recombination expression plasmid PBCAS was constructed. And then it was transferred into tobacco genome via Agrobacterium,and Southern-blotting analysis was used for detecting transgenic plants. [Result] CAS19 gene was integrated into the tobacco genome and highly expressed. [Conclusion] This study had provided theoretical basis for further exploring the expression mechanism of cold resistant gene CAS19 in tobacco.

低钾胁迫对水稻(Oryza sativa L.)化感潜力变化的影响

研究以国际公认的化感水稻PI312777和非化感水稻Lemont为供体,稗草(Echinochloa crus-galli L.)为受体,采用稻/稗共培体系,研究低钾胁迫对水稻化感潜力变化的影响及其机制。受体稗草的形态指标分析结果表明,低钾胁迫促使化感水稻PI312777对共培稗草的根长、株高和干重的抑制率均升高,增幅远大于非化感水稻Lemont。受体稗草生理生化指标分析结果表明,低钾胁迫下化感与非化感水稻对受体稗草保护酶系(SOD、POD、CAT)及根系活力的抑制作用增强,但化感水稻PI312777比非化感水稻Lemont的抑制程度大,且达极显著差异。实时荧光定量PCR分析结果表明,低钾胁迫下,化感水稻PI312777根部与叶部中酚类代谢的关键酶——苯丙氨酸解氨酶、肉桂酸-4-羟化酶、羟化酶、O-甲基转移酶的基因均上调表达,而非化感水稻根部相应酶均下调表达,叶部除苯丙氨酸解氨酶上调,其余酶也下调表达。而萜类代谢途径关键酶——HMG-CoA还原酶、角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶的基因,在两种水稻根部中呈现出相同或相似的表达方式(上调或下调),即HMG-CoA还原酶上调表达,角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶下调表达;而在水稻叶部,非化感水稻Lmont相应酶基因表达方式仍然不变,化感水稻PI312777除了角鲨烯合酶下调表达,其余4个酶均上调表达。水稻根系分泌物中酚类物质的HPLC分析结果表明,低钾胁迫下,化感水稻PI312777根系分泌物中,所检出的酚酸类物质总量是正常营养条件下的2.30倍,而非化感水稻Lemont则是正常营养条件下的0.91倍。综合分析认为低钾胁迫下,化感水稻PI312777抑草能力增强主要是由于酚类代谢途径关键酶基因表达上调,导致酚类代谢途径旺盛,分泌出更多的酚类物质,进而破坏受体稗草保护酶系统,抑制了稗草的正常生长。

不同化感潜力水稻(Oryza sativa L.)苗期的氮素养分效率及相关基因的表达

为了阐明养分水平引起水稻(Oryza sativaL.)化感抑草潜力变化的生理生态机制,研究了不同N素营养处理下,不同化感潜力水稻苗期对N素营养逆境的响应特性及N素养分效率的差异,并运用实时荧光定量RT-PCR技术(FQ-PCR)检测与N素代谢和次生代谢关键酶的基因表达。结果表明:弱化感水稻品种Lemont对N素营养胁迫较敏感,强化感水稻品种PI312777对资源波动的适应性较强,N素养分效率较高。FQ-PCR分析结果显示,在低N条件下Lemont中的亚硝酸还原酶基因(nir),谷氨酰胺合成酶基因(gs)相对表达量均有不同幅度的下调,PI312777分别下调了1.2倍和1.4倍,而Lemont分别下调了3.0倍和1.8倍,Lemont下调的幅度分别是PI312777的2.5倍和1.3倍,但对于苯丙氨酸解氨酶基因(pal)与3-羟基-3甲基戊二酰辅酶A还原酶基因(hmgr)而言,PI312777叶组织中的pal和hmgr均上调表达,与对照相比上调了6.0倍和1.6倍,而Lemont中对应的基因均下调表达,分别下调了1.3倍和6.8倍,佐证了上述差异的分子生态学特性。

Isolation, Expression Characteristics and Chromosomal Locations of Three cDNA Fragments Under Salt Stress in Rice

cDNA libraries were constructed from the leaves of a rice (Oryza sativa L.) salt tolerancevariety Tesan抋i 2 growing in solutions with 150 mmol/L NaCl for 3 h or without salt stress. Three salt-responsive cDNA clones, Ts1, Ts2 and Ts3 were isolated by differential screening. Northern blottinganalysis showed that the transcription levels of Ts1 and Ts2 increased within 3 h salt stress and kept onincreasing within 24 h, while the transcription level of Ts3 reached its peak within 3 h. Sequence analysisindicated that there were no homologies between the three cDNA clones and any known gene. The threecDNA clones were mapped using a doubled haploid (DH) population derived from an indica variety ZYQ8,which was a salt tolerance parent of Tesan抋i 2, with a japonica variety JX17. Ts1, Ts2 and Ts3 werelocated on chromosomes 1, 3 and 7, respectively. It was noted that Ts1, Ts2, and Ts3 were in or near theregions of major or minor salt tolerance quantitative trait loci (QTLs), which were mapped in the same DHpopulation in a parallel study.

Comparative mapping of QTLs for Al tolerance in rice and identification of positional Al-induced genes

Aluminum (Al) toxicity is the major factor limiting crop productivity in acid soils. In this study, a recombinant inbreed line (RIL) population derived from a cross between an Al sensitive lowland indica rice variety IR1552 and an Al tolerant upland japonica rice variety Azucena, was used for mapping quantitative trait loci (QTLs) for Al tolerance. Three QTLs for relative root length (RRL) were detected on chromosome 1, 9, 12, respectively, and 1 QTL for root length under Al stress is identical on chromosome 1 after one week and two weeks stress. Comparison of QTLs on chromosome 1 from different studies indicated an identical interval between C86 and RZ801 with gene(s) for Al tolerance. This interval provides an important start point for isolating genes responsible for Al tolerance and understanding the genetic nature of Al tolerance in rice. Four Al induced ESTs located in this interval were screened by reverse Northern analysis and confirmed by Northern analysis. They would be candidate genes for the QTL.

水稻OsRLCK167基因的组织表达模式和生物信息学分析

类受体胞质激酶(receptor-like cytoplasmic kinases,RLCKs)家族是一类特殊蛋白激酶,在植物生长和病原菌防御中发挥着重要的生物学功能。在对水稻RLCKⅥ家族成员OsRRK1基因的研究中发现水稻4号染色体上存在一个与其同源性特别高的基因,即OsRLCK167(LOC_Os04g56060)。为了丰富类受体胞质激酶家族并探究水稻胞质受体激酶基因的作用,本研究利用ExPASy-Protparam、Protscale、CD-search等在线工具和DNAMAN软件对OsRLCK167基因进行生物信息学分析;采用qRT-PCR技术对OsRLCK167基因做组织表达模式分析。结果显示:OsRLCK167蛋白的分子量为43.77776 kD,为疏水性、不稳定的酸性蛋白;对该蛋白的二级结构进行预测,显示其主要由无规则卷曲(41.94%)、α-螺旋(35.29%)、延伸链(15.35%)和β-转角(7.42%)组成;该基因在水稻不同组织均有表达,且在叶鞘中的表达量最高。结果表明,OsRLCK167基因在进化过程中具有高度的保守性,在水稻中具有功能多样性。

Expression of PIN Genes in Rice （Oryza sativa L.）： Tissue Specificity and Regulation by Hormones

Twelve genes of the PIN family in rice were analyzed for gene and protein structures and an evolutionary relationship with reported AtPINs in Arabidopsis. Four members of PIN1 （designated as OsPINla-d）, one gene paired with AtPIN2 （OsPIN2）, three members of PIN5 （OsPIN5a-c）, one gene paired with AtPIN8 （OsPIN8）, and three monocot-specific PINs （OsPIN9, OsPINIOa, and b） were identified from the phylogenetic analysis. Tissue-specific expression patterns of nine PIN genes among them were investigated using RT-PCR and GUS reporter. The wide variations in the expression domain in different tissues of the PIN genes were observed. In general, PIN genes are up-regulated by exogenous auxin, while different responses of different PIN genes to other hormones were found.

Molecular cloning,in vitro expression and enzyme activity analysis of violaxan-thin de-epoxidase from Oryza sativa L.

The violaxanthin de-epoxidase gene was cloned from rice (Oryza sativa subsp. japonica). The full length of the cDNA is 1887 bp, encoding a 446-amino acids protein with the transit peptide of 98 amino acids. The bacterial expression vector pET-Rvde was constructed and the expression quantity of the exogenous protein increased with the induction time by 0.4 mmol/L IPTG. Its molecular weight was similar with that of the native VDE. Western blotting indicated that the expressed protein has immu-nological reaction with the VDE polyclonal antibody. The absorbance spectrum together with xanthophyll pigments quantification by HPLC demonstrated that the expressed VDE has its enzyme activity, which can de-epoxidate violaxanthin into antheraxanthin and zeaxanthin in vitro.

逆境处理下水稻叶角质层蜡质积累及其与蜡质合成相关基因OsGL1表达的关系

植物角质层蜡质在抵抗各种生物和非生物胁迫中起着非常重要的作用。本试验以水稻(Oryza sativa L.)幼苗为材料,分别以200mmol L-1 NaCl、12%PEG、1.0%H2O2、40℃高温和8℃低温为逆境,研究叶角质层蜡质的积累情况以及其与水稻蜡质合成相关基因OsGL1表达的关系。扫描电镜观察以及叶角质层蜡质总量测定结果表明,12%PEG、1.0%H2O2和8℃低温处理下水稻幼苗叶角质层蜡质的积累明显增加,而200mmol L-1 NaCl和40℃高温处理下叶角质层蜡质覆盖量略有下降。RT-PCR分析显示,逆境处理下水稻蜡质合成相关基因OsGL1的表达量变化与水稻幼苗叶角质层蜡质的积累存在相关性。

利用CSSL群体研究稻米AC和PC相关QTL表达稳定性

利用以Asominori为遗传背景具IR24染色体片段的置换系（CSSL）群体，在“2年4点”8个环境对稻米直链淀粉含量（AC）和蛋白质含量（PC）进行QTL定位和表达稳定性分析。结果共检测到8个AC和PC相关QTL，其中2个QTL在8个环境中都能重复出现，即影响AC的qAC-8和控制PC的qPC-8，平均贡献率分别为21．0％和26．9％。qAC-8和qPC-8对应置换系与背景亲本Asominori在8个环境中相应性状的表现型都达到极显著差异（P〈0．01）；都仅与8个环境中的2个环境之间存在显著的互作效应；说明qAC-8和qPC-8的效应显著且稳定性较高。此外，qAC-8和qPC-8都被定位在第8染色体R727～G1149区间，IR24的等位基因可同时提高AC和PC，这为研究水稻籽粒直链淀粉和蛋白质形成途径之间的相互关系以及碳氮代谢协同调控的遗传机制提供了新的信息。

水稻Dof基因家族的组织表达谱及胁迫诱导表达特征分析

基因的表达与调控是一个复杂的分子网络,受许多转录因子的时空表达调节。Dof蛋白是一类植物特有的转录因子,在多种植物特有生物学过程中起着关键的调控作用。水稻全基因组中发现含30个Dof基因,但对其绝大部分基因的功能尚缺少了解。本研究对30个水稻Dof基因及启动子区进行生物信息学分析,发现OsDof基因上游1.5kb启动子区含有大量的光反应、组织器官表达、植物激素反应及逆境反应等重要的顺式作用元件。进一步通过荧光定量RT-PCR技术分析,结果表明了大部分OsDof基因呈多器官较低丰度表达,少部分为器官优势或高丰度表达。另外,与未胁迫处理的水稻幼苗相比较,28个OsDof基因的表达受光、ABA、NaCl或PEG等胁迫处理的调节,其中27个OsDof基因可以对2种或2种以上的胁迫条件产生响应,表明这些成员可能在生理水平上参与不同胁迫相关信号途径之间的交互作用。

水稻RR基因家族生物信息学及表达模式分析

水稻反应调节因子(response regulators,RR)家族在植物生长发育中扮演着重要角色,探究RR基因在水稻中的进化及生物学特征具有重要意义。水稻RR基因家族共有22个成员,包含15个A型和7个B型基因。分析发现,水稻RR蛋白的理化性质存在差异,其中B型均为亲水蛋白(GRAVY<0);水稻RR基因有5个旁系基因对,分别为OsRR8和OsRR15、OsRR5和OsRR11、OsRR9和OsRR10、OsRR1和OsRR2、OsRR20与OsRR18,预测其与已知功能基因具有相似的功能;保守基序Motif 1与Motif 3均属于REC超级家族,作为响应调节器接收来自双组分磷酸转移系统中的信号传导,在22个OsRR蛋白中均有分布。调控元件与表达模式分析表明,RR基因具有调控脱落酸、水杨酸、茉莉酸、赤霉素与生长素等多种信号转导的潜能,参与植物防御低温、干旱等胁迫及光和厌氧等信号响应过程,同时也具有调控水稻不同组织生长发育的功能。通过对水稻RR基因进行生物信息学分析,为后续深入研究水稻RR家族成员的功能提供理论依据,也为进一步揭示植物生长发育及信号传导机制奠定了基础。

野生稻高产QTL导入晚稻恢复系的增产效果

以含有野生稻高产QTLyld1.1和yld2.1的测交材料(V20A/O.rufipogon∥V20B///V20B////测64-7)为基因供体(作父本),以中熟晚稻恢复系测64-7为受体(作母本),连续回交3代再多代自交,每代采取分子标记辅助选择和田间选择相结合的方法,育成携带野生稻高产QTLyld1.1和yld2.1的籼型晚稻新恢复系远恢611。以远恢611及受体亲本测64-7作父本,分别与21个不同类型的不育系配制21对杂交稻组合,同时以威优46、汕优63、金优207为生产对照,分析了野生稻高产QTL的增产效果。结果表明,远恢611系列组合平均理论产量比测64-7系列组合(CK)增产15.5%,21对组合中有17对表现增产,增产组合数约占81.0%;实收产量有16对组合(占76.2%)表现比CK增产,平均增幅7.8%;单位面积颖花量有19对组合(占90.5%)表现比CK增加,平均增幅14.5%;这3项指标的差异均达到极显著水平。对供试45个组合的理论产量、实收产量、单位面积颖花量进行排名,远恢611系列组合分别有15个、12个和15个进入前20位,其中部分组合显示了超高产潜力。从产量结构来看,穗大粒多是远恢611系列组合最显著的特点,其平均穗总粒数达244.7粒,比CK增加61.3%;穗实粒数平均145.5粒,比CK增加74.0%,差异均达极显著水平;而有效穗数和千粒重相对较低,抗倒性相对较强。说明野生稻高产QTL在新恢复系远恢611及其系列组合中得到了较好的表达,具有显著的增产效果和重要的育种价值。此外,还对野生稻高产QTL及远恢611进一步利用的途径进行了分析。

水稻蜡质合成相关基因OsGL1-5的表达模式及逆境响应特征分析

通过生物信息学方法对水稻蜡质合成相关基因OsGL1-5启动子序列特点进行分析;并以水稻中花11不同组织和发育时期的穗子为材料,通过半定量RT-PCR及mRNA原位杂交技术分析OsGL1-5的时空表达特征;同时,分别以200mmol·L-1NaCl、100μmol·L-1ABA和1.0%H2O2处理水稻幼苗,通过半定量RT-PCR分析逆境胁迫下OsGL1-5的表达模式。生物信息学分析表明,OsGL1-5启动子中包括大量与干旱、冷、盐等多种逆境胁迫相关的调控序列。时空表达特征分析表明,OsGL1-5主要在不同发育时期的穗子中表达,且表达部位集中在稃片的毛状体和花药绒毡层中,叶中表达量相对较低,而茎和根中没有检测到表达。NaCl、ABA以及H2O2等逆境胁迫下,OsGL1-5表达量在不同处理时间有所增加。