Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice, We combined the RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice. We believe that the findings described here will help to elucidate the molecular mechanism(s) underlying the developmental processes of rice leaf.     

磷胁迫对不同杉木无性系酸性磷酸酶活性的影响

 缺磷是限制目前农林业产量的一个重要因子,传统的农林业生产主要通过施肥和土壤改良来满足植物对磷的需求,近年来人们开始发掘磷高效利用植物来替代传统方法提高磷的利用效率。杉木(Cunninghamia lanceolata)是我国亚热带地区最重要的造林树种之一,生长快、材质好、产量高,在中国人工林经营中占有重要地位。为揭示磷素胁迫条件下杉木无性系酸性磷酸酶的变化规律和筛选磷高效利用杉木无性系,通过土培试验,设计4种磷素处理水平(正常供磷16 mg•kg－1、轻度磷胁迫8 mg•kg－1、中度磷胁迫4 mg•kg－1、重度磷胁迫0 mg•kg－1),进行磷素胁迫条件下不同杉木无性系酸性磷酸酶(APA)活性的比较研究。结果表明：缺磷条件下1年生杉木叶片和根际的APA活性明显高于正常供磷处理。随缺磷时间的延长、不同杉木无性系酸性磷酸酶的变化规律不同,其中8号、24号、37号无性系叶片和根际的APA活性明显高于正常供磷处理;5号、9号无性系根际APA活性虽然增幅较大,但其叶片酶活性变化较小;3号、23号、34号无性系整体而言对磷胁迫不敏感,缺磷条件不对其叶片APA及根系APA活性造成显著影响。在磷胁迫条件下,杉木无性系可通过叶片及根际酸性磷酸酶活性的增强来适应环境磷缺乏,但不同杉木无性系对磷缺乏的适应性存在明显差异,因此能否将APA活性作为杉木无性系磷效率的评价指标之一仍需作进一步研究。     

Root and shoot traits responses to phosphorus deficiency and QTL analysis at seedling stage using introgression lines of rice

Phosphorous （P） deficiency is a major restraint factor for crop production and plants have developed several mechanisms to adapt to low P stress. In this study, a set of 271 introgression lines （ILs） were used to characterize the responses of seedlings to low P availability and to identify QTLs for root traits, biomass, and plant height under P-deficiency and P-sufficiency conditions. Plant height, total dry weight, shoot dry weight, and root number were inhibited under P-deficiency, whereas maximum root length （MRL） and root-shoot ratio （RS） were induced by P-deficiency stress. Relative MRL （RMRL, the ratio of MRL under P-deficiency to MRL under P-sufficiency con- dition） and relative RS （RRS） were used to evaluate P-deficiency tolerance at the seedling stage. A total of 24 additive QTLs and 29 pairs of epistatic QTLs were detected, but only qRN4 was detected in both conditions. This suggested that different mechanisms may exist in both P supply levels. QTLs for adaptive traits （RMRL, RRS, RRV, and RRDW） and qRN4 consistently expressed to increase trait stability may contribute to P-deficiency tolerance. Twelve intervals were cluster regions of QTLs for P-deficiency tolerance, and one QTL （qRRSS） showed pleiotropic effects on P-deficiency tolerance and drought tolerance. These interesting QTLs can be used in marker-assisted breeding through the target ILs.     

Identification of major quantitative trait loci for root diameter in synthetic hexaploid wheat under phosphorus-deficient conditions

Synthetic hexaploid wheat (SHW) possesses numerous genes for resistance to stress, including phosphorus (P) deficiency. Root diameter (RDM) plays an important role in P-deficiency tolerance, but information related to SHW is still limited. Thus, the objective of this study was to investigate the genetic architecture of RDM in SHW under P-deficient conditions. To this end, we measured the RDM of 138 F₉ recombinant inbred lines derived from an F₂ population of a synthetic hexaploid wheat line (SHW-L1) and a common wheat line (Chuanmai32) under two P conditions, P sufficiency (PS) and P deficiency (PD), and mapped quantitative trait loci (QTL) for RDM using an enriched high-density genetic map, containing 120,370 single nucleotide polymorphisms, 733 diversity arrays technology markers, and 119 simple sequence repeats. We identified seven RDM QTL for P-deficiency tolerance that individually explained 11–14.7% of the phenotypic variation. Five putative candidate genes involved in root composition, energy supply, and defense response were predicted. Overall, our results provided essential information for cloning genes related to P-deficiency tolerance in common wheat that might help in breeding P-deficiency-tolerant wheat cultivars.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

Genome-Wide Functional Analysis of Cotton (Gossypium hirsutum) in Response to Drought

Cotton is one of the most important crops for its natural textile fibers in the world. However, it often suffered from drought stress during its growth and development, resulting in a drastic reduction in cotton productivity. Therefore, study on molecular mechanism of cotton drought-tolerance is very important for increasing cotton production. To investigate molecular mechanism of cotton drought-resistance, we employed RNA-Seq technology to identify differentially expressed genes in the leaves of two different cultivars (drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6) of cotton. The results indicated that there are about 13.38% to 18.75% of all the unigenes differentially expressed in drought-resistant sample and drought-sensitive control, and the number of differentially expressed genes was increased along with prolonged drought treatment. DEG (differentially expression gene) analysis showed that the normal biophysical profiles of cotton (cultivar J-13) were affected by drought stress, and some cellular metabolic processes (including photosynthesis) were inhibited in cotton under drought conditions. Furthermore, the experimental data revealed that there were significant differences in expression levels of the genes related to abscisic acid signaling, ethylene signaling and jasmonic acid signaling pathways between drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6, implying that these signaling pathways may participate in cotton response and tolerance to drought stress.     

水稻穗发育功能基因对穗期氮肥的响应

为探讨氮肥对水稻(Oryza sativa)穗发育的调控作用, 使用高通量测序技术检测氮肥处理前后水稻叶片和幼穗组织中转录组的变化, 并从中筛选到大量差异表达基因。这些基因的功能涉及转录调控、激素代谢和信号转导、物质代谢和转运、胁迫响应、信号转导(受体)和蛋白质降解等。同时对目前克隆得到的穗发育相关基因进行分析, 发现在氮素穗肥的作用下, 部分重要功能基因的表达量发生了明显变化, 其中一些基因还参与调控水稻株高、抽穗期、分蘖和结实率等性状。对这些差异表达基因的功能研究有助于揭示氮素穗肥调控水稻每穗颖花数的分子机制。     

Mapping QTLs for Phosphorus-Deficiency Tolerance at Wheat Seedling Stage

Soil phosphorus (P) deficiency is one of the major limiting factors to crop production throughout the world. It is an important strategy to breed varieties with improved P-deficiency tolerance for sustainable agriculture. The objective of this study was to map QTLs for P-deficiency tolerance in wheat, and develop molecular marker assisted selection in breeding wheat with improved P-deficiency tolerance. A doubled haploid (DH) population, consisting of 92 DH lines (DHLs) derived from P-deficiency tolerant wheat variety Lovrin 10 and P-deficiency sensitive variety Chinese Spring, was developed for mapping QTLs for P-deficiency tolerance. A genetic linkage map consisting of 34 linkage groups was constructed using 253 SSR markers. Shoot dry weight (SDW), tiller number (TN), shoot P uptake (SPU), and shoot P utilization efficiency (PUE) were investigated at seedling stage under P deficiency (−P) and sufficiency (+P) condition in two pot trials in 2002 and 2003, respectively. All traits segregated continuously in the population under either −P or +P condition. Significant positive correlations were found in between TN, SDW and SPU, whereas significant negative correlations were observed between PUE and SPU and between PUE and TN. Twenty and 19 QTLs were detected under −P and +P condition, respectively. The 39 QTLs were distributed on 21 chromosomal regions. There were three clusters of QTLs, which were associated with Xgwm25l (on chromosomes 4B), Xgwm271.2 (on chromosome 5A), and Xgwm121 (on chromosome 5D), respectively. Compared to the DHLs with all Chinese Spring alleles at the three loci, those with all Lovrin 10 alleles had, on average, much higher SPU, SDW and TN under −P condition in both trials, suggesting the importance of the three loci in controlling P-deficiency tolerance. It was interesting to find that two of the above three loci were closely linked with vernalization requirement genes VRN-A1 (on chromosome 5A) and VRN-D1 (on chromosome 5D). Potential implication of the linkage between P-deficiency tolerance and VRN genes was discussed.     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

基于RNA-Seq转录组测序技术揭示肉兔生长发育调控的分子机制

为研究肉兔肌肉生长发育调控的分子机制,本试验以84日龄齐卡巨型白兔和齐兴肉兔为研究对象,屠宰后取背最长肌组织并提取总RNA,反转录建立c DNA文库,利用Illumina HiSeq 2500测序平台进行测序,并进行序列分析和注释。筛选与肉兔肌肉生长发育相关的信号通路及差异表达基因,最后进行荧光定量PCR验证。结果表明:齐卡巨型白兔和齐兴肉兔背最长肌中显著差异表达基因总数为833个,其中齐卡巨型白兔中显著上调基因325个,显著下调基因508个。KEGG功能注释发现差异基因显著富集到PI3K-AKT通路、癌症通路和黏着斑通路,最终筛选出4个可能与生长发育显著相关的差异表达基因,为进一步对肉兔进行遗传改良提供了理论基础。