Differential Gene Expression Between Wheat Hybrids and Their Parental Inbreds in Primary Roots

To provide an insight into the molecular basis of heterosis, differential display of mRNA was used to analyze the difference of gene expression between wheat (Triticum aestivum L.) heterotic hybrid A, nonheterotic hybrid B and their parental inbreds in the primary roots. By using 5′ end random primers in combination with three one-base-anchored primers, it was found that 22.5% and 22.9% of 877 total displayed cDNAs were differentially expressed between hybrid A, B and their parents, respectively. Both quantitative and qualitative differences in gene expression between hybrids and their parental inbreds were obvious, indicating that the patterns of gene expression in hybrids alter significantly as compared to their corresponding parents. On the other hand, by using MADS-box gene specific 5′ end primer for DDRT-PCR, we found that nearly all of the displayed cDNA fragments were polymorphic between hybrids and their parents, and major difference occurred in qualitative level, in which hybrid specific-expressed and silenced genes are the major two patterns, suggesting that MADS-box gene may be important for manifestation of differential gene expression and wheat heterosis. In comparison with our previous results by using seedling leaves, it is indicated that differential gene expression between hybrids and parents is dependent on the tissues tested, and more differentially expressed genes were observed in the primary roots than in the seedling leaves. Therefore, it is concluded that the expressions of both randomly displayed cDNAs and transcription factor genes, such as MADS-box, alter significantly between hybrids and their parents, which might be responsible for the observed heterosis.

DDRT-PCR Analysis of Wheat Roots Under Iron-Deficient Condition and Differential Expression of ABC Gene

Differential expression of gene in iron-efficient wheat cultivar Jing411 and iron-inefficient cul-tivar SanshumaiS under iron-deficiency and iron-sufficiency conditions was revealed by differential display reverse transcript PCR (DDRT-PCR) method. Northern blotting was carried out using ATP-binding transporter (ABC) cDNA obtained from DDRT-PCR products of the cultivar Jing411 as probe. Our results suggested that ABC gene expression was suppressed under iron-deficiency condition.

Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar(Jimai 20) during grain development using the Gene Chip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis(DPA) was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves.Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes, and Map Man analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by q RT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

Characterization of Ppd-D1 alleles on the developmental traits and rhythmic expression of photoperiod genes in common wheat

Photoperiodic response is an important characteristic that plays an important role in plant adaptability for various environments. Wheat cultivars grow widely and have high yield potential for the strong photoperiod adaptibility. To assess the photoperiodic response of different genotypes in wheat cultivars, the photoperiodic effects of the Ppd-D1 alleles and the expressions of the related TaGI, TaCO and Ta FT genes in Liaochun 10 and Ningchun 36 were investigated under the short-day（6 h light, SD）, moderate-day（12 h light, MD） and long-day（24 h light, LD） conditions. Amplicon length comparison indicated that the promoter of Ppd-D1 in Ningchun 36 is intact, while Liaochun 10 presented the partial sequence deletion of Ppd-D1 promoter. The durations of all developmental stages of the two cultivars were reduced by subjection to an extended photoperiod, except for the stamen and pistil differentiation stage in the Liaochun 10 cultivar. The expression levels of the Ppd-D1 alleles and the TaGI, TaCO and TaFT genes associated with the photoperiod pathway were examined over a 24-h period under SD and MD conditions. The relationships of different photoperiodic responses of the two cultivars and the expression of photoperiod pathway genes were analyzed accordingly. The photoperiod insensitive（PI） genotype plants flower early under SD; meanwhile, the abnormal expression of the Ppd-D1 a allele is accompanied with an increase in Ta FT1 expression and the TaCO expression variation. The results would facilitate molecular breeding in wheat.

Biotic and abiotic stress-responsive genes are stimulated to resist drought stress in purple wheat

Triticum aestivum L. cv. Guizi 1(GZ1) is a drought-tolerant local purple wheat cultivar. It is not clear how purple wheat resists drought stress, but it could be related to anthocyanin biosynthesis. In this study, transcriptome data from droughttreated samples and controls were compared. Drought slightly reduced the anthocyanin, protein and starch contents of GZ1 grains and significantly reduced the grain weight. Under drought stress, 16 682 transcripts were reduced, 27 766 differentially expressed genes(DEGs) were identified, and 379 DEGs, including DREBs, were related to defense response. The defense-response genes included response to water deprivation, reactive oxygen, bacteria, fungi, etc. Most of the structural and regulatory genes in anthocyanin biosynthesis were downregulated, with only Ta DFR, Ta OMT, Ta5,3GT, and Ta MYB-4 B1 being upregulated. Ta CHS, Ta F3H, TaCHI, Ta4CL, and TaF3’H are involved in responses to UV, hormones, and stimulus. Ta CHS-2D1, Ta DFR-2D2, Ta DFR-7D, TaOMT-5A, Ta5,3 GT-1B1, Ta5,3GT-3A, and Ta5,3GT-7B1 connect anthocyanin biosynthesis with other pathways, and their interacting proteins are involved in primary metabolism, genetic regulation, growth and development, and defense responses. There is further speculation about the defense-responsive network in purple wheat. The results indicated that biotic and abiotic stress-responsive genes were stimulated to resist drought stress in purple wheat GZ1, and anthocyanin biosynthesis also participated in the drought defense response through several structural genes.

Genetic dissection of the powdery mildew resistance in wheat breeding line LS5082 using BSR-Seq

Powdery mildew of wheat is a destructive disease seriously threatening yield and quality worldwide.Comprehensive dissection of new resistance-related loci/genes is necessary to control this disease.LS5082 is a Chinese wheat breeding line with resistance to powdery mildew.Genetic analysis,using the populations of LS5082 and three susceptible parents(Shannong 29,Shimai 22 and Huixianhong),indicated that a single dominant gene,tentatively designated PmLS5082,conferred seedling resistance to different Blumeria graminis f.sp.tritici(Bgt)isolates.Bulked segregant RNA-Seq was carried out to map PmLS5082 and to profile differentially expressed genes associated with PmLS5082.PmLS5082 was mapped to a 0.7 cM genetic interval on chromosome arm 2BL,which was aligned to a 0.7 Mb physical interval of 710.3–711.0 Mb.PmLS5082 differs from the known powdery mildew(Pm)resistance genes on chromosome arm 2BL based on their origin,chromosome positions and/or resistance spectrum,suggesting PmLS5082 is most likely a new Pm gene/allele.Through clusters of orthologous groups and kyoto encyclopedia of genes and genomes analyses,differentially expressed genes(DEGs)associated with PmLS5082 were profiled.Six DEGs in the PmLS5082 interval were confirmed to be associated with PmLS5082 via qPCR analysis,using an additional set of wheat samples and time-course analysis postinoculation with Bgt isolate E09.Ten closely linked markers,including two kompetitive allele-specific PCR markers,were confirmed to be suitable for marker-assisted selection of PmLS5082 in different genetic backgrounds,thus can be used to detect PmLS5082 and pyramid it with other genes in breeding programs.

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

: To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) near-isogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg2+ transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways. The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.

mRNA differential display on gene expression in settlement metamorphosis process of Ruditapes philippinarum larvae

The mRNA differential display (DDRT-PCR) technique was adopted to find out the genes related tosettlement metamorphosis development process of Ruditapes philippinarum larvae.In this study,we haveobtained three hundred and forty-six amplification bands in total from pediveliger larvae,veliger larvae,eye spot larvae and post-larvae.Sixty-five out of three hundred and forty-six bands are distinctly differen-tial display from band pattern,which can be put into four groups,standing for different expression char-acters.Sixteen differential display bands were cloned,sequenced and analyzed and nine different se-quences are obtained in the study.Three sequences have higher similarity to the cDNAs deposited indatabase and three are very similar to the rDNA of other species,considered as the rDNA of Ruditapesphilippinarum.The rest three sequences are found to be novel sequences after analyzed.Their accessionnumbers are AY916799,AY916798,and AY916797 respectively.We thought the novel sequences arepossibly relevant to the early embryo development of Ruditapes philippinarum larvae and can provide somefundamental understandings that are helpful for the improvement of scallop seed raising industry.

Primarily screening and analyzing ESTs differentially expressed in rats' primary liver cancer

Objective： To screen and analyze key express sequence tags （ESTs） which were differentially displayed in every period of SD rats＇ primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Methods： Using diethylnitrosamine （DENA） as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique （DDRT-PCR）. After the fragments were sequenced, bioinformatics were .used to analyze the results. Results： Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments （EST-1, EST-3 and EST-5） had extremely low identity to any genes registered in GENBANK databases. Conclusions： BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-I, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.

Comparative genomic and transcriptomic analyses uncover the molecular basis of high nitrogen-use efficiency in the wheat cultivar Kenong 9204

Studying the regulatory mechanisms that drive nitrogen-use efficiency(NUE)in crops is important for sustainable agriculture and environmental protection.In this study,we generated a high-quality genome assembly for the high-NUE wheat cultivar Kenong 9204 and systematically analyzed genes related to nitrogen uptake and metabolism.By comparative analyses,we found that the high-affinity nitrate transporter gene family had expanded in Triticeae.Further studies showed that subsequent functional differentiation endowed the expanded family members with saline inducibility,providing a genetic basis for improving the adaptability of wheat to nitrogen deficiency in various habitats.To explore the genetic and molecular mechanisms of high NUE,we compared genomic and transcriptomic data from the high-NUE cultivar Kenong 9204(KN9204)and the low-NUE cultivar Jing 411 and quantified their nitrogen accumulation under high-and low-nitrogen conditions.Compared with Jing 411,KN9204 absorbed significantly more nitrogen at the reproductive stage after shooting and accumulated it in the shoots and seeds.Transcriptome data analysis revealed that nitrogen deficiency clearly suppressed the expression of genes related to cell division in the young spike of Jing 411,whereas this suppression of gene expression was much lower in KN9204.In addition,KN9204 maintained relatively high expression of NPF genes for a longer time than Jing 411 during seed maturity.Physiological and transcriptome data revealed that KN9204 was more tolerant of nitrogen deficiency than Jing 411,especially at the reproductive stage.The high NUE of KN9204 is an integrated effect controlled at different levels.Taken together,our data provide new insights into the molecular mechanisms of NUE and important gene resources for improving wheat cultivars with a higher NUE trait.

Transcriptome analysis of wheat grain using RNA-Seq

With the increase in consumer demand,wheat grain quality improvement has become a focus in China and worldwide.Transcriptome analysis is a powerful approach to research grain traits and elucidate their genetic regulation.In this study,two cDNA libraries from the developing grain and leaf-stem components of bread wheat cultivar,Nongda211,were sequenced using Roche/454 technology.There were 1061274 and 1516564 clean reads generated from grain and leaf-stem,respectively.A total of 61393 high-quality unigenes were obtained with an average length of 1456 bp after de novo assembly.The analysis of the 61393 unigenes involved in the biological processes of the grain showed that there were 7355 differentially expressed genes upregulated in the grain library.Gene ontology enrichment and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that many transcription products and transcription factors associated with carbohydrate and protein metabolism were abundantly expressed in the grain.These results contribute to excavate genes associated with wheat quality and further study how they interact.

用DDRT-PCR技术对太谷核不育小麦基因差别表达的研究

采用DDRT-PCR技术对太谷核不育小麦的一对近等基因系进行了差别表达分析,以找出与太谷核不育基因Ta1表达有关的基因并研究其引起雄性不育的机制.共用30对随机引物进行差异显示,从展示的近1000条cD-NA片段中找出30条特异表达的cDNA片段,其中包括可育特异和不育特异,这些片段可能与Ta1基因的表达有关.

用改进的DDRT-PCR技术进行小麦穗和花药特异表达基因的分析

本研究采用改进的DDRT-PCR技术对小麦穗和花药发育的不同阶段进行了差别表达分析,以找出穗和花药的特异表达基因。用30个随机单引物进行RAPD扩增,从展示的近1000条带中找出14条差别cDNA片段,然后以cDNA为探针进行反向Northern-Blot,结果得到一条在花药内特异表达增强的阳性片段,命名为S1176450。

不同磷胁迫处理转OsPHR2小麦的转录组学分析

PHR基因是磷信号调控体系的核心转录因子,负责启动下游部分应对磷饥饿的适应性反应。本课题前期获得了磷高效转OsPHR2小麦纯系,但OsPHR2提高小麦磷吸收利用效率的分子机制尚不清楚。为揭示该机制,本研究以前期获得的磷高效转OsPHR2小麦纯系为研究材料,采用水培试验,小麦长至四叶一心时进行低磷胁迫处理,分别在低磷胁迫0、6、24和72 h,利用RNA-Seq进行转录组测定,分析转基因小麦与对照之间根部和叶片的差异表达基因(differentially expression gene, DEG),并分别对根部和叶片DEG进行GO和KEGG功能富集分析。结果显示:低磷胁迫处理0、6、24和72 h转基因系与对照根部有22个共同的DEG,叶片有9个共同的DEG。转基因小麦与对照根部DEG数量在低磷胁迫处理0 h最多,其次为6 h。GO和KEGG富集分析显示,低磷胁迫0 h和6 h,根部DEG主要富集在糖代谢、苯丙素生物合成等生物学过程,以及养分贮存器活性、ATP酶活性等分子功能。转基因小麦与对照叶片DEG数量在低磷胁迫72 h最多,主要富集在糖代谢、有机酸生物合成等生物学过程,以及与糖基转移酶活性、纤维素合酶活性等有关的分子功能。与对照相比,转基因系OsT5-28根部血红素过氧化物酶、谷胱甘肽S-转移酶等防御系统关键酶基因,以及叶片磷酸丙糖转运体家族基因在低磷胁迫前后均上调表达。转OsPHR2小麦与对照对低磷胁迫的响应程度具有一定的差异性,低磷胁迫下转基因小麦较对照具有较强的磷素吸收利用能力,主要是OsPHR2调控了小麦中相关基因表达。

小麦BNS雄性不育系及其转换系花药差异蛋白鉴定与分析

BNS是一个新发现的温敏核型小麦雄性不育系,它有良好的不育性和转换性,被认为在小麦杂种优势利用上有重要价值。为探讨BNS的不育机制,以不育系及其转换系的单核早期、单核晚期到二核期花药为材料,用2-DE和MALDI-TOF-MS方法分离鉴定2个系的差异蛋白。结果发现,在转换系中,ATP合酶α和β亚基、胞质苹果酸脱氢酶、线粒体醛脱氢酶亚基、Rubisco亚基等呼吸、光合能量代谢蛋白表达丰富,但在不育系中这些蛋白缺失或下调。在不育系中还分离出山梨醇脱氢酶、组蛋白H2B.2、Harpin诱导子1、延伸因子TU等非小麦正常代谢蛋白。根据这些差异蛋白的功能,推测ATP合酶α、β亚基蛋白最可能是BNS不育的源头蛋白,它的表达可能受其上游的温度感应子调控。BNS的温度感应子隐性突变后转录启动温度阈值上调,在较低温下,α、β亚基基因不表达或表达量小,导致ATP合酶数量减少,继而影响ATP合成。ATP数量减少,使小孢子ATP供应短缺,进一步影响到小孢子的发育,使代谢异常,花粉败育。

普通小麦不同优势杂交种及其亲本之间基因表达差异比较研究

为探讨小麦杂种优势形成的分子机理 ,本研究选用普通小麦品种 (系 ) 3338,6554和 2 4 10 TD及其强优势杂种 (3338× 6655)和弱优势杂种 (2 4 10 TD× 6555) ,采用 m RNA差异显示技术 ,对生长至三叶一心(即分蘖初期 )和产生二级分蘖时 (即分蘖盛期 )的幼苗叶片基因表达差异进行了比较研究。结果表明 ,小麦杂种一代苗期基因表达较亲本明显不同 ,表现为数量水平和质量水平上的差异 ,但与分蘖初期相比 ,小麦杂交种与亲本之间在分蘖盛期的基因表达差异更明显 ,并且在分蘖初期主要为量的表达差异 ,而生长至分蘖盛期时 ,质的表达差异比例显著增高 ,这表明小麦杂交种与亲本间的基因表达差异与发育时期有关。分析发现 ,强优势杂种 与其亲本间的差异表达基因比例明显高于弱优势杂种 。另外 ,无论分蘖初期或盛期 ,在强优势杂种组合 中 ,增强型和沉默型所占比例均明显高于弱优势杂种组合 ,而单亲表达减弱型比例则较杂种组合 低。本文还对 m

小麦杂交种与亲本之间穗下节间基因差异表达分析

以株高和穗下节间长度表现杂种优势的小麦杂交组合矮9×冀矮8号的杂种及其亲本为材料,应用cDNA-AFLP技术分析杂种、亲本的穗下节间基因差异表达情况。分离差异表达基因段后,对其克隆、测序并在GenBank进行Blast-x分析和功能推测,发现差异表达的基因包括分别受赤霉素和细胞分裂素诱导表达、与细胞分裂有关的基因cdc2和PAS1基因,它们在小麦杂种节间中分别特异表达和偏高亲表达。另有一个与细胞快速膨大有关的液泡质子泵H+-PPase基因在杂种中偏高亲表达。其他差异表达的基因包括蛋白激酶等与植物信号传递相关基因、与物质代谢相关的基因、参与基因转录调控的基因、与泛素参与的蛋白降解相关的基因等。小麦杂交种与亲本的穗下节间,大量基因涉及细胞分裂、膨大和物质代谢、转录调控等过程,初步推测它们的差异表达可能与穗下节间及株高杂种优势形成有关。

NaCl 胁迫下小麦突变体和野生型叶片中一些有机溶质累积和基因表达差异

盐胁迫下突变体和野生型叶片中的脯氨酸累积量均有显著的增加，野生型的增加幅度不及突变体。至９６ ｈ ，两者含量均下降，但突变体的脯氨酸含量仍高于野生型。１００ｍ ｍｏｌ／Ｌ的ＮａＣｌ 胁迫７２ ｈ ，突变体叶片中可溶性糖的含量有显著的增加，增加量随盐浓度增加而降低。至９６ ｈ，各个盐浓度处理的突变体可溶性糖的含量基本恢复到其对照的水平；除１００ ｍｍｏｌ／Ｌ 盐胁迫处理组外，野生型叶片中可溶性糖含量均大幅度下降。盐胁迫下突变体和野生型叶片细胞可溶性蛋白组分有明显的差异。ｍＲＮＡ 差异显示结果表明，突变体有６ 个差异性的ｃＤＮＡ

不同面团强度小麦品种差异表达贮藏蛋白基因分析

以2个高分子质量麦谷蛋白亚基(HMW-GS)组合完全一致但籽粒蛋白质含量和面团强度有较大差异的小麦品种(郑麦158:低蛋白质含量、高面团强度;郑麦119:高蛋白质含量、低面团强度)为试验材料,分别在开花后14、21、28 d,对其籽粒进行转录组测序,分析3个时间点2个品种间差异表达的贮藏蛋白基因、面粉中硫和巯基含量及差异表达基因编码蛋白质的半胱氨酸和自由巯基含量,以探讨HMWGS组合相同情况下面粉质量差异的可能原因。结果表明,郑麦158的籽粒总蛋白质含量较低,但面筋指数、面团稳定时间较高;2个品种的HMW-GS组合完全一致,而且高、低分子质量麦谷蛋白亚基基因表达量无显著差异;筛选出28个显著差异表达的贮藏蛋白基因,其中,5个基因(3个类燕麦贮藏蛋白和2个醇溶蛋白基因)在郑麦158中表达量显著上调,23个基因(16个醇溶蛋白基因、5个类燕麦贮藏蛋白基因和2个球蛋白基因)在郑麦158中表达量显著下调;郑麦158面粉中硫元素含量较低,但自由巯基、总巯基和二硫键含量较高,且郑麦158中上调表达贮藏蛋白基因编码蛋白质的半胱氨酸、自由巯基含量均较高。综上,HMW-GS组合可能不是决定面团强度的主要因素,降低醇溶蛋白基因的表达量是提高面团强度的有效措施,个别醇溶蛋白和类燕麦贮藏蛋白可能与面团强度密切相关,高自由巯基含量是高面团强度的分子基础。

atp1基因在小麦BNS雄性不育系和自身转换系中的差异表达

atp1基因在植物中是线粒体基因组编码,其产物ATP1是线粒体ATP合酶F1的α亚基,在小麦BNS的雄性不育系和它的转换系中差异表达。为了检测该基因的表达丰度,探讨与BNS不育性的联系,以BNS不育系和它的转换系的幼穗、花药和穗轴等组织为材料,利用实时荧光定量PCR方法,测定和比较该基因在不同组织中的表达水平,结果表明,该基因在各组织中的总表达量比内参甘油醛-3-磷酸脱氢酶基因表达量高3-4个数量级;在幼穗及各穗轴营养组织中表达量一致;花药与营养组织比较,在不育系四分体和单核期花药中表达均上调;在不育系中,该基因表达量在单核期花药中显著下调。这些结果说明,线粒体atp1基因在小麦中是一个高水平表达基因,在BNS营养组织中组成型表达,在花药中特异性上调表达,在不育系中表达受到抑制,表现显著下调,表明atp1基因的下调表达与BNS不育性有关。