基于WGCNA的马铃薯根系抗旱相关共表达模块鉴定和核心基因发掘

权重基因共表达网络分析(Weighted Gene Co-expression Network Analysis, WGCNA)是系统生物学皀一种研究方法,在多样本转录组数据中挖掘与目标性状相兲皀基因模块有较广泛皀应用。为了深入探究马铃薯应对干旱胁迫皀分子机制,本研究以国际马铃薯中心引迚栻培种C16 (CIP 397077.16)和C119 (CIP 398098.119)为试验材料,将其无菌组培苗利用甘露醇模拟干旱胁迫,处理0 h、2 h、6 h、12 h和24 h,取其根系迚行转录组测序,每样设3个生物学重复,共30个样本。基于以上转录组数据,利用WGCNA构建与抗逆生理性状相兲联皀权重基因共表达网络,得到15个与根系抗旱密切相兲皀基因共表达模块,幵从4个与目标性状兲联度最高皀模块中収掘到数个核心基因,功能注释表明其中大部分参与干旱胁迫调控通路。这些结果为迚一步研究马铃薯根系抗旱皀分子遗传机制提供了线索。     

转录组在植物抗盐育种中的应用

盐胁迫是影响植物种子萌发、生长发育以及开花结实的主要非生物胁迫之一,能够导致农业生产力降低甚至造成植物死亡。植物在盐胁迫下的响应策略一直是抗盐分子机制的研究热点。转录组不仅可以更好地研究植物盐胁迫条件下的基因结构和基因功能,而且能发掘抗盐基因。目前,基于转录组技术的种质资源鉴定及精确育种已广泛地应用于植物抗盐育种研究工作中。本综述总结了转录组技术在植物抗盐育种上的应用,浅谈不同植物材料对盐胁迫的响应,为植物抗盐育种提供理论基础。     

普通小麦颖壳蜡质缺失突变体glossy1的转录组分析

为了明确突变体颖壳蜡质含量显著变化的分子机制,本研究对源自济麦22颖壳蜡质缺失突变体glossy1与野生型进行了转录组分析。结果表明,在glossy1突变体中,共筛选到12,230个差异表达基因,其中5811个基因在突变体中上调表达,6419个下调表达。GO(gene ontology)功能富集分析发现,差异基因主要富集在蜡质合成和转运途径,具体分布在酰基转移酶活性、脂质结合和水解酶活性等条目,由此推测这些途径与小麦穗部蜡质缺失性状是紧密相关的。我们还利用RT-qPCR检测了参与蜡质代谢途径部分基因的表达,结果与转录组结果是一致的。本研究为今后探究小麦蜡质代谢的分子机制和基因调控网络提供了数据支持,同时也为抗逆小麦育种奠定了理论基础。     

Genetic variation in root development responses to salt stresses of quinoa

Soil salinity has become a serious environmental abiotic stress limiting crop productivity and quality. The root system is the first organ sensing the changes in salinity. Root development under elevated salinity is therefore an important indicator for saline tolerance in plants. Previous studies focused on varietal differences in morphological traits of quinoa under saline stresses; however, variation in root development responses to salinity remains largely unknown. To understand the genetic variation in root development responses to salt stress of quinoa, we conducted a preliminary screening for salinity response at two salinity levels of a diverse set of 52 quinoa genotypes and microsatellite markers were used to link molecular variation to that in root development responses to salt stresses of represented genotypes. The frequency distribution of saline tolerance index showed continuous variation in the quinoa collection. Cluster analysis of salinity responses divided the 52 quinoa genotypes into six major groups. Based on these results, six genotypes representative of groups I to VI including Black quinoa, 2-Want, Atlas, Riobamba, NL-6 and Sayaña, respectively, were selected to evaluate root development under four saline stress levels: 0, 100, 200 and 300 mM NaCl. Contrasts in root development responses to saline stress levels were observed in the six genotypes. At 100 mM NaCl, significant differences were not observed in root length development (RLD) and root surface development (RSAD) of most genotypes except Black quinoa; a significant reduction was observed in this genotype as compared to controls. At 200 mM NaCl, significant reduction was detected in RLD and RSAD in all genotypes showing this as the best concentration to discriminate among genotypes. The strongest inhibition of root development was found for all genotypes at 300 mM NaCl as compared to lower saline levels. Among genotypes, Atlas of group III shows as a saline-tolerant genotype confirming previous reports. Variation in root responses to salinity stresses is also discussed in relation to climate conditions of origins of the genotypes and reveal interesting guidelines for further studies exploring the mechanisms behind this aspect of saline adaptation.     

基于高通量测序技术的杭白芷(Angelica dahurica)根转录组数据分析

为获得杭白芷转录组信息特征,本研究利用Illumina HiSeqⅩTen测序平台对杭白芷根进行高通量转录组测序,获得高质量序列(Clean reads)47742445条,Trinity denovo组装后得到47044条Unigenes,平均长度1164.20 nt。BLAST分析显示分别有32208(68.46%)、23049(48.99%)、10479(22.27%)、17883(38.01%)、28201(59.95%)、20731(44.07%)、55(0.12%)条Unigenes在数据库NR、Swiss-Prot、KEGG、KOG、eggNOG、GO、Pfam中获得注释,可归为GO分类的生物过程、细胞组分和分子功能3大类57分支,涉及205个KEGG代谢通路,其中包括27个次生代谢通路。蛋白编码框序列32303个,高等植物转录因子58个家族,借助MISA软件发现10020个SSR,其中二碱基重复最丰富,有4336个,出现频率为43.27%;五碱基重复SSR最少仅占0.37%。本研究获得了大量基因序列信息以及SSR信息,为今后开展相关分子机制研究提供了数据资源和理论基础。     

香蕉根系转录组SSR位点信息分析

分析香蕉根系转录组中的SSR位点信息，并设计SSR引物，为开发新的分子标记奠定基础。利用MISA工具对香蕉根系转录组unigene序列进行SSR检索。从25158条unigene中共发现4663个SSR，分布在3820条unigene序列中，出现频率为18.53%，平均每7.77 kb含有1个SSR位点，共有58种重复基元。香蕉根系转录组中，二、三核苷酸重复基元所占比例最大，分别为40.50%和40.63%；二者分别以AG/CT和AGG/CCT重复基元为主，分别占该重复基元的88.03%和30.83%。SSR重复次数以5~9次为主，基序长度主要分布于12~20 bp，平均长度为18.80 bp。香蕉根系转录组SSR位点频率、密度较高，类型多样，在香蕉遗传多样性研究及分子标记辅助育种中有较大应用潜能。     

假禾谷镰孢侵染小麦后3种植物激素相关基因的差异表达分析

假禾谷镰孢(Fusarium pseudograminearum)是我国近年新发现的小麦病原真菌,本研究目的是揭示植物激素信号传导途径中相关基因表达对假禾谷镰孢侵染的响应。利用假禾谷镰孢野生菌株WZ2-8A侵染小麦品种周麦24,对接种后5 d和15 d样品进行转录组测序,分析差异表达基因,并对候选基因进行q RT-PCR验证。假禾谷镰孢侵染后,小麦幼苗生长受到明显抑制,根长、株高、根重和地上部分鲜重均显著降低。转录组分析结果表明,植物激素信号传导途径中有29个基因差异表达,涉及到生长素、细胞分裂素和脱落酸3种植物激素。在接种后5 d有11个差异表达基因,其中2个上调,9个下调;在接种后15 d共有25个差异表达基因,其中8个上调,17个下调。在生长素信号传导途径中,生长素输入载体AUX1差异表达,影响生长素的极性运输,从而影响小麦根部的细胞伸长。在细胞分裂素信号传导途径中,起正调控作用的B-ARR上调表达,推测其促进细胞分裂素的信号传导,从而抑制细胞分裂,与生长素协同作用,造成小麦的长势减弱。在脱落酸途径中,脱落酸受体PYR/PYL下调表达;起到负调控作用的PP2C相关基因均上调表达。脱落酸使植物对真菌和细菌的抗性起到负调控作用,其信号传导途径与茉莉酸/乙烯途径相互拮抗,脱落酸信号传导途径的阻遏可能会使茉莉酸/乙烯途径信号通路打开。q RT-PCR结果基本能够和转录组测序结果相拟合,说明在假禾谷镰孢侵染胁迫下,脱落酸的信号传导被抑制可能是中抗品种周麦24对假禾谷镰孢产生一定的抗性的生理基础。     

Drought responses of above‐ground and below‐ground characteristics in warm‐season turfgrass

Water shortages have become more chronic as periodic droughts prolong and water demand for urban and agricultural use increases. Plant drought responses involve coordinated mechanisms in both above‐ and below‐ground systems, yet most studies lack comparisons of root and canopy responses under water scarcity and recovery. This is particularly true of research focused on warm‐season turfgrasses in sandy soils with extremely low water holding capacity. To address the lack of examination of coordinated stress and recovery responses, this study compared the above‐ and below‐ground plant responses during a dry‐down period of 21 days and recovery among four warm‐season turfgrass species in the field. Canopy drought responses and recovery were quantified using digital image analysis. In situ root images were captured using a minirhizotron camera system. Common bermudagrass [Cynodon dactylon (L.) Pers.] endured the entire drought period without losing 50% green cover while other species lost 50% green cover in 11–34 days predicted from the regression. The interspecific differences in drought resistance were mainly due to root characteristics. Other drought mechanisms appear to be responsible for differences identified in drought resistance between “Zeon” and “Taccoa Green” manilagrass [Zoysia matrella (L.) Merr.]. Recovery was delayed for up to 2 weeks in the second year, warranting further evaluation for turfgrass persistence under long‐term drought. Three‐week drought posed no threat to the survival of zoysiagrass. Species and genotypic variations were found in achieving full post‐recovery, which can be used to develop water conservation strategies and to adjust consumer expectations.     

马铃薯StIgt基因家族的鉴定及其对干旱胁迫的响应分析

干旱胁迫是影响马铃薯产量和品质的主要非生物胁迫因素之一。马铃薯在抵御干旱胁迫的过程中,根系的生长发育和构型分布发挥着重要作用。Igt基因家族是普遍存在于植物中的一类功能基因,在调控植物根系构型和提高植株抗逆性等方面效果显著。本研究以马铃薯双单倍体‘DM-v4.03’高质量基因组为参考,在全基因组范围内分析鉴定了StIgt基因家族的成员,并采用多种生物信息学软件对其进行了系统进化树构建、染色体定位、保守蛋白结构域、基因结构和顺式元件预测。同时,利用本课题组前期对马铃薯四倍体品系在不同干旱条件下的转录组测序结果,分析了StIgts响应干旱胁迫的表达谱。结果表明,在马铃薯中共鉴定获得10个StIgt家族成员,其中StIgt1由本课题组前期克隆获得。除StIgt1位置信息不明外,其余基因不均匀地分布在1、2、5、7、10和11号染色体上。StIgt家族蛋白长度为110~283个氨基酸,分子量介于13.136~32.542 kD之间,预测等电点为3.82~9.86。系统进化树分析发现,该基因家族可分为3个亚族,亚族间的基因结构、蛋白保守域和顺式作用元件差别明显。干旱胁迫下的表达谱分析表明,StIgt6、StIgt7、StIgt9和StIgt10响应早期干旱胁迫,在干旱2 h时即迅速上调表达。这些结果为阐明StIgt基因家族的进化关系和进一步研究其成员的功能特性提供了理论基础。     

Meta-QTL analysis and identification of candidate genes related to root traits in maize

Maize root system architecture determines key functions of uptake of water and nutrients in plants. A large number of quantitative trait loci (QTLs) of root-related traits have been found in different populations of maize. Identification of consistent QTLs across diverse genetic backgrounds could be instrumental on marker-assisted selection of traits and identification of candidate functional genes. In this study, 20 published papers were investigated regarding on reported results of QTLs related to root traits of maize, and in total 428 individual QTLs for 23 root-related traits were used for meta-analysis, resulting in 53 Meta-QTLs (MQTLs) retrieved over ten maize chromosomes. Among these MQTLs regions, in total 45 maize homologs were considered as candidate genes affecting maize root traits by comparing with 7 genes from rice and 36 genes from Arabidopsis. Three maize genes (GRMZM5G813206, GRMZM2G167220 and GRMZM2G467069) identified from MQTL_8-5 could play important roles on lateral root and crown root development of maize. Two of the MQTLs, i.e. MQTL_7-2 and MQTL_9-1, involved in nitrogen (N) and phosphorus (P) stress responses and both of them with small physical distance (less than 3 Mb), could be used for abiotic stress improvement of maize root traits. These MQTLs and candidate genes will be helpful for future gene cloning and marker-assisted selection in maize.     

转录组学及其在蔬菜学上应用研究进展

介绍了转录组学的主要技术方法及其原理,并对转录组学在蔬菜及其他植物上的应用做了分析。认为研究转录组学可克服蔬菜基因组数据匮乏的障碍,弄清楚蔬菜特定基因的结构及其在抗逆、抗病等过程中的作用。研究表明,当前转录组学方法主要基于杂交技术或者测序技术,代表性方法有基因芯片、SAGE(serial analysis of gene expression)、MPSS(massively parallel signature sequencing)、RNA-Seq(RNA-sequencing)等;转录组学可用于了解蔬菜抗逆、抗病分子机制,证明特定基因表达情况,但是如何快速测序以及科学分析成为制约其发展的瓶颈因素。因此,笔者认为转录组学在论证分子机制时需要谨慎,更需要借助蛋白组学、生物信息学等方法综合验证;但是,以转录组学为代表的前沿生物学理论应更多地应用于蔬菜学这门古老学科。     

牡丹DNA甲基结合域蛋白基因PsMBD5的克隆和表达特性分析

前期通过转录组结合表达谱分析发现一类MBD片段在低温解除牡丹花芽内休眠中的差异表达,为了进一步研究MBD基因特征及其在牡丹休眠解除中的作用,利用RACE扩增获得1204bpPsMBD5全长cDNA,其中编码框1056bp,推测编码351个氨基酸。生物信息学分析结果显示,PsMBD5蛋白分子量约为38.1274kDa,等电点为5.14,不稳定系数为44.27,推测该蛋白为不稳定蛋白。同源性分析表明,牡丹PsMBD5与梅PmMBD5的同源性最高,为38.1%,与亚麻荠CsMBD5的同源性最低,为25.8%,与13种拟南芥MBD蛋白同源性最高的是AtMBD5。实时荧光定量PCR分析表明,PsMBD5基因在牡丹不同组织中均有表达,在初花期牡丹的根中转录水平最高,在花瓣、心皮和萼片中次之,在雄蕊中最低;PsMBD5基因在牡丹花芽中受低温诱导,且转录水平在低温处理14d时达到最大值,之后维持较高的转录水平。研究结果为进一步解析PsMBD5基因对牡丹花芽的休眠解除的调控机制奠定基础。     

石榴NAC转录因子家族的生物信息学分析

NAC (NAM-ATAF1/2-CUC2)是植物特有的转录因子大家族之一,其参与植物叶片的衰老、花的形成、种子的发育、根的发育、次生细胞壁的合成、激素的信号转导、果实的成熟及着色等生长发育过程。本研究基于石榴基因组数据库,从中鉴定了73个NAC基因;运用生物信息学方法分析NAC基因家族的蛋白理化性质、基因结构、保守结构域、进化和基因表达。结果表明,NAC基因可分为9个亚族。基于不同组织器官的转录组数据,分析了Pg NAC基因的表达模式,发现Pg NAC30有可能在石榴根系的发育中起重要作用;Pg NAC3和Pg NAC13有可能参与调控石榴种子大小,Pg NAC32有可能调控石榴果实成熟发育,Pg NAC49参与调控石榴种子木质素的生物合成。该研究结果为石榴NAC家族基因功能研究,探索NAC调控石榴果实发育及品质形成提供参考,为石榴的分子育种提供科学依据。     

绞股蓝皂苷生物合成后修饰酶基因的cDNA克隆和组织表达特征

从绞股蓝转录组挖掘可能与绞股蓝皂苷生物合成相关修饰酶基因细胞色素P450(cytochrome P450,CYP)及UDP-糖基转移酶(UDP-dependent glycosyltransferase,UGT)基因的cDNA进行克隆并开展了以下研究。根据课题组前期绞股蓝转录组数据,筛选CYP及UGT Unigenes,利用RT-PCR克隆ORF全长,并结合生物信息学对其编码蛋白进行功能分析,最后通过荧光定量PCR验证其在根、茎、叶的差异性表达。结果克隆得到两个ORF序列,分别命名为CYP94A1和UGT91A1;CYP94A1序列包含1509 bp的开放阅读框架,编码一条含503个氨基酸残基的肽链,具有CYP保守结构域;UGT91A1序列包含1383 bp的开放阅读框架,编码一条含461个氨基酸残基的肽链,具有UGT保守结构域。这两个基因的转录表达均具有组织特异性,在叶或茎中的表达均高于根。CYP94A1和UGT91A1的克隆、转录表达及生物信息学分析为进一步挖掘绞股蓝中CYPs、UGTs及功能验证提供了帮助,增加对CYP和UGT两个超基因家族的认识。     

洋甘菊WRKY转录因子的鉴定及表达分析

为了系统分析德国洋甘菊WRKY基因家族,本研究利用生物信息学方法,从德国洋甘菊转录组共鉴定出42个WRKY转录因子,包括12个Ⅰ型基因,1个Ⅱ-a型基因,5个Ⅱ-b型基因,11个Ⅱ-c型基因,5个Ⅱ-d型基因,3个Ⅱ-e型基因和5个Ⅲ型基因。保守基序分析显示,同类型的基因拥有特异性的保守基序。表达谱数据分析表明,德国洋甘菊WRKY基因至少在一个组织中表达,并具有器官特异性。5个MrWRKY基因只在根中表达,1个MrWRKY基因只在茎和根中表达,2个MrWRKY基因只在茎和叶中表达,2个MrWRKY基因只在根和叶中表达。这一研究结果为进一步解析德国洋甘菊WRKY转录因子的功能提供了科学基础。     

Breeding of rutabaga (Brassica napus var. napobrassica L. Reichenb.) based on biomarker selection for root maggot resistance (Delia radicum L.)

Cabbage root maggot (Delia radicum) is the most devastating and persistent pest for rutabaga (Brassica napus var. napobrassica) in all production areas in Canada. With the deregistration of terbufos (Counter^?) to combat maggot attack, only chlorpyrifos (Lorsban^?), an organophosphorous pesticide, remains and extensive use could lead to insecticide resistance. An unprotected crop would lead to serious domestic and export losses. Root maggot resistance from canola, that originated from the weedy crucifer, Sinapis alba, was transferred to rutabaga by standard hand crossing. A population of doubled haploids was developed from the F1s and screened in a high pressure root maggot rutabaga production field. Resistant and susceptible isolines were identified from different crossing groups and these isoline pairs were used to develop a biochemical selection protocol based on HPLC profiles where glucosinolates can be present as an aid to resistance breeding. Fourteen peaks in the HPLC profile were identified as markers and predictably varied between the more resistant and more susceptible lines. The 3–4 leaf stage was identified as the ideal stage for tissue extraction for profiling which is close to the stage when gravid female maggot flies seek host plants for oviposition utilizing olfactory signals from the host. Olfactory signals for Delia commonly are isothiocyanates which are volatile break down products of glucosinolates. The peaks in the HPLC profiles identified as markers for resistance contain glucosinolates and may be partially responsible for the plant-insect interaction. A predictive model is proposed as an aid to breeders for the development of root maggot resistant rutabaga lines.     

长期定位施肥对冬小麦后期根系衰老和产量的影响

利用连续28年长期定位施肥试验,研究了长期施肥对冬小麦生育后期根系衰老及产量的影响,结果表明,施用有机肥特别是有机肥与一定量的氮肥配合施用能显著抑制根系的膜脂过氧化作用,使小麦根系中SOD活性提高、MDA含量降低、从而延缓了根系的衰老,使小麦生育后期仍能维持较高的根系生理活性,提高了粒重,增加籽粒产量。     

缺磷胁迫对黑籽南瓜幼苗根系生长和根系分泌物的影响

采用营养液培养方法,以1/2单位日本园试配方为基本营养液,在温室内培养黑籽南瓜(Cucurbita ficifolia B.)幼苗,测定正常供磷处理和缺磷胁迫下黑籽南瓜幼苗在根系生长、根系形态和根系分泌物等方面的差异,以期为南瓜磷高效种质创新和耐低磷机制研究提供理论依据.结果表明,缺磷胁迫后21 d,黑籽南瓜幼苗地上部生物量显著降低,根系生物量与正常供磷处理相比没有明显差异,根冠比增大;总根长和根表面积显著增加,根数、根横径和根体积与正常供磷处理相比没有明显的差异;根系吸磷量和磷转运率下降,根系吸磷量占总吸磷量的百分数增加,根系磷利用效率显著增高.总根长和根表面积增加可能与其磷吸收效率密切相关,是黑籽南瓜幼苗对缺磷胁迫的适应性变化.与正常供磷处理相比,缺磷胁迫后21 d,黑籽南瓜幼苗根系分泌的化合物种类组成类似,主要由酚类、芳香烃类、酯类、胺类、烯烃类和烷烃类化合物组成.但缺磷胁迫后根系分泌物中的化学成分多于正常供磷处理,邻苯二甲酸二(1丁基2异丁基)酯等8种化合物是缺磷胁迫下黑籽南瓜根系分泌物的特有成分.缺磷胁迫后黑籽南瓜的自毒作用增强.     

玉米氮素敏感性差异自交系的表达谱分析

玉米材料的氮肥敏感性存在显著差异,但基因表达模式尚不清楚。基于此,本研究以玉米氮敏感型自交系 B73和钝感型自交系Mo17为材料,对足氮(sufficient nitrogen,简称SN)和低氮(limiting nitrogen,简称LN)条件下苗期叶片组织的转录组进行分析。对于叶片总氮含量,敏感型B73在足氮和低氮条件下存在显著差异,而钝感型Mo17的差异小且不显著。基因表达差异分析显示Mo17在两种氮环境下差异基因的数目达13 867个,在低氮环境下基因上调比例高于下调比例1.9倍;B73差异基因的数目为10 028个,低氮环境基因上调比例低于下调比例。基因聚类分析也显示低氮环境下,钝感型Mo17基因表达上调或下调的幅度高于敏感型B73。差异基因双尾方差分析表明受氮环境和基因型共同影响的差异基因为342个,功能主要集中在与氨基酸代谢、光合作用、次级代谢及基因复制表达等相关途径。综上所述,在低氮条件下氮钝感型Mo17较敏感型B73激活更多的基因来提高植株对氮的吸收和同化能力,被激活的基因可能与玉米氮肥转运和利用有关。