谷子PEPC基因的鉴定及其对非生物逆境的响应特性

磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvatecarboxylase,PEPC)是C4植物光合作用关键酶,并在植物多种代谢途径及逆境信号应答过程中起重要作用。本研究通过序列比对,从谷子基因组中筛选出6个SiPEPC候选基因。SiPEPC蛋白特征参数相似度很高,序列非常保守,都含有PEPC基因特征功能域PEPcase Motif。SiPEPC成员主要被定位在细胞质、细胞核和线粒体。在SiPEPC成员启动子序列中含大量有光、激素、逆境以及其他生长调控相关的顺式应答元件。苗期逆境qRT-PCR表达谱分析表明,5个SiPEPC基因(SiPEPC1、SiPEPC2、SiPEPC3、SiPEPC5、SiPEPC6)不同程度受ABA、PEG、高盐和低温诱导表达,表明其参与了苗期对非生物逆境的响应。5个SiPEPC基因表达量在正常生长条件下随着谷子的生长呈增强趋势,且在不同生育时期干旱胁迫下明显增加,表明其参与了拔节、抽穗、灌浆期的干旱胁迫应答。拔节期弱光可诱导5个SiPEPC基因的表达,而在拔节期中等强度光照以及抽穗期和灌浆期的中等光照和弱光照下表达量均急剧降低,揭示光照强度严重影响...     

谷子糖转运蛋白基因SiSTPs的鉴定及其参与谷子抗逆胁迫响应的研究

糖转运蛋白(sugar transporter proteins, STPs)是一类主要转运己糖的单糖转运蛋白, 在作物的生长发育和抗逆过程中发挥着重要作用。谷子是绿色旱作农业的主栽作物, 也是C₄光合作用机理和禾本科抗逆基因挖掘的模式植物, 目前有关谷子STP基因功能的研究还鲜有报道。因此, 本研究利用生物信息学方法, 对包括谷子在内的6种禾本科作物的STPs基因进行全基因组鉴定, 重点对谷子SiSTPs基因的理化性质、染色体定位、系统进化、基因结构、保守结构域、组织表达模式, 并对其参与谷子干旱和低磷胁迫以及白发病抗性响应进行了深入分析。在谷子、狗尾草、高粱、玉米、小麦和水稻中分别鉴定出STP基因家族成员24、26、23、22、33和27个, 系统进化分析聚为4类。谷子的24个SiSTP基因不均匀的分布在7条染色体上, 其编码氨基酸的大小为499~581 aa, 这些SiSTP蛋白都具有Sugar_tr (PF00083)保守结构域, 并且在启动子区存在大量的光响应以及逆境响应元件, 分析发现谷子SiSTPs基因在进化过程中受到了强烈的纯化选择压力, 存在明显的组织表达特异性, 且受光诱导, 不同成员在低磷胁迫、干旱胁迫和白发病侵染过程中表达存在差异。本研究为阐明谷子STP蛋白的功能以及响应逆境胁迫机理提供理论基础。     

谷子PPDK2在非生物逆境胁迫下的表达分析

谷子是具有抗旱、耐瘠、抗逆性强、适应性广等特点的C₄禾本科作物,发掘谷子高光效、逆境相关基因,旨在揭示谷子在光照和逆境胁迫下的基因表达特征。采用生物信息学方法,在谷子中鉴定出一个PPDK基因,命名为SiPPDK2;该基因位于谷子3号染色体,含有18个内含子,有3个转录本,原始转录本编码945个氨基酸。亚细胞定位预测结果显示,该基因位于叶绿体中。功能域分析和多序列比对发现,SiPPDK2蛋白与玉米、高粱和水稻PPDK蛋白的亲缘关系较近。Real-time PCR分析表明,PEG、ABA、盐和低温胁迫对苗期SiPPDK2基因表达有不同程度的诱导。进一步研究表明,SiPPDK2基因在拔节期、抽穗期和灌浆期均参与了对干旱和光照的胁迫响应,其中抽穗期和灌浆期在干旱条件下及拔节期和灌浆期在弱光条件下其表达量显著上调。顺式元件分析结果表明,在SiPPDK2启动子区域主要包括激素类应答、逆境应答、光应答以及其他类生长调控相关的顺式元件。结果推测,SiPPDK2基因可能参与了谷子对非生物逆境胁迫的响应。     

砂藓RcRab基因响应非生物胁迫的表达分析及载体构建

为了研究砂藓(Racomitrium canescens)中的小G结合蛋白基因RcRab在非生物胁迫下发挥的作用,以砂藓为材料,利用实时荧光定量PCR,对砂藓的RcRab基因的表达情况进行了分析.结果表明,RcRab在不同的逆境胁迫处理下的表达不相同;与对照相比,RcRab在干旱和高温处理下表达量均升高,在低温和水渍处...     

谷子PP2C基因家族的特性

PP2Cs (2C type protein phosphatases)是一种单体丝氨酸/苏氨酸蛋白磷酸酶,在真核生物中,PP2Cs在脱落酸(ABA)、茉莉酸(JA)、水杨酸(SA)等激素信号传导途径中起着重要的调控作用。本研究通过序列比对,从谷子基因组中筛选出80个PP2C候选基因,聚类分析将其分为12个亚族(A、B、C、D、E1、E2、F1、F2、G、H、I、J)。与拟南芥PP2C基因家族比对表明,A~I为2个物种共有的亚族,J亚族只存在于谷子基因组中,L亚族只存在于拟南芥中。将谷子A亚族的10个成员命名为SiPP2CA1-10。基因表达谱分析表明,A亚族基因不同程度受ABA、干旱、高盐、低温和低氮诱导表达,其中,SiPP2CA6、SiPP2CA8在5种处理下诱导表达量都高。对10个A亚族成员的启动子分析发现,在这些基因的启动子序列中含有多种参与逆境胁迫应答的顺式作用元件,其中,SiPP2CA5、SiPP2CA6、SiPP2CA7、SiPP2CA8的启动子中含有参与低氮胁迫响应的元件。进一步研究发现,SiPP2CA8主要在根部表达,且在低氮胁迫下一直有较高的表达水平。亚细胞定位结果显示SiPP2CA8定位在细胞膜、细胞质、细胞核中;双分子荧光互补试验(BiFC)结果表明,SiPP2CA8与一个ABA受体类似蛋白SiRCAR3(基因号Si018317m.g)在细胞膜、细胞质及细胞核上互作,表明SiPP2CA8在谷子中可能参与ABA信号传导过程。     

马铃薯StSnRK2.4基因的序列分析及其在非生物胁迫下的表达分析

SnRK2是植物特有的一种丝氨酸/苏氨酸类蛋白激酶,在ABA信号转导和植物抵御逆境中起着重要作用.为探究马铃薯SnRK2在非生物胁迫下的调控作用,本研究以'青薯9号'为试验材料,克隆得到一个SnRK2家族基因StSnRK2.4,利用PlantCARE、ExPASy、 Cell-PLoc 2.0等软件对其进行序列分析,并...     

番茄SlETR6基因的克隆及非生物胁迫下的表达分析

为解析番茄乙烯受体基因SlETR6在非生物胁迫过程中的功能。以番茄为材料,克隆了番茄SlETR6基因,利用MEGA 5. 0软件对SlETR6基因编码的蛋白进行了聚类分析,并利用实时荧光定量PCR分析了SlETR6基因在番茄根、叶、花、果实不同发育时期的组织表达情况及对其在高盐、高温(40℃)、低温(4℃)、干旱胁迫条件下的表达模式。结果表明,SlETR6基因开放阅读框(Open reading frame,ORF)为2 265 bp,编码754个氨基酸,蛋白质分子质量为85. 05 ku,等电点为7. 28,与马铃薯St ETR-like蛋白的同源性最高;启动子分析显示,SlETR6基因含有热胁迫、干旱胁迫、低氧胁迫、光响应、乙烯、水杨酸及赤霉素应答等相关的顺式作用元件。实时荧光定量PCR分析表明,SlETR6在番茄不同组织中均有表达,且在花和转色期果实中有显著高表达。高盐胁迫6 h后,SlETR6基因呈现上调表达,12 h后达到峰值,而后回复正常表达水平;高温胁迫后,SlETR6基因表达量有明显的上升趋势;干旱胁迫早期SlETR6基因应答强烈,在胁迫处理1 h后表达量达到峰值。因此,推测SlETR6基因可能在番茄适应高盐、高温、干旱等逆境胁迫过程中发挥重要作用,可能参与番茄生长发育过程中的逆境调控。为番茄抗逆研究提供了新的候选基因资源。     

烟草转录因子NtNAC080在非生物胁迫下的表达分析及功能鉴定

NAC作为植物特有的转录因子,广泛参与植物生长发育、衰老及胁迫响应等生物学过程。为探究烟草NtNAC080在非生物胁迫响应中的功能,利用qRT-PCR技术分析了不同胁迫处理下NtNAC080的表达模式,结果表明,NtNAC080的表达受干旱、高盐胁迫以及ABA、MeJA和SA激素的诱导;以NtNAC080基因的敲除突变体及野生型(K326)烟株为材料,分析敲除株系在高盐和干旱胁迫下抗逆表型。试验表明,与野生型相比,2个敲除株系的耐盐抗旱能力均明显增强;干旱和盐胁迫下敲除株系的抗氧化酶(SOD、POD、CAT)活性以及可溶性蛋白、脯氨酸含量显著高于野生型,而丙二醛含量显著低于野生型。相反地,异源表达NtNAC080的转基因拟南芥与野生型(Col-0)相比对盐和干旱的耐受性明显减弱。qRT-PCR分析发现在干旱和盐处理后胁迫相关基因(NtDREB1A、NtKAT2、NtNHX1等)在NtNAC080基因敲除株系中表达水平显著高于野生型。以上结果表明,NtNAC080在烟草的非生物胁迫响应中起负调控作用,这可能是通过调控抗氧化酶活性及胁迫相关基因的表达来实现的。     

小麦幼苗活性LTR反转录转座子筛选及其对非生物胁迫的响应

LTR (长末端重复, long terminal repeat)反转录转座子占小麦基因组的60%以上, 筛选小麦基因组中具有转座活性的LTR反转录转座子, 并分析其在非生物胁迫下的响应, 对研究反转录转座子在小麦抗逆境胁迫中的作用具有重要意义。本研究通过生物信息学分析, 从转座子数据库(TREP database)中筛选出4个具有完整结构的LTR反转录转座子Fatima、Wis、Angela和Babara; 同时利用实时荧光定量PCR (qRT-PCR)、甲基化特异PCR (methylmion specific PCR, MSP)和转座子展示(transposon display, TD)技术分别分析了它们在盐、ABA、H₂O₂和干旱等处理的小麦幼苗期(二叶一心)叶和根中的表达水平、甲基化水平和转座活性变化。结果表明, 这4个反转录转座子在正常条件下均存在基础水平的转录, 并且能够响应上述4种胁迫而发生转录水平的变化, 且在相同胁迫条件下表达水平变化趋势一致。Fatima、Angela和Babara在非生物胁迫处理下表达水平的提高与其甲基化水平的降低有关, Wis则相反。反转录转座子LTR序列含有胁迫响应顺式作用元件, 但在非生物胁迫条件下顺式作用元件对这4个反转录转座子的调控作用不显著。与叶相比, 这4个反转录转座子在根中对胁迫的响应程度更高, 且在盐和ABA处理下转座活性更强。本研究将有助于进一步揭示LTR反转录转座子对非生物胁迫的响应规律, 为进一步研究利用反转录转座子进行小麦抗逆育种的遗传改良积累资料。     

Inheritance of sethoxydim resistance in foxtail millet, Setaria italica (L.) Beauv.

Interspecific hybridization between foxtail millet cultivars (Setaria italica) and a green foxtail (S. viridis) resistant to the herbicide sethoxydim were undertaken to breed foxtail millet for improved herbicide resistance. Parents, reciprocal F₁ hybrids, F₂ selfed derived populations and BC₁ backcross progeny were produced and analysed for mortality and fresh weight over a range of dosages. All resistant progeny were 700 times more resistant than susceptible cultivars and was symptom free under current field dosages. Segregations of resistant and susceptible progeny (3:1 in F₂ and 1:1 in BC₁) were not dependent upon dosage. Heterozygous individuals displayed the same magnitude of resistance as homozygous plants at twelve times the recommended field dosage. Results suggested that sethoxydim resistance in foxtail millet was controlled by a single, completely dominant, nuclear gene. This revised version was published online in July 2006 with corrections to the Cover Date.     

Traditional maintenance and multiplication of foxtail millet (Setaria italica (L.) P. Beauv.) landraces in China

Summary Foxtail millet (Setaria italica L. P. Beauv.) has been one of the most important cereals in northern China. A large number of landraces have formed under natural and man-made selection since domestication. Farmers in China have developed various traditional methods of maintenance and multiplication of foxtail millet seeds for next generation. Panicle selection was often done in fields. The selected seeds were generally stored at dry places. Archaeological and historical evidence of the origin of foxtail millet in China and the geographical distribution of landraces is also presented.     

Mitochondrial DNA variation in foxtail millet, Setaria italica (L.) P. Beauv.

Mitochondrial DNA (mtDNA) wascharacterized by RFLPs in 94 strains offoxtail millet, Setaria italica (L.)P. Beauv. Three RFLP patterns wereobserved by using rice atp6 as aprobe, and were designated as types I-III. Difference between types I and II seems tobe attributed to recombination between twoatp6 genes. In East and SoutheastAsia and Afghanistan, both of types I andII were found, while type I was predominantin India, Central Asia and Europe. InChina, type III was also found. Chinesestrains showed higher gene diversity thanthose from other regions. This resultcoincided the previous studies on isozymesand nuclear RFLPs.     

Development and utilization of novel SSRs in foxtail millet [Setaria italica (L.) P. Beauv.]

Although the foxtail millet [Setaria italica (L.) P. Beauv.] is recently regarded as a model crop for studying functional genomics of biofuel grasses, its genetic improvement to some extent was limited due to the non‐availability of molecular markers, particularly the microsatellite markers and the saturated genetic linkage map. Considering this, we attempted to generate a significant number of microsatellite markers in cultivar ‘Prasad’. Two hundred and fifty‐six clones were sequenced to generate 41.82‐kb high‐quality sequences retrieved from genomic library enriched with dinucleotide repeat motifs. Microsatellites were identified in 194 (76%) of the 256 positive clones, and 64 primer pairs (pp) were successfully designed from 95 (49%) unique SSR‐containing clones. The 67.4% primer designing ability, 100% PCR amplification efficiency and 45.3% polymorphic potential in the parents of F₂ mapping population established the efficacy of genomic microsatellites. All the 64 microsatellite markers displayed high level of cross‐species amplification (~67%) in 10 millets and non‐millets species. These experimental findings suggest the utility and efficacy of SSRs in diverse genotyping applications, resolving QTLs, phylogenetic relationships and transferability in several important grass species.     

Indentification of true genetic dwarfing sources in foxtail millet (Setaria italica Beauv.)

Summary True genetic dwarfs hither-to not reported have been located in foxtail millet. The dwarfs as a group distinguished themselves from the talls in having altered constellation of characters. The morphological differences were highly significant for plant height, internodal length, and tillering potential. The talls had elongated internodes compared to dwarfs while there was no difference for node number on the main stem suggesting that short internodal length was primarily responsible for dwarfism. The dwarfs also showed slightly higher leaf number per plant, leaf area and harvest index compared to talls. The dwarfs were insensitive to exogenous GA₃ application indicating that GA₃ synthesis is not impaired. This suggests that dwarfing gene sources presently identified are true genetic dwarfs and their behaviour is similar to dwarfs derived from Norin 10 in wheat and Dee-geo-woo-gen in rice. The superior morphological frame makes these dwarfs ideal as far as plant type is concerned and offers immense potentialities in breeding high yielding foxtail millets.     

Breeding strategy in foxtail millet,Setaria italica (L.P. Beauv.), following interspecific hybridization

Summary An interspecific cross between foxtail millet Setaria italica and its wild relative Setaria viridis was undertaken in order to introduce from the latter triazine resistance in the cultivated plant. Four backcross generations obtained with S. italica as recurrent parent were studied. Results show that only two backcross generations associated with selection are enough to eliminate weedy characters and to return to the cultivated type. S. viridis could thus be an interesting source for improvement of foxtail millet without a complex and time consuming breeding strategy.     

Thresholds,sensitive stages and genetic variability of finger millet to high temperature stress

Finger millet [Eleusine coracana (L.) Gaertn.] is an important coarse cereal crop grown in the arid and semi‐arid regions and often experiences high temperature (HT) stress. The objectives of this research were (i) to quantify effects of season‐long HT stress on physiological and yield traits, (ii) to identify the developmental stages most sensitive to HT stress and (iii) to quantify the genetic variability for HT stress tolerance in finger millet. Research was conducted in controlled environment conditions. HT stress decreased the chlorophyll index, photosystem II activity, grain yield and harvest index. Maximum decrease in number of seeds per panicle and grain yield per plant was observed when stress was imposed during booting, panicle emergence or flowering stages. Maximum genotypic variation was explained by panicle width and number of seeds per panicle at optimum temperature (OT) and grain yield per plant at HT and number of seeds at HT. Based on the stress response and grain yield, tolerant or susceptible genotypes were identified. Finger millet is sensitive to HT stress during reproductive stages, and there was genotypic variability among the finger millet genotypes for number of seeds per panicle and grain yield under HT, which can be exploited to enhance stress tolerance.     

Genetic variability in foxtail millet, Setaria italica (L.) P. Beauv. - RFLP using a heterologous rDNA probe

Ribosomal DNA was surveyed in a world collection (43 lines) of Setaria italica for variation in repeat unit length and restriction enzyme site variability using a non-radioactive wheat rDNA probe. Only two lengths of repeat units of about 7.9 or 7.6 kb were observed. The fact that these two repeat unit lengths were not randomly dispersed throughout the world collection suggests the existence of three genetic groups. The central European lines and most western European accessions have only the 7.6 kb repeat unit and most Asiatic lines have a 7.9 kb repeat unit. Lines originating from the north or the south of Asia showed different numbers of Bam HI fragments. The fact that the difference between the Asiatic and European pool is not continuous (7.9 or 7.6 kb) excludes the hypothesis of domestication being based on the spread of an initial population over Eurasia.     

Genetic variability in foxtail millet, Setaria italica (L.) P. Beauv.: Identification and classification of lines with RAPD markers

The DNA of 37 lines of Setaria italica, representative of Eurasian diversity, were used for the detection of polymorphism using the random amplified polymorphic DNA (RAPD) method. Four 10-mer primers, differing from each other by one or two G-C inversions, were used. Each one gave specific RAPD products. Twenty-five bands were polymorphic and allowed the identification of 33 different genotypes. A factorial analysis of correspondence was performed on the presenc-absence data, through which three genetic groups could be identified. These genetic groups were closely related to the geographical origin of the different lines: one central European and two Asiatic groups (the first Asiatic lines originating in latitudes below 35°N and the second comprising the Asiatic lines originating in latitudes above 35° N). The lines originating from western Europe were very variable and were dispersed in the three genetic groups described previously.