烟碱降解细菌Ochrobactrum intermedium DN2生长培养基及培养条件的优化研究

在烟碱生物降解的研究和实际应用中,需要在较短的时间内获得大量O.intermediumDN2菌体。为了提高菌株DN2的菌体浓度,缩短其培养时间,本研究采用PlackettBurman设计对影响O.intermediumDN2生长的内在和外在相关因素进行了评价。结果表明,影响菌株DN2生长的显著因素有胰蛋白胨、MgSO4·7H2O、初始pH值、温度、装液量和培养时间。在此基础上,采用响应曲面法分别对该菌生长培养基的组成和培养条件进行优化,得到最佳培养基组成为(g/L):胰蛋白胨11.34、牛肉膏3.00、NaCl5.00、MgSO4·7H2O3.71,pH值7.23;最佳培养条件为温度32℃、转速120r/min、装液量88ml/250ml三角瓶、培养时间34h。5L发酵罐验证实验表明,菌株DN2达到最大生长量的时间为36h,与预测值34h基本一致,比优化前缩短约12h;最大菌体浓度为6.49×109cfu/ml,与预测值6.73×109cfu/ml接近,比优化前高近一个数量级。

Ochrobacterum intermedium DN2新型烟碱降解酶的分离纯化

中间苍白杆菌(OchrobacterumintermediumDN2)是一株新发现的可以降解烟碱的菌株。本研究对O.intermediumDN2烟碱降解酶的合成动态进行了分析,确定其酶的合成模式为同步合成型。另外,对它的烟碱降解酶进行了分离纯化。粗酶经硫酸铵沉淀、SephadexG-100分子筛层析、DEAE-SephadexA50离子交换层析等步骤后获得电泳纯的烟碱降解酶,纯化倍数35.24倍,酶活回收率6.4%。经SDS-PAGE电泳确定为电泳纯,测得该酶的分子量约为40.06kDa。由中间苍白杆菌产生的烟碱降解酶的分离纯化在国内外还是首次报道。

小冰麦(Triticum aestivum-Agropyron intermedium)对盐胁迫和碱胁迫的生理响应

将两种中性盐(NaCl和Na2SO4)和两种碱性盐(NaHCO3和Na2CO3)按摩尔质量比1∶1混合,在60～300mmolL-1盐浓度内模拟出5种强度的盐胁迫条件,在30～180mmolL-1盐浓度内模拟出6种强度的碱胁迫条件,并以此对小冰麦苗胁迫处理12d。测定相对生长率(RGR)、含水量、丙二醛(MDA)、电解质外渗率、叶绿素、类胡萝卜素6项胁变指标和Na+、K+、脯氨酸、甜菜碱、有机酸5种溶质含量。结果表明,碱胁迫下小冰麦的各项胁变反应均明显大于盐胁迫下。在本试验条件下,小冰麦可耐受的最高盐胁迫浓度为300mmolL-1,而碱胁迫仅为150mmolL-1。碱胁迫造成小冰麦光合色素含量急剧下降,可能是其危害甚于盐胁迫的原因之一。碱胁迫下有机酸大量积累可能是小冰麦响应碱胁迫的特殊生理机制。试验结果证明盐、碱胁迫是两种性质不同的胁迫,不仅对植物的作用机制不同,而且植物的适应机制也不同。

Heterosis and Combining Ability for Major Yield Traits of a New Wheat Germplasm Shannong 0095 Derived from Thinopyrum intermedium

The wheatgrass, Thinopyrum intermedium （Host） Barkworth ＆ DR Dewey, shows many beneficial characteristics, such as big spikes and high resistance to many diseases. To transfer the beneficial genes of this species, many wheat- Thinopyrum intermedium alien chromosome lines were developed. Of them, Shannong 0095 （SN0095）, a disomic substitution, has long spikes and flag-leaves, and thus may be an important genetic resource for wheat yield improvement. In order to realize its heterosis and combining ability on major yield traits, a 7 ×7 complete diallel design was made according to Griffing＇s Method-1. The results showed that heterosis for spike length （SPL）, flag-leaf area （FLA）, number of spikes per plant （NSP）, number of spikelets per spike （NSL）, kernels per spike （KPS）, 1 000-kernel weight （TKW） and grain yield per plant （GYP） existed in all the crosses by SN0095, but heterobeltiosis occurred only for KPS, TKW, and GYP. The relative mid-parent heterosis （RMH） and relative high-parent heterosis （RHH） for GYP, which valued as high as 35.32 and 29.92% respectively, were the highest among all the traits mearsured. Though additive and non-additive gene effects and cytoplasmic effects （or cytoplasmic-nuclear interaction effects） were found in governing all the traits measured above, additive gene action played a predominant role. The results also showed that SN0095 was the best-general combiner for SPL and FLA, and high-general combiner for NSP amongst all the parents. Estimates of specific combining ability （SCA） showed that SN0095 could also make high-SCA combinations for GYP, such as SN0095 × Jimai 19 （JMI9）. SN0095 could be a unique and important parent in hybrid wheat breeding programs.

phlb基因在中国春Tal kr phlb基因综合体与Agropyron intermedium杂交中的作用

本文对中国春Tal kr phlb基因综合体,Tal中国春与Ag.intermedium杂种F_1花粉母细胞减数分裂中期Ⅰ染色体配对情况进行了比较研究。 证明phlb基因可以诱导普通小麦与Ag.intermdium间部分同源染色体配对、交换,从而有可能以染色体易位的方式把Ag.intermedium中的有益基因转移到普通小麦中。在Ag.intermedium(葡萄牙),Ag.intermedtam(伊朗)中都不含有Ph1基因。但在Ag.intermedium(伊朗)中可能存在Ph-like基因,其作用比Ph1小。

Molecular cytogenetic characterization of a novel wheat-Thinopyrum intermedium introgression line tolerant to phosphorus deficiency

Thinopyrum intermedium has been used as a resource for improving resistance to biotic and abiotic stresses and yield potential in common wheat. Wheat line SN304 was derived from a cross between common wheat cultivar Yannong 15 and Th. intermedium. Genomic in situ hybridization(GISH) produced no hybridization signal in SN304 using Th. intermedium genomic DNA as a probe, but fluorescence in situ hybridization(FISH) using oligonucleotides AFA-3, AFA-4, pAs1-1, pAs1-3, pAs1-4, pAs1-6, pSc119.2-1,and(GAA)10 as probes detected hybridization signals on chromosomes 2 A, 7 A, 2 B, 3 B, 6 B, and 7 B in SN304 that differed from Yannong 15. Results of specific markers also indicated that there were Th. intermedium chromatin introgressions on different chromosomes in SN304. In a hydroponic culture experiment, SN304 not only produced more biomass and higher stem and leaf dry weight but also accumulated more phosphorus than Yannong 15 under phosphorus-deficiency stress. Moreover, SN304 produced a lower pH and released more organic acids, especially oxalic acid, than Yannong 15, which suggests that SN304 exudates enabled more absorbance of P than Yannong 15 under comparable conditions.The results indicate that SN304 is a wheat-Th. intermedium introgression line with tolerance to phosphorus-deficiency stress.

Ochrobactrum intermedium培养条件优化及其对含蜡原油作用效果分析

利用微生物对原油蜡组分的降解作用,解决含蜡原油开采与输送过程中的蜡沉积问题。从大庆油田受原油污染土壤中筛选出一株Ochrobactrum intermedium H6,考察菌H6对含蜡原油的作用效果。实验结果表明,菌H6最优培养条件为温度34℃、培养基初始pH为6。菌H6可产生脂肽类生物表面活性剂,在与大庆原油作用10 d后,原油蜡含量降低5.74%,当温度为菌株最优培养温度34℃时,降黏率达到49.37%。菌H6可对原油蜡组分进行降解,具有在油田清防蜡过程中发挥作用的潜力。

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.

小冰麦(Triticum aestivum-Agropyron intermedium)碱胁迫响应机制的研究

植物生长过程中受多种胁迫的影响,盐、碱胁迫是影响作物产量的重要因素,对作物的危害仅次于干旱。本文阐述了小冰麦抗盐碱的机制,为提高小冰麦耐盐碱能力及抗盐碱的育种提供依据。

COLONIZATION OF VIGNA RADIATA ROOTS BY CHROMIUM RESISTANT BACTERIAL STRAINS OF OCHROBACTRUM INTERMEDIUM,BACILLUS CEREUS AND BREVIBACTERIUM SP.

The present study deals with colonization potential of plant growth promoting bacterial strains (Ochrobactrum intermedium, Bacillus cereus and Brevibacterium sp.) on Vigna radiata roots. The roots were heavily colonized with O. intermedium and B. cereus as compared to Brevibacterium sp. O. intermedium mainly colonized rhizoplane while B. cereus occurred both on the rhizoplane and near root zone. O. intermedium and B. cereus were found to be present both on the rhizoplane and near root zone, while Brevibacterium only in the rhizosphere in the form of groups. The cells of B. cereus were found more in the sites where root exudates were existed. From the above results it was observed that the number of O. intermedium cells were large at root exudate site. Fig 2, Tab 1, Ref

响应面法优化羽毛降解菌Ochrobactrum intermedium JY-25的降解培养基

【目的】作为构成羽毛主要成分的角蛋白,因其存在大量相互交联的二硫键、氢键和盐键等化学键,导致羽毛结构稳定难以被常规手段降解。相较于物理、化学方法降解废弃羽毛,利用微生物产生的角蛋白酶使废弃羽毛变成可再生资源,具有高效、环保的优点。课题组前期从家禽养殖场的土壤中分离、筛选到一株羽毛降解菌,经鉴定该菌株为中间苍白杆菌JY-25。研究旨在进一步提高中间苍白杆菌JY-25的羽毛降解率,为今后工业化降解废弃羽毛提供依据。【方法】依次采用单因素试验、Plackett-Burman试验设计和响应面法对中间苍白杆菌JY-25的羽毛降解培养基进行优化。【结果】通过单因素试验确定了在降解培养基中外加碳源(糊化淀粉)和氮源(玉米粉)时,都能提高羽毛的降解率。Plackett-Burman试验设计发现降解培养基的8种成分中,糊化淀粉、硫酸亚铁和羽毛量是影响中间苍白杆菌JY-25对羽毛降解率的显著因素。最后通过响应面法确定了最佳降解培养基的组分为:羽毛2 g/L、糊化淀粉15 g/L、玉米粉10 g/L、硫酸亚铁0.02 g/L、磷酸氢二钾1.5 g/L、氯化钠0.3 g/L、硫酸镁0.025 g/L、氯化钙0.025 g/L,在该优化的培养基配方下,菌株JY-25的羽毛降解率高达76.31%,相较于初始的降解率提高了24.4%。【结论】经过多种优化方式,显著地提高了中间苍白杆菌JY-25的羽毛降解率,为菌株用于工业化降解废弃羽毛提供了理论基础。

Thinopyrum ponticum and Th. intermedium: the promising source of resistance to fungal and viral diseases of wheat

Thinopyrum ponticum and Th. intermedium provide superior resistance against various diseases in wheat （Ttricum aestivum）. Because of their readily crossing with wheat, many genes for disease resistance have been introduced from the wheatgrasses into wheat. Genes for resistance to leaf rust, stem rust, powdery mildew, Barley yellow dwarf virus, Wheat streak mosaic virus, and its vector, the wheat curl mite, have been transferred into wheat by producing chromosome translocations. These genes offer an opportunity to improve resistance of wheat to the diseases; some of them have been extensively used in protecting wheat from damage of the diseases. Moreover, new resistance to diseases is continuously detected in the progenies of wheat-Thinopyrum derivatives. The present article summaries characterization and application of the genes for fungal and viral disease-resistance derived from Th. ponticum and Th. intermedium.

Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers

The wheat line H960642 is a homozygous wheat-Thinopyrum intermedium translocation line with resistance to BYDV by genomie in situ hybridization (GISH) and RFLP analysis. The genomie DNA of Th. intermedium was used as a probe, and eonunon wheat genomie DNA as a blocking in GISH experiment. The results showed that the chromosome segments of Th. intermedium were transferred to the distal end of a pair of wheat chromosomes. RFLP analysis indicated that the transloeation line H960642 is a T7DS·7DL-7XL translocation by using 8 probes mapped on the homoeologous group 7 in wheat. The tranalocation breakpoint is located between Xpsr680 and Xpsr965 about 90—99 cM from the centromere. The RFLP markers psr680 and psr687 were closoly linked with the BYDV resistance gene. The gene is located on the distal end of 7XL around Xpsr680 and Xpsr687.

Breeding and molecular cytogenetic identification of wheat-Thinopyrum intermedium addition lines resistant to powdery mildew

Wheat-related species Th. intermedium was used to cross with common wheat Yannong 15. In the self progenies of the hybrid, two addition lines, II -1-7-1 and II -3-3-2, stable in cytology, were developed by cytology and powdery mildew resistance identification. Their chromosome number were 2n = 44 and formed 22 bivalents at PMC MI. In F1 of the two addition lines crossing with Yannong 15, there appeared about one univalent at PMC MI, respectively. Resistance identification in greenhouse and field using the No. 15 and mixed strains of E. gramnis f. sp. tritici showed that they were immune to powdery mildew. Chromosome number and resistance identification using the F2 single plants of the addition line crossing with Yannong 15 indicated that the resistant gene was located on the alien chromosomes. In situ hybridization using St and E genomic DNA as probe showed that the added chromosome in the two addition lines probably came from the E genome of Th. intermedium, which indicated that a pair of E genome

Molecular Cytogenetic Identification of a Wheat-Thinopyron intermedium （Host） Barkworth ＆ DR Dewey Partial Amphiploid Resistant to Powdery Mildew

Wide cross and molecular cytogenetic methods were used to transfer the powdery mildew resistance gene from Thinopyron intermedium(Host) Barkworth & DR Dewey to wheat. Among the progeny of crossing common wheat (Triticum aestivum L.) Yannong 15 with Th. intermedium, a partial amphiploid E990256, with resistance to powdery mildew, was developed. It had 56 chromosomes and could form 28 bivalents in pollen mother cells at metaphase I of meiosis. Resistance verification by race 15 at the seedling stage and by mixed strains of Erysiphales gramnis DC. f. sp. tritici Em. Marchal at the adult stage showed it was immune to powdery mildew at both stages. Gene postulation via 21 isolates of E. gramnis f. sp. tritici and 29 differential hosts showed it was nearly immune to all the isolates used, and its resistance pattern was different from all the mildew resistance genes used, which indicated it probably contained a new resistance gene to powdery mildew. Biochemical verification showed it might convey different Th. intermedium chromosomes from those of the wheat- Th. intermedium partial amphiploids Zhong 1–5. Genomic in situ hybridization analysis by using St genomic DNA as the probe showed E990256 contained a recombination genome of St and E.

Chromosomal Constitutions and Reactions to Powdery Mildew and Stripe Rust of Four Novel Wheat-Thinopyrum intermedium Partial Amphiploids

As a staple food crop for one-third of the world＇s population, common wheat （Triticum aestivum L., 2n = 6x = 42, AABBDD） plays an important role in humans＇ food security. However, the genetic variation of cultivated wheat has been dramatically narrowed by genetic erosion under the modem cultivation system, resulting in vulnerability to biotic and abiotic stresses （Jiang et al., 1994; Friebe et al., 1996）. The wild relatives of wheat represent a large reservoir of superior genes, and transferring these alien genes to modem cultivars through chromosome engineering is a successful method of broadening the genetic diversity of wheat （Chen et al., 2003;

一株分离自地层水的石油降解菌的特性研究:Ochrobactrum intermedium环境分离菌株与临床分离菌株的比较基因组分析（英文）

目的:对一株地层水分离的石油降解菌Ochrobactrum intermedium 2745-2进行生理生化特性的研究、全基因组测序以及比较基因组研究。创新点:首次对一株分离自地层水的石油降解菌O.intermedium 2745-2进行了生理生化特性研究以及基因组测序,从基因组角度解释菌株2745-2对石油的降解能力。通过菌株2745-2与同种其他临床分离菌株的比较基因组学分析,表明2745-2仍具有多种与致病性相关的基因。方法:通过微生物富集培养的方法从油井的地层水中分离石油降解微生物,通过聚合酶链反应(PCR)扩增16S核糖体RNA(r RNA)序列进行比较和分析确定菌株的分类地位属于O.intermedium(图1)。采用Illumina Hi Seq2000对菌株2745-2进行高通量测序,采用Velvet 1.2.07和RAST server分别进行数据组装和注释(表1)。PHAST寻找基因组中的噬菌体相关序列(图4和表2)。通过BLAST+和BRIG对环境分离菌株(2745-2)和临床分离菌株(M86、229E和LMG3301T)进行基因组比较(表3、表4和图5)。结论:首次对一株环境分离的O.intermedium(2745-2)进行全基因组测序,揭示具有多种与石油降解相关的基因。通过环境分离菌株(2745-2)与临床分离菌株(M86,229E和LMG3301T)的基因组比较分析,表明2745-2仍具有多种致病性相关的基因。

Characterization of Three Novel Wheat LThinopyrum intermedium Addition Lines with Novel Storage Protein Subunits and Resistance to Both Powdery Mildew and Stripe Rust

Wide hybridization is an effective approach for enhancing the resistance of bread wheat （Triticum aestivum L.） to biotic and abiotic stresses by introducing favorable alien genes （Sepsi et al., 2008）. Wheatgrass, Thinopyrum intermedium （Host） Barkworth ＆ D.R. Dewey or Agropyron intermedium （Host） Beauvoir （2n = 42; genome formula JJjSjSstst）, is a perennial species in the tribe Triticeae and an important source of wheat improvement for biotic and abiotic stress resistance and quality-related traits, such as high grain protein concentration （Chen et al., 1998; 2001; 2003; Han et al., 2004; Li and Wang, 2009）. In addition, the ready crossing ability of wheatgrass with various Triticum species has made it popular in germ- plasm development.

Cytogenetic identification and molecular marker development for the novel stripe rust-resistant wheat- Thinopyrum intermedium translocation line WTT11

Stripe rust,caused by Puccinia striformis f.sp.tritici(Pst),is one of the most destructive diseases of wheat(Triticum aestivum L)worldwide.Xiaoyan 78829,a partial amphidiploid developed by crossing common wheat with Thinopyrum intermedium,is immune to wheat stripe rust.To transfer the resis-tance gene of this excellent germplasm resource to wheat,the tr anslocation line WTT11 was produced by pollen irradiation and assessed for immunity to stripe rust races CYR32,CYR33 and CYR34.A novel stripe rust-resistance locus derived from Th.intermedium was confirmed by linkage and diagnostic marker analyses.Molecular cytogenetic analyses revealed that WTT11 carries a TTh 2DL translocation.The breakpoint of 1B was located at 95.5 MB,and the alien segments were found to be homoeologous to wheat-group chromosomes 6 and 7 according to a wheat660K single-nucleotide polymorphism(SNP)array analysis.Ten previously developed PCR-based markers were confirmed to rapidly trace the alien segments of WTT11,and 20 kompetitive allele-specific PCR(KASP)markers were developed to enable genotyping of Th.intermedium and common wheat.Evaluation of agronomic traits in two consecutive crop seasons uncovered some favorable agronomic traits in WTT11,such as lower plant height and longer main panicles,that may be applicable to wheat improvement.As a novel genetic resource,the new resistance locus may be useful for wheat disease-resistance breeding.

Breeding of T.aestivum-Ag.intermedium translocation lines with T.timopheevii cytoplasm and characterization by GISH

A male-sterile T. aestivum-Ag. intermedium partial amphiploid with cytoplasm of T. timopheevii as a female parent was crossed to common wheat. The hybrid was backcrossed to the male parent several times continually and self-crossed at last. Two stable lines with common wheat phenotype, H96269-2 and H96278, have been obtained. The chromosome numbers of the two lines are all 2n = 42 in somatic cells. By inoculation test, the two lines show a high level of resistance to yellow rust. Through genomic in situ hybridization (GISH) with Ag. intermedium total genomic DNA as a probe, it is demonstrated that the two stable lines are all small segmental translocation lines, and the translocated chromosome segments from Ag. intermedium are located on the short arm terminals of wheat chromosomes. Genetics analysis suggests that the yellow rust resistance gene(s) are probably located on the translocated chromosome segments of Ag. intermedium.