Molecular identification of asymmetric somatic hybrids between wheat and Italian ryegrass

Sexual incompatibility between common wheat and Italian ryegrass was an obstacle for transferring useful traits from italian ryegrass to wheat. In order to use those desirable genetic resources to improve wheat and to create new cytoplasmic germplasm, the protoplasts of wheat and Italian ryegrass were successfully electrofused and the somatic hybrid plants were regenerated. Examination with 6 restriction enzymes, 13 probes including 9 mtDNA probes (H454, Pst24, B30, Pro I, 490, B342, pHJ2-7-1, B376, 7), 3 cpDNA probes (pHvc p1, pHvc p5 and pHvc p8) and one nuclear DNA probe-- pTA71 (rDNA) in total 73 enzyme/ probe combinations revealed rich polymorphism between the fusion partners. RFLP analysis indicated that approximately 93.4% of the regenerated plants were true somatic hybrids. AFLP analysis implied that the somatic hybrids were highly asymmetric. The RFLP analysis using mt- and cpDNA specific probes also demonstrated the non-coexistence of mitochondria and chloroplasts from the fusion partners in the somatic hybrid cells.     

青苗碱谷与高冰草的体细胞杂交及杂种性质鉴定

将高冰草（Agropyrom elongahan Host Nevski）原生质体用强度为380μW／cm^2的紫外线分别照射0s、30s、1min、2min后作为融合供体;而未经射线处理的青苗碱谷（Setaria italica Beaur）原生质体作为融合受体,利用PEG方法诱导融合．对再生克隆进行形态学和染色体观察,并用同工酶、RAPD、5SrDNA间隔序列和叶绿体微卫星DNA（SSR）进行杂种性质鉴定．结果证明融合产物具有双亲基因组,高频率地形成了体细胞杂种。     

利用原生质体融合技术筛选红霉素优良菌株

以红霉素高产菌HE-08-6及低产优组分菌HA-08-20为出发菌株,其原生质体经紫外诱变后在适宜条件下进行融合,再生培养后挑取融合子89株,经筛选得到3株具有二亲本优良性状的高产菌株HEA-08-01,HEA-08-02,HEA-08-03.其化学效价较出发菌株HA-08-20提高约25%,产品的红霉素A组分含量较出发菌株HE-08-6提高约26%.传代培养证明该菌株具有一定的遗传稳定性,在50L-Fus智能发酵罐上连续3批发酵结果表明,该优良菌种在提高红霉素产品质量及效价上取得较大进展,具有推广应用价值.     

香菇菌丝原生质体制备及融合条件的研究

讨论不同菌龄、酶解时间、酶解温度、渗透压稳定剂对香菇原生质体产率的影响。结果表明 ,香菇菌丝制备原生质体时最适菌龄为 6天 ,酶解时间为 3h ,酶解温度为 34℃ ,渗透压稳定剂用 0 .6mol/L甘露醇为最佳。在此基础上 ,以 35 %的PEG为融合诱导剂进行香菇B0 1、L2 6种间原生质体融合 ,并用显微镜观察到融合子的形成     

木本植物原生质体培养与融合研究进展

为了全面了解模板植物原生质体培养和融合的现状，找出原生质体培养的规律，概述了原生质体的分离、培养及再生植株的条件和影响因素，介绍了国内外原生质体培养和融合及无性系变异筛选及遗传转化的研究动态。     

复合诱变和原生质体融合选育S-腺苷甲硫氨酸酿酒酵母高产菌株

对生产S-腺苷甲硫氨酸（SAM）的酿酒酵母（S.cerevisiae）菌株R1进行复合诱变和原生质体融合。采用酶解法获得出发菌株S.cerevisiae R1的单倍体菌株hR10,对其进行UV和DES复合诱变,筛选出了4个单倍体正突变株DR1-3、NM14、NM39和NM45。制备了单倍体正突变株的原生质体,采用热灭活和UV灭活法灭活双亲原生质体,在聚乙二醇（PEG）的诱导下进行原生质体融合,筛选得到融合子F35,其生产 SAM的产量为22.5 mg/L,与出发菌株R1（11.1 mg/L）相比,提高了103%,并且能够稳定遗传。采用正交试验优化了F35发酵生产SAM的培养基和发酵时间,优化了的培养基配方为麦芽糖100 g/L,蛋白胨10 g/L,NH₄H₂PO₄4g/L,NH₄Cl₅g/L,MgSO₄0.1g/L,KH₂PO₄6 g/L,发酵时间为72h。     

林肯链霉菌原生质体形成、再生和融合的研究

林肯链霉菌生长在0.5%甘氨酸的S培养基中,能较好地被溶菌酶破壁形成原生质体。原生质体能在RB培养基上再生,但不能在R_2培养基上再生,这和已报道过的其他链霉菌不同。78-11菌株的再生频率约为25.7%,S-3菌株的再生频率约为20.5%。PEG(聚乙二醇)1000对诱导融合的效果较好,重组频率最高可达10~(-2)。重组子和亲本在培养特性、孢子形态方面无特殊差异,但经C_O~(60)诱变后重组子的正变率超过亲本。     

绿棉/棉混纺纱中绿棉定性鉴别与混纺比测定

研究天然绿棉/白棉混纺纱线的混纺比测定方法.用氨水对不同绿棉含量的绿棉/白棉混纺纱进行化学预处理,将处理后的纱线用显微镜观察并计数绿棉和白棉纤维根数,测量两种纤维的直径和密度,计算绿棉混纺比.利用回归分析处理实验数据,得到天然绿棉/棉混纺纱混纺比的测定方法.     

用流式细胞仪和RAPD快速鉴定柑橘体细胞杂种

利用流式细胞仪和随机扩增多态DNA（RAPD）方法对获得的3例柑橘体细胞杂种进行鉴定,结果表明,两者相结合可快速有效地鉴定体细胞杂种．所检测的3个组合共9株再生植株,有8株是四倍体体细胞杂种,1株为二倍体叶肉亲本型杂种．体细胞杂种一般表现为具有双亲的特征带,且综合双亲的所有带,但在部分杂种中检测到了双亲都没有的新带,也发现有丢失亲本的特征带或共有带的现象,说明融合后染色体发生了重组和交换;有的引物只检测到叶肉亲本的特征带,根据柑橘叶肉细胞无论单独培养还是共培养均不能再生的事实,可推测其为体细胞杂种或二倍体叶肉亲本型胞质杂种．对这两种方法相结合用于体细胞杂种鉴定的可行性进行了讨论．     

Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion

Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. In-traspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveDˉ and olmAˉ (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, re-spectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the produc-tion of other avermectin components and oligomycin will make F23 and F29 useful strains for aver-mectin production. Strain F29's tolerance of fermentation conditions will also make it suitable for in-dustrial applications.     

Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

The mechanisms of cotton fiber development and somatic embryogenesis have been explored sys-tematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples,with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression re-sults,normalization of real-time PCR data against one or several internal control genes is required,which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes,including 18S rRNA,Histone3,UBQ7,Actin,Cyclophilin,Gbpolyubiquitin-1 and Gbpolyubiquitin-2,in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene(arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quanti-fication should be an efficient and convenient method for the fiber developmental series. The expres-sion of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3,UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.     

Enhancement of monacolin K production via intergeneric protoplast fusion between Aspergillus terreus and Monascus anka

Intergenric protoplast fusion between Aspergillus terreus CA99 and Monascus anka M-3, the high and low producers of monacolin K respectively,was performed for enhancement of monacolin K production. The 24-hour-old mycelia of A. terreus CA99 and M. anka M-3 were treated with 0.5% lywallzyme, 0.3% snailase and 0.3% cellulase at 34℃ for 5 h and at 30℃ for 3.5 h, and their protoplasts formation reached 1.76×107/mL and 1.68×107/mL respectively. Parental protoplasts were irradiated with a 30 W UV light away from 30 cm for 3 min and then mixed. The mixture was incubated with 30% PEG 6000 for 15 min. The reviving fusants were isolated on the regeneration plates. Of the 363 fusants isolated, over 100 showed enhanced monacolin K production compared with the parental strain M. anka M-3. Ten of them produced monacolin K about 1.6-fold of that M.anka M-3 does and the monacolin K titer of two fusants (F49 and F104) increased by about 1-fold. The monacolin K yields of F49 and F104 were 460 μg/mL and 457 μg/mL respectively. In optimized fermentation medium, the monacolin K titer of F49 reached 1216 μg/mL.     

Enhancement of monacolin K production via intergeneric protoplast fusion between Aspergillus terreus and Monascus anka

Intergenric protoplast fusion between Aspergillus terreus CA99 and Monascus anka M-3, the high and low producers of monacolin K respectively，was performed for enhancement of monacolin K production. The 24-hour-old mycelia of A. terreus CA99 and M. anka M-3 were treated with 0.5% lywallzyme, 0.3% snailase and 0.3% cellulase at 34℃ for 5 h and at 30℃ for 3.5 h, and their protoplasts formation reached 1.76×10⁷/mL and 1.68×10⁷/mL respectively. Parental protoplasts were irradiated with a 30 W UV light away from 30 cm for 3 min and then mixed. The mixture was incubated with 30% PEG 6000 for 15 min. The reviving fusants were isolated on the regeneration plates. Of the 363 fusants isolated, over 100 showed enhanced monacolin K production compared with the parental strain M. anka M-3. Ten of them produced monacolin K about 1.6-fold of that M.anka M-3 does and the monacolin K titer of two fusants (F49 and F104) increased by about 1-fold. The monacolin K yields of F49 and F104 were 460 μg/mL and 457 μg/mL respectively. In optimized fermentation medium, the monacolin K titer of F49 reached 1216 μg/mL.     

Induction of phenylalanine ammonia-lyase and lipoxygenase in cotton seedlings by mechanical wounding and aphid infestation

Plants have developed a multitude of inducibledefense mechanisms against aggressive biotic and abi otic agents[1]. Wound and herbivory induced plantresponses can negatively affect herbivore’s physiologydirectly by stimulating the synthesis of toxic metabo lites[2—4]. In addition to such direct defense mecha nisms, plants can also emit specific blends of volatilesthat attract carnivorous enemies to defend themselvesagainst herbivores[5—9].Herbivory induced plant volatiles can n…     

应用滋养和包埋技术进行籼稻原生质体培养和植株再生

从几个籼稻品种不同发育阶段的幼穗分离出密度平均为２２×１０５个／ｍＬ～４６×１０５个／ｍＬ的原生质体。将这些原生质体包埋在藻酸钠颗粒里并分别在粳稻愈伤组织滋养下培养和没有愈伤组织滋养。在这两种培养条件下原生质体稳定,分裂频率分别达到４８４％和３８８％,群体存活率达到５３％和４７％,愈伤组织绿苗分化率达到７１６％和６２３％。这两种方法优于常用的琼脂糖包埋法和琼脂糖颗粒法。     

基于数据融合的无人机影像碎屑岩岩性识别

不同类型岩性影像纹理相似性高,基于单一的二维影像进行岩性识别精度较低。本文针对这一问题,开展了顾及影像深度信息的岩性智能识别方法研究。利用无人机影像具有多模态的特性,采用通道叠加、IHS变换、小波变换以及多模态融合四种影像融合方式,将深度信息融入到影像数据中,运用深度卷积神经网络DeepLabv3+进行碎屑岩岩性识别。经人工解译结果对比分析,结果表明,实验区内基于多模态融合影像的岩性识别精度最高,Kappa系数可达76.17%,总体识别精度可提升到91.05%;分析认为,顾及影像深度信息的岩性智能识别方法针对岩层表面不平整,高差落差大的砾岩识别效果有明显提升,但表面平整、高差表现不明显的泥岩和砂岩地层识别效果有待提升,总体为野外碎屑岩露头岩性快速识别提供了新思路。     

大鼠谷氨酰胺转运蛋白EGFP-SNAT3融合蛋白的表达与鉴定

SLC38基因家族编码的Na+偶联的中性氨基酸转运蛋白3(SNAT3)是System N中的一员.它在大脑谷氨酸-谷氨酰胺循环以及肝细胞质膜的谷氨酰胺转运等生理过程中发挥着重要作用.为了方便检测SNAT3在细胞膜上的表达与定位,本研究采用PCR扩增和酶切连接技术将增强型绿色荧光蛋白(EGFP)与SNAT3的N末端连接,通过菌液PCR、酶切和DNA测序验证得到pBK-CMV(Δ[1098-1300])-EGFP-SNAT3重组真核表达质粒.用脂质体转染法将构建好的质粒瞬时转染进入人体胚肾细胞(HEK293T cells),利用激光扫描共聚焦显微镜和Western blot技术检测EGFP-SNAT3融合蛋白的表达和亚细胞定位.结果表明,EGFPSNAT3重组质粒在细胞中正确表达并定位于细胞膜上.EGFP-SNAT3重组质粒的成功构建将有助于日后进一步研究SNAT3的结构和功能.     

Development and characterization of interspecific hybrids between Oryza sativa and O. latifolia by in situ hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chro- mosome pairing was identified and most of the chromosomes were univalent, which resulted in com- plete male sterility in the hybrid.     

水稻籼粳杂种多倍体的研究

以秋水仙碱诱导的水稻籼粳杂多倍体植株中，部分植株的体细胞呈现具有整倍体和非整倍体染色体数细胞嵌合现象，其中四倍体细胞最高可占35．3％，其余均为染色体数低于48条的各种细胞，与二倍体植株相比，这些植株的叶表皮气孔增大，气孔数减少，花粉粒直径增大都是十分显的，与多倍体植株无明显区别，并对这类植株根尖体细胞中存在具各种染色体数目细胞嵌合现象可能的原因进行了分析。     

钝齿棒杆菌AS1.542原生质体形成和再生条件的研究

本文研究了钝齿棒状杆菌(Corynebacterium crenatum AS1.542)原生质体形成和再生的条件。结果表明:菌体培养至对数生长期,用1.0 μg/ml青霉素G处理4小时,再以1000μg/ml蛋清溶菌酶在37℃下处理14小时原生质体形成率达99.0％,在含0.5M蔗糖的高渗完全基上,它的再生率为20.0～50.0％。高渗缓冲液DF比NSM和SMM更适合于原生质体的再生,高渗稳定剂0.5 mol/L蔗糖优于0.5 mol/L NaCl。用电镜观察了菌体和原生质体的形态结构。