3个玉米黄叶突变体的光合特性研究

在农业生产中光合作用是作物积累生物量的主要方式,其主要依赖于多种光合色素和完整的叶绿体结构与功能。而玉米叶色突变体对于研究叶绿体发育、提高玉米光合作用能力和产量具有重要意义。以两个玉米自交系郑58（Z58）和B73为对照,对从甲基磺酸乙酯（ethyl methanesulphonate,EMS）处理后的不同玉米诱变群体中筛选到的2株黄叶突变体yl?1（yellow leaf?1,Z58背景）、yl?2（yellow leaf?2,B73背景）以及从玉米自交系Z58中发现的1株自然黄叶突变体yl?3（yellow leaf?3）等3个表型相似的玉米黄叶突变体的形态特征、光合色素含量、叶绿素合成前体物质含量进行了比较研究。结果表明,与对照相比,3个突变体在整个生长周期内均呈现不同程度的黄叶表型、不复绿、植株矮小、发育迟缓;叶片总叶绿素、叶绿素a和叶绿素b含量均显著降低（P<0.05）,叶绿素a/叶绿素b比值显著升高（P<0.05）;不同突变体的各类叶绿素合成前体物质含量有不同程度的降低。3个突变体的黄叶表型可能是由不同基因的突变导致相关四吡咯化合物合成异常引起的。研究结果为定位引起3个突变体黄叶表型的突变基因和进一步探讨其利用潜力奠定了理论基础。     

矮化早熟高粱突变体的RAPD分析

以随机扩增多态DNA(RandomAmplifiedPolymorphicDNA,RAPD)标记方法对航天搭载得到的矮化早熟高粱突变体进行遗传特性分析,利用适合玉米的引物对高粱进行引物筛选,从31种随机引物中共扩增出4条多态性片段,反映出突变体的遗传差异性。     

诱发玉米抗小斑病突变体的研究——Ⅳ.从玉米单倍体胚性细胞无性系筛选抗玉米小斑病突变体

将经γ射线和EMS诱变处理的玉米八趟白单倍体胚性细胞无性系为试验材料,以玉米小斑病菌Helminthosporium maydis单胞系342号菌株产生的致病毒素为选择剂,采用正选择法进行筛选,已获得抗玉米小斑病的突梦体。这个突变体脱离选择剂5代(5个月)后性状稳定,用小斑病菌的分生孢于直接接种鉴定仍具有很强的抗病力。过氧化物酶同工酶的分析,抗病突变体与不抗病对照间出现明显差异。     

利用氧化剂甲基紫精筛选拟南芥寿限延长突变体

在模式动物中的研究表明,寿限延长与氧化胁迫耐受能力密切有关;在模式植物拟南芥中也发现晚花突变体具有更强的抗氧化胁迫能力,但迄今为止未见有关拟南芥寿限延长突变体的研究报道。本文建立了用氧化剂甲基紫精筛选寿限延长突变体的方法,并对用该方法从经快中子诱变的拟南芥Columbia生态型M_2代群体中筛选获得的突变体SFNA-9-4进行分析,发现该突变体的抗氧化胁迫能力和寿限均显著增加。由此说明,用该方法筛选拟南芥寿限延长突变体是可行的。     

高通量筛选工具的设计与应用

定向进化方法作为新兴的高效蛋白质工程手段,其内容包括蛋白质突变体文库的构建和有效突变体的快速筛选。高通量筛选方法是定向进化方法的重要组成部分,是成功获得有效突变体的关键。筛选的突变体数量越多,获得有效突变体的几率越大。以下介绍了目前已经成功应用于或有潜力应用于定向进化改造蛋白质的几种高通量筛选工具。高通量筛选工具的不断设计与开发将推动蛋白质工程领域的技术革新。     

利用氧化剂甲基紫精筛选拟南芥寿限延长突变体

在模式动物中的研究表明,寿限延长与氧化胁迫耐受能力密切有关;在模式植物拟南芥中也发现晚花突变体具有更强的抗氧化胁迫能力,但迄今为止未见有关拟南芥寿限延长突变体的研究报道。本文建立了用氧化剂甲基紫精筛选寿限延长突变体的方法,并对用该方法从经快中子诱变的拟南芥Columbia生态型M2代群体中筛选获得的突变体SFNA-9—4进行分析,发现该突变体的抗氧化胁迫能力和寿限均显著增加。由此说明,用该方法筛选拟南芥寿限延长突变体是可行的。     

基于柔性区域的计算设计改造玉米赤霉烯酮水解酶热稳定性

来源于粉红螺旋聚孢霉Clonostachys rosea的玉米赤霉烯酮水解酶 (ZHD101) 可以有效降解谷物农副产品和饲料中的霉菌毒素玉米赤霉烯酮 (Zearalenone,ZEN),但是,该酶的热稳定性较低,限制了其在工业中的应用。由于水解ZEN的反应没有吸光值的变化,不适合高通量筛选。本文以ZHD101为模式酶,进行计算虚拟突变并结合实验验证。通过比对不同温度下的分子动力学模拟轨迹,选取32个柔性位点;再通过位置特异性评分和酶构象自由能计算,从32个柔性位点上的608个虚拟饱和突变体中筛选出12个突变体。经实验验证,其中3个突变体N156F、S194T和T259F的热熔融温度有一定程度的提升 (ΔTm>4 ℃),且酶活性与野生型类似甚至更高 (相对酶活性为95.8%、131.6%和169.0%)。分子动力学模拟分析显示,导致3个突变体热稳定性提高的可能作用机理分别为NH-π作用力、盐桥重排和分子表面空穴填充。将3个突变体进行迭代组合突变,N156F/S194T表现出最高的热稳定性 (ΔTm=6.7 ℃)。这项工作表明基于柔性区域的虚拟饱和突变在酶稳定性改造上的可行性,探索计算虚拟改造结合实验验证的酶改造策略。     

理性设计盐桥构建伯克霍尔德菌脂肪酶热稳定突变体

【目的】借助蛋白质工程技术提高伯克霍尔德菌ZYB002脂肪酶LipA的热稳定性,以期更好地将其应用于工业生产中。【方法】利用YASARA、Fold X、Rosetta、Gromacs等生物信息学软件,构建1个脂肪酶Lip A的热稳定性提高的微型突变体电子文库;通过对突变体的结构信息和自由能变化进行评估,筛选出潜在的有价值的突变体。继而利用基因定点突变技术,构建上述候选突变体的突变基因文库,通过实验筛选出热稳定性提高的脂肪酶LipA突变体。【结果】利用上述方法,从构建的20个脂肪酶LipA突变体中,筛选到热稳定性有显著提高的3个突变体LipA-Asn~(125)Asp、LipA-Asn~(125)Glu和LipA-Gln~(262)Glu。经55°C处理12 min后,上述3个突变体的T1250值较野生型分别提高4.0°C、5.5°C和4.4°C;在55°C下的半衰期较野生型分别提高了2.2倍、3.8倍和2.6倍。【结论】利用生物信息学软件,构建脂肪酶LipA突变体电子文库,结合蛋白质的结构信息,可以快速筛选到热稳定性提高的脂肪酶LipA突变体。     

球孢白僵菌T-DNA突变体库的构建及高温和高渗敏感型突变体的筛选

摘要：【目的】筛选对高温和高渗等逆境胁迫敏感的球孢白僵菌T-DNA随机插入突变体,并克隆相关基因,为研究杀虫真菌适应逆境的分子机理奠定基础。【方法】利用逆境胁迫的方法从球孢白僵菌的T-DNA随机插入突变库中筛选对高温和高渗敏感的突变体,进而利用YADE (Y-shaped adaptor dependent extension)技术克隆相关基因。【结果】筛选得到5个对高温和高渗敏感的突变体。其中2个（212和2550）对高温敏感,在32oC下生长完全受抑;其它3个突变体对高渗胁迫敏感。突变体212的分生孢     

水稻雄性不育系及雄性不育突变体筛选研究进展

 为了获得新细胞质源的水稻雄性不育系及雄性不育突变体,以解决目前杂交水稻生产上所面临的不育系来源单一的问题,人们采用了常规育种、诱变育种、组织培养和离体诱变等方法,其中离体筛选尤显其优势性,但也有不少问题有待解决,如能通过离体培养定向地筛选出雄性不育系及雄性不育突变体,无疑将大大提高工作效率。此外,对影响离体筛选雄性不育突变体的因素进行了探讨。     

Biochemical and Cytological Studies During Seed Development in Somaclones of Lathyrus sativus

Soluble proteins in six somaclones and the parent at three different stages of development of Lathyrus sativus seeds showed polypeptides of mol wts ranging from 20–165 kD on SDS-PAGE. Immature seeds showed eight distinct esterase bands and two peroxidase bands. Pattern for both the enzymes among somaclones was almost similar. During maturation a new isoform of peroxidase with high mobility appeared in all the somaclones, while one new isoform of intermediate mobility was visible in somaclones Bio L-08 and Bio I-08. Cytological analysis of all the somaclones confirmed the stability of the chromosome count of 2n=14. At metaphase I seven distinct bivalents and at anaphase I and II univalents showing complements of the seven chromosomes were visible. No structural or chromosomal aberration were observed among somaclones.     

Gliadin and Glutenin Variation in Seeds of Somaclones of Triticum aestivum

Gliadin and glutenin of seeds of a pollen-derived pure line “H-33” and its 9 stable somaclones of wheat (Triticum aestivurn L.) from inflorescence callus were investigated using SDSPAGE. Among the 9 somaclones 6 were identical to the “H-33” in SDS-PAGE patterns of of gliadin and glutenin. The other 3 somaclones were identical with each other, but different from “H-33”. The varied electrophoretic bands of seed protein showed that somaclonal variation had occured at gene level during tissue culture in which the somaclones were produced.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

RAPD Analysis of Lathyrus sativus Somaclones

RAPD patterns were studied in seven somaclones of Lathyrus sativus having contrasting characteristics alongwith the parent cultivar P-24. Out of 81 decamer random primers used, 5 did not amplify and 24 revealed DNA polymorphism while the rest generated monomorphic banding patterns. Eight unique bands were amplified with different primers in four different somaclones. With most of the informative primers differences were observed between somaclones and also between some of the somaclones and parent cultivar P-24. More than 90% similarity in the RAPD patterns was evident among the somaclones and the parent cultivar P-24. Though it was not possible to identify a particular somaclone with a single primer, a combination of two or more primers could be employed to identify a somaclone.     

RAPD Analysis of Maize Somaclones

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 17 decanucleotide primers used each allowed amplification of 2–17 fragments ranging 200–2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64–74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.     

RAPD-analysis of corn somaclones

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 15 out of 17 decanucleotide primers used each allowed amplification of 2-17 fragments ranging 200-2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64-74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.     

A comparative analysis of the development and quality of nursery plants derived from somatic embryogenesis and from seedlings for large-scale propagation of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Plants of Coffea arabica L. derived via somatic embryogenesis, namely, somaclones, were evaluated with C. arabica seedlings grown in the nursery. At the time of their transfer to the nursery, somaclones of C. arabica cvs. Caturra and Costa Rica 95 (Catimor) were smaller and less vigorous than seedlings of the same cultivars. Following an initial slow growth for a period of 10 weeks, somaclones began to grow faster than seedlings until both groups of plants were equal in size at 21 weeks (entire duration of growth in the nursery). Comparisons of aerial and root systems of 30-cm long somaclones and seedlings of two cultivars revealed that plants of somaclones were more vigorous than seedlings, based on the higher number of leaves (13–16 vs. 9), larger leaf area (1060–1280 vs. 730–890 cm²), and greater dry weight of aerial organs (8.5–12 vs. 7.0–7.5 g). For cv. Caturra, the root dry weight of somaclones was significantly greater than that of seedlings (2.7 vs. 1.9 g) and was attributable to the large diameter roots (>0.5 mm). Analysis of 176,000 F₁ hybrid somaclones revealed that these exhibited more heterogeneous growth than did the seedlings derived from zygotic embryos; moreover, there was a genotype effect. Almost 9–20% of somaclones required an additional 3–4 months of growth in the nursery, and 8–12% were culled for other undesirable horticultural attributes. Only 0.10–0.23% of somaclones displayed variant phenotypes. The observed somaclone vigor in the nursery was carried over to field performance as these plants were more precocious than seedlings and yielded coffee beans 1 year earlier than seedlings.     

In vitro induction, screening and detection of high essential oil yielding somaclones in turmeric (Curcuma longa L.)

Leaf of turmeric contains an essential oil used extensively in perfumery, pharmaceuticals and aromatherapy. Five somaclones were induced in turmeric on MS media with varying amounts of plant growth regulators. All somaclones were subsequently transferred to the field. Essential oil was extracted from leaves of in vitro and ex vitro grown plants and subjected to quantitative and qualitative evaluation. A positive correlation was established between the leaf oil content and oil constituent of in vitro grown and field transferred somaclones. Somaclones (C2, C4, C5) containing 0.16–0.18 % oil in vitro retained normal oil content (0.48–0.5 %) in the field. Similarly in vitro grown somaclones C3 and C7 with 0.36 and 0.25 % oil content retained proportionately increased oil yields of 1 % and 0.76 under ex vitro condition. GC–MS analysis of the oil revealed similar spectrum of constituents both among in vitro and ex vitro grown plants with alpha-phellandrene as major one. Thus the novel method of in vitro screening could be applied for rapid identification of high essential oil yielding turmeric genotypes thereby reducing labour, cost and time required in conventional ex vitro screening of somaclones.     

运用致病毒素筛选抗稻瘟病细胞突变体

本文研究报道了抗稻瘟病细胞突变体筛选方法、程序与结果,并对有关问题进行了讨论。毒素对水稻成熟胚愈伤组织的诱导与分化有明显的抑制作用,抑制程度随毒素浓度提高和处理时间延长而加强,但抗感品种之间差异显著。以含毒素的诱导培养基或诱导与分化培养基均含毒素的筛选处理效果较好,而且简便易行。5年来接种筛选了29个品种25000多个外植体,在已鉴定的349个R_1个体中,有183株表现抗病。经多次接种鉴定与选择,已获得12个高抗稻瘟病的株系和1个抗病丰产品系,并对部分抗病突变体的抗性基因进行了遗传分析。     

The regeneration and screening of watercress somaclones for resistance to Spongospora subterranea f. sp. nasturtii and measurement of somaclonal variation

A range of watercress (Rorippa nasturtium-aquaticum) explants (stems, hypocotyls, true-leaves, cotyledons and petioles) were tested for their capacity to regenerate adventitious shoots from callus formed using Murashige and Skoog medium containing different concentrations of thidiazuron and 2,4-dichlorophenoxyacetic acid. The highest shoot regeneration rate was a mean of 18 shoots per responding explant from stem callus formed on medium containing 5 μM thidiazuron and 0.05 μM 2,4-dichlorophenoxyacetic acid. A histological study confirmed that shoots originated directly from callus tissue. Twenty five percent of somaclones exhibited somaclonal variation in leaf shape, plant height, axillary branching or ploidy. The variation in 6% of somaclones was heritable to the first selfed generation. A screening protocol was developed to permit the identification of somaclones with increased resistance to the economically damaging watercress root pathogen, Spongospora subterranea f. sp. nasturtii. Although 883 somaclones were screened using this protocol, no significant increase in disease resistance was detected. This revised version was published online in June 2006 with corrections to the Cover Date.