利用离体诱变培育花生新品种宇花4号

为了丰富花生遗传资源,开拓新的育种方法,本研究对离体诱变创造花生新种质、培育花生新品种进行了研究。利用花生品种花育20号胚小叶作为外植体,平阳霉素(PYM)作为诱变剂进行离体诱变培养,然后在含有羟脯氨酸(HYP)的培养基上进行定向筛选,最终获得了15个再生小苗。再生小苗经嫁接移栽田间,从后代中获得了23个高油株系,3个产量显著提高的品系,其中一个高产高油品系2015年通过了安徽省新品种登记鉴定,定名为宇花4号,在参试的品种中名列第一,比对照白沙1016增产16.63%。宇花4号为早熟、小粒、高油花生品种,经农业部油料及制品质量监督检验测试中心(武汉)化验,籽仁含油率达56.10%,达到高油标准,比诱变亲本花育20号(含油率49.50%)高6.6个百分点,荚果产量比花育20号增产15%以上。本研究结果表明,离体诱变结合离体定向筛选是创造花生新种质、培育新品种的有效途径。     

快中子辐照结合组织培养培育花生新品种宇花7号

为开拓新的花生育种方法,对辐照诱变结合组织培养创造花生新种质、培育新品种进行了研究。以我国北方地区主栽花生品种鲁花11号成熟种子为试材,经快中子辐照处理后取种子胚小叶进行组织培养,通过胚胎发生途径获得再生苗。再生苗经嫁接驯化后移栽田间,83个单株获得种子。后代按系谱法进行选育,从83个再生植株后代中获得了107份突变体,分别在主茎高、分枝数、荚果形状和大小、种皮颜色、内种皮颜色、含油率、蛋白含量等性状上发生了明显变异。从突变体后代中选育出了低油早熟耐涝大花生新品种宇花7号,其产量比亲本鲁花11号增产14%以上;其含油率(47.0%)比鲁花11号低5.1个百分点。宇花7号2016年参加辽宁省新品种登记试验,比对照品种白沙1017平均增产13.8%。2018年通过了国家非主要农作物品种登记,登记号为"GPD花生(2018) 370105"。研究结果说明,辐照结合组织培养是创造花生新种质、培育新品种的有效方法。     

花生叶片离体培养花器官分化及其开花结果研究

以花生幼叶为外植体进行离体培养,研究BA浓度对花器官分化的影响并进一步观察试管内花器官的发育.结果表明:经MSB 1mg/LBA 0.5mg/LKIN 2mg/LIAA培养基诱导的愈伤组织,转接到附加1～3mg/LBA的MSB培养基上培养,均能直接诱导分化花器官,但2mg/LBA的诱导效率最高达21.13%;诱导分化的花器官转接到MSB培养基继续培养,部分花器官可以在试管内开花、受精、成针、结实.试验实现了以花生幼叶为外植体,在试管内完成诱导花芽、开花、受精、形成果针、子房膨大,直至形成荚果等过程,为离体条件下研究花生花器官分化、荚果及种子发育提供了技术体系和材料.     

高油酸花生新品种宇花91的选育

宇花91是青岛农业大学选育的高油酸花生新品种。以普通油酸含量品种鲁花11号为母本,F435型高油酸花生品种开农1715为父本配置杂交组合。利用PCR产物测序法筛选获得F_1代真杂种,对F_2代单株提取叶片基因组DNA,利用PCR产物测序法筛选基因型纯合的单株个体。对当代收获的单株籽粒利用近红外法多粒模型测定油酸、亚油酸含量,筛选油酸含量在80%以上且油酸亚油酸比值在10.0以上的单株种植成株行,随后利用系谱法进行选择育种。宇花91荚果为普通型小果,网纹较细、较明显,百果重148.06 g,百仁重63.31 g,果皮薄,出米率75.15%。籽仁长椭圆形,种皮粉红色、无裂纹,内种皮白色。籽仁蛋白质含量26.57%,脂肪含量52.72%,油酸含量80.40%,亚油酸含量2.50%,棕榈酸含量5.57%,油酸亚油酸比值32.16。苗期生长旺盛,封垄早,结果集中,中抗叶斑病和青枯病。2017年参加山东省夏播多点试验,平均荚果产量215.79 kg/667 m~2,比对照花育20号增产15.27%;平均籽仁产量157.33kg/667m~2,比对照花育20号增产21.64%。2018年通过国家花生品种登记,登记号:GPD花生(2018) 370210,适于在山东花生产区种植。     

花生胚离体发育及渗调物质的影响

在无外源激素培养基上花生胚能继续发育．渗调物质如甘露醇可抑制胚早萌,维持胚性发育,促进贮藏蛋白质合成和累积．渗调物质对胚离体发育的调控与其提高胚内源ＡＢＡ含量有关．     

平阳霉素对小菊品种离体培养的影响

利用0、1、2、4、8 mg/L平阳霉素（PYM）对小菊品种‘意大利红’、‘银星’离体培养的叶片和茎段进行诱变处理20d后,再转移到不添加PYM的培养基中进行愈伤组织的诱导和分化.结果表明：PYM对‘意大利红’、‘银星’叶片和茎段愈伤组织的诱导和分化具有明显的抑制作用;随着PYM浓度的增加,2个品种叶片与茎段愈伤组织的诱导和分化均呈明显的下降趋势,8 mg/L PYM处理后的愈伤组织诱导率和分化率均为最低;2种不同来源的外植体相比,茎段的愈伤组织诱导率和分化率均高于叶片;2个品种相比,‘银星’的愈伤组织诱导率和分化率高于‘意大利红’;品种与PYM浓度的互作、外植体类型与PYM浓度的互作以及品种、外植体、平阳霉素浓度三者的互作也对愈伤组织的诱导率和分化率产生不同的影响,但品种与外植体类型互作对愈伤组织的诱导率和分化率的F测验结果无显著性差异.     

同源四倍体灯盏花离体再生及其倍性鉴定

以四倍体灯盏花的无菌苗叶柄为外植体进行离体培养,研究其再生体系,并对再生植株进行染色体倍性鉴定。结果表明:叶柄外植体在MS+0.05 mg·L-1NAA+0.5 mg·L-16-BA+0.65%琼脂+3%蔗糖的培养基上不定芽的再生频率可达87.3%;继代培养(MS+0.1 mg·L-1NAA+0.3 mg·L-16-BA+0.65%琼脂+3%蔗糖)增殖系数为7.8,诱导(1/2MS+1.0 mg·L-1NAA+1.0 mg·L-1IBA+0.65%琼脂+3%蔗糖)生根率100%,移栽成活率为90%。细胞学鉴定结果表明,再生植株的染色体数为2n=4x=36,而原二倍体的染色体数目为2n=2x=18,基数x=9,因此,再生植株为四倍体。     

单花吊钟花的离体培养与植株再生

1植物名称单花吊钟花（Enkianthus pauciflorusWils.）,别名少花灯笼花。2材料类别枝段。     

观赏向日葵丛生芽增殖、生根及离体成花

本文以观赏向日葵品种‘富阳'为供试材料,通过MS基本培养基添加不同植物生长调节剂试验,初步建立了适宜观赏向日葵无菌苗增殖、生根和离体成花培养条件.结果表明,MS+低浓度BA对无菌苗增殖效果较佳,丛生芽增殖最适培养基为MS+0.05 mg/L BA;无菌苗生根率受营养水平影响,最适生根培养基为1/2MS;在离体条件下,低浓度BA可延缓植株衰老促进开花,最适开花培养基为MS+0.05 mg/L BA.花芽分化受GA3和PP333所抑制.本研究可为观赏向日葵遗传转化体系建立及转基因植株性状鉴定提供基础.     

离体植物花芽和花器官的发育研究进展

在近二十几年,尤其是近十年由于离体培养技术的完善和进一步向精确的层次的发展,已使得许多种植物花序、花芽和单独花器官以及部分的花器官在体外成功地进行了培养和尝试,并且对花芽及花器官的离体发育有了更深入的认识。不同植物的花发育需要不同的植物生长调节剂以及它们的不同配比,并且不同植物在其花发育所需要的营养因子也存在着相当大的差异性。这种差异性又随植物器官及花发育的不同阶段而受到加大或缩小。通过对正常花及突变体花进行的离体培养实验研究已经对一些花器官发生过程中的调节程序有了新的了解。利用离体培养技术,包括刚发展起来的薄层细胞培养技术在阐明花发育的机理、形态建成的分子机制以及成花梯度的物质基础等问题上具有广阔的潜力。     

Effects of soil moisture stress on the growth and yield of Spanish variety peanut

Summary Greenhouse grown Spanish variety peanut was subjected to soil moisture stress during the early flowering and pod formation growth stages. Vegetative growth was not significantly reduced by the moisture stress. Primary root length and the number of pods produced were significantly reduced. However, there was no difference between the two stress treatments.     

Application of in vitro mutagenesis to identify the gene responsible for cold agglutination phenotype of Streptococcus mutans

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold-agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold-agglutination-negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR-based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N-terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.     

Diversification and specialization of HIV protease function during in vitro evolution

Our goal is to understand how enzymes adapt to utilize novelsubstrates. We and others have shown that directed evolutiontends to generate enzyme variants with broadened substrate specificity.Broad-specificity enzymes are generally deleterious to livingcells, so this observed trend might be an artifact of the mostcommonly employed high throughput screens. Here, we demonstratea more natural and effective screening strategy for directedevolution. The gene encoding model enzyme HIV protease was randomlymutated, and the resulting library was expressed in Escherichiacoli cells to eliminate cytotoxic broad-specificity variants.The surviving variants were screened for clones with activityagainst a reporter enzyme. The wild-type human immunodeficiencyvirus type I protease (HIV PR) is cytotoxic and exhibits nodetectable activity in reactions with beta-galactosidase (BGAL).In contrast, the selected variants were nontoxic and exhibitedgreater activity and specificity against BGAL than did the wild-typeHIV PR in reactions with any substrate. A single round of wholegene random mutagenesis and conventional high-throughput screeningdoes not usually effect complete inversions of substrate specificity.This suggests that a combination of positive and purifying selectionengenders more rapid adaptation than positive selection alone.     

油田采出水石油烃降解菌分离、鉴定及高通量选育复合诱变菌株

【目的】为解决石油烃类物质(Total petroleum hydrocarbons,TPHs)引起的环境污染问题,提高野生型菌株对TPHs的降解率。【方法】基于微生物修复技术,通过富集培养、初筛和复筛从油田采出水中分离TPHs优势降解菌株,经形态学、生理生化以及16S rRNA基因序列分析确定其种属。采用紫外线与等离子体复合诱变技术,以96孔板发酵法替代摇瓶培养发酵,采用以多功能酶标仪双波长紫外光谱法测定为基础的高通量筛选方法选育TPHs高效降解菌株,并通过方差分析检验突变株的遗传稳定性。【结果】经鉴定,分离自采出水的野生型菌株PW04为多食鞘氨醇杆菌(Sphingobacterium multivorum)。通过复合诱变获得了3株能够高效降解TPHs的突变菌株：S. multivorum PW04-H10、S. multivorum PW04-G9、S. multivorum PW04-A6,降解率分别为85.1%、82.7%、82.9%。遗传稳定性分析结果表明这3株菌遗传性能均稳定。【结论】经分离、诱变选育出的TPHs高效降解菌株其降解率最高达85.1%,与野生型菌株相比提高了48%,可显著降低环境中TPHs含量,有效修复原油污染环境。     

用于蛋白质体外分子进化研究的DNA随机突变技术

蛋白质体外分子进化是模拟自然的进化过程,利用基因随机突变和定向筛选（选择）技术,以获得具有预期新功能的突变体分子。虽然体外进化近几年才产生,但已成为医药和工业领域中筛选具有特殊催化性质的酶的最重要的方法之一。ＤＮＡ随机突变技术是蛋白质体外分子进化研究的基础,本文将对几种最重要的突变方法：倾向错误的ＰＣＲ、ＤＮＡ重排、模板交错延伸反应和随机延伸突变的原理和应用等加以介绍。     

Development of NaCl-tolerant strain in Chrysanthemum morifolium Ramat. through in vitro mutagenesis

One NaCl-tolerant chrysanthemum (Chrysanthemum morifolium Ramat.) variant (E2) has been developed in a stable form through IN VITRO mutagenesis using ethylmethane sulfonate (EMS) as the chemical mutagen. Salt tolerance was evaluated by the capacity of the plant to maintain both flower quality and yield under stress conditions. Enhanced tolerance of the E2 variant has been attributed to the increased activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR), and, to a lesser extent of membrane damage than NaCl-treated control plants. Isoform analysis revealed that an increase in total SOD activity in the E2 variant was solely due to significant activation of the Cu/Zn isoform. Elevated levels of carotenoids and ascorbate in E2 leaves have been reflected in their higher free radical scavenging capacity (RSC) expressed in terms of DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging ability. Data reflect that a proper balance between enzymatic and non-enzymatic defence systems is required for combating salinity stress in chrysanthemum. Better performance of the E2 progeny under same salinity stress condition, even in the second year, confirms the genetic stability of the salt-tolerance character. On the whole, the E2 variant, developed through 0.025 % EMS treatment, might be considered as a NaCl-tolerant strain showing positive characters towards NaCl stress.     

Cloning,expression and directed mutagenesis of the genes for ribulose bisphosphate carboxylase/oxygenase

The dominant natural form of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of large (L) 55-kDa and small (S) 15-kDa subunits. This enzyme (as the L₈S₈ form) is widely distributed among oxygenic photosynthetic species and among chemosynthetic bacteria. Another form lacking small subunits is found as an L₂ dimer in Rhodospirillum rubrum or an L oligomer of uncertain aggregation state from Rhodopseudomonas spharoides. The present article reviews two basically different approaches in cloning the R. rubrum gene for RuBisCO. One results in high level expression of this gene product fused with a limited aminoterminal stretch of -galactosidase and the other results in expression of wild-type enzyme in Escherichia coli. Also reviewed are a number of reports of cloning and assembly of the L₈S₈ enzyme in using E. coli L and S subunit genes from Anacystis nidulans, Anabaena 7120, Chromatium vinosum and Rps. sphaeroides.In vitro oligonucleotide-directed mutagenesis has been applied to the gene for RuBisCO from R. rubrum. In terms of contributing new information to our understanding of the catalytic mechanism for RuBisCO, the most significant replacement has been of lys 166 by a number of neutral amino acids or by arg or his. Results establish that lys 166 is a catalytically essential residue and illustrate the power of directed mutagenesis in understanding structure-function correlates for RuBisCO.Oligonucleotide-directed mutagenesis has also been applied to the first and second conserved regions of the S subunit gene for RuBisCO from A. nidulans. In the latter region, corresponding amino acid changes of trp 55 and trp 58 to phe, singly or together, had little or no effect upon enzyme activity. In contrast, mutagenesis in the first conserved region leading to the following pairs of substitutions: arg10 arg 11 to gly 10 gly11; thr14 phe 15 ser 16 to ala 14 phe 15 ala 16; ser 16 tyr 17 to ala 16 asp 17; or pro 19 pro 20 to ala 19 ala 20, are all deleterious.Advances are anticpated in the introduction and expression of interesting modifications of S (and L) subunit genes in plants. A new method of introducing and expressing foreign genes in isolated etiochloroplasts is identified.Abbreviations RuBisCO ribulose bisphosphate carboxylase/oxygenase - 2-CABP 2-carboxyarabinitol-1,5-bisphosphate - 4-CABP 4-carboxyarabinitol-1,5-bisphosphate     

Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content

SUMMARY: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.