碳离子辐照对菘蓝药性品质和分子水平的诱变效应

以野生菘蓝种子为材料,以不同剂量的碳离子(12 C6+)进行辐照处理(辐照剂量分别为30Gy、60Gy、90Gy、120Gy),分析12 C6+辐照对菘蓝种子萌发、幼苗生长、主要药用成分含量及其基因和蛋白质多态性变化的影响,为菘蓝品质育种、分子生物学研究和重离子辐照诱变的应用提供依据。结果显示:(1)12 C6+辐照处理后菘蓝的成苗率和根鲜重均随辐照剂量增加而逐步显著降低,其中30Gy处理对菘蓝生长抑制程度最小,但处理后菘蓝根中的主要药效成分4(3H)喹唑酮和靛玉红的含量增加幅度最大且最高,分别为野生型的2.2倍和2.3倍。(2)SRAP分子标记分析表明,菘蓝基因组的变异度随着辐照强度的增强而增大,其中30Gy处理的突变体与野生型相比有33.59%的多态性变异。(3)SDS-PAGE考马斯亮蓝染色和磷酸化染色分析表明,菘蓝的总蛋白和磷酸化蛋白表达水平均随辐照剂量变化出现了不同程度的改变,但并不与辐照强度呈正相关,说明植物在防御重离子辐照伤害时存在补偿机制。研究发现,30Gy的12 C6+辐照是菘蓝诱变的最佳剂量,能够显著提高其有效成分的含量,为优质菘蓝诱变育种奠定了基础。     

碳离子辐照对四尾栅藻光合色素及抗氧化活性的影响

为了探讨重离子辐照对微藻的生物学效应,实验研究了不同剂量碳离子辐照（10~80 Gy）对四尾栅藻（Scenedesmus quadricauda）光合色素及抗氧化活性的影响,分别测定了辐照后短期内其叶绿素a（Chl a）、叶绿素b（Chl b）和类胡萝卜素含量、脂质过氧化物丙二醛(MDA)含量及超氧化物歧化酶（SOD）活性。结果显示：(1)较低剂量（10~20 Gy）辐照后,光合色素含量变化较小或无显著变化,中等剂量（40~60 Gy）辐照后,光合色素含量显著升高,之后又回落,恢复至正常水平,高剂量（80 Gy）辐照后,光合色素含量明显降低,不能恢复正常;(2)低剂量（10 Gy）辐照后,丙二醛（MDA）含量显著上升,8 h后出现回落,到24~48 h时,回升至正常水平,较低剂量（20 Gy）辐照后,MDA含量瞬时有所下降,到24~48 h时,回升至正常水平,中等至高剂量（40~80 Gy）辐照后,MDA含量降低,24~48 h时显著升高,不能恢复正常;(3)低剂量（10 Gy）辐照后,超氧化物歧化酶（SOD）活性显著上升,8 h后出现回落,恢复正常,中等剂量（20~60 Gy）辐照后,SOD活性显著上升,到48 h时回落至正常水平,高剂量（80 Gy）辐照后,SOD活性无明显上升,到48 h时,活性明显降低,不能恢复正常。     

60Coγ射线辐照花魔芋球茎的早期诱变效应研究

本试验开展了花魔芋早期诱变效应研究。通过^60Coγ射线辐照后的植株苗期发育观察和根尖细胞学检测分析,结果表明魔芋辐射诱变效应显著：辐照诱发核畸变和染色体畸变,且变异频率与剂量呈二次曲线关系;低剂量时对细胞分裂有刺激作用;辐照抑制芽体发育、苗期生长,抑制效应随剂量增加而加大,直至产生致死作用。依据花魔芋的辐射敏感性,建立了魔芋辐照诱变体系,以催芽球茎为诱变材料,诱变剂量范围为0Gy～50Gy,剂量率为1Gy／min,中等适宜剂量为7Gy～10Gy,致死剂量50Gy。     

12C6＋离子束辐照对大青叶生理生化特性的影响

利用辐射能量为100 MeV/u的12C6＋ 重离子束辐照大青叶种子,辐照剂量分别为20、40、50和80 Gy研究其对大青叶M1代的生物学效应.研究结果表明,随着辐照剂量的增大,大青叶的根系活力和叶绿素含量显著降低,发现辐照损伤主要抑制了根的生长及营养吸收;酚酸类化合物含量随着辐照剂量的增大而升高的趋势,表明辐照可提高大青叶的抗氧化和抗病虫害能力;可溶性糖、丙二醛、超氧化物歧化酶（SOD）和过氧化物酶（POD）含量变化的总体趋势为随着辐照剂量的增大先升后降.表明重离子辐照能改变大青叶的一些生理生化特征,其中40 Gy的12C6＋离子束辐照有利改善大青叶抗逆性和一些有效化学成分的积累.     

60Co-γ射线辐照孤挺花诱变效应研究

用不同剂量(5 Gy、10 Gy、15 Gy、20 Gy)60Co-γ射线辐照处理3年生孤挺花鳞茎,并设置对照组,对其成活率及幼苗性状进行田间观测。结果表明:成活率、叶长、叶宽随辐照剂量的增加呈递减趋势。运用聚丙烯酰胺凝胶电泳(PAGE)方法对不同辐照剂量下的植株叶片做同工酶分析,结果表明,在5 Gy剂量辐照下,过氧化物酶和酯酶的活性最强。     

重离子辐照诱变阿佛曼链霉菌的实验观察

目的：对阿佛曼链霉菌采用新的诱变手段,以获得稳定高产的优良菌株。方法：采用重离子束辐照阿佛曼链霉菌,研究了0.25Gy、0.5Gy、3Gy、5Gy、10Gy和15Gy剂量的12C＋粒子束辐照阿佛曼链霉菌菌株后,菌落特性的变化及对菌株产素能力的影响。结果：重离子辐照阿佛曼链霉菌后,在其各个辐照剂量区都存在变异菌株,诱变后阿佛曼链霉菌的菌落形态多样,小山状,火山口状、彗星尾状、车轮状、边缘放射状等;菌落大小不一,有的直径达4～5mm,有的小如针尖状。效价提高到7298μg/mL,获得了高产菌株。结论：重离子束辐照阿佛曼链霉菌菌株后,阿佛曼链霉菌的产素能力显著提高,可得到高产的菌株。     

80MeV/u20Ne10+离子对SMMC-7721细胞DNA损伤及细胞周期的影响

从辐照剂量和修复时间两个角度研究了重离子辐照对肿瘤细胞DNA损伤及细胞周期的影响,为重离子治癌的临床应用积累基础数据。不同剂量的80MeV／u^20Ne^10 辐照SMMC—7721细胞样品,利用单细胞凝胶电泳技术(Single Cell Gel Electrophoresis,SCGE)对细胞DNA损伤进行了检测,利用流式细胞技术(Flow Cytometry Methods,FCM)对细胞周期变化进行了分析。80MeV／u^20Ne^10 辐照后4小时内,SMMC—7721细胞的DNA损伤与辐照剂量呈线性关系,在0小时组其线性相关因子r为0．9621,4小时组为0．914;随着修复时间的增加,DNA损伤与辐照剂量不再线性相关,但0．5Gy,1Gy和2Gy三个剂量点的DNA损伤程度极为相近。另外,重离子辐照后SMMC—7721细胞发生S期和G2／M期阻滞现象,其随剂量变化及时间变化的规律不同于X、γ等低LET(Linear Energy Transfer)射线辐照。     

~(60)Co-γ辐照对中国仓鼠卵巢成纤维细胞DNA和组蛋白合成的影响

本文介绍了~(60)Co-γ辐照对同步的和非同步的CHO细胞的DNA合成和组蛋白合成关系的影响的研究,用~3H-胸腺嘧啶核苷和~(14)C-丙氨酸双标记,未经辐照的和经4Gy～60Gy ~(60)Co-γ辐照的CHO细胞,通过~3H和~(14)C的参入来估价DNA和组蛋白的合成,并用聚丙烯酰胺凝胶电泳鉴定辐照前后组蛋白各组分的变化情况,实验表明: 1)、在4～60Gy剂量范内,无论是同步的还是非同步的CHO细胞其DNA合成和组蛋白合成都受到不同程度的抑制。2)、在辐照后1—3小时,DNA合成和组蛋白合成都受到不同程度的抑制,但辐照后4小时,DNA合成被进一步抑制而组蛋白的合成却逐渐恢复正常,到辐照后48小时组蛋白的合成几乎接近对照水平。3)、16Gy ~(60)Co-γ辐照后8小时,非同步的CHO细胞的DNA合成被抑制的情况比G_1期CHO细胞更为严重。4)、16Gy ~(60)Co-γ辐照S期细胞,在辐照后1—24小时中DNA合成被明显抑制的同时,组蛋白的合成也受到相应的抑制。5)、从未经辐照的和经6、16和60Gy~(60)Co-γ辐照的CHO细胞分别提取全组蛋白,进行聚丙烯酰胺凝胶电泳,从电泳图谱的变化清楚地看到组蛋白H_1和H_3受辐照影响大于组蛋白H_4和H_(2B)+H_(2A),因此我们推测DNA合成和组蛋白H_1和H_3的关系较之组蛋白H_4和H_(2A)+H_(2B)更为密切。     

安化红茶辐照杀菌工艺研究

采用~(60)Co-γ射线辐照处理安化红茶,研究不同吸收剂量的杀菌效果及对其主要品质成分和感官品质的影响。结果表明,辐照处理杀菌效果显著,吸收剂量为5.93 k Gy的辐照处理可将其菌落总数和霉菌数都控制在100 CFU·g~(-1)以内;吸收剂量为10.13 k Gy以内辐照对其主要品质成分及感官品质无明显影响。综合试验结果,确定了安化红茶辐照杀菌的适宜工艺剂量范围为4.0~6.0 k Gy。     

~(60)Co-γ射线辐照对南瓜实蝇的不育效果及对南瓜品质的影响

为研究~(60)Co-γ辐照处理对南瓜实蝇的影响及其对南瓜品质的影响,分别用不同剂量的~(60)Co-γ射线(150、250、300、350、400 Gy)处理不同龄期的南瓜实蝇(包括卵、1龄、3龄幼虫和蛹),观察在不同辐照剂量处理下,南瓜实蝇F1代的致死作用和不育作用;并用最高剂量400 Gy处理南瓜果实,探究对南瓜品质和生理的影响。采用250-350 Gy的~(60)Co-γ处理处于卵、1龄和3龄幼虫和蛹期的南瓜实蝇后,均可实现F1完全不育;以400 Gy处理处于各龄期的南瓜实蝇,均可实现F1完全致死。以400 Gy剂量辐照南瓜,其蛋白质、总糖、还原糖、维生素、淀粉以及可溶性固形物等主要营养成分与对照无明显差异,南瓜主要保护酶SOD和POD处理10 d内有上升,但10 d后会恢复至与对照无明显差异。结果表明~(60)Co-γ射线可在不影响南瓜品质的基础上,有效控制南瓜实蝇的存活率,作为南瓜实蝇的检疫处理措施值得进一步深入研究。     

Detection of genomic instability in offspring of male mice chronically exposed to gamma-radiation by the "adaptive response" test

The genomic instability (GI) in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-dose gamma-radiation was studied by comparative analysis of chromosome damage. BALB/C male mice exposed to 0.1 Gy (0.01 Gy/day) and 0.5 Gy (0.01 and 0.05 Gy/day) were mated with unirradiated females 15 days after irradiation. For comparison of radiosensitivity, two-month-old males, the descendants of irradiated and unirradiated animals, were subjected to irradiation with a dose of 1.5 Gy (0.47 Gy/min) from a 60Co source. GI was revealed by the standard scheme of adaptive response. The experiments indicated that, by using the test "adaptive response", it is possible to detect the transition of gamma-radiation-induced genomic instability in sex cells of male parent into somatic cells of mice (F1 generation) either from changes in radiosensitivity or by the absence of the adaptive response induced by a standard scheme.     

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.     

Induction of cytogenetic adaptive response in spermatogonia and spermatocytes by pre-exposure of mouse testis to low-dose (12)C(6+) ions

To investigate the effects of pre-exposure of mouse testis to low-dose (12)C(6+) ions on cytogenetics of spermatogonia and spermatocytes induced by subsequent high-dose irradiation, the testes of outbred Kun-Ming strain mice were irradiated with 0.05Gy of (12)C(6+) ions as the pre-exposure dose, and then irradiated with 2Gy as challenging dose at 4h after per-exposure. Poly(ADP-ribose) polymerase (PARPs) activity and PARP-1 protein expression were respectively measured by using the enzymatic and Western blot assays at 4h after irradiation; chromosomal aberrations in spermatogonia and spermatocytes were analyzed by the air-drying method at 8h after irradiation. The results showed that there was a significant increase in the frequency of chromosomal aberrations and significant reductions of PARP activity and PARP-1 expression level in the mouse testes irradiated with 2Gy of (12)C(6+) ions. However, pre-exposure of mouse testes to a low dose of (12)C(6+) ions significantly increased PARPs activity and PARP-1 expression and alleviated the harmful effects induced by a subsequent high-dose irradiation. PARP activity inhibitor 3-aminobenzamide (3-AB) treatment blocked the effects of PARP-1 on cytogenetic adaptive response induced by low-dose (12)C(6+) ion irradiation. The data suggest that pre-exposure of testes to a low dose of heavy ions can induce cytogenetic adaptive response to subsequent high-dose irradiation. The increase of PARP-1 protein induced by the low-dose ionizing irradiation may be involved in the mechanism of these observations.     

Alterations of DNA copy number and expression in genes involved in cell cycle regulation and apoptosis signal pathways in gamma-radiation-sensitive SX9 cells and -resistant SR-1 cells

In the present study, genomic differences related to sensitivity to radiation were examined by comparative genomic hybridization and GeneChip 45K microarray in SX9 cells (radiation-sensitive) and their parental line, SR-1 (radiation-resistant). SX9 cells have defective DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity. DNA-PKcs is a DNA double-strand break repair protein that maintains chromosomal stability through nonhomologous end joining. However, the molecular basis of the radiation sensitivity of SX9 cells is unclear. Flow cytometry analysis showed that SR-1 and SX9 cells had a larger G2/M-phase population at 12 h after 4 Gy gamma irradiation, while only SR-1 cells progressed to G1/S at 24-36 h. SX9 and SR-1 cells had similar patterns of DNA copy number alteration, but the gains were observed on chromosome 9 (cent-E2), 11 (cent-A3), and 12 (C1-E) only in SX9 cells. Expression of genes located on those regions is higher in SX-9 cells than in SR1 cells, and the regions include genes associated with apoptosis and cell cycle regulation. Time-course data for gene expression at 0, 1, 3, 6 and 12 h after 4 Gy gamma irradiation revealed that the genes whose expression was altered in SX9 cells but not in SR-1 cells are in 16 clusters. Three of these clusters included genes for cell cycle regulation: JNK, PKC (PRKC) and ceramide cascade protein. These results suggest that amplification and altered expression of genes associated with cell cycle and apoptosis regulators in DNA-PK-deficient SX9 cells affect the differences in response to gamma radiation between SX9 and SR-1 cells.     

SNP discovery using Paired‐End RAD‐tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae)

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost‐effective approaches to uncover genome‐wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD‐PE (Restriction site Associated DNA Paired‐End sequencing) approach. RAD tags were generated from the PstI‐digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired‐end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N₅₀ = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD‐PE as an inexpensive genome‐wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.     

60Co-γ射线辐照毛竹种子的过氧化物同工酶和RAPD分子检测的研究

利用6种不同剂量的60Co-γ射线辐照毛竹种子,对辐照后的毛竹种子进行了过氧化物酶（POD）同工酶检测和RAPD分子检测.结果表明,过氧化物酶的活性在30 Gy和120 Gy时明显增强,其原因是毛竹在60Co-γ射线辐照胁迫下的保护效应和伤害效应.RAPD分析表明毛竹不同辐照剂量之间的DNA存在明显差异,高剂量γ 射线辐照对DNA分子的影响较大,能引起碱基序列的改变;对碱基序列差异片段测序并比对,发现部分片段与已知序列有高达83 %的同源性,部分差异片段与GeneBank中的已知序列没有较高的同源性,应该属辐照引起的DNA序列变异.同工酶和RAPD分子检测为毛竹的诱变育种参数的制定提供了理论依据.     

用RAPD技术研究我国苹果果实轮纹病菌的遗传变异(英文)

为了从分子水平上明确近年来我国苹果生产上发生的A、B、D和F 4种不同症状型苹果果实轮纹病的遗传分化现象及其不同致病菌的遗传变异程度。本研究对来自我国三大苹果产区的26个相关病原菌株的DNA指纹图谱进行了分析(Table 1)。结果表明 (1) 上述4种不同症状型轮纹病原菌株在基因组DNA水平上具有明显的遗传差异(Fig.1&Table 2),并在所分析的11个多态性引物中有4个引物的扩增图谱(Table 4)均可使上述4种不同致病型菌株相互区别(Table 5)。统计分析结果显示,这4种不同致病菌相互之间的遗传距离为0.1314~0.2541(Table 6);(2) 相同症状型的不同来源致病菌之间具有较高的遗传相似性 (88.4~100%)(Table 3),由此说明苹果果实轮纹病菌的遗传分化与地理环境条件差异无明显的相关性。 以上研究结果对于进一步阐明苹果果实轮纹病害的遗传演化规律及该病害监测与防治技术的改进具有重要的理论与实际意义。     

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增,共获扩增片段１０３ 条,分子量在０．３ － ３ｋｂ 之间,其中５ 种引物ＯＰＪ－ １ ,７ ,９,１１ ,１２ 扩增出差异片段１２ 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变;ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响,以及氮离子注入诱变效应的可能机制。