高含固率木质纤维素厌氧发酵物总DNA的4种提取方法比较

不同的样本特性和提取方法对获得微生物总DNA的质量有重要影响。文章基于高含固率木质纤维素厌氧发酵物腐殖酸、酚类物质含量高、质地均一性差、微生物浓度低的特点,研究了4种方法提取不同高含固率粪秸厌氧发酵物中微生物总DNA的效果。结果表明,常规的十二烷基磺酸钠法(sodium dodecyl sulfate,SDS)、十二烷基磺酸钠和溴化十六烷基三甲铵结合法(sodium dodecyl sulfate and cetyltrimethyl ammonium bromide,SDS-CTAB)和商业的粪便试剂盒法提取的DNA质量均较差,SDS法和试剂盒法未能获得聚合酶链式反应(polymerase chain reaction,PCR)扩增目的条带,SDS-CTAB法得到的条带较模糊;改进SDS-CTAB法获得的DNA杂质少、纯度高,具有较好的稳定性,A260/A280和A260/A230值分别为1.74~1.86和1.65~1.86,每克样品的DNA浓度在50 ng·μL^-1以上,电泳条带单一齐整、清晰明亮,PCR扩增的目的条带清晰度高,适宜后续分子生物学技术的分析。林格氏液洗脱、聚乙烯吡咯烷酮-40(Polyvinyl Pyrrolidone-40,PVP-40)洗涤液除杂以及裂解液和多种酶联合破壁是改进SDS-CTAB法获得该类专一性样本高质量微生物总DNA的关键步骤。     

利用广亲和基因S5-n的功能标记鉴定特殊配组类型杂交种纯度研究

“粳不育系/广亲和恢复系”配组方式在浙江省得到了越来越多的应用。广亲和基因S5-n的功能标记可快速检测种子纯度。为验证水稻广亲和基因S5-n功能标记鉴定粳不育系/广亲和恢复系配组方式杂种一代的效果,我们分别将长粳1A和6个恢复系获得的F₁代进行大田统计和分子标记鉴定,结果显示,由于广亲和基因S5-n分子标记不能特异性检测出串粉形成的异形株,导致其鉴定结果较大田统计鉴定结果高1.0~2.0百分点。未来还需要进一步优化广亲和基因分子标记技术的特异性,进而提高分子检测的准确性,这将有助于杂交稻的安全生产。     

利用简化的SDS法提取杨树基因组DNA

以杨树组培苗幼嫩叶片为材料,在室温下采用简化的SDS法快速制备其基因组DNA,并对制备DNA过程中的影响因子进行了初步研究。结果表明,采用此方法制备的基因组DNA在数量和纯度上可以满足聚合酶链式反应(PCR)的要求。另外,DNA提取缓冲液中添加一定浓度的PVP、β-巯基乙醇,有利于DNA提取。     

Germination of <Emphasis Type="Italic">Juniperus procera</Emphasis> seeds in response to stratification and smoke treatments,and detection of insect-damaged seeds with VIS + NIR spectroscopy

Seeds of Juniperus procera collected from five provenances across its geographic range in Ethiopia were subjected to cold-moist stratification at 5°C or 10°C for 6–12 weeks. The effect of aqueous smoke solution in overcoming the light requirement for germination, and the potential of visible (VIS) and near infrared reflectance (NIR) spectroscopy for sorting sound and insect-damaged seeds were also investigated. Highly significant differences in germination were detected among provenances (P < 0.0001) and stratification periods (P < 0.0001), but not between temperature regimes (P=0.111). Seeds from the south and southeast distribution ranges had higher percentage germination after 6 weeks of stratification than seeds collected from north, northwest and central ranges of distribution. The smoke treatment did not affect germination regardless of whether the seeds were exposed to light. Exposure to light increased germination capacity three fold. Sound and damaged seeds were distinguished with 90% accuracy using VIS + NIR spectroscopy. It can be concluded that dormancy in juniper seeds varies with provenances, and cold stratification for 6 weeks alleviates dormancy in some seed lots. Tentatively, smoke treatment seems ineffective in overcoming photo-dormancy in juniper seeds. VIS + NIR spectroscopy has demonstrated a great potential for sorting damaged seeds, thereby upgrading seed lot purity.     

SSR标记对甜瓜杂交种‘众天翠雪’纯度的鉴定

旨在筛选能够用于甜瓜杂交种纯度和真实性鉴定的SSR引物。利用公认的18对多态性较好的甜瓜SSR(Simple Sequence Repeats)引物对甜瓜杂交种"众天翠雪"及其亲本进行多态性筛选,并在杂交群体中进行验证。13对引物在亲本间具有多态性条带,经过多次重复,筛选到2对引物在亲本间具有多态性、子代中兼具双亲条带,并且在子代群体中检测结果与田间形态鉴定结果一致。引物RF151和RF144能够作为‘众天翠雪’杂交种纯度鉴定的特异引物进行纯度检测。作为共显性标记特性的SSR分子标记可以作为杂交种纯度鉴定的重要工具。     

The use of seed acid phosphatases in the determination of the purity of F1 hybrid brussels sprout seed

Summary Starch gel electrophoresis of single seeds of five different F₁ hybrids of Brussels sprouts showed that they possessed three cathodal acid phosphatases. By comparison with cathodal acid phosphatases present in their inbred parents these have been interpreted as the two parental types plus a hybrid enzyme. All of the parental material could be classified into two groups depending upon whether or not their cathodal acid phosphatase was fast or slow moving. It was shown that these acid phosphatases are suitable for the determination of sibs in F₁ hybrid sprout seed provided that one of the parents possesses the slow moving cathodal acid phosphatase and the other the fast moving one. A survey of 35 different F₁ hybrids showed that 18 could be analysed for sibs using this method, those which could not were assumed to have had parents who possessed cathodal acid phosphatases of the same mobilities.     

利用SSR标记区别小麦品种种子混杂和SSR位点不纯的研究

在用SSR标记检测小麦种子纯度时,种子混杂和SSR位点不纯都会造成株间SSR带型的差异,本文提出了SSR位点不纯的概念,并分析了存在这一现象的原因,指出这一现象普遍存在于小麦品种中。为此在检测小麦种子纯度时,需要注意区别种子混杂和SSR位点不纯,以免将SSR位点不纯造成的株间带型差异误认为是种子混杂,其原则是:(1)必须进行多个SSR位点的检测;(2)某单株的多个SSR位点的带型与被检测品种不同,可确认为混杂植株;(3)剔除混杂植株后,某单株的个别SSR位点的带型与被检测品种不同,将被认为是SSR位点不纯所致。     

A simple method to control the seed purity of japonica hybrid rice varieties using PCR-based markers

Cytoplasmic male sterility (CMS) by the cms‐bo cytoplasm and its restoration by the nuclear restorer gene, Rf‐1, are used for seed production of japonica hybrid rice varieties. To produce pure hybrid seeds, a prerequisite is to properly manage the seed purity of parental lines, especially CMS lines. In this study, three dominant polymerase chain reaction (PCR)‐based markers (M1, M2 and M3) were developed to detect mutual contamination in seed batches of CMS lines, maintainer lines, restorer lines and hybrids. M1 detected the mitochondrial sequence that was present in the cytoplasm of common japonica varieties and absent in the cms‐bo cytoplasm. M2 and M3 detected the chromosomal sequence related to the Rf‐1 allele in restorer lines and the rf‐1 allele in common japonica varieties, respectively. By the strategic use of these markers, japonica hybrids and their parental lines could be efficiently distinguished from each other. Furthermore, sensitivity tests for the three markers with a series of crude DNA samples prepared from polished grains demonstrated that these markers could detect one contaminating grain among 500 or 1000 grains. Therefore, the bulk PCR analyses with the markers developed here probably make it possible to control the seed purity of japonica hybrids properly by selecting appropriate seed batches of their parental lines quickly and efficiently.     

过氧化物酶及种子蛋白分析鉴定甜椒杂交种纯度

用聚丙烯酰胺均匀胶和等电聚焦电泳技术，分析了甜椒种子及种苗的过氧化物同功酶谱和种子水溶蛋白图谱。在过氧化物酶谱带的两个位点ＰＲＸ－１和ＰＲＸ－２上，自交系亲本与Ｆ１杂交种有明显的差异。水溶蛋白的一个位点ＰＲＯＴ亲本与Ｆ１有明显差异。供试甜椒组合的过氧化物酶和蛋白的图谱，分别具有这些位点。     

蜂蜜流变特性及其质量鉴定

对紫云英蜂蜜和白砂糖溶液以及二者的混和液进行流变试验,建立了流变体模型(New-ton)。并就温度和浓度对粘度的影响进行分析,提出采用测粘度的方法鉴别紫云蜜是否掺水和掺糖。