浅析福建省水稻种子纯度鉴定技术现状

本文通过分析杂交水稻种子纯度鉴定在种子工作中的重要性和存在的各种问题,以及为解决这些问题 所采取的各种传统鉴定方法与日益发展变化的生化鉴定方法和分子标记技术,根据实际工作需要对各种鉴定方 法的可行性和局限性进行评价,为福建省水稻种子纯度鉴定技术提出新的发展思路。

Screening of SSR Core Primers for Purity Identification of Pepper(Capsicum) Hybrids

[Objective] This study aimed to screen a set of SSR core primers suitable for purity identification of pepper （Capsicum） hybrids. [Method] DNA fingerprint of 100 pepper hybrids was analyzed using 17 SSR primers. [Result] According to the polymorphism and heterozygosity, Hpms1-214, Es395 and Hpmsl-5 were determined as three preferred core primers for purity identification of pepper hybrids. By using these three preferred core primers, 97 pepper hybrids （accounting for 97%） had heterozygous band pattern with at least one primer. Es330, Es363, Epms923, Es120 and Es64 were determined as candidate core primers for purity identification of pepper hybrids. Specific primers of 14 varieties were obtained, which could be used to further screen parent-complementary primers of each pepper hybrid. [Con- clusion] This study laid the foundation for constructing standard DNA fingerprints for purity identification of pepper hybrids.

Purity Identification of Xinshikui 6 Using SSR Marker Technique

[Objective] SSR molecular marker technique was used to determine the purity of sunflower seed with the aim to provide accurate, convenient method for the identification of the purity of hybrid seeds in production and processing. [Method] With the DNA of Xinshikui 6 and its parents as template, about 100 pairs of SSR molecular markers were screened after DNA extraction, PCR amplification and electrophoresis production. [Results] SSR polymorphic primer marker 532 produced a specific band of 469 bp in the female parent, and a specific band of 451 bp in the male parent; primer marker 574 produced a specific band of 364 bp in the female parent, and a specific band of 384 bp in the male parent. The indoor molecular purity identification and field purity identification were consistent with each other. The primer marker 532 and 574 could be obtained from the SSR molecular marker method to distinguish the male parent, female parent and hybrid of Xinshikui 6, and both of the 2 primer markers can effectively identify the purities of the hybrid seeds of Xinshikui 6, as well as the authenticity of the seeds. [Conclusion] The proposed method was simple, fast, accurate to operate with the advantages of high reproducibility, and it had become the major method in the identification of sunflower varieties.

A Method for Rapid Identification of Ningza 11 Seeds Purity with SSR Markers

[Objective] This study aimed to establish a method for rapid identification of Ningza 11 seeds purity with SSR markers. [Method] Taking Ningza 11 hybrid seeds as experimental materials, a method for rapid identification of hybrid rape-seeds was established with SSR molecular markers; meanwhile, the test seeds were planted in the field for comparison and verification. [Result] A method for rapid identification of Ningza 11 seeds purity with SSR molecular markers was estab-lished： DNA from seeds germinated in the night was extracted by alkaline lysis method; the PCR amplification was performed for 2 h, and electrophoresis for 1.5 h, and a silver staining for 10 minutes. It took less than one day to from obtaining sampling seeds to obtaining the purity identification result, so a skil ed professional can complete the detection of at least 6 ×96 = 576 seeds per weekday. By using this set of detection system, the measured purity of seeds from nine samples was extremely significantly positively correlated to the actual purity identified in the field test, with a correlation coefficient of up to 0.984 （P〈0.01）. [Conclusion] This SSR-PCR molecular identification system can be applied for rapid and accurate identifi-cation of Ningza 11 hybrid seeds.

RAPD Appraisal of Purity of the Hybrid Rice SY63

使用纯度低或真实性差的杂交种子会给农业生产造成极大损失。然而杂交种子的纯度无法单纯从种子形态上鉴别．本文首次采用ＲＡＰＤ特异扩增谱带做为分子标记对水稻杂交种纯度进行了鉴定。汕优６３杂交水稻种子由中国水稻研究所从某制种单位随机取样获得。珍汕９７Ａ不育系和明恢６３恢复系等品种由中国水稻研究所提供。在做形态观察的同时，分别取上述三种材料叶片制备ＤＮＡ作为初始模板ＤＮＡ。对３００个ＲＡＰＤ随机引物，经过三次多态性初筛复筛，发现随机引物Ｐ１８可以稳定地扩增出一条来源于父本明恢６３的０．８ｋｂ的特异条带。用Ｐ１８引物对１００株汕优６３杂交单株进行ＲＡＰＤ扩增，其中８３个获得了０．８ｋｂ特异条带（Ｆｉｇ．１ｕｐｐｅｒ）。分子杂交证明其结果是可靠的（Ｆｉｇ．１Ｌｏｗｅｒ）。以ＲＡＰＤ扩增结果为依据，对这１００株材料进行植株形态比较，发现那１７个不能扩增出特异条带的单株均为假杂种。说明该制种单位汕优６３的假种率为１７％。大大超过农业部的有关规定。应对其原因和后果进行追究。用Ｐ１８引物对１８种分别为籼粳及其中间型的常用稻种进行ＲＡＰＤ扩增鉴定，除明恢６３的亲本──圭６３０和中间型Ｐｃｃｏｓ，Ａｕｓ３７３具有０．８ｋｂ的特异条带?

Structure identification for compound I separated and purified from taxoids-produced endophytic fungi (Alternaria. alternata var. taxi 1011)

Endophytic fungi are widely found in almost all kinds of plants. Many endophytic fungi can produce some physio-logical active compounds, which are same to or analog to those isolated from their hosts. Producing physiological active com-pounds through microbial fermentation can give a new way to resolve resource limitation and to find out alternative source. Through the methods of organic solvent extraction, thin layer chromatography (TLC) and column chromatography, compound I was isolated, purified from the liquid fermentation metabolites of the taxoids-produced endophytic fungi (Alternaria. alternata var. taxi 1011 Y. Xiang et LU An-guo) that was screened from the bark of Taxus. cuspidata Sieb.et Zucc.. Compound I was identified as one kind of taxoids type III, based on the analyzing results by using the methods of ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). This study provides a com-pleted method for separation and purification of the endophytic fungi as well as structure identification of its fermentation me-tabolite

Purification and Characterization of Angiotensin I Converting Enzyme Inhibition Peptides from Sandworm Sipunculus nudus

Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography(RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC_(50) values of the purified peptides for ACE inhibition activity were 34.72 μmol L^(-1), 20.55 μmol L^(-1) and 22.77 μmol L^(-1), respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.

RAPD Technique Used to Determine the Purity of Hybrid Hot Pepper Seeds

Two hybrid hot pepper varieties Xiangyan 5 and Xiangyan 10, and their parents were analyzed the polymerase chain reaction with MJ /PT 200 Peltrier Themal Cycler and DS 800 White-ultravilot Transilluminator to set up a RAPD system adaptable to the purity determination of the hy-brid seeds. Among the 39 random primers, 2 and 4 primers were found to be used effectively in Xiangyan 5 and Xiangyan 10 respectively.

Analysis on the Different Results of Seed Purity Identification for the Same Hybrid Rice Variety Using Different SSR Primers

Different results of seed purity identification for Gangyou 158, Ⅱ You 808, Wuyou 308 and Tianfengyou 316 were obtained using different SSR primers in our early work. To find out the reasons, the four hybrid combinations were grown in field to identify their purity according to their phenotypic traits. Then, the results of field identification were compared with that of laboratory tests using different SSR primers. The comparison revealed that only sterile lines （female parent） were distin- guished from true hybrids using the primers RM208, RM264, RM242 and RM164 for the purity identification of Gangyou 158, II You 808, Wuyou 308 and Tianfengyou 316, so the results were higher than that of field identification. In contrast, the primers RM341, RM297, RM21 and RM110 were able to distinguish not only the sterile plants but also the cross-pollinated ones from the true hybrids of Gangyou 158,Ⅱ You 808, Wuyou 308 and Tianfengyou 316, and the results of purity identifi- cation using them were close that of field identification, in summary, several pairs of primers should be used for the purity identification of rice hybrids to distinguish all the off-type plants and thus improve the accuracy.

Comparison of methods of purification and identification of Verticillium dahliae toxins

The complexes excreted by VerticiUium dahliae are phytotoxins, which are responsible for most of the symptoms associated with Verticillium wilt disease. Verticillium dahliae toxins （VD-toxins） can be purified by different methods. In the present study, we reported a simpler, more effective method to purify VD-toxins. The supematant of V. dahliae culture was frozen, lyophilized and dialyzed by 1 kDa Dialysis Membranes （MWCO）. We also partially identified the characteristics of the purified VD-toxins. The results showed that the components of VD-toxins include glycoprotein within 35.8-83.2 kDa. The phytotoxic activity of VD-toxins was remained after VD-toxins were pretreated by high temperature, Concanavalin-A, and proteinase E, respectively. These data suggest that VD-toxins are heat-stable, and the protein fraction and glycosyl are both important contributors to the phytotoxic activity. VD-toxins purified effectively from the culture filtrates of V. dahliae may help in further understanding the mechanisms of interactions between V. dahliae and plants.

Perspectives in application of biosensors for the modernization of traditional Chinese medicine

Biosensor is an instrument which is sensitive to biological material and converts its concentration into electrical signals.Organisms such as enzymes, antibodies, tissues, cells and so on can selectively identify specific substances. Thesemolecular recognition functions can be combined with the target, such as the binding of antibodies and antigens, and thebinding of enzymes to the substrate through the recognition process. Biosensor has the advantages of high specificity,high sensitivity, fast reaction speed, low cost and easy operation. It has wide application prospect in food, pharmacy,chemical industry, clinical examination, biomedicine, environmental monitoring and so on, especially as a newtechnology means, in the field of modern herbal medicine research influence. Studies have demonstrated that thebiosensing technology has been applied to, traditional Chinese medicine （TCM） targets, isolation and purification ofTCM, the mechanism of TCM, quality control of TCM, the active ingredients detection of TCM and other basicresearches. Biosensor technology has made an important contribution to the research of modern herbal medicine, and hasbecome a Hot-spot in future research.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Chemical constituents from the seeds of Capparis masaikai

A total of 21 compounds were isolated from the seeds of Capparis masaikai and identified as oxazolidine-2-thione(1), succinimide(2), catechol(3), octathiocane(4), monoethanolamine(5), 3-hydroxypropanenitrile(6), L-arabinose(7), 1,2,3-propanetriol(8), drummondol(9), spionoside B(10), adenosine(11), corchoionoside C(12), coniferin(13), syringin(14), cis-syringin(15), dihydrosyringin(16), indole-3-carboxylic acid(17), β-D-glucopyranosyl indole-3-carboxylic acid(18), 6-hydroxyindole-3-carboxylic acid β-D-glucopyranosyl ester(19), linoleis monoglyceride(20), and triolein(21). Their structures were identified based on physicochemical property and spectroscopic analysis, including MS, NMR, and single-crystal X-ray crystallographic data. Compounds 2–10 and 12–21 were isolated from C. masaikai for the first time.