大片段DNA克隆载体的研究进展

DNA克隆技术是分子生物学和基因工程研究中非常重要的一项技术。自第一个质粒载体pSC101作为第一个克隆载体以来,随着分子生物学技术的迅猛发展克隆载体的整体结构、容载能力和转化效率都有了很大的改善。通过综述克隆载体的发展概况,以及大片段DNA文库的构建和应用,对大片段DNA遗传转化的发展做了展望。

片段

一 课间 “如果大海能够带走我的哀愁……” “加菲，唱得不错嘛!” “如果大海能够带走我的哀愁……” “加菲，后面一句呢？”

浅谈“片断”与“片段”的使用

"片断"与"片段"在使用与教学中常令人迷惑。通过微观上的词义比对分析和宏观上的统计,可以发现它们在使用上的异同及其发展趋势。这些认识对异形词的规范有一定的意义。

片段也精彩

提起伏阳，我从心底里佩服，因为他刻苦好学、努力的精神，深深地打动了我。

片段组合呈现真我

看！那个镜子里的小破孩儿是谁？那就是我。

Using Subtracted AFLP to Efficiently Mark an Alien Chromosome Fragment in Wheat Background

为解决AFLP中样品间共同片段对多态性片段识别的干扰 ,作者建立了一种“减法AFLP”标记技术 ,并运用这种技术成功对小麦中的外源染色体片段进行了标记。运用该技术 ,样品间的共同扩增片段显著减少 ,在非变性聚丙稀酰胺凝胶上带型差异十分明显 ,多态性片段很容易分辨。其真实性通过将其中两条成功转换成外源染色体片段特异的SCAR标记得到了验证。减法AFLP标记技术的建立为标记作物中的外源染色体片段提供了一种有效手段。

Cloning and Sequence Analysis of Actin Gene from Rehmannia glutinosa

[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.

Bioleaching of Pb-Zn-Sn chalcopyrite concentrate in tank bioreactor and microbial community succession analysis

The variation of microbial community structure was investigated for the tank bioleaching process of Pb-Zn-Sn chalcopyrite concentrate in the presence of mixed moderately thermophilic bacteria. The parameters, such as pH value, solution potential and concentrations of metal ions, were determined by the method of polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） to analyze the succession of microbial community. The results showed that a final copper extraction rate of 85.6% could be obtained after tank bioleaching for 30 d. The Acidithiobacillus caldus was the dominant population with abundance of about 73.80%in the initial stage, then Sulfobacillus thermosulfidooxidans dominated from the 18th day to the end of bioleaching, while the abundance of Leptospirillum ferriphilum changed slightly. A higher solution potential within a certain range and appropriate concentration of ferric ions were essential for this tank bioleaching of chalcopyrite.

Mapping of QTLs Related to Grain Weight Using Chromosome Single Segment Substitution Lines in Rice

Grain weight, one of the major factors determining rice yield, is a typical quantitative trait control ed by multiple genes. With Guangluai 4 as recipient and Nipponbare as donor, a population of 119 chromosome single segment substitution lines had been developed. Correlation analysis between grain weight and grain shape by SPSS revealed that 1 000-grain weight shared extremely significant posi-tive correlation with grain length and length-width ratio, but no significant correlation with grain width and thickness. The QTL analysis of grain weight was carried out using one-way analysis of variance and Dunnett＇s test. Nineteen stable QTLs re-sponsible for grain weight were identified over two years. Al 19 QTLs were identi-fied on al chromosomes except for chromosome 10 and 12 at a significance level of P≤0.001. Among them, 10 QTLs had a positive effect and were derived from the Nipponbare al ele, the additive effect of these QTLs ranged from 0.49 to 2.74 g, and the contributions of the additive effects ranged from 2.00% to 11.05%. Another 9 QTLs had a negative effect and were al derived from Guangluai 4 al ele, the ad-ditive effect of these QTLs ranged from 0.60 to 2.35 g, and the contributions of the additive effects ranged from 2.40% to 9.84%. The results provide a basis for the fine mapping and gene cloning of novel locus associated with rice grain weight.

DGGE Analysis on Mitochondrial Cyt b Gene of Eriocheir sinensis and Eriocheir hepuensis

[Objective] The aim was to investigate the possibility to analyze the genetic diversity of Eriocheir sinensis and Eriocheir hepuensis by using the technique denaturing gradient gel electrophoresis（DGGE）.[Method] Mitochondrial cyt b gene fragment was amplified from 180 individuals of five populations of E.sinensis and a population of E.hepuensis and then analyzed by using DGGE.[Result] All PCR products showed two kinds of electrophoretic mobility on DGGE.The PCR products of all individuals from E.hepuensis showed the same mobility with that of the individuals from 46.7% of Jiangdu population,23.3% of Yizheng population and 20.0% of Wenzhou population of E.sinensis,while the rest of the individuals from the three populations of E.sinensis mentioned above as well as all the individuals of Nanjing and Panjin populations showed the same mobility,which was higher compared with that of E.hepuensis.The results indicated that there was the same genetic marker in E.sinensis populations as that of E.hepuensis population,which was consistent with previous studies.[Conclusion] DGGE technique could be used to analyze the genetic diversity of Chinese mitten crab.

Discussion on AFLP Molecular Markers in Piper methysticum and Pepper

The aim of the research was to discuss the genetic relationships between Piper methysticum, Pepper and other wild species in Pepper genus. DNA was extracted from leaves which belonged to 28 germplasms including 6 materials of P. methysticum, 21 maerials of cultivated and wild Pepper, 1 material of Peperomia pellucida belonged to different genus. Premiers with good band-type and high polymorphism and resolution were selected from 64 pairs of primers for AFLP amplification and the clustering analysis was conducted with MVSP3.13f software. 191 bands were amplified by 4 pairs of premiers, 189 of which had polymorphism, being 98.6%. 28 germplasms were classified into 6 different groups at the genetic similarity coefficient of 0.52 by silver staining AFLP, in which 6 materials of Piper methysticum were clustered into a single group, indicating that P. methysticum belonged to Pepper family of Pepper genus but were distantly related to the others. The research provided the basis for selecting rootstocks for P. methysticum graft, molecular identification of P. methysticum and the fingerprint construction of P. methysticum.

Cloning and Sequencing of Two Acetylcholinesterase cDNA Fragments from Cotton Aphid,Aphis gossypii Glover

Two acetylcholinesterase (AChE) genes cDNA fragments,Ag.acel and Ag.ace2,have been cloned from cotton aphid,Aphis gossypii Glover using degenerate primers with RT-PCR technique.Ag.acel gene cDNA fragment is of 282?bp encoding 94 amino acids,and Ag.ace2 gene cDNA fragment is of 264?bp encoding 88 amino acids.Both two putative AChE genes cDNA fragments share numerous similarities with those cloned from other insects.This is the first report of two AChE cDNA fragment sequences in the insect species,which provided the direct evidence of multiple AChE existence in insects.

A Method for Detecting Adhesive Related-Factors of Streptococcus suis Serotype 2 by Real-time PCR

[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.

Effects of Fragmentation Intensity of Perennial Roots and Their Burial Depth on Sprouting and Early Growth of Alternanthera philoxeroides(Mart.)Griseb

[Objective] The aim was to provide theoretical basis for the prevention and control of the invasion of Alternanthera philoxeroides（Mart.）Griseb.[Method] Effects of fragmentation intensity of fresh roots and their burial depth on sprouting and early growth of A.philoxeroides were studied by control test.[Result] More sprouts of A.philoxeroides emerged when the fragmentation intensity of fresh roots was higher,while if the fragmentation intensity of fresh roots was lower,the early growth of A.philoxeroides was more rapid.The soil buried depth had significant effect on fresh root sprouts＇ emergence,but once fresh root sprouts could reach the soil surface and were given enough growth time,even if the fresh roots were buried in different depths,soil buried depth had no significant effect on its young plant growth.[Conclusion] If different fragmentation intensities of fresh roots present,there is a kind of trade-off strategy between root sprouts＇ emergence and plant＇ early growth,by which A.philoxeroides can invade new habitat successfully.To control the invasion of A.philoxeroides,it is critical to prevent its fresh root sprouts from emerging to soil surface,that is,to bury the fresh roots at a further soil depth.

Expression, Purification and Insecticidal Activity Analysis of Cry1Ac without Helix α-1

[Objective] This study aimed to analyze the expression, purification and insecticidal activity of Cry1Ac without helix α-1 of domain I in E. coli. [Method] Plasmid of B. thuringiensis HD-1 was used as the template for amplification of Cry1Ac gene, which was linked with pET28a vector to construct the recombinant plasmid. After transformation and IPTG induction, expression of target protein was detected by using SDS-PAGE method. Target protein was extracted and coated on the surface of feed for H. armigera to determine the insecticidal activity. [Result] Cry1Acdela1 gene could normally express the target protein in E. coli. However, the target protein existed in the form of inclusion body. Results of bioassay analysis showed that under the same concentration, fatality rate of activated Cry1Ac toxin reached above 75%, while that of Cry1Acdelα1 was only about 10%. [Conclusion] E. coli-expressed Cry1Acdelα1 had no insecticidal activity against H. armigera.

Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti

[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.

Mapping QTLs Associated with Sheath Blight Resistance Using Chromosome Segment Substitution Lines of Rice(Oryza sativa L.)

In this study, a population of 119 chromosome segment substitution lines （CSSLs） derived from backcross between indica 9311 and japonica Nipponbare was employed to map quantitative trait loci （QTL） associated with sheath blight resis-tance in rice with toothpick inoculation method. A total of three sheath blight resis-tance-associated QTLs （qsb8-1, qsb8-2 and qsb8-3） were identified, which were lo-cated on adjacent molecular markers RM3262, RM5485 and RM3496 of chromo-some 8; the genetic interval was 81.7cM-91.7cM, 91.7cM-108.1cM and 108.1cM-119.6cM, respectively. The additive effect of qsb8-2 was negative, indicating that sheath blight resistance of susceptible parent harboring qsb8-2 fragment was en-hanced; additive effects of qsb8-1 and qsb8-3 were positive, indicating that sheath blight resistance of susceptible parent harboring qsb8-1 and qsb8-3 fragments was reduced.

Studies on the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer

AIM To study the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer.

QTLs Mapping for Salinity Tolerance at Seedling Stage or Rice(Oryza sativa L.) Using Chromosome Segment Substitution Lines

In this study, a population of chromosome segment substitution lines （CSSLs） derived from the cross between 9311 （indica） and Nipponbare （japonica） was employed to map the quantitative trait loci （QTLs） for salt tolerance under the salt stress simulated with 0.5% NaCI, using survival rate as the index. The data were analyzed by QTL IciMapping v3.1, and the results showed that one QTL （QSsr3） related to salt tolerance was located in the vicinity of the marker RM1350 on chromosome 3, into a genetic interval of 113.2-132.8 cM, with a contribution rate of 17.75%. The additive effect was 10.9, indicating that the QTL derived from the parent Nipponbare improved the salt tolerance of rice at seedling stage. This study will provide a theoretical basis for the selection of salt tolerant rice germplasm.

Location and Analysis of Introgressed Segments in the Parthenogenetic Progenies of Zea mays×Z. diploperennis by GISH

用来自玉米 (ZeamaysL .)与二倍体多年生类玉米 (Z .diploperennisIltis,DoebleyandGuzm偄ｎ)杂交的孤雌生殖后代同一抗病个体的 4个株系进行了基因组原位杂交 ,用改进的杂交技术获得了近 10 0 %的检出率 ,每一检出片段在同源染色体两成员和每两个姊妹染色单体上均有清晰的信号。