The techniques used to detect genetically modified organisms (GMO), including qualitative polymerase chain reaction (PCR), quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and many others, are systematically described and discussed. The application progress of GMO in species-specific detection, endogenous genes, standard substances and restraining factors influencing detection are reviewed. The ongoing problems and development prospects of detection techniques of GMO are also pointed out.

Mature porcine oocytes containing first polar bodies (Pb I) were obtained by in vitro culture of follicle oocytes from ovaries obtained from a local abattoir, and zygotes with second polar bodies (Pb II) were grown after in vitro fertilization of the mature oocytes. Extrusion, biological activity and morphology of Pb I and Pb II were statistically analysed. Polar bodies were isolated and collected from oocytes by enzyme digestion or micromanipulation. Their vigour under different preservation conditions was analysed and evaluated using a Trypan blue staining method. The results showed that 66.7% of the oocytes extruded Pb I after 40 h of in vitro mature culture of oocytes, and 49.7% of the zygotes extruded Pb II 20 h after in vitro fertilization. The efficiency of isolation of Pb II by micromanipulation significantly exceeded that by enzyme digestion, the Pb I and Pb II isolated by micromanipulation presenting with good vigour and normal morphology (95.3% versus 58.9%). The survival rates of Pb I and Pb II were 63.3% and 93.1% for 4 h at 39°C, 85.0% and 72.9% for 40 h at 4°C, and over 95.0% and 84.6% for less than 7 days at −20°C. In comparison with the above preservation conditions for Pb I and Pb II, the results for cryopreservation were best, with rates of survival as high as 89.1% for Pb I and 87.9% for Pb II for preservation periods of over a month, and rates of normal morphology of 97.8% and 95.7%, respectively. The Pb I and Pb II could be isolated and preserved effectively, for use in further research on the recombination of oocytes and zygotes.

Three pairs of primers were designed for amplification of the adiponectin gene in geese (Anser demeatica) based on the published sequences of chicken, duck and other adiponectin homologues. The complete 1062 bp sequence of the adiponectin gene in geese, obtained by polymerase chain reaction (PCR) amplification and splicing, contained two exons and one intron. The coding sequence was 738 bp long and included codes for 245 amino acids (GenBank Accession No. EU370686). The coding sequence and the corresponding protein of goose adiponectin shared the highest homology with ducks, 94.85% and 95.51%, respectively; homology with chicken adiponectin was next highest, while the lowest homology was to be found with mammals. The predicted molecular weight (MW) and isoelectric point (pI) were 26,603.3 Da and pH 5.19, respectively, which were highly similar to those of species considered above. Reverse transcription-PCR revealed that goose adiponectin mRNA was highly expressed in skeletal muscle, fatty tissue, heart and stomach muscles. Medium levels of expression were detected in the small intestine, stomach glandular tissue, kidney and lung, while it was only weakly expressed in the liver, spleen, ovaries and diencephalon.

Amplified fragment length polymorphism (AFLP) markers linked to the stem nematode resistance gene were developed in sweet potato (Ipomoea batatas (L.) Lam.). Using bulked segregant analysis (BSA), 800 AFLP primer combinations were screened in the resistant and susceptible bulked DNA from the 186 progeny of an F1 single-cross population of Xu781 (resistant parent)×Xushu18 (susceptible parent), and 245 of these AFLP primers showed polymorphic bands between resistant and susceptible DNA. Primer combinations detecting polymorphism between the two bulks were used to screen the parents and eight individuals from each of the bulks. The results showed that E2M23 and E33M20 produced a specific band of about 500 bp and 200 bp in length, respectively, in the resistant plants but not in the susceptible plants, suggesting that the markers named E2M23500 and E33M20200 linked to a gene for stem nematode resistance. Amplified analysis of the 186 F1 individuals indicated that the genetic distance between these two markers and the stem nematode resistance gene was 6.9 cM and 11.1 cM, respectively, measured with Mapmaker 3.0. These two AFLP markers were used to identify ten sweet potato varieties planted widely in China and the results were consistent with those of conventional resistance identification, indicating that the two markers can be used in molecular marker-assisted breeding for stem nematode resistance in the sweet potato.

The potential relationship between the establishment of Foot-and-mouth disease virus (FMDV) persistent infection and gene variation was identified by investigating the variation of VP1 and 3ABC genes from yellow cattle persistent infection isolates. Five yellow cattle were inoculated on their tongue with 1.0×104 ID50/ml of FMDV O/Akesu/58 strain. After displaying clinical or subclinical signs, they probably became asymptomatic carriers. Oesophageal–pharyngeal fluids were collected monthly from the carriers with a probang and inoculated into a baby hamster kidney cell line (BHK-21); 12 FMDV isolates were obtained. The VP1 and 3ABC genes of the 12 isolates were then amplified by reverse-transcriptase polymerase chain reaction (RT-PCR). Cloning and sequencing revealed that the homology of the VP1 nucleotide and amino-acid sequence of all the isolates was above 98%, with no base deletion or insertion. When compared with the O/Akesu/58 FMDV strain, the homology of the VP1 nucleotide sequence of the isolates was only 85%, and that of the deduced amino-acid sequence only 90%.There were several nucleotide mutations in the VP1 gene of the isolates, including 16 consistent nucleotide mutations, with only two of them leading to a change in amino acid (I56→T, A210→E). Moreover, it was found that four nucleotide points and three amino-acid points had transversions among all isolates. The 3ABC gene had only 13 nucleotide transversions and five amino-acid mutations. It was presumed that persistent FMDV infection might have little connection with variation in the VP1 and 3ABC genes, and was probably related to other structural protein gene and key factors.

The plant expression vectors pCAMBIA1301PMI and pBIPMI were constructed by substituting the Escherichia coli phosphomannose-isomerase (PMI) gene for the hpt gene of pCAMBIA1301 and gus gene of pBI121, respectively. Epicotyl explants of the Xuegan sweet orange (Citrus sinensis L. Osbeck) were inoculated with Agrobacterium tumefaciens EHA105- pCAMBIA1301PMI and EHA105-pBIPMI and subsequently selected in a medium supplemented with a combination of 25 g/l mannose and 5 g/l sucrose as the carbon source. The transformation efficiency rate was 27.7% when transformed by pCAMBIA1301PMI and 12.7% by pBIPMI. Genetic transformation was confirmed by chlorophenol red assay and polymerase chain reaction (PCR). A new method for obtaining transgenic Xuegan sweet orange plants was developed using the PMI/mannose selection system.

N6 medium, Murashige and Skoog (MS) medium and BNBN medium were adopted in tissue cultures of Nipponbare (japonica) scutellum for performance comparison. Results indicated that, among the three kinds of culture media, the NB medium worked best in callus induction and gene transformation and was most suitable for tissue culture of Nipponbare scutellum. Based on this, the elicitor-encoding gene pemG1 from Magnaporthe grisea was introduced into the genome of Nipponbare via the Agrobacterium-mediated method. Transgenic rice plants were obtained. The integration, transcription and expression of pemG1 in rice were confirmed by polymerase chain reaction (PCR), Northern blot and Western blot, respectively. Genetic analysis showed that the transgenes were segregated normally in the progenies.

Seven isoenergetic semi-purified test diets containing graded levels of protein ranging from 20 to 50% were formulated using fish meal and casein as the protein sources. Test diets were fed to triplicate groups of Barbodes caldwelli juveniles with initial body weights of 1.26±0.02 g for eight weeks. The results indicated no significant effect of dietary protein levels on survival rate, relative weight of the viscera and relative weight of the liver in the juvenile fish. The weight gain and specific growth rate of the fish were found to be greater as dietary protein levels increased from 20 to 35%, but were not affected significantly as dietary protein level increased from 35 to 50%. Feed efficiencies did not differ significantly when fish were fed diets with protein levels from 30 to 50%, but were significantly higher than those of fish fed diets with protein levels of 20 and 25%. The protein efficiency ratio (PER) was negatively correlated with diet protein level (x) (PER=3.006−0.03251x, R=0.9366). There was no significant effect of dietary protein levels on carcass moisture, crude protein and ash. However, carcass lipid levels (L) decreased with an increase in dietary protein level (x) (L=8.2169−0.0458x, R=0.8551). There was no significant variation in hepatopancreas protease activity among the tests. Intestine protease activity and hepatopancreas amylase activity were increased to some extent, and then decreased as dietary protein levels continued to rise. The intestine amylase activity (IAA) of the juveniles was negatively correlated with dietary protein level (x) (IAA=84.625−0.9147x, R=0.8463). It was estimated that the suitable protein level for the B. caldwelli juvenile is 34% (the broken-line model was used to regress the relationship of the weight gain of the juvenile and dietary protein level).

Four bioengineered bacteria having the fasG gene were constructed using two Escherichia coli expression strains (BL21 (DE3) and Rossetta (DE3)) with different tRNA profiles and two E. coli expression vectors (pET28a(+) and pGEX-KG) to examine the influence of rare codons on the expression of the fasG gene. The induction expression result of bioengineered bacteria showed that the expression levels of target proteins in Rossetta (DE3), which contained the tRNAs of the rare codons, were higher than in BL21 (DE3), either using pET-fasG or pKG-fasG.

The expression of the YUCAA1 gene and the amount of endogenous indole acetic acid (IAA) in rice (Oryza sativa subsp. japonica) plants and rice suspension cells infected by Rice stripe virus (RSV) were investigated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and high-performance liquid chromatography, respectively. The results showed that the expression of the YUCAA1 gene and the amount of endogenous IAA increased at various times (16, 32, 48 and 64 h) after infection of rice suspension cells by RSV. In rice plants infected with RSV, the expression of the YUCAA1 gene and the amount of endogenous IAA increased, in comparison with healthy rice plants, at 4–8 days after infection, and decreased at 12 and 16 days. These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally, the expression of the RSV gene CP increased 2.9 times in rice plants after they were treated with a KPSC buffer to deplete the endogenous auxins, and decreased 45% after treatment with 30 μmol/l IAA. All of these results suggest that auxin may play a role in RSV replication in rice plants.

Domestic yak (Bos grunniens) metallothionein-I (MT-I) and metallothionein-III (MT-III) cDNA 3′-end full-length sequences (331 and 378 bp; GenBank accession MT-I no. AY758557, MT-III no. DQ492300) from the total RNA of liver and brain tissues were amplified and cloned by reverse transcription polymerase chain reaction (RT-PCR) and 3′-rapid amplification of cDNA ends (RACE) using the primers YMT-I SP and M13 primers M4, and YMT-III SP and M13 primers M4, respectively, which included the MT-I (183 bp) coding sequence, MT-III (207 bp) cDNA coding sequences, and also the tailing signal AATAAA and Poly(A) at the 3′-ends of the MT-I and MT-III, respectively. The analyses showed that the yak cDNA sequence coding for MT-I protein was composed of 61 amino acids, including 20 cysteines having a conserved tripeptide structure, for example C–X–C, C–C–X–C–C, C–X–X–C and so on. The yak cDNA sequence coding for MT-III protein was composed of 68 amino acids, including 19 cysteines having both MT-III-specific conserved tripeptides and the same conserved tripeptides as MT-I, including T, CPCP, GEGAEA and so on. They were all comparatively conservative in molecular evolution. These structures indicated that MT-III had the same physiological functions regarding heavy metals (detoxification and so on) as MT-I, and also different specific functions from MT-I, including inhibition of neuron growth.

Based on conserved homologous amino-acid sequences of the Gq protein α subunit in arthropods, a pair of degenerate primers were designed to amplify the gene from the English grain aphid (Sitobion avenae), using reverse transcriptase polymerase chain reaction (RT-PCR) and (3′/5′)-rapid amplification of cDNA ends (3′/5′ RACE) techniques. A Gqα protein was obtained from the alate adult aphids. The open reading-frame was 1062 bp, encoding 352 amino-acid residues with a calculated molecular weight of 40.8 kDa. The cDNA sequence was deposited in GenBank with accession no. EF638906. The deduced amino-acid sequence of Gqα shared a high identity (≥82.17%) with reported Gqα from other insects and even vertebrates, and had the typical characteristics of Gqα protein. In order to explore the function of the Gqα gene, a eukaryotic expressional system (baculovirus expression vector system, BEVS) was constructed by TOPO and Gateway techniques. After the recombinant reaction occurring between pUC-Gqα and the Gateway-adapted baculovirus DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV), the construct recombinant viruses containing V5-His6Gqα were transfected singly into the insect cell line of Tn-5B1-4. After collecting the infected cell, detection was conducted by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The result showed that the system comprising recombinant baculovirus and Tn could express Gqα protein successfully.

Epidermal growth factor receptor (EGFR) is becoming a perfect target for killing carcinoma cells, especially because of its overexpression on the surface of these cells. Cationic antimicrobial peptides (CAP) have their own special mechanism of membrane-lytic cytotoxicity. In this study, a membrane-lytic immunotoxin (IT), chimeric protein MEGFMEL, was constructed to kill carcinoma cells with EGFR overexpression. This protein is composed of mouse (Mus musculus) epidermal growth factor (MEGF), as the target part, and melittin (MEL), as the cytotoxic part. Using Escherichia coli BL21 and pET30a as expression strain and vector, respectively, 63.45 μg/ml of MEGFMEL (68% purity) was obtained through low-temperature induction of expression and a thawing-freezing purification procedure (without cytolysis). In vitro activity measurement showed that this MEGFMEL significantly induced a lethal effect on A431 carcinoma cells overexpressing EGFR on the surface, with an LD50 value 52.6 μg/ml. The results suggest that the use of CAP as the toxin in the construction of unique membrane-lytic ITs aimed at EGFR is feasible.

Seventeen special loci were selected from 77 microsatellite markers to distinguish three varieties of tilapias, including the six differential loci UNH636, UNH117, UNH172, UNH738, UNH878 and UNH896 in Oreochromis aureus; five differential loci UNH913, UNH907, UNH222, UNH980 and UNH880 in O. niloticus; and six differential loci of UNH876, UNH899, UNH853, UNH932, UNH933 and UNH773 in O. mossambicus. Any one of the 17 loci could amplify particular bands to distinguish one tilapia from the other two. The genetic structure of O. aureus, O. niloticus and O. mossambicus stocks and their phylogenetic relationships were also analysed using these 17 loci. In total 142 alleles were detected, and the average number of alleles per locus was 8.35. Additionally, a clustering analysis was performed based on the result of the Popgen32 software package and phylogenetic trees were constructed by MEGA4 using the unweighted pair group method using arithmetic average (UPGMA). The results showed that the mean value of observed heterozygosity was 0.0941, 0.5490 and 0.2588, the mean value of expected heterozygosity was 0.1089, 0.7230 and 0.1965, and the polymorphism information content was 0.0869, 0.7149 and 0.1643, in O. aureus, O. niloticus and O. mossambicus, respectively. The UPGMA tree demonstrated that O. aureus was more closely related to O. mossambicus than to O. niloticus.

Single-nucleotide polymorphisms (SNPs) of exon 8 of the GHR gene were detected in Chinese Holstein cows by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The results showed that exon 8 of the GHR gene digested by TasI could be divided into two kinds of alleles and three kinds of genotypes. The frequencies of allele A and T were 0.6339 and 0.3661, respectively. The frequencies of genotypes AA, AT and TT were 0.459, 0.350 and 0.191, respectively. Sequencing showed one single nucleotide mutation T→A at 4962 bp of the gene in genotype TT when compared with genotype AA, and this mutation resulted in an amino acid change of phenylalanine (TTT)→tyrosine (TAT). The result of χ2 testing indicated that the genotypic frequency of the GHR gene digested by TasI did not fit with Hardy–Weinberg equilibrium in this population (P<0.05). The least-square mean of the fat percentage of this population was higher for genotype TT than for genotype AA (P<0.05).

The testosterone-inducible regulator (teiR) gene was cloned from Comamonas testosteroni chromosomal DNA, and introduced into plasmids pKtac2 (containing a tac promoter) and pK18 to yield plasmids pKtac2-teiR and pKteiR100. The recombinant plasmids were transformed into competent Escherichia coli HB101 and total protein was extracted to detect the TeiR protein expression level using enzyme-linked immunosorbent assay (ELISA). E. coli transformed by pKtac2-teiR and pKteiR100 produced 6.65 and 5.93 μg/mg of TeiR protein, respectively. Recombinant plasmids were also co-transformed into competent E. coli HB101 with plasmid p6 [containing hsdA gene (3α-HSD/CR, 3α-hydroxysteroid dehydrogenase/carbonyl reductase encoding gene)] to reveal the relationship between 3α-HSD/CR and TeiR by ELISA. The amounts of TeiR protein expressed by E. coli containing pKtac2-teiR and pKteiR100 were 5.94 μg/mg and 5.33 μg/mg, respectively, and these increased up to 6.81 μg/mg and 6.10 μg/mg after inducing with 1 mmol/l isopropyl-β-d-thiogalactoside (IPTG). Interestingly, 3α-HSD/CR protein expression level, after co-transformation with plasmids pKtac2-teiR and p6, was lower than that observed in the co-transformation with pKteiR100 and p6. The first co-transformation induced 1.20 μg/mg 3α-HSD/CR protein and the second 1.71 μg/mg. These values rose to 1.42 and 1.80 μg/mg, respectively, after treatment with 1 mmol/l IPTG. Our results proved that the tac promoter was more efficient than the lacZ promoter and that the teiR gene could act as an activator for hsdA gene expression.

Simple sequence repeat (SSR) analysis was performed on five Secale species, four Triticum species and a Triticale line Fenzhi-1 using 102 pairs of microsatellite primers. A 387-bp specific DNA fragment FZ387 (GenBank accession no. EF179137) was obtained from the Triticale Fenzhi-1 with primer Xgwm614, without amplification in Secale. NCBI BLAST revealed that this FZ387 sequence had 94% and 95% similarity to part of the Gypsy Ty3-LTR retrotransposon Fatima in Triticum monoccocum (AY485644) and Triticum turgidum (AY494981), respectively. A pair of specific polymerase chain reaction (PCR) primers, FaF and FaR, was designed based on the conserved region of this FZ387 sequence. The amplification of primer pair Xgwm614F and FaR revealed that a specific 350-bp band (designation as A350) was obtained from the species containing A chromosomes. Furthermore, PCR on Langdon Chinese Spring substitution lines was performed, and the results found that this segment was located on both long and short arms of all A chromosomes. However, the amplification of primer pair FaF and Xgwm614R gave rise to a specific DNA band of about 350 bp (designated AB350) from materials containing A and/or B chromosomes. The wild species of wheat and the relatives were amplified using the two pairs of primers, and revealed that only A350 and AB350 were found in Chinese Spring (CS). Sequence comparison and variation of SSR primers binding regions of FZ387 indicated that significant diversity might exist in the internal sequence of this Fatima-like element among triticeae genomes. Meanwhile, both A350 and AB350 can be used as molecular markers for the detection of A and AB genomes.

A pair of degenerate primers was designed based on a conserved domain of GA 20-oxidase reported in other plants. The full-length (1179 bp) carnation (Dianthus caryophyllus L. cv. Master) GA 20-oxidase cDNA (named Dc20ox) was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). BLAST analysis revealed that the deduced amino acid sequence had high homology (66–75%) with the GA 20-oxidase sequences from other plants. An RNAi vector (pART400) was constructed from a 400 bp fragment representing a highly conserved region of GA 20-oxidase.

An efficient AHL (N-acyl-homoserine lactone) bioassay strain, JZA1, of Agrobacterium tumefaciens was used to detect the AHL production from Acidovorax avenae subsp. citrulli [the pathogen causing bacterial fruit blotch (BFB) of melons], and the results showed that A. avenae subsp. citrulli produced a 3-O-C8-homoserine (HSL) type signal molecule. Gene aiiA, which could degrade AHL molecules, was transformed into A. avenae subsp. citrulli strain NJF10, creating strain NJF10-aiiA. The AHL production from NJF10-aiiA was significantly reduced compared with wild-type NJF10. Inoculation tests showed that NJF10-aiiA had an obvious reduction of virulence on watermelon fruits. Our finds showed that AHL production by A. avenae subsp. citrulli was related to its pathogenicity. This work might provide a novel way to control BFB by QS (quorum sensing) interference.

In exploring an effective and reliable karyotyping method in Brassica crop plants, Brassica oleracea was successfully karyotyped using genomic in situ hybridization (GISH). B. oleracea genomic DNA was labelled as probe using DIG-high prime mix kit, with B. rapa genomic DNA acting as blocking agent. Specific fluorescent signals were detected on each pair of homologous chromosomes, and nine pairs of chromosomes of B. oleracea were clearly identified according to the signal characteristics. A practical and accurate method for conducting karyotyping of small chromosomes has been demonstrated.