[Development research of the genetics textbook in Chinese universities].

Textbook construction is an important part of course construction. The development of the Chinese genetics teaching has been full of ups and downs, demonstrating its specificity compared with other subjects of life science. Through the investigation upon the developmental course of genetics textbooks in China from before liberation to the 21st century, we hope that we can provide valuable reference for composing new textbooks that fit the characteristic of undergraduates teaching, and keep close to the genetics front, bringing valuable reference to the cultivation of application and study talents with basic genetics knowledge and innovation ability.

Cloning and characterization of a novel PI-like MADS-box gene in Phalaenopsis orchid.

The specification of floral organ identity during development depends on the function of a limited number of homeotic genes, which are grouped into three classes. Most of these genes belong to the MADS-box gene family. The PISTILLATA (PI) family of MADS-box genes plays important roles in controlling the development of the petal and stamen of flowering plants. In an attempt to understand the molecular mechanisms behind floral development in the orchid, a MADS-box gene, PhPI10 was cloned from Phalaenopsis orchid. We provide phylogenetic evidence that PhPI10 is closely related to PI-like genes of angiosperms, which are required for establishing petal and stamen identity. In addition, there is a PI-motif in the C-terminal of the putative amino acid sequence of PhPI10. Southern analysis showed that a single copy of PhPI10 was present in the Phalaenopsis orchid genome. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that its transcription was only detectable in the top of the floral bud and undetectable in other vegetative organs. In the floral organs its expression was limited to the lip of the Phalaenopsis flower.

Cloning, expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1, a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers. The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene. In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves. The peak expression signal was observed in mesophyll cells under low phosphorus (P) induction. A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris. At the meantime, the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters deficient. Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa.

Use of green fluorescent protein as molecular marker for tagging Bacillus brevis in soil under the control of a novel constitutive promoter F1.

A constitutive expression vector pHY300-Flgfp was constructed to test the function of promoter F1 subcloned from a rice epiphyte Bacillus brevis strain DX01. The DX01 cells harboring plasmid pHY300-F1gfp were detected to produce bright green fluorescence. Subsequently, the gfp-tagged B. brevis strain was released into the soil and its survival was investigated by PCR and the detection of green fluorescence. The spatial location of in situ gfp-tagged bacterial cells on the root surface of rice seedlings was visualized. All these results indicated that green fluorescent protein is an ideal molecular marker for the detection of the activities of promoter F1, and it is also a reliable probe to monitor specific B. brevis bacteria in the environment.

Expression of green fluorescent protein in Bacillus brevis under the control of a novel constitutive promoter F1 and insertion mutagenesis of F1 in Escherichia coli DH5alpha.

The constitutive expression vector pHY300-F1gfp was constructed to test the function of a promoter, F1, cloned from the rice epiphyte Bacillus brevis strain DX01. The DX01 cells harboring the plasmid pHY300-F1gfp were shown to produce bright green fluorescence. The results were confirmed by Western blot analysis and fluorescence-activated cell sorting. Expression of the F1 promoter was constitutive. To improve the activity of F1, insertion mutagenesis of F1 based on in vitro transposition reaction was performed. Seven mutants with enhanced transcription activity in Escherichia coli DH5alpha were obtained. The enhanced promoters showed similar high activities in B. brevis strain DX01.

Cloning and prokaryotic expression of a salt-induced cDNA encoding a chloroplastic fructose-1,6-diphosphate aldolase in Dunaliella salina (Chlorophyta).

A salt-induced fructose-1,6-diphosphate (FDP) aldolase cDNA (DsALDP) in Dunaliella salina was cloned by suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis of DsALDP revealed that the 1520 bp cDNA had an open reading frame (ORF) of 327 amino acid residues. BLAST Search showed that DsALDP shared an amino acid identity (73-66%) with AldP in other plants. Alignment with homologues in other plants indicated that all the conserved substrate-specific binding sites could also be found in DsALDP. Phylogenetic analysis further confirmed the deduced amino acid sequence of the D. salina DsALDP gene belonged to the same subfamily to AldP of other green algae. Southern blot analysis suggested possible presence of the D. salina DsALDP gene as a few copies and Northern blot analysis confirmed salt-induced expression pattern at the transcriptional level. A 62 kDa fusion protein generated by adding a Trx-His.tag at the N-terminal of DsALDP was induced by IPTG in Escherichia coli BL21. An improvement of salt tolerance in E. coli expressing DsALDP fusion protein was observed.

Isolation and characterization of a salt-induced PsaG of photosystem I in Dunaliella salina.

Some cDNA sequences of subunit V (PsaG) of Photosystem I (PS I) reaction center in several species have been cloned, and PsaG has been characterized as a connection of light-harvesting complex I (LHC I) protein to photosystem reaction center. Here we present the isolation and characterization of its salt-induced homologue (DsPsaG) in Dunaliella salina. Sequence alignment shows that there is a significant similarity between the deduced amino acid sequence of DsPsaG and PsaG protein in Chlamydomonas reinhardtii, as well as between the deduced amino acid sequence of DsPsaG and other PsaG gene products. The differential expression of DsPsaG at different time points after salt stress reveals that DsPsaG mRNA was absent without salt stress and was indeed salt-induced. Its expression reached its maximum level 5 days after stress. Our study suggests that DsPsaG should be a compensation of PsaG in D. salina when the alga was in a hyperosmotic condition. It can also be useful to effectively transfer light energy from LHC I to PS I reaction center.

Cloning and characterization of a novel avirulence gene (arp3) from Xanthomonas oryzae pv. oryzae.

A novel avirulence gene was cloned from Xanthomonas oryzae pv. oryzae strain PX0339, which is the standard representative of the Philippines race 9a. The full-length gene spans 2118 bp and encodes a protein of 705 amino acids. BLAST search in NCBI indicated that the gene belongs to avrBs3 gene family, and designated arp3 (AvrBs3-related protein 3, arp3). The central region of the arp3 contains only 5.5 copies of 102bp repeats, the smallest copy number of repeats found in avrBs3 gene family by now. Together with the repeats is heptad repeats, resembling leucine zippers. Three functional nuclear localization signals and an acidic activation domain are also found in the C-terminal region. However, the arp3 lacks of two segments in its N-terminal region, which is unique in avrBs3 gene family. Southern blotting data showed that the arp3 is present as a single-copy in genomic DNA of PX0339 and locus in plasmid clone. The arp3 could be expressed in vitro in Escherichia coli BL21 and a 128kDa fusion protein was detected by Western analysis.

SQUA-like genes in the orchid Phalaenopsis are expressed in both vegetative and reproductive tissues.

The SQUA family (AP1/FUL family) of MADS-box genes plays an important role in the transition from the vegetative to the reproductive development of angiosperms, and its origin might be concurrent with fixation of floral structure in angiosperms. Here, we isolated two Phalaenopsis MADS-box genes designated ORAP11 and ORAP13, both of which belong to the monocot FUL-like clade of the SQUA family. RT-PCR showed that both genes are strongly expressed in the floral bud, and also detected in the vegetative organs. During later stages, ORAP11 was only detected in the column, but ORAP13 signal was absent from all of the floral organs. In-situ hybridization experiments detected both genes in the tips and margins of developing petals and lips, the developing column, and ovule. Over-expression of both genes in tobacco induced early flowering and changed plant architecture. Our results suggest that in Phalaenopsis, both genes might share partly redundant activities and play important roles in the process of floral transition and morphological architecture.

Flavonoid-3',5'-hydroxylase from Phalaenopsis: a novel member of cytochrome P450s, its cDNA cloning, endogenous expression and molecular modeling.

A novel cDNA for the flavonoid-3',5'-hydroxylase (F35H) gene was cloned from petals of Phalaenopsis, and designated as Phf35h (accession number DQ148458 in GenBank/EMBL/DDBJ). The genomic clone of Phf35h was isolated by a PCR-based strategy. Nucleotide sequence analysis revealed that its genomic clone contains one intron and an open reading frame encoding a polypeptide of 507 amino acid residues. Southern hybridization analysis indicated the presence of a single gene coding for Phf35h. RT-PCR analysis showed that the Phf35h mRNA is transcribed in late phase of petal development, which is concomitant with the appearance of anthocyanins in petal tissue. The transcript is abundant in the purple petals but not in leaves or roots. The three-dimensional model of PhF3'5'H protein is classified into an alpha-domain which contains most of the alpha-helices with three small beta-sheets, a beta-domain that contains the larger beta-sheets with three small alpha-helices by homology modeling. The substrate-binding site for dihydrokaempferol on PhF3'5'H protein was determined by molecular docking algorithm. A highly conserved HPPTPLSLPH sequence was predicted to contact the aromatic ring of dihydrokaempferol.

Expression and localization of recombinant human EDG-1 receptors in Pichia pastoris.

The receptor for human endothelial differentiation gene-1 protein (EDG-1) was C-terminally tagged with green fluorescent protein and expressed in the methylotrophic yeast, Pichia pastoris. EDG-1 expression was driven by the highly inducible alcohol oxidase 1 promoter. Expression of EDG-1 recombinant protein was detected by Western blot analysis and confocal microscopy. The recombinant EDG-1 receptor protein was located in the plasma membrane. Radioligand binding assays demonstrated that the EDG-1 receptors expressed in Pichia pastoris have specific and saturation binding of (32)P-labeled sphingosine 1-phosphate.

Molecular evolution and functional specialization of chalcone synthase superfamily from Phalaenopsis orchid.

Plant genomes appear to exploit the process of gene duplication as a primary means of acquiring biochemical and developmental flexibility. The best example is the gene encoding chalcone synthase (CHS, EC2.3.1.74), the first committed step in flavonoid biosynthesis. In this study, we examined the molecular evolution of three CHS family members of Phalaenopsis including a novel chs gene (phchs5), which is slowly evolved. The inferred phylogeny of the chs genes of Phalaenopsis with other two orchid plants, Bromoheadia finlaysoniana and Dendrobium hybrid, suggested that gene duplication and divergence have occurred before divergence of these three genera. Relatively quantitative RT-PCR analysis identified expression patterns of these three chs genes in different floral tissues at different developmental stages. Phchs5 was the most abundantly expressed chs gene in floral organs and it was specifically transcribed in petal and lip at the stages when anthocyanin accumulated (stage1-4). Phchs3 and phchs4 were expressed at much lower levels than phchs5. Phchs3 was expressed in pigmented tissue (including lip, petal and sepal) at middle stages (stages 2-4) and in colorless reproductive tissue at late stage (stage 5). Phchs4 was only expressed in petal at earlier stages (stage 1-3) and in lip at middle stage (stage 4). These results present new data on differentiation of gene expression among duplicate copies of chs genes in Phalaenopsis.

Functional evaluation of a novel constitutive promoter F1 of Bacillus pumilus, as a rice epiphytic strain, and construction of an efficient expression and secretion system under the control of F1.

To establish a constitutive, high-efficiency expression and secretion system for Bacillus pumilus, the function of a promoter and the abilities of three signal peptides in B. pumilus DX01 were tested. F1, cloned from the rice epiphyte B. pumilus strain DX01, had strong transcription activity and was a vegetative-phase constitutive promoter. The signal sequences of Bacillus subtilis levansucrase (sacB) and subtilisin, as well as B. pumilus DX01 RNase signal sequence could drive the secretion of E. coli beta-lactamase from B. pumilus DX01 efficiently, among which the signal sequence of B. subtilis sacB was the most effective. Likewise, they could also direct the secretion of green fluorescence protein (GFP) from DX01.

Characterization of a rice (Oryza sativa L.) gene encoding a temperature-dependent chloroplast omega-3 fatty acid desaturase.

A cDNA, designated Osfad8, encoding a chloroplast omega-3 fatty acid desaturase responsible for trienoic fatty acid formation, was isolated from the leaves of Oryza sativa L. by RT-PCR. Southern blot hybridization indicated that a small gene family composed of two copies or closely linked genes exists. RNA in situ hybridization showed that the accumulation of Osfad8 mRNA was abundant in leaves but hardly detectable in roots. The Osfad8 transcript level in leaves was much higher at 15 degrees C than at normal temperature (25 degrees C). In situ hybridization also showed particularly prominent expression of Osfad8 in the palisade layer and spongy parenchyma cells of leaves when exposed to 15 degrees C conditions for 5 days and 10 days. Two transgenic lines (8S-52 and 8S-101) harboring the Osfad8 ORF in sense orientation under the control of the CaMV 35S promoter contained increased levels of hexadecatrienoic (16:3) and linolenic (18:3) fatty acids. When exposed to 2 degrees C for 7 days, the damage observed to the control plants was significantly alleviated in the 8S-52 and 8S-101 lines. The amounts of trienoic fatty acids in an Osfad8 antisense line (8A-35) declined 40.2% compared to the control plants. The 8A-35 plants survived after growth at 44 degrees C for 3 days while the control plants died. These data suggest that Osfad8 encodes a temperature-dependent chloroplast omega-3 fatty acid desaturase.

Cloning of a NaCl-induced fructose-1, 6-diphosphate aldolase cDNA from Dunaliella salina and its expression in tobacco.

Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA encoding a NaCl-induced fructose-1, 6-diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homology (66%-73%) to chloroplast fructose-1, 6-diphosphate aldolase (AldP) in many plants according to their amino acid sequences. The phylogenetic analysis further confirmed that AldP in alga is the nearest to DsALDP. As to its expression pattern, DsALDP was de novo synthesized by NaCl induction. Its expression level was significantly changed with inducing time. After the selected DsALDP cDNA subcloned into a binary vector pBI121, the new construct was introduced into tobacco by Agrobacterium tumefaciens. The results of Southern blot and RT-PCR analysis of four transgenic T1 plants indicated that DsALDP was integrated into genome of these transgenic plants and effectively expressed. Aldolase activities have been detected in T1-1, T1-2 and T1-3 plants by bioassay under 100-200 mmol/L NaCl. It was also observed that proline contents in them were differentially increased.

Phylogenetic analysis reveals that a dwarfing disease on different cereal crops in China is due to rice black streaked dwarf virus (RBSDV).

A viral disease with dwarfing symptoms is associated with severe damage of different cereal crops including rice, maize, wheat and sorghum grown in China. It is believed that the pathogenic agent of the disease on rice and sorghum is rice black streaked dwarf virus (RBSDV), however, the cause of maize dwarf disease in China is still inconclusive. In this report, dsRNA was isolated from virus particles obtained from the diseased plants of rice, maize, wheat and sorghum from two Chinese provinces. Full-length cDNAs of genome segments 9 (S9) and 10 (S 10) were obtained through a RT-PCR approach. Sequence analysis showed that the S9 sequences of Chinese isolates and Japanese RBSDV isolate were very similar to each other (89.1-89.6% identity at the nucleotide level, 92.3-92.9% and 95.8-98.6% identity at the amino acid level for ORF1 and ORF2, respectively). In addition, the S10 sequences of Chinese isolates and Japanese RBSDV were very similar to each other (93.0-95.4% identical nucleotides and 96.2-97.0% identical amino acids, respectively). However, there were lower similarities for S9 and S10 sequences between Chinese isolates and an Italian Maize Rough Dwarf Virus (MRDV) isolate. Phylogenetic analysis indicates that Chinese viral isolates found to infect rice, maize, wheat and sorghum and leading to similar cereal dwarfing manifestations could be grouped to the same virus species, RBSDV.