A newly identified cluster of glutathione S-transferase genes provides Verticillium wilt resistance in cotton.

As the largest cultivated fiber crop in the world, cotton (Gossypium hirsutum) is often exposed to various biotic stresses during its growth periods. Verticillium wilt caused by Verticillium dahliae is a severe disease in cotton, and the molecular mechanism of cotton resistance for Verticillium wilt needs to be further investigated. Here, we revealed that the cotton genome contains nine types of GST genes. An evolutionary analysis showed that a newly identified cluster (including Gh_A09G1508, Gh_A09G1509 and Gh_A09G1510) located on chromosome 09 of the A-subgenome was under positive selection pressure during the formation of an allotetraploid. Transcriptome analysis showed that this cluster participates in Verticillium wilt resistance. Because the Gh_A09G1509 gene showed the greatest differential expression in the resistant cultivar under V. dahliae stress, we overexpressed this gene in tobacco and found that its overexpression resulted in enhanced Verticillium wilt resistance. Suppression of the gene cluster via virus-induced gene silencing made cotton plants of the resistant cultivar Nongda601 significantly susceptible. These results demonstrated that the GST cluster played an important role in Verticillium wilt resistance. Further investigation showed that the encoded enzymes of the cluster were essential for the delicate equilibrium between the production and scavenging of H2 O2 during V. dahliae stress.

[Characterization of Plasma Induced by Laser Effect on Coal Sample].

With the output of an OPG/OPA pumped by the third harmonic output 355 nm of a pulsed Nd·YAG laser as radiation source, the emission spectrum of laser induced coal sample plasma is created. The emission spectral line shows the character of Lorenz profile. So Stark broadening is the main widening way of this plasma system. The spatial distribution of the plasma temperature and electron density is measured from the intensity and Stark broadening of the spectral lines. It is found that in the direction from vertical to plasma luminous flame, both plasma temperature and electron density are symmetrically relative to the center. While in the direction of parallel to plasma luminous flame, they are asymmetrically relative to the center. Plasma temperature and electron density is maximized in the centre of the flame, and the emission intensity of the plasma in the centre is also strong. So we ought to collect the emission spectrum in the plasma centre when using the technique of spectroscopy for the diagnosis of plasma characteristics. It is also found that there is a dip in the centre of some spectral lines. This indicates that there exists strong self-absorption in the plasma. The appearance of self-absorption varies with laser wavelength. It is most obvious when the wavelength is near to the center of the profile, because the transition probability is the largest at the center of the profile. Both emission intensity and self-absorption increase with laser energy. These experimental results can be interpreted as the increase of the particle density with laser energy. Thus we ought to select spectral lines with no self-absorption when measuring the parameters of the plasma with the technique of laser spectroscopy. This can ensure higher detection accuracy.

[Laser induced breakdown spectra of coal sample and self-absorption of the spectral line].

The LIBS of one kind of household fuel coal was obtained with the first harmonic output 532 nm of an Nd·YAG laser as radiation source. With the assignment of the spectral lines, it was found that besides the elements C, Si, Mg, Fe, Al, Ca, Ti, Na and K, which are reported to be contained in coal, the presented sample also contains trace elements, such as Cd, Co, Hf, Ir, Li, Mn, Ni, Rb, Sr, V, W, Zn, Zr etc, but the spectral lines corresponding to O and H elements did not appear in the spectra. This is owing to the facts that the transition probability of H and O atoms is small and the energy of the upper level for transition is higher. The results of measurement also show that the intensity of spectral line increases with the laser pulse energy and self-absorption of the spectral lines K766.493 nm and K769.921 nm will appear to some extent. Increasing laser energy further will make self-absorption more obvious. The presence of self-absorption can be attributed to two factors. One is the higher transition rate of K atoms, and the other is that the increase in laser intensity induces the enhancement of the particle number density in the plasma.

Transcriptome profiling of Gossypium barbadense inoculated with Verticillium dahliae provides a resource for cotton improvement.

BACKGROUND: Verticillium wilt, caused by the fungal pathogen Verticillium dahliae, is the most severe disease in cotton (Gossypium spp.), causing great lint losses worldwide. Disease management could be achieved in the field if genetically improved, resistant plants were used. However, the interaction between V. dahliae and cotton is a complicated process, and its molecular mechanism remains obscure. To understand better the defense response to this pathogen as a means for obtaining more tolerant cultivars, we monitored the transcriptome profiles of roots from resistant plants of G. barbadense cv. Pima90-53 that were challenged with V. dahliae. RESULTS: In all, 46,192 high-quality expressed sequence tags (ESTs) were generated from a full-length cDNA library of G. barbadense. They were clustered and assembled into 23126 unigenes that comprised 2661 contigs and 20465 singletons. Those unigenes were assigned Gene Ontology terms and mapped to 289 KEGG pathways. A total of 3027 unigenes were found to be homologous to known defense-related genes in other plants. They were assigned to the functional classification of plant-pathogen interactions, including disease defenses and signal transduction. The branch of "SA→NPR1→TGA→PR-1→Disease resistance" was first discovered in the interaction of cotton-V. dahliae, indicating that this wilt process includes both biotrophic and necrotrophic stages. In all, 4936 genes coding for putative transcription factors (TF) were identified in our library. The most abundant TF family was the NAC group (527), followed by G2-like (440), MYB (372), BHLH (331), bZIP (271) ERF, C3H, and WRKY. We also analyzed the expression of genes involved in pathogen-associated molecular pattern (PAMP) recognition, the activation of effector-triggered immunity, TFs, and hormone biosynthesis, as well as genes that are pathogenesis-related, or have roles in signaling/regulatory functions and cell wall modification. Their differential expression patterns were compared among mock-/inoculated- and resistant/susceptible cotton. Our results suggest that the cotton defense response has significant transcriptional complexity and that large accumulations of defense-related transcripts may contribute to V. dahliae resistance in cotton. Therefore, these data provide a resource for cotton improvement through molecular breeding approaches. CONCLUSIONS: This study generated a substantial amount of cotton transcript sequences that are related to defense responses against V. dahliae. These genomics resources and knowledge of important related genes contribute to our understanding of host-pathogen interactions and the defense mechanisms utilized by G. barbadense, a non-model plant system. These tools can be applied in establishing a modern breeding program that uses marker-assisted selections and oligonucleotide arrays to identify candidate genes that can be linked to valuable agronomic traits in cotton, including disease resistance.

[Theoretical study of 1+2+1 double-resonance multiphoton ionization probability].

The technique of resonance enhanced multiphoton ionization (REMPI) spectroscopy has already become an important method for studying the energy level structure of atoms and molecules. Analytic expression of 1+2+1 double REMPI probability of four-level system has been deduced with the theory of rate equation. Based on the expression, the present paper simulated the variations of ionization probability versus laser intensity, laser pulse duration and collision relaxation rate. It was found that in the 1+2+1 ionization mechanism the ionization probability increases with laser intensity, until saturation phenomenon appears in one and two excitation steps. If laser intensity increases further, the ionization probability will oscillate around the saturation value, and the oscillation amplitude will increase with laser intensity. With regard to the influence of laser pulse duration and collision relaxation rate on the ionization probability, the results show that the ionization probability increases from zero to the saturation value 1 with the increase in laser pulse duration, while it decreases linearly with the increase in collision relaxation rate.

[Investigation on internal energy transfer and relaxation kinetics of NO2 by photoacoustic and fluorescence emission spectra].

With 532 nm laser as excitation source, the excitation and relaxation process of NO2 molecule was investigated by the technique of photoacoustic and fluorescence emission spectra. The results show that NO2 molecules will be pumped to the first excited electronic state by laser photon. When the sample pressure is lower, some of the excited molecules relax to the ground state by radiation process directly; the other parts are redistributed to a few of the excited rovibronic energy levels by the process of fast internal energy transfer. With the increase in the sample pressure, continual collisions dominate the relaxation process gradually. This makes the excited molecules to be redistributed to many excited rovibronic energy levels. Emission from these excited levels forms a continuous spectrum. Just then, the efficiency of fluorescence emission from laser excited level decreases and the fluorescence intensity on the long wavelength side increases. The intensity of PA signals increases also. These phenomena indicate that besides the relaxation process of radiation, there is a strong relaxation process of continual collision under the condition of higher sample pressure. It converts vibration energy of the excited molecules into translation one. This induces the increase in gas temperature and a sound wave is produced.

[Theoretical analysis of photoacoustic spectrum of NO molecule].

The technique of photoacoustic (PA) spectrum is based on the conversion of photon to acoustic energy by collision quenching of the excited molecules. It holds the characteristic of higher detection sensitivity, wide detection spectral region, no damage to the sample etc. It is used in many scientific observation areas such as gas composition analysis, research on chemistry and biology, environmental monitor and so on In the present paper, the analytic formula of the PA signal produced from the in teraction of intense laser with gas system was deduced by solving the dynamic rate equation about the interaction of photon and material. The results show that the magnitude of the PA signal depends on the factors of molecular absorption cross-section, laser intensity, photon number absorbed by the molecule and collision relaxation rate. With the aid of the relation of the PA signal versus laser intensity, the PA spectrum of NO molecule in the wavelength region of 420.0-470.0 nm is ascribed to the transition of X 2 pi (v" = 0) --> A 2 sigma (v' = 0, 1) and X 2 pi (v" = 0) --> E 2 sigma (v' = 2, 3, 4), F 2 sigma (v' = 1, 2, 3) and R 2 sigma (v' = 0, 1). These transitions are realized via two or three-photon process. The vibration constants of NO A 2 sigma, E 2 sigma, F 2 sigma and R 2 sigma electronic states were calculated from the wavelength of the spectral peaks. They are 2 346, 2 342, 2 397 and 2 381 cm(-1) respectively. The results are consistent with the one of other method. The phenomenon of saturation appears when the buffer gas pressure is high enough. This is owing to the finite excited molecules.

[Time decay spectrum of NO2 fluorescence radiation].

Investigation of the optical absorption and fluorescence of NO2 molecule has long been of interest because it is not only one of the key substances of air pollution, but also a stable molecule of nonzero spin and has many special properties such as that the vibronic levels of the first excited state are coupled strongly to the high vibration levels of the ground state, so that once NO2 molecules are excited, they must undergo complicated quenching process. The quenching mechanism influences the lifetime of the excited molecule severely. In the present paper, the fluorescence lifetime of NO2 excited electronic states are observed experimentally by the technique of LIF time decay spectroscopy and with an optical parameter generator and amplifier pumped by a Nd:YAG laser as excitation source. The results show that the fluorescence lifetime of excited NO2 molecules depends on the excitation wavelength and sample pressure. The time decay curves present a property of bi-exponential when the excitation wavelength is selected as 429.0, 452.0, 509.0 and 532.0 nm, respectively. This indicates that the fluorescence is composed of two components. One has a long lifetime, while the other has a short one. The short-lived component comes from the radiation of the molecules excited by A2B2, B2B1<--X2 A1 transition And the long one is owing to the radiation of the molecules excited to the high rovibronic levels of the ground electronic state. These levels are correlated with A2B2 state. The de-excitation mechanism of the excited molecules is investigated by measuring the variation in fluorescence lifetime versus the sample pressure. The conclusion is that the excited molecules corresponding to the short lifetime quench mainly through the process of radiation and fast inner conversion. As to the excited molecules with long lifetime, the de-excitation process is not only radiation, but also the non-radiation process of collision.

[Self-absorption of NO fluorescence induced by two-photon].

The two-photon LIDFS of NO excited with 452.4 nm output of an optical parameter generator and optical parameter amplifier pumped by a Nd: YAG laser was obtained. The vibration frequency and the inharmonic coefficients of the ground electronic state of NO were calculated from the results of the spectral ascription. They are omega"e = (1 904. 7 +/- 7. 3) cm(-1), omega"e chi"e = (14.2 +/- 1.2) cm(-1), and omega"e y"e = -(0.021 8 +/- 0.009 1) cm(-1) respectively. It was found for the first time that the intensity of the spectral lines departs from the Frank-Condon principle due to the self-absorption of NO molecules. The spectral line of A 2sigma (v' = 0) --> X 2 II (v" = 0) transition disappears in the fluorescence spectra. But it appears gradually with decreasing the sample pressure and the distance from the laser-gas interaction volume to the receiving window. All of these provide an important reference for monitoring NO by the technique of laser-induced fluorescence.

[Study on the relationship between 22q11 microdeletion and congenital heart disease].

To investigate if microdeletion of chromosome 22q11 is an epidemiologically important cause of congenital heart disease (CHD), we studied 25 cases with CHD phenotypes. Venous blood samples were tested by fluorescence in situ hybridization (FISH) for microdeletion of 22q11. Among 23 cases with simple CHD, 19 were shown not to have microdeletion of 22q11 and the other 4 cases were shown to have 22q11 microdeletion. Microdeletion of 22q11 was found in 2 cases with Tetralogy of Fallot (TOF) accompanied by multiple malformations. The results suggested that microdeletion of 22q11 was associated with CHD.

[The 440-495 nm laser-induced fluorescence excitation spectrum of NO2].

Under the conditions of room temperature and low pressure, laser-induced fluorescence excitation spectrum of NQ2 in the wavelength region of 440-495 nm was obtained by using optical parameter generator (OPG) and amplifier (OPA) as an excitation source. The OPG/OPA was pumped by a Nd:YAG laser. The peaks of the spectrum are attributed to B2B1 <-- X2A1 transition. The angle-vibration frequency of B2B1 electronic state was calculated. The fluorescence lifetime of B2B1 (0, 9, 0) vibration state under the pressure of 15 Pa was also deduced by measuring the fluorescence time decay spectrum of this state. It is about 49 micros. Fitting the curve the fluorescence radiant lifetime versus pressure, the spontaneous radiant lifetimes tau0 and the rate coefficient kq of non-radiant transition relaxation of B2B1 (0, 9, 0) state are deduced. The results are: tau approximately 55 micros and k(q) = 1.2 x 10(-9) cm3 molecule(-1) s(-1).

[Laser induced dispersed fluorescence spectra of NO2 in the range of 550-740 nm].

Laser induced dispersed fluorescence spectra (LIDFS) of NO2 molecules, excited by second harmonic lines (the output wavelength is 532 nm) of a pulsed Nd: YAG laser at room temperature and at low and high NO2 pressures, were obtained and analyzed. The authors got vibronic progressions in the range of 550-740 nm at low pressure and ascribed them to the transitions from the first excitation electronic state A(2)B2 to the vibrational levels of the ground electronic state X(2)A1, and then calculated the harmonic frequencies of symmetry stretch and bond stretch: omega1 = 1300.72 cm(-1) and omega2 = 744.14 cm(-1), respectively. On the basis of what was above-mentioned, the authors compared the spectra at low pressure with those at high pressure and pointed out the difference between them. Then the authors also interpreted the obvious red shift of fluorescence spectra at high NO2 pressures in terms of a stepladder model of vibrational deactivation and obtained significative results.

[Study on the emission spectrum of B (3)pig state of N2 molecule].

DC glow discharge was used to study N2 molecule at low pressure (4 Torr). The emission spectrum was examined in the range of 320-470 nm in the discharge plasma of N2, which showed that the emission was composed of a series of spectral lines equidistant, and the relative emission intensity became weaker with longer wavelength, whose distribution accorded with Frank-Condon rule. It is attributed to the transition of C 3pi(u)-->B3pi(g). On this base, the authors have calculated the vibrational frequency of B 3pig state to be 1738.50 cm(-1). The Frank-Condon factor about C3pi(u) (v' = 0)-->B3H(g) (v" = 0-5) was calculated, which is consistent with the results of experiment.

[Study on the genetic alterations in MTX-resistant cells by differential polymerase chain reaction].

Gene amplification is a common mechanism that contributes to the drug resistance. To explore the molecular genetic background related to the MTX resistance in the mouse MTX-resistant cells, differential PCR was used to determine the amplification and overexpression of DHFR gene. In addition, the correlations between c-myc, p53 status and dhfr amplification were studied. Amplification and overexpression of dhfr suggested its role in MTX-resistant cells. However, no amplification and overexpression of c-myc were detected. On the other hand, no alteration of p53 copy number was found. The increased mRNA level of p53 suggested the normal function of p53. These results implicated the status of c-myc and p53 had no correlation with dhfr amplification, therefore some other molecular genetic alterations may exist to permit the dhfr amplification in MTX-resistant cells.

[Application of atomic force microscopy in the study of morphology of double minute chromosomes].

Atomic force microscopy (AFM) has many advantages in the study of biological samples, such as the convenient specimen preparation and the high resolution. In the present study, AFM was used to observe the double minute chromosomes (DMs) in mouse methotrexate-resistant cell line 3T3R500. AFM images were obtained by tapping mode, contact mode and later force mode of AFM. DMs were composed of two compact spheres connected with fibers. The number of DMs in the 3T3R500 cells increased with increasing levels of methotrexate (MTX) resistance. The data of the height and the underside diameter of the DMs were also obtained. The details of specimen preparation and scan mode selection of AFM were discussed. Our results show that AFM is a powerful method in the study of DMs.

[AFLP analysis of cotton with fusarium and verticillium wilts from the Huanghe and Changjiang valleys].

Genetic diversity among 101 cottons varieties, including 72 cultivars from the Huanghe valley and 29 cultivars from the Changjiang valley, was investigated using AFLP markers. In total, 20 primer combinations revealed 200 polymorphic bands among the Huanghe valley cottons and 127 polymorphic bands among the Changjiang valley, respectively. Euclidean distance values were calculated using SPSS (11.0) software. Average Euclidean distance value was 4.356 (the Huanghe valley) and 4.391 (the Changjiang valley), respectively. When the value was 15.2, 72 varieties from the Huanghe valley were divided into four groups (the Huanghe valley groups, abbrevate HVGs), including HVG1 (27), HVG2 (19), HVG3 (10) and HVG4 (16). 29 varieties from the Changjiang valley were classified into four groups (the Changjiang valley groups, abbreviate CVGs), including CVG1 (14), CVG2 (4), CVG3 (5) and CVG4 (6). Compared with Euclidean distance matrices and frequency distribution of pairwise Euclidean distances of cottons from the Huanghe and Changjiang valleys, it was showed that there was similar genetic diversity between the cotton cultivars from Huanghe valley and those from Changjiang valley.

[Mapping and expression analysis of a different expression cDNA fragment from lung adenocarcinoma cell line].

Lung cancer is one of the most common malignant tumors in humans. Metastasis is the basic biological feature of malignant tumors, which is the main cause of death. Molecular mechanism of metastasis is still unclear, although lots of studies have been done in tumor metastasis. To study and explore the molecular basis of metastasis in lung cancer, and isolate tumor metastasis-related genes, two human lung adenocarcinoma cell lines AGZY 83-a and Anip 973 were chosen as research materials. The Anip973 was derived from AGZY83-a, but manifested much higher metastasis potential than the parent line. Using mRNA differential display technique, an unknown cDNA fragment, OPB7-1, which is over-expressive in Anip973 cell line, was obtained. It was used as a template to isolate its corresponding cDNA through dbEST searching and PCR. To search and clone lung adenocarcinoma metastasis-related candidate gene, and to explore the molecular basis of development of lung carcinoma, differential expression of OPB7-1 cDNA fragment among 9 human lung adenocarcinoma cell lines and 12 normal human tissues were detected using cell culture, cDNA clone, Northern blot analysis and bioinformation technology. Results showed that there were significant differences in OPB7-1 expression among 9 human lung adenocarcinoma cell lines. High expression tendency was observed in Anip973 cell line with high metastasis potential, TKB-18 cell line with high invasion potential and GLC-82 cell line with low differentiation potential. Besides, a bigger fragment can be found in Anip973 cell line on the Northern blot hybridization. The 3.0 kb transcriptions were found in various tissues. Over-expression in heart and skeletal muscle could be observed, whereas expression in spleen, liver, kidney, placental and lung could be found except colon, thyroid gland and small intestine. These manifests indicate that OPB7-1 gene has a wide-rage expression in human multiple tissues. A 1.0 kb cDNA fragment was acquired by linking up EST fragments homologous match 5' end and PCR. BLAST analysis revealed that OPB7-1 gene has extremely low sequence identity with any known genes from GenBank and any sequences from EST database. The chromosomal localization of it was determined by RH location method. The OPB7-1 fragment was localized to chromosome 1p31-34. That OPB7-1 gene has an extensive expression pattern, may be a novel tumor gene related to lung carcinoma. Further research needs to be done to obtain the full-length cDNA of OPB7-1 gene. It will be helpful to investigate the expression in lung cancer cases and other tumor tissues for further determining the function of OPB7-1 gene in development of tumor.

[Studies of TGF-beta/Smads expression in lung cancer].

Smad proteins transduce signals from transforming growth factor beta superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. TGF-beta/Smads signal pathway not only has transforming potential but can also drive tumourigenesis, malignant progression, invasion and metastasis of human cancers. Using the immuno-histochemistry, we investigate the expression and location of TGF-beta R II, Smad2, Smad4 and Smad7 in 20 lung cancer specimens and 8 lung cancer cell lines. The results suggest that aberrant smads protein expression is significantly related to lung cancer tumoruigenesis and progression. Interestingly, TGF-beta R II and Smad7 strongly express in high metastasis cell lines. High expression of TGF-beta R II and smad7 in the cell lines with high-metastatic potential showed a conceivable TGF-beta signal pathway independent Smads in the lung cancer, and that might mediate invasion and metastasis of lung cancer.

[Y chromosome variations and hyplogroups from 15 biallelic markers in six Chinese populations].

By using 15 biallelic markers, 342 male individuals from six populations in China were genotyped with ASPCR (allele specific PCR). The 15 biallelic markers included M1 (YAP), M15 (9 bp insertion), M89 (C-->T), M9 (C-->G), M119 (A-->C), M50 (T-->C), M110 (T-->C), M103 (C-->T), M95 (C-->T), M88 (A-->G), M111 (2-bp deletion), M45 (G-->A), M122 (T-->C), M7 (C-->G) and M134 (1 bp deletion). The distribution of variation frequencies of 15 biallelic markers in six populations showed that with the extremely high frequencies of M9G (96.20% & 96.43%) and Han nationality displayed higher diversity than the four minority populations. It's noteworthy that M95T (82.14%) in Sichuan Han and M45A (18.57%) in Hui gave prominace to the two populations. The six populations displayed 34 (Fujian Han), 21 (Sichuan Han), 14 (Mongol), 26 (Hui), 10 (Xibo) and 8 (Hezhe) haplogroups respectively with 2, 1, 2, 1, 2 and 2 prominent haplogroups among them. Furthermore, the haplogroup analysis revealed that one predominant haplogroup was shared in the four minority populations and even two predominant haplogroups were shared in Mongol, Hezhe and Xibo. Unlike Han populations, the minority populations showed strikingly different haplogroups which were close to the ancestral pattern. However, the two Han populations exhibited divergence between them with the distinct frequencies of M89T and M95T. With the comparison of the number of people sharing the common haplogroups between any two of the four minority populations, relative genetic distance among them was deduced.

[Population genetic analysis of Y-chromosomal single nucleotide polymorphism in six Chinese populations].

OBJECTIVE: To analyze the genetic polymorphism of 15 single nucleotide polymorphism (SNP) loci on the nonrecombining portion of the Y chromosome in 6 populations in China. METHODS: Allelic specific polymerase chain reaction and 2% agarose gel electrophoresis and 6% PAGE were used to analyze the genetic polymorphism of 343 unrelated males, representing 6 populations in China, including Fujian Hans, Sichuan Hans, Mongolian, Hezhen, Sibo and Hui from the South, Northeast and Northwest. RESULTS: Thirty haplogroups were observed, and 3 of them (H15, H16, H18) were seen in all of the six populations. Although the heterozygosity levels of the Hezhen, Mongolian, Sibo populations are similar and those of the other 3 populations (Fujian Hans, Sichuan Hans, Hui) are similar, the pairwise differences among haplogroups are significant. Analysis of molecular variance (AMOVA) and principal component (PC) analysis of the haplogroup distributions suggested highly different allele diversity between group I including Hezhen, Mongolian, Sibo and group II including Hui, Fujian Hans, Sichuan Hans. CONCLUSION: The above analyses show more significant variance components in Northeast/South populations and clearly reveal the geographic genetic relationship among the six populations in the Northeast/Northwest/South. These results confirm the complexity of the genetic structure of Chinese populations and make a significant contribution for constructing the contemporary human gene pool and tracing genetic dispersal trail from Chinese populations.