In recent years, Tomato yellow leaf curl China virus (TYLCCNV) is spreading continuously from south to north in China, which caused the yield reduction, even no yield and serious economy loss. We aimed to obtain transgenic tobacco to immune to TYLCCNV. In this study, we constructed inverted-repeat dsRNA (double-strand RNA)(pBIN19-2CP) with TYLCCNV partial of coat protein gene(187 bp) using RNA interference technology to silence the expression of the target gene. In order to get the transgenic tobacco, the dsRNA had been transformed into Nicotiana benthamiana. Restriction endonuclease Kpn I and Xho I were introduced to amplify the reverse fragment, and restriction endonuclease BamHI and Hind Ⅲ were introduced to amplify the forward fragment. pBIN19 was used as plant expression vector. The inverted-repeat dsRNA recombinant was carried out by PCR analysis, restriction enzyme digestion and DNA sequencing, the results were consistent with expected. The recombinant was transferred into Agrobacterium tumefaciens LBA4404 with the freeze-thaw method, then recombinant A. tumefaciens was transformed into tobacco by leaf discs method. And the infection medium was MS, the co-cultivation medium was MS+6-BA (1 mg/L)+NAA (0.1 mg/L), the selection medium was MS+6-BA(1 mg/L)+ NAA(0.1 mg/L)+Kn(50 mg/L)+Cef(250 mg/L), the rooting medium was MS+NAA(0.2 mg/L)+Kn(50 mg/L)+ Cef (250 mg/L). We had obtained the transgenic tobacco plants. To evaluate the transgenic tobacco, PCR screening, Southern blot and Northern blot were carried out. The PCR screening results of the transgenic plants showed that the positive plants were obtained among the transgenic plants. Some positive plants were chose randomly to conduct Southern blot, the result showed that the target gene had been integrated into tobacco genome. When transgenic tobacco plants were inoculated with TYLCCNV, symptom observation and PCR screening demonstrated that among 41 transgenic tobacco plants, 6 plants (14.6%) were immune type, 5 (12.2%) were resistant type and 30 plants (73.2%) were susceptible type. Northern blotting analysis indicated that the levels of target mRNA accumulation varied among transgenic N. benthamiana lines, and inverse correlation between target mRNA accumulation and virus resistance was found. The transcript accumulation in the immune type transgenic tobacco plants was little, but the level of transcript accumulation in the resistant type and the susceptible type transgenic tobacco plants were obviously high. This study has certain theoretical directive significance for controlling TYLCCNV using RNA interference technology.

Gene expression or transcription profile of plant cells changes after inoculation with Agrobacterium tumefaciens. To understand the dynamic transcription and the modulation of metabolism pathways in plant infected with Agrobacterium tumefaciens, transcriptomic data of Arabidopsis thaliana inflorescence stalks from 3 h and 6 d after wounding or inoculation with GV3101 were analyzed by performing the statistical method RAM and the pathway analysis program MapMan. The results showed that 4 651 genes were differentially transcribed in the inflorescence stalks after inoculation with GV3101 while 3 885 genes changed in transcription after wounding, among which 1 045 genes were common. When inoculated with GV3101, the number of differently transcribed genes related to primary metabolism was reduced in comparison with the reference. Subcategorization of genes in stress category showed that 60 genes coding for PR proteins (pathogenesis-related proteins) were transcribed differentially after inoculation with GV3101, among which 37 genes coding for NB-LRR (nucleotide-binding site, leucine-rich repeat) disease resistance proteins were down-regulated. While in reference stalks, only 18 PR proteins-encoding genes were differentially transcribed, among which 6 genes coding for NB-LRR proteins were transcribed negatively. These results suggested that Agrobacterium GV3101 modulates gene transcriptional profile in Arabidopsis thaliana and suppresses disease resistance of host plant cells, which might facilitate the successful integration of T-DNA into host genome.

In order to study the enzymatic properties of purified pectate lyase, the pectate lyase gene (pel) from Dickeya sp. DCE-01, an efficient strain for bast fiber bio-extracting, will be cloned, and its prokaryotic expression system will be constructed. Primers were designed by the potential pel G403 annotated from the whole genome sequence of Dickeya sp. DCE-01. The pel was cloned, linked to pEASY-E1, and expressed in Escherichia coli BL21(DE3). The extracellular pectate lyase (Pel) was purified by the two-step process involving ultrafiltration and gel filtration (Sephadex G-100) and its enzymatic properties were studied. The results showed that a pel gene (GenBank accession No. JX964998) coding for pectate lyase (Pel) was cloned from the genome of Dickeya sp. DCE-01 and expressed successfully in Escherichia coli BL21 (DE3). The pel gene contained an ORF of 1 164 bp, encoding 387 amino acids. The pectate lyase deduced by the nucleotide sequences included a signal peptide of 35 amino acids, and a calculated molecular mass of 37.8 kD for the mature protein, and 41.4 kD for the precursor. Using polygalacturonic acid sodium as substrate, a maximum activity of 27.82 IU/mL was obtained from fermentation filtrate of E. coli harboring the pectate lyase gene. After the two-step process purification, the pectate lyase exhibited one prominent band at about 38 kD on SDS-PAGE gel. With minor impurities, the purity of pectate lyase was approximate 98.6% determined by Gel Analyzer 2010 software. The optimal reaction temperature of the pectate lyase was at 55℃ and was stable at no more than 60℃ after being incubated for 100 min. The optimal reaction pH of the pectate lyase was pH 9.5 and was stable at pH 9.0 to 10.0. Pectin from apple was the optimum substrates for this Pel comparing with pectin from citrus and polygalacturonic acid sodium. Ca2+ was indispensable for the Pel catalytic activity and the maximal activity of Pel was obtained at Ca2+ concentration of 1.5 mmol/L. However, the Pel activity was inhibited seriously by Mn2+, Pb2+ and EDTA. In summary, a pectate lyase, encoding by the pel cloned from the Dickeya sp. DCE-01, was successfully expressed in E. coli BL21(DE3) and its thermophilicity and alkalophilicity may be valuable for biomass biorefinery.

Adiponectin(Adp) is an adipocytokine maily secreted by adipocytes. Evidence suggests adiponectin action on the reproductive system. Recent researches in roden showed that Adp inhibits LH secretion mediated by AdpR in pituitary. The aims of this study were to observe the correlation and developmental patterns of adiponectin receptors and reproduction-related factors mRNA expressions in pituitary of Wannahua pigs. The test animals were sampled on 0, 30, 45, 90, and 180 d of age, including 5 boars and 5 sows at each different days of age The development patterns of adiponectin receptors(AdpR1, AdpR2), Gonadotropin Releasing Hormone Receptor(GnRHR), Luteinizing Hormone(LH-β), and Follicle Stimulating Hormone(FSH-β) mRNA in pituitary were investigated and their correlation were analyzed by Real-time fluorescent quantitative PCR(RT-PCR) while the concentration of LH and FSH in serum at different days of age were detected by ELISA. The results showed that: 1) The expression of AdpR2 mRNA were significantly changed with age increasing(P<0.05); The mRNA expression of AdpR1 in different days were significantly higher than that of AdpR2 at any days of age, while the expression of AdpR1 at birth、30 and 180 d of boars were significantly higher than that of those of sows. 2) The expression of LH-β at different growth stages and FSH-β mRNA in different days of sows were significantly changed with age increasing(P<0.05); The expression of FSH-β at birth of sows was significantly higher than that of boars(P<0.05) and GnRHR at 90 d of boars was significantly higher than that of sows(P<0.05). The LH and FSH level in serum were significantly changed with age increasing(P<0.01). The FSH contents in different days of sows were higher than those of boars. 3)The expression of AdpR1 in different days of boars were negatively correlated with GnRHR, FSH-β and LH-β respectively(P<0.05). The expression of AdpR2 of boars were negatively correlated with GnRHR(P<0.05). The expression of AdpR2 in different days of sows were negatively correlated with GnRHR、LH-β mRNA and FSH level in serum respectively(P<0.01 or P<0.05). Furthermore, the mRNA expression of AdpR1 and AdpR2 were significant positive correlation(P<0.01 or P<0.05)both in boars and in sows and GnRHR were significantly positive correlated with FSH-β(P<0.05)and LH-β(P<0.01) respectively. Our results indicated that the adiponectin may be mediated by AdpR and inhibit secretion of FSH and LH by GnRHR or directly in pituitary.

Melatonin is a hormone secreted by the pineal gland and retina, which has a wide range of biological functions. To investigate the effects of the melatonin receptor gene (MTNR1A) on laying traits of Shouguang chickens, the single nucleotide polymorphisms (SNPs) of the 5' regulatory region of MTNR1A gene were detected by direct sequencing and their associations with laying traits were analyzed in Shouguang chickens(Gallus gallus). Eight SNPs loci were found in the 5' regulatory region of MTNR1A gene. The three loci of -34, -151 and -617 with lower polymorphism information content (PIC) values of 0.13, 0.12 and 0.05 respectively, and the other loci of -100, -110, -126, -329 and -538 with intermediate PIC values of 0.37, 0.37, 0.37, 0.37 and 0.36 respectively. The loci of -100, -110, -126, and -329 significantly associated with the age at first egg and the egg production during 20~25 weeks of age (P<0.05). Haplotype analysis showed that the three loci of -100, -110 and -126 closely linked together, three haplotypes (TTT, TTC and GCT) and six dipoltypes were constructed. Compared with haplotype GCT, hens with TTC had earlier ages at first egg by 2.6 d and increased 1.4 eggs during 20~25 weeks of age (P<0.05). In conclusion, the 5' regulatory region of MTNR1A gene are highly polymorphic and the haplotype TTC can be used as candidate molecular markers for improving the sexual maturity in Shouguang chickens.

Ochratoxin A (OTA) is a mycotoxin contaminating food and feedstuffs. OTA causes nephrotoxic and hepatotoxic in experimental animals. In the present study, we investigated the oxidative damage and DNA damage induced by OTA. The results showed that OTA inhibited cell proliferation in a time and dose-independent manner, and increased the release of lactate dehydrogenase (LDH), glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). We had also observed a dose-independent production of reactive oxygen species (ROS) and the decrease of superoxide dismutase (SOD) activity in HepG2 cells. OTA-induced DNA strand break, as detected by comet assay, was demonstrated in all group tested. Our work was the first time to research the effect of OTA on the global DNA methylation. The detection of 5-methyl-deoxycytidine (5mdC) indicated that OTA lowered the level of DNA methylation, but there was no significant difference among the groups. These results show that OTA induces oxidative damage, and disrupts the stability and integrity of DNA, which may be involved in OTA-induced hepatotoxicity.

Functional analysis of the pathogencity related genes during the colonization process of rice blast fungus Magnaporthe oryzae will contribute to uncovering its molecular pathogenesis mechanism. In this study, the function of putative phenylacetone monooxygenase gene (MoPAMO1) in M. oryzae was analyzed through bioinformatics and molecular genetics. The results showed that MoPAMO1 encoded a putative phenylacetone monooxygenase, which is most close to the ortholog of filamentous fungi Verticillium dahliae according to the phylogenetic analysis. The expression of MoPAMO1 was induced during the infection process of M. oryzae. MoPAMO1 deletion mutants displayed fewer conidia, while the vegetative growth and patheogencity showed as normal as the wild type strain(P<0.01)．Our results indicated that MoPAMO1 was involved in conidiation of M. oryzae, and its relationship with vegetative growth and pathogenicity was still undefined since there were other 5 putative phenylacetone monooxygenase genes, which may be functional redundancy. Our research establishes a foundation for further clarifying the biological function of MoPAMO1.

Ribosomal protein has been proved to play an important role in protein synthesis, cell proliferation, apoptosis, development and some kinds of regulation. In order to investigate the physiological role of ribosomal protein gene in sea cucumber, full-length cDNA sequence of ribosomal protein L17(RPL17) gene was cloned from the body wall of sea cucumber (Apostichopus japonicus) using molecular cloning method in this study. The results showed that the cDNA (GenBank accession No. JQ922561) length was 643 bp, including the 33 bp of the 5'-UTR and 58 bp of the 3'-UTR. The open reading frame was 522 bp, which encoding polypeptides of 183 amino acids, with a calculated relative molecular mass of 21.5010 kD and pI of 10.29. The RPL17 was a kind of hydrophilic and unstable protein, with grand average of hydropathicity (GRAVY) of －0.964. By Blastn and Blastx searching in NCBI, the sea cucumber homologies of gene sequence and deduced amino acid sequence of RPL17 with other kinds of animals were over 70%, and the homologies between the sea cucumber and sea urchin (Strongylocentrotus purpuratus) were 75% and 81%, respectively. The predicted secondary structure of RPL17 was consisted of four recondary structure elements, including 36.61% of alpha helix, 7.65% of beta turn, 42.08% of random coil and 13.66% of extended strand. The RPL17 contained 1 functional site (RPL22 signature site)and 9 phosphorylated sites (3 cAMP- and cGMP-dependent protein kinase phosphorylation sites, 4 Protein kinase C phosphorylation sites and 2 casein kinase II phosphorylation sites). Phylogenetic relationship analysis, which was similar to the results of the biological classification, indicated that A. japonicus was firstly clustered with S. purpuratus, and also closely related with Ciona intestinalis and Xenopus laevis. And all vertebrates were clustered together. Real-time PCR was performed to analyze the expression profiling of RPL17, results showed that mRNA of RPL17 could be expressed in 5 different tissues in sea cucumber. The highest expression levels occurred in coelomocytes(P<0.01), and then in the intestine(P<0.05), body wall and longitudinal muscle. There were no significant differences in the following two tissues. The lowest expression was detected in the respiratory tree. The relative expression level in coelomocytes was 5.67, 21.63, 43.25 and 346 folds of those in intestine, body wall, longitudinal muscle and the respiratory tree. The sequences and mRNA expression level of RPL17 can be a necessary basis of study in mechanism of protein synthesis, regenerate and other physiological regulation in sea cucumber.

The major histocompatibility complex (MHC) gene in chicken, which has a high degree of polymorphism and stability, and closely associated with resistance and immune response, is the preferred marker gene for disease resistance research in poultry breeding. In order to explore the distribution of polymorphism in MHC B-LβⅡ(MHC class Ⅱ) gene exon 2 and the effects of its polymorphism on immune function, PCR-RFLP technique was applied to analyze the polymorphism of MHC B-LβⅡ gene exon 2 Hin1Ⅰ locus and its impact on some immune traits (lymphocyte proliferation, antibody response kinetics to Newcastle disease(ND) vaccine, concentration of Interferon-γ(IFN-γ), Interleukin-4(IL-4) and Interleukin-12(IL-12)) in 170 Liangfeng Cyan-shank partridge chickens(Gallus gallus). The results showed that two alleles (A, B) and three genotypes (AA, AB and BB) were detected by digestion of restriction endonuclease Hin1Ⅰ, and these frequencies were 0.553, 0.447 and 0.235, 0.636 and 0.129, respectively; and the lymphocyte proliferation, concentration of ND antibody, IFN-γ and IL-4 of genotype AA, AB were higher than that of genotype BB, while there were no significant differences between AA, AB and BB type in the concentration of IL-12. The results indicated that MHC B-LβⅡ gene Hin1Ⅱ locus polymorphism has a certain influence on the immune function of chicken，and it can be treated as a candidate gene for disease resistance.

Salicornia bigelovii Torr. is one of the most salt-tolerant plant species in the world. In order to study the mechanism of salt tolerance and isolate new genes related to salt tolerance in S. bigelovii Torr., a normalized full-length cDNA library in S. bigelovii Torr. was constructed by SMART TM(switching mechanism at 5’end of RNA transcript) technique. The primary library had a titer of 5.46×105 pfu/mL, and it’s recombination rate reached 96.7%. The average size range of cDNA inserts was 0.5~2 kb, 60％ of them more than 1.0 kb. 100 clones were randomly selected and sequenced, and then preliminary analysis was performed using bioinformatics software, 65 gene sequences were generated from the cDNA library. By Blast searches, we found that 35 genes showed significant similarity to the annotated genes, and the remaining 30 genes represented novel genes without any annotation. In addition, we analyzed SbX and SbSAMS mRNA level by semi-quantity RT-PCR in S. bigelovii Torr. under 400 mmol/L NaCl. We found that SbX was up-regulated by salt stress, and the expression of SbSAMS was opposite. The results may provide basic information about the mechanism of salt tolerance in plants, improvement and germplasm enhancement of crop genetic traits.

Naringin is a bittering agent of kinds of hydrotestosterone flavonoids and the rhamnosyltransferase is a key enzyme in the synthesis of naringin. In this study we cloned a 1,2rhamnosyltransferase gene(Cm1,2RhaT) which wa from Citrus maxima(Burm.) Merr. cv. Liangpin Yu. The Cm1, 2RhaT gene expression vector pET28a-CmRhaT was constructed by the gene fragment cloned into the prokaryotic expression vector pET-28a(+), which was then transformed into Escherichia coli BL21(DE3). The objective fusion protein was induced and expressed. Further fusion protein was induced to express and purified. The SYBR Green Real-time qRT-PCR was employed to analyze the expression differences of Cm1,2RhaT in the fruits and leaves's different developmental stages of Citrus maxima (Burm.) Merr. cv. Liangpin Yu and other four citrus varieties.The results showed that Cm1,2RhaT was isolated and characterized from Citrus maxima (Burm.) Merr. cv. Liangpin Yu. The accessed cDNA (GenBank accession No. JQ217381) was 1 436 bp in length encoding 453 deduced amino acids with a predicted molecular mass of 51.2 kD. We obtained the fusion protein after isolation and purification. Its concentration and purity were 0.526 mg/mL and 95%, respectively. Real-time fluorescence qPCR(RT-PCR)showed that the Cm1,2 RhaT expressed in the Liangpingyou's fruits and leaves. The expression level of Cm1,2 RhaT was reduced gradually with the fruit developing from 30~80 d after flowering. Its homologous genes in other species also showed the same as volatility expression patterns as in Liangpingyou. Cm1, 2RhaT which was amplified from Citrus maxima (Burm.) Merr. cv. Liangpin Yu expressed a kind of enzyme with typical glycosyl transfer group. It is a key enzyme in the synthesis of fiavanone which is one of Citrus′s bitter substances. The enzyme involves in secondary metabolism process of Citrus. To sum up, the research and results has certain significance to not-bitter citrus's varieties improvement.

Severe crop yield losses are caused by the rhizotoxicities of aluminum (Al3+) and protons (H+) in acid soil. Some plant species and cultivars have evolved mechanisms for detoxifying Al3+ and H+, but little is known about molecular mechanisms of their tolerance. Here, we reported a gene, MsSTAR2(GenBank accession no. KC430619), encoded a transmembrane domain of a bacterial-type ABC transporter in Medicago sativa L., and shared a high similarity with rice(Oryza sativa) OsSTAR2 and Arabidopsis thaliana AtALS3, which had been implicated in aluminum tolerance. MsSTAR2 was constitutively expressed in all tissues and it was predominantly expressed at leaves. Different with OsSTAR2 and AtALS3, either Al3+ or H+ increased the expression of MsSTAR2, but the gene didn't respond to other metal ions, such as Cd and Mn. Furthermore, we found that there was no positive correlation between the amount of gene expression and aluminum resistance in Medicago Sativa L. All these results indicated that MsSTAR2 is an aluminum-activated gene in Medicago Sativa L., and it may induce the protection mechanism caused by aluminum damage, which probably functions as a bacterial-type ABC transporter by forming a complex with a nucleotide binding domain protein.

Gene silencing caused by small RNAs provides new opportunities to explore pathogen-host interactions and potential strategies for disease control over the past years. Plant pathogenic fungi(PPF) are major disease agents responsible for crop losses and represent a serious threat to the future of food stocks and global security. Therefore, knowledge of the pathogenetic mechanism of PPF as well as the resistance mechanism of host plant, development of new and improved strategies to control diseases caused by these large group of PPF are primary challenges for the scientific community. Host-induced gene silencing (HIGS), developed on the basis of RNAi, is a new technology which has been shown to be an auspicious strategy for the development of transgenic plants to control fungal diseases and proved a novel tool to address gene function in obligate biotrophic pathogens. More and more studies have shown that the expression of silencing constructs in plants designed on fungal genes can specifically silence their targets in invading pathogenic fungi. This review described the molecular mechanisms, development, application prospects, and the advantages or disadvantages of HIGS in functional verification of pathogenic fungi genes. In addition, the prominent basic questions needed to be solved of HIGS are discussed.

Several countries and authorities have established relevant regulations on labeling for the surveillance of genetically modified organisms (GMOs). Therefore, it is imperative to establish appropriate methods to detect transgenic products. In this study, a loop-mediated isothermal amplification method (LAMP) was established for the rapid detection of genetically modified (GM) maize(Zea mays L.) LY038. Four LAMP primers were designed based on the cordapA gene. The LAMP assay was performed under isothermal conditions at 63℃ for 50 min. The specificity of the four primers was verified using eight other maize samples, in combination with enzymatic analysis using EcoR V to digest the LAMP product. All the results showed that the specificity of the primers was satisfying. To compare the detection limit of LAMP assay with PCR assay, seven kinds of different LY038 contents samples (100%, 10%, 1%, 0.5%, 0.1%, 0.05% and 0.01%) were prepared. The limit of detection of LAMP assay was 0.01%, which was 5-fold of traditional PCR assay. The established LAMP method has high specificity, high sensitivity and stability. Besides, it doesn’t need special equipment, and it is simple, which shows a good application prospect in primary and field use.

Tetraploid plants usually grow more vigorously and produce larger flowers than their diploids. To breed tetraploid poted Asiatic lilies, the present study carried out the crosses among Asiatic lily(Lilium) cultivars Petit Brigitte, Orange Pixie, Black Bird, and Pollyanna with normal artificial pollination, then, the scales of the progenies obtained from three crossing combination were treated 4 h with 0.001%, 0.002%, 0.003% and 0.005% Oryzalin, and the regenerated bulblets were analyzed with chromosome preparation and fluorescence in situ hybridization(FISH) technique. The results showed that the chromosome doubling rates were 19% and 23% with 0.003% and 0.005% Oryzalin respectively, and FISH not only confirmed the chromosome doubled plants, but indicated that some particular chromosomes might not be doubled in some plants. The present result conformed that Oryzalin is effective to double lily chromosomes, and that cytological method, combined with FISH technique, enable individual chromosomes whether to be doubled or not more accurately.

The simultaneous transfer of multiple genes into plants helps researchers to improve complex traits in plants. In this study, a spacer sequence cloned from pGWB605 was inserted into pXCS-HAStrep, and the co-expression vector named pXCS-Dgene-HAStrep was constructed. HbWRKY11 and HbWRKY7 were cloned into pXCS-Dgene-HAStrep. The plasmid was then introduced into Agrobacterium strain GV3101 (pMP90RK) and transformed into WT Arabidopsis thaliana (Col-0). Eighty-four Basta resistant lines were obtained, and eight of which were randomly selected for semi-quantitative RT-PCR and qRT-PCR analysis. The results showed that HbWRKY11 and HbWRKY7 could express simultaneously, and the expression level of HbWRKY7 was higher than that of HbWRKY11, but the difference was not significant (Pr=0.4867>0.05). The different expression levels of HbWRKY11 and HbWRKY7 were observed among transgenic lines. Among 8 transgenic lines, the highest expression levels of HbWRKY11 and HbWRKY7 were detected in two single-copy T-DNA-transformed lines, S2 and S5. Four single-copy transgenic Arabidopsis lines (L3, L4, J1, J5) had been constructed by overexpressing HbWRKY11 and HbWRKY7 in previous study, respectively. T3 seedlings of L3, L4, J1, J5, S2 and S5 were investigated under cold and jasmonic acid (JA) stress. The results showed that cold- responsive AtCOR47 was strongly induced in all of six lines and wild type genotype under cold stress, and the highest expression level of AtCOR47 appeared in line J5 when all of six lines were treated under cold stress for 24 h. The increase level of AtCOR47 in four single-gene overexpressing lines was higher than that in wild type genotype under cold stress; otherwise the increase level in double-gene co-overexpressing lines was lower than that in wild type genotype. The increase level of soluble sugar content in double-gene co-overexpressing lines was also lower than other lines. Although JA-responsive AtCOI1 was induced under JA stress, the increase level of AtCOI1 in sigle-gene overexpressing lines was lower than that in wild type genotype, and the highest increase level was in double-gene co-overexpressing lines when they were treated under JA stress for 6 h. All the results indicated that two genes can express seperately under one ORF cassette in transgenic plant.