西洋参转基因冠瘿组织培养及人参总皂苷含量

高压纸层析电泳证实了根癌农杆菌C58对西洋参茎的转化成功,得到西洋参冠瘿组织。考察了冠瘿组织生长及皂苷合成的最佳培养基和收获期,以及光照、温度、培养基初始pH值、碳源、氮源等因子的影响。

荔枝转基因胚性愈伤组织原生质体分离条件的优化

对荔枝品种下番枝转基因胚性愈伤组织原生质体分离条件进行了优化.结果表明:转基因胚性愈伤组织抗性细胞系Q811培养16-18 d后,转入含8 g.L-1纤维素酶Cellu lose R-10和4 g.L-1离析酶M acrease R-10的CPW+130 g.L-1甘露醇酶液中,在(25±2)℃、黑暗条件下,连续振荡(30-40 r.m in-1)10-11 h进行酶解,转基因原生质体产量达到18.2×106个.g-1,存活率达97.5%.

小麦非组织培养转基因技术研究及展望

小麦是重要的粮食作物,其转基因技术体系的研究一直是学界的热点和难点。目前,通过组织培养方法为基础的小麦转基因技术,已获得了大量的转基因材料,但由于组织培养方法具有较强的基因型依赖性,且操作复杂、耗时较长、易产生体细胞变异等原因,限制了该技术的普及。鉴于此,作为重要的备选方案,非组织培养方法受到学界的关注,小麦非组织培养转基因技术也有了广泛的研究。本文总结了小麦非组织培养转基因技术的研究现状,通过分析不同方法的潜在受体位点,并与其他植物的相关研究进行比较,探讨了小麦非组织培养转基因技术的未来发展方向。

Isolation of a Genomic DNA for Gastrodia Antifungal Protein and Analyses of Its Promoter in Transgenic Tobacco

A genomic DNA containing 5'-upstream region and complete open reading frame of a Gastrodia antifungal protein was isolated by screening of a genomic library from Gastrodia elata B1. To investigate the promoter activity, the 5'-flanking region - 1 157 lip upstream from the putative transcription start site was fused to the coding sequence of beta-glucuronidase (GUS) gene and transformed into Nicotiana tabacum. The strongest GUS activity was detected in the roots of transgenic tobacco, followed by stems. The leaves only showed a low GUS activity. Furthermore, the promoter established inducible expression pattern in transgenic tobacco upon fungus Trichoderma viride inoculation and jasmonic acid and salicylic acid treatments.

Insect Resistance of Different Tissues of Transgenic Cotton to Spodoptera exigua(Hbner)

[Objective] The aim was to learn the resistance of different tissues and organs of transgenic cotton to Spodoptera exigua （Hbner）. [Method] Flowers,the 1st,the 3rd,the 6th and the 14th leaves from the top of 33B,GK12 and SGK321 were used to feed S. exigua neonates respectively. Survival larvae and dead ones were counted on the 3rd,the 7th,the 10th,the 16th and the 19th day; meanwhile,the pupae amount was recorded,and the pupae weight was measured at the 24th h after pupation. [Result] The survival curves,pupation rates and pupae weights of S. exigua feeding on different tissues of transgenic cotton were not significantly different from those of S. exigua feeding on the corresponding tissues of conventional cotton; pupation rate of S. exigua feeding on different leaves of the same cotton variety were not significantly different from each other,but all higher than that of S. exigua feeding on the flowers of that cotton; and there were no differences among pupation weights of S. exigua feeding on different leaves or flowers of the same cotton variety. [Conclusion] Transgenic cotton showed weak resistance to S. exigua. Hence,in the transgenic cotton fields,more attention should be paid to occurrence trend of S. exigua and its control.

Lymph node micrometastasis and its correlation with MMP-2 expression in gastric carcinoma

AIM： To examine matrix metalloproteinase-2 （MMP-2） expression in gastric cancer tissues and to evaluate its relationship with lymph node micrometastasis. MATERIALS： The authors studied 850 lymph nodes resected from 30 patients with gastric carcinoma who underwent gastrectomy with lymphadenetomy using reverse transcription polymerase chain reaction （RT-PCR） assay in addition to H-E staining. MMP-2 expression of the tumor tissues was detected by immunohistochemical technique （EliVision^TM plus）. RESULTS： MMP-2 expression was positive in 21 （70%） cases and negative in g （30%） cases. No significant correlations were found between MMP-2 expression and other variables such as age, gender, tumor location, tumor diameter, Lauren classification and lymphatic invasion. In contrast, MMP-2 expression correlated significantly with depth of tumor infiltration （P = 0.022）, lymph node metastasis （P = 0.030） and tumor differentiation （P = 0.043）. Lymph node micrometastases were detected in 77 （12.5%） lymph nodes of 14 （46.7%） gastric carcinoma patients. MMP-2 expression was positive in 12 （85.7%） of the 14 patients with lymph node micrometastasis, and in g （56.3%） of the 16 patients without lymph node micrometastasis （P = 0.118）. CONCLUSIONS： Our results demonstrate that MMP-2 expression has significant correlation with tumor invasion, tumor differentiation and lymph node metastases. MMP-2 expression may be an important biological characteristics and significant prognostic parameter of gastric carcinoma. We also conclude that MMP-2 may participate in the development of lymph node micrometastasis of gastric carcinoma. Further investigations are needed to draw a conclusion.

A Preliminary Study on Tissue Culture and Transformation of Casuarina equisetifolia L.

[Objective] The paper aimed to study the tissue culture and transformation of C. equisetifolia. [Method] C. equisetifolia were used as experimental materials to explore the effects of three conditions including callus induction, adventitious bud dif- ferentiation and Agrobacterium-mediated transformation on transformation rate of C. equisetifolia. [Result] The appropriate plant growth regulator combination on induction and differentiation of C. equisetifolia adventitious buds was DCR＋5.0 mg/L of 6-BA＋ 0.5 mg/L of NAA; hygromycin was selected for the selective pressure and co-culture time was 3 d; 94 stains of transgenic C. equisetifolia were obtained with the initially- established transgene system via Agrobacterium-mediated method, and 61 stains were PCR-positive plants according to the results of PCR detection. [Conclusion] The study had laid the foundation for tissue culture and transgene research of C. equi- setifolia.

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcat- egory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed fre- quently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post- transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

Down-regulation of miR-622 in gastric cancer promotes cellular invasion and tumor metastasis by targeting ING1 gene

AIM:To evaluate the biological and clinical characteristics of miR-622 in gastric cancer. METHODS:We analyzed the expression of miR-622 in 57 pair matched gastric neoplastic and adjacent non-neoplastic tissues by quantitative real-time polymerase chain reaction. Functional analysis of miR-622 expression was assessed in vitro in gastric cancer cell lines with miR-622 precursor and inhibitor. The roles of miR-622 in tumorigenesis and tumor metastasis were analyzed using a stable miR-622 expression plasmid in nude mice. A luciferase reporter assay was used to assess the effect of miR-622 on inhibitor of growth family,member 1 (ING1) expression. RESULTS:Expression of miR-622 was down-regulated in gastric cancer. MiR-622 was found involved in differentia-tion and lymphatic metastasis in human gastric cancer. Ectopic expression of miR-622 promoted invasion,tumorigenesis and metastasis of gastric cancer cells both in vitro and in vivo. ING1 is a direct target of miR-622. CONCLUSION:These findings help clarify the molecular mechanisms involved in gastric cancer metastasis and indicate that miR-622 modulation may be a bona fide treatment of gastric cancer.

Expressions of chemokine receptor CXCR4 and its ligand CXCL12 in salivary adenoid cystic carcinoma

Objective: To examine expressions of chemokine receptor CXCR4 and its ligand CXCL12 in primary focus and lymphogenous metastasis of salivary adenoid cystic carcinoma (ACC) with lung metastasis. Methods: Using immunohistochemical hypersensitivity catalyzed signal amplification (CSA), expressions of chemokine receptor CXCR4 and ligand CXCL12 were detected in tissue specimens from 20 cases of primary cancer focus and lymphogenous metastasis of salivary adenoid cystic carcinoma, of which 7 cases were associated with lung metastasis and 3 with lympogenous metastasis. Twenty cases of tongue carcinoma (including 10 cases with lymphogenous metastasis) and 15 cases of mucoepidermoid carcinoma (including 5 cases with lymphogenous metastasis) were used as the malignant control group;and salivary mixed tumor (n=10), tongue leukoceratosis (n=10) and cervical lymph node reactive hyperplasia (n=10) were used as the benign control group. Results: Expression of CXCR4 in the tissues and lymph metastases of oral and maxillofacial salivary ACC, mucoepidermoid carcinoma and tongue carcinoma was significantly higher than that of the benign control group (P<0.05); expression of CXCR4 in the primary focus of ACC was significantly higher than that of the malignant control group; and expression of CXCR4 in the ACC with lung metastasis was 87.1% (6/7), significantly higher than that without lung metastasis(P<0.01). There was evident positive expression of CXCL12 in endotheliocytes of microvessels within cancer and paracancer tissues and significantly high expression of CXCL12 in lymphogenous metastasis(P<0.05). Conclusion: Chemokine receptor CXCR4 and its ligand CXCL12 may be associated with local invasion and lymphogenous metastasis of oral and maxillofacial cancer, especially with lung metastasis of salivary ACC.

Establishment of Plant Acceptor System for Hyperosmosis Transformation

Calli and adventitious buds induced from different explants such as young stems and leaves of tomato variety Moneymaker,and mature embryos and young spikelets of rice variety TP309 were used to establish hyperosmotic transformation system at various osmotica treatments.The results revealed that the calli induced from tomato young leaves and rice young spikelets were the ideal transfomation acceptor.The cells of calli were still vigorous when treated with 0.75 mol/L hyperosmotic solution for 4 hours.The differentiation rates of calli varied from 7.5% to 93.3% in different media.The bud differentiation was apparently inhibited by hyperosmotic treatments.0.75mol/L sucrose hypertonic solution and 0.2mol/L CaCl2 solution were favorable hyperosmoticum and hypcosmoticum respectively.

The expression of Elf-1 and Ki-67 and their correlations in non-small-cell lung cancer

Objective: Elf-1 is a member of the proto-oncogenes Ets-related transcription factor family and over-expressed in many human tumors, Ki-67 is an important nuclear antigen associated with cell proliferation. This study investigated the expression of Elf-1 and Ki-67 in non-small-cell lung cancer(NSCLC) and studied their correlation with the clinicopathological features. Methods: Tissue microarray from 64 cases lung cancer tissue and 10 cases normal lung tissue was constructed, immunohistochemical method was used to evaluate the protein expressions of Elf-1 and Ki-67, correlations of the expression of Elf-1 and Ki-67 to clinicopathological features of NSCLC were analyzed. Results: Expression of Elf-1 and Ki-67 in NSCLC tissues were significantly higher than in normal lung tissues(P < 0.05), the positive rate of Elf-1 and Ki-67 was 73.44% and 64.06% in NSCLC group, Overexpression of Elf-1 in NSCLC was significantly related to histopathological grading, different clinical staging and the intensity of ELF-1 expression was significantly higher in the group with lymph node metastasis than that without(P < 0.05). Overexpression of Ki-67 was also closely related to tumor differentiation, clinical stages and lymph node metastasis(P < 0.05). In addition positive correlation was found between the expressive intensity of Elf-1 and Ki-67(τ = 0.295, P = 0.018). Conclusion: The high expression and positive correlation of Elf-1 and Ki-67 in NSCLC suggest that they probably play a role in onset and progression of lung cancer, united detecting their expression could be used as an valuable molecular biological index for predicting the malignant behavior and early diagnosis of NSCLC.

Transgenic Date Palm Containing Endotoxin Cry3Aa Gene

Date palm, like all other crops, is very sensitive to the injury by many insect pests, which may lead to the death of the affected plant and causes decrease in yield. In the present study, an efficient Agrobacterium for genetic transformation was successfully achieved for well known date palm （Phoenix dactylifera L.） cv. Medjool and Khalas using callus as explant. Embryogenic callus were recorded 100% mortality when cultured on MS medium containing 100 mg/L kanamycin with different cultivars, thus it was chosen for the selection of transformed explants. Embryogenic callus of Medjool and Khalas were incubated with Agrobacterium tumefaciens strain LBA 4404 for 0.5, 1, 2, 4 and 24 h on LB medium. After the incubation periods, embryogenic callus was transferred to MS medium with 0.1 mg/L NAA, 0.05 mg/L BA, 250 mg/L carbenicillin and 100 mg/L kanamycin for detection of transgenic embryogenic callus. Polymerase chain reaction （PCR） was used for the rapid screening of Cry3Aa gene. For screening, total genomic DNA was isolated from transformants. Using primer specific to Cry3Aa gene （forward and reverse）, a PCR product with a size of about 2,000 bp was amplified when all nucleic acid from the transformants were utilized as templates. PCR analysis confirmed the appearance of the transgene of 2,000 bp in one individual plantlet. Presence and integration of foreign Cry3Aa gene in regenerated kanamycin resistant embryogenic callus was also confirmed by Southern blot hybridization. It was found that one transgenic embryogenic callus for both Medjool and Khalas showed a single copy of gene integration. These results signify the successful transfer of Cry3Aa gene into date palm plant.

Isolation of a pollen-specific promoter in tritordeum

The promoter is a cis-acting element in regulating gene expression. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic tritordeum was carried to examine tissue-specific expression of GUS(beta-glucuronidase) activity. The pollen-specific promoter was trapped and identified successfully in a transformant line. PCR(polymerase chain reaction) method was used to isolate this pollen-specific promoter. By sequencing and analyzing the amplified fragment from PCR, a part of UidA gene and a flanking sequence were obtained. Some essential elements of plant promoters were found in the sequence. To determine the function of it, the cloned fragment was fused with UidA gene, then cloned and transformed into Triticum durum. The transgenic plant transformed by this vector showed GUS expression only in pollen. Therefore a pollen-specific promoter was isolated successfully.

ATP-binding cassette transporter enhances tolerance to DDT in Tetrahymena

The reuse of dichlorodiphenyltrichloroethane(DDT) as an indoor residual spray was permitted by the World Health Organization in 2007, and approximately 14 countries still use DDT to control disease vectors. The extensive exposure of insects to DDT has resulted in the emergence of DDT resistance, especially in mosquitoes, and the mechanism for this resistance in mosquitoes has been widely reported. Spraying can also introduce DDT directly into surface water, and DDT can subsequently accumulate in microorganisms, but the mechanism for the resistance to DDT degradation in microorganisms is unclear. Using whole-genome microarray analysis, we detected an abcb15 gene that was up-regulated in a specific manner by DDT treatment in T. thermophile. The deduced ABCB15 peptide sequence had two transmembrane domains(TMDs) and two nucleotide-binding domains(NBDs) to form the structure TMD-NBD-TMD-NBD, and each NBD contained three conserved motifs: Walker-A, C-loop, and Walker-B, which indicated the T. thermophila abcb15 was a typical ABC transporter gene. The expression of ABCB15 fused with a C-terminal green fluorescent protein was found to be on the periphery of the cell, suggesting that ABCB15 was a membrane pump protein. In addition, cells with abcb15 partially knocked down(abcb15-KD) grew slower than wild-type cells in the presence of 256 mg L-1 DDT, indicating the tolerance of abcb15-KD strain to DDT exposure was decreased. Thus, we suggest that in Tetrahymena, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance to DDT and protect cells from this exogenous toxin by efficiently pumping it to the extracellular space.

Identification and analysis of mouse non-coding RNA using transcriptome data

Transcripts are expressed spatially and temporally and they are very complicated, precise and specific; however, most studies are focused on protein-coding related genes. Recently, massively parallel c DNA sequencing(RNA-seq) has emerged to be a new and promising tool for transcriptome research, and numbers of non-coding RNAs, especially linc RNAs, have been widely identified and well characterized as important regulators of diverse biological processes. In this study, we used ultra-deep RNA-seq data from 15 mouse tissues to study the diversity and dynamic of non-coding RNAs in mouse. Using our own criteria, we identified totally 16,249 non-coding genes(21,569 non-coding RNAs) in mouse. We annotated these non-coding RNAs by diverse properties and found non-coding RNAs are generally shorter, have fewer exons, express in lower level and are more strikingly tissue-specific compared with protein-coding genes. Moreover, these non-coding RNAs show significant enrichment with transcriptional initiation and elongation signals including histone modifications(H3K4me3, H3K27me3 and H3K36me3), RNAPII binding sites and CAGE tags. The gene set enrichment analysis(GSEA) result revealed several sets of linc RNAs associated with diverse biological processes such as immune effector process, muscle development and sexual reproduction. Taken together, this study provides a more comprehensive annotation of mouse non-coding RNAs and gives an opportunity for future functional and evolutionary study of mouse non-coding RNAs.