Construction and Identification of HSP70 Antisense RNA Expression Vector for Genetic Engineering Male Sterility in Plant

[ Objective] In order to study the relation between the HSPTO gene and male sterility of plant further. [ Methods ] Anther specific expression promoter Osg6B of rice was coloned by PCR then connected with HSP70 antisense fragment to construct HSPTO antisense expression vector. The expression vector was identified by PCR experiment and enzyme digestion. [ Result] The sequence of coloned Osg6B promoter had 97% homology to the published sequence, and the cis-regulatory element in promoter area was integrated. HSP70 antisense expression vector driven by the promoter Osg6B was confired by colony PCR and enzyme digestion. [ Conclusion] The construction of expression vector would lay solid foundation for utilization of genetic engineering male sterility of plant.

Gene engineering in swine for agriculture

Domestic pigs are the second most important source of meat world-wide, and the genetic improvement of economic traits, such as meat production, growth, and disease resistance, is a critical point for efficient production in pigs. Through conventional breeding and selection programs in pigs, which are painstakingly slow processes, some economic traits, such as growth and backfat, have been greatly improved over the past several decades. However, the improvement of many polygenetic traits is still very slow and challenging to be improved by conventional breeding strategies. The development of reproductive knowledge and a variety of techniques, including foreign gene transfer strategies, somatic cell nuclear transfer（SCNT） and particularly, recently developed nuclease-mediated genome editing tools, has provided efficient ways to produce genetically modified（GM） pigs for the dramatic improvement of economic traits. In this review, we briefly discuss the progress of genomic markers used in pig breeding program, trace the history of genetic engineering, mainly focusing on the progress of recently developed genome editing tools, and summarize the GM pigs which have been generated to aim at the agricultural purposes. We also discuss the specific challenges facing application of gene engineering in pig breeding, and future prospects.

GENE ENGINEERING EB VIRUS MEMBRANE ANTIGEN IN DETECTION OF MA-IgA ANTIBODY(COMPARISON WITH VCA-IgA AND EA-IgA ANTIBODIES)

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluo-rescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pretreatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.

Neurodegenerative diseases and gene engineering

Totally three articles focusing on ＂molecular biological mechanism by which gene modification and RNA interference techniques interfere Parkinson’s disease and Alzheimer’s disease＂ are published in three issues. We hope that our readers find these papers useful to their research.

Identification of a gene engineering antibody against cystic echinococcosis in liver

OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selection technique with the recombinant antigen on solid surface. The positive clones were demonstrated by ELISA and their DNA sequences were also determined. RESULTS: The DNA sequence data showed that the antibody gene is composed of 768bp. In addition, a specific combination capacity with recombinant Echinococcus granulosus antigen B (r-EgB) was demonstrated by ELISA. CONCLUSION: The obtained gene engineering antibody against r-EgB may have potential implications in immunological treatment and drug targeting delivery.

A remarkable progress in breeding new rice line through gene engineering

Much progress has been made in the research of breeding new rice line through gene engineering by Life Science College of Fudan Univ, Shanghai, and the Plant Science Res Inst of Shanghai Acad of Agri. It was the first time internationally that the research adopted a man-combined gene, taking agrobacterium as car-

Status and Advances of Molecular Genetic Improvement of Poplar Species in China

Poplars are among the most important deciduous tree species in China. China is replete with natural resources of poplars. Poplars have a number of good characteristics, including fast growth rate, high yield, many uses, easiness of tissue culture and small gene group that make them well suited as a model system for the application of genetic engineering in forest trees. In the last decade, much progress has been made in genetic improvement of poplar species in China. Modern biotechnology is an important tool for genetic improvement in forest trees, and its applications to genetic improvement in poplars, which covers genetic transformation, gene expression, construction of genetic linkage map, QTLs (quantitative trait loci) identification and molecular assisted selection are reviewed in this paper. At the same time, the existing problems and outlook about the application of modern biotechnology to genetic improvement in forest trees are also discussed.

Molecular Cloning of a Cytosolic G6PDH Gene from Populus Suaveolens and Its Expression to Improve the Cold Resistance of Tobacco Plants

Glucose-6-phosphate dehydrogenase (G6PDH,EC 1.1.1.49) is the first and main regulated enzyme of oxidative pentose phosphate pathway (OPPP),catalyzing the conversion of glucose-6-phosphate to 6-phospho-gluconolactone and playing important roles in the growth and development of plants. It is preciously reported that the enhancement of freezing resistance of Populus suaveolenscuttings is clear related to the distinct increase in cytosolic G6PDH activity. Here,a 1697 bp cDNA fragment (PsG6PDH) is amplified by RT-PCR from cold-induced total RNA of the freezing-tolerant P. suaveolens. A sequence analysis showed that PsG6PDH coding region had 1 530 bp and encoded 510 predicted amino acid residues. Genomic Southern analysis revealed that the isoform is encoded by a few copies of the gene in the poplar genome. The cloned gene PsG6PDHis cloned into binary vector pBI121 and used to transform tobacco. PCR and Southern blotting results verified integration of this gene into the genome of tobacco. Moreover,cold treatment experiments and membrane defense enzymeactivity analysis confirmed that overexpression of the PsG6PDHgene could enhance the tolerance to cold or frigid stresses in transgenic plants.

Construction of a Tapetum-Specific and Tetracycline-Inducible System

A regulated gene expression system would offer the unique opportunity to study the gene physiological functions at different developmental stages. For realizing gene special expression in plant anther at given time, we constructed a new system that combined tetracycline- inducible elements with TA29 promoter, a tapetum-specific promoter of tobacco. The system was tested in transient GUS assay system by electroporation (gene gun) transformation of tobacco ( Nicotiana tabacum L. cv. Winsconsin 38) anther. In the absence of tetracycline as the inducer, no GUS activity was detected. However, strong GUS expression was observed in tapetum. tissue upon tetracycline induction, and little GUS activity was found outside the tapetum. Our results suggested that gene expression can be restricted to a specific tissue at the given time under the control of this new system, and this system would be a very useful tool for both basic plant biology research and biotechnological applications.

Creation of Male Sterile Line in Tobacco With HSP70 Anti-sense Fragment

In order to create the Male Sterile Line in tobacco, the anti-sense fragment of HSP70 gene was linked to anther specific expression promoter TA29 and the reconstructed vector was transformed into tobacco by Agrobacterium mediated transformation, and the transformants were then screened. Gus and spot blotting hybridization analysis of the transformants indicated that anti-sense fragment of HSF70 gene had been integrated into tobacco genome and expressed, thus the male sterile tobacco line was obtained. Microscope observation of anther and pollen showed that pistils of transgenic tobacco were normal, whereas anthers and pollens were fairly abortive in the same transgenic tobacco flower, comparing with pistils and stamens in control plants. The ratio of HSI：＇70 protein before and after heat shock in mitochondrial was found to be 1.39 in control tobacco plants and 1.01 in transgenic tobacco sterile lines. This is suggested that the anti-sense gene fragment of HSP70 can effectively inhibit the expression of HSP70 protein and lead to transgenic male sterility in tobacco flowers. The assay provided a new genetic engineering method for male sterility creation in plants.

Gene engineered exosome reverses T cell exhaustion in cancer immunotherapy

Cancer patients by immune checkpoint therapy have achieved long-term remission,with no recurrence of clinical symptoms of cancer for many years.Nevertheless,more than half of cancer patients are not responsive to this therapy due to immune exhaustion.Here,we report a novel gene engineered exosome which is rationally designed by engineering PD1 gene and simultaneously enveloping an immune adjuvant imiquimod(PD1-Imi Exo)for boosting response of cancer immune checkpoint blockage therapy.The results showed that PD1-Imi Exo had a vesicular round shape(approximately 139 nm),revealed a significant targeting and a strong binding effect with both cancer cell and dendritic cell,and demonstrated a remarkable therapeutic efficacy in the melanoma-bearing mice and in the breast cancer-bearing mice.The mechanism was associated with two facts that PD1-Imi Exo blocked the binding of CD8^(+)T cell with cancer cell,displaying a PD1/PDL1 immune checkpoint blockage effect,and that imiquimod released from PD1-Imi Exo promoted the maturation of immature dendritic cell,exhibiting a reversing effect on the immune exhaustion through activating and restoring function of CD8^(+)T cell.In conclusion,the gene engineered exosome could be used for reversing T cell exhaustion in cancer immunotherapy.This study also offers a promising new strategy for enhancing PD1/PDL1 therapeutic efficacy,preventing tumor recurrence or metastasis after surgery by rebuilding the patients’immunity,thus consolidating the overall prognosis.

Molecular breeding of hybrid rice

This paper describes in a general way the objectives, contents and methods of molecular breeding, and the application of molecular breeding in hybrid rice.

Progress in the Study of Molecular Genetic Improvements of Poplar in China

The poplar is one of the most economically important and intensively studied tree species owing to its wide application in the timber industry and as a model material for the study of woody plants. The natural resource of poplars in China is replete. Over the past 10 years, the application of molecular biological techniques to genetic improvements in poplar species has been widely studied in China. Recent advances in molecular genetic improvements of poplar, including cDNA library construction, gene cloning and identification, genetic engineering, gene expression, genetic linkage map construction, mapping of quantitative trait loci （QTL） and molecular-assisted selection, are reviewed in the present paper. In addition, the application of modern biotechnology to molecular improvements in the genetic traits of the poplar and some unsolved problems are discussed.

TALENs:Customizable Molecular DNA Scissors for Genome Engineering of Plants

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.

Gene-guided OX40L anchoring to tumor cells for synergetic tumor“self-killing”immunotherapy

The low objective response rates and severe side effects largely limit the clinical outcomes of immune checkpoint blockade(ICB)therapy.Here,a tumor“self-killing”therapy based on gene-guided OX40L anchoring to tumor cell membrane was reported to boost ICB therapy.We developed a highly efficient delivery system HA/PEI-KT(HKT)to co-deliver the OX40L plasmids and unmethylated CG-enriched oligodeoxynucleotide(CpG).On the one hand,CpG induced the expression of OX40 on T cells within tumors.On the other hand,OX40L plasmids achieved the OX40L anchoring on the tumor cell membrane to next promote T cells responses via OX40/OX40L axis.Such synergistic tumor“self-killing”strategy finally turned“cold”tumors to“hot”,to sensitize tumors to programmed cell death protein 1/programmed cell death ligand 1(PD-1/PD-L1)blockade therapy,and promoted an immune-mediated tumor regression in both B16F10 and 4T1 tumor models,with prevention of tumor recurrence and metastasis.To avoid the side effects,the gene-guided OX40L anchoring and PD-L1 silencing was proposed to replace the existing antibody therapy,which showed negligible toxicity in vivo.Our work provided a new possibility for tumor“self-killing”immunotherapy to treated various solid tumors.

Gene editing nuclease and its application in tilapia

Gene editing nucleases including zinc-finger nucleases(ZFNs), transcription activator like effector nucleases(TALENs) and clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated(Cas) system(CRISPR/Cas9) provide powerful tools that improve our ability to understand the physiological processes and their underlying mechanisms. To date, these approaches have already been widely used to generate knockout and knockin models in a large number of species. Fishes comprise nearly half of extant vertebrate species and provide excellent models for studying many aspects of biology. In this review, we present an overview of recent advances in the use of gene editing nucleases for studies of fish species. We focus particularly on the use of TALENs and CRISPR/Cas9 genome editing for studying sex determination in tilapia.