Plants are sessile organisms suffering severe environmental conditions. Drought stress is one of the major environmental issues that affect plant growth and productivity. Although complex regulatory gene networks of plants under drought stress have been analyzed extensively, the response mechanism in the early stage of drought stress is still rarely mentioned. Here, we performed transcriptome analyses on cotton samples treated for a short time (10 min, 30 min, 60 min, 180 min) using 10% PEG, which is used to simulate drought stress. The analysis of differently expressed genes (DEGs) showed that the number of DEGs in roots was obviously more than that in stems and leaves at the four time points and maintained > 2000 FDEGs (DEGs appearing for the first time) from 10 min, indicating that root tissues of plants respond to drought stress quickly and continuously strongly. Gene ontology (GO) analysis showed that DEGs in roots were mainly enriched in protein modification and microtubule-based process. DEGs were found significantly enriched in phosphatidylinositol signaling system at 10 min through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, implying the great importance of phosphatidylinositol signal in the early stage of drought stress. What was more, two co-expression modules, which were significantly positively correlated with drought stress, were found by Weighted Gene Co-expression Network Analysis (WGCNA). From one of the co-expression modules, we identified a hub-gene Gohir. A07G058200, which is annotated as "phosphatidylinositol 3-and 4-kinase " in phosphatidylinositol signaling system, and found this gene may interact with auxin-responsive protein. This result suggested that Gohir. A07G058200 may be involved in the crosstalk of phosphatidylinositol signal and auxin signal in the early stage of drought stress. In summary, through transcriptome sequencing, we found that phosphatidylinositol signaling system is an important signal transduction pathway in early stage in response to drought stress, and it may interact with auxin signal transduction through phosphatidylinositol 3-and 4-kinase.

The Gossypium harknessii background cytoplasmic male sterility (CMS) system has been used in cotton hybrid breeding in China. However, the mechanism underlying pollen abortion and fertility restoration in CMS remains to be determined. In this study, we used RNA-seq to identify critical genes and pathways associated with CMS in G. harknessii based CMS lines (588A), the near isogenic restorer lines (588R), and maintainer lines (588B). We performed an assembly of 80,811,676 raw reads into 89,939 high-quality unigenes with an average length of 698 bp. Among these, 72.62% unigenes were annotated in public protein databases and were classified into functional clusters. In addition, we investigated the changes in expression of genes between 588A and 588B (588R); the RNA-seq data showed 742 differentially expressed genes (DEGs) between 588A and 588B and 748 DEGs between 588A and 588R. They were mainly down-regulated in 588A and most of them distributed in metabolic and biosynthesis of secondary metabolites pathways. Further analysis revealed 23 pollen development related genes were differentially expressed between 588A and 588B. Numerous genes associated with tapetum development were down-regulated in 588A, implicating tapetum dysplasia may be a key reason for pollen abortion in CMS lines. Also, among DEGs between 588A and 588R, we identified two PPR genes which were highly up-regulated in restorer line. This study may provide assistance for detailed molecular analysis and a better understanding of harknessii based CMS in cotton.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants. Most PPR genes are localized in mitochondria and chloroplasts functioning in regulation of plant growth and development, fertility restoration for cytoplasmic male sterility (CMS), and stress defense. In this study, using in silico cloning and PCR amplification with degenerate primers based on Arabidopsis PPR genes, we cloned eight new full-length PPR genes encoding protein sequences ranging from 458 to 875 amino acids, with 8 to 16 repetitive PPR elements in upland cotton and all of them lack introns. Expression analysis revealed that eight PPR genes were differently expressed in roots, stems, leaves, and floral buds. As for GhI12, its expression in floral buds at days 3–5 was significantly higher in line 777R (restorer line) than in line 777A (CMS line). Further tests with real-time PCR showed that GhI12 expression peaked at day 3 in 777R, followed by a gradual decline, while its expression fluctuated in 777A, peaking at day 5 and day 13. In addition, Gh155c17 and GhI12 were upregulated under salt stress. This is the first report of upland cotton PPR genes involved in salt stress response.

鸡α干扰素在防御病毒感染及治疗病毒性疾病中起着重要的作用.旨在利用家蚕杆状病毒表达系统研制有活性的鸡α干扰素.首先对鸡α干扰素基因(ChIFN-α)进行了优化与合成,将其克隆到杆状病毒转移载体pVL1393中,与ORF1629缺损的亲本病毒BmBcmid共转染,纯化重组病毒,再感染家蚕幼虫,收集蚕血淋巴以检测α干扰素基因的表达.Western blot分析结果显示,成功表达出分子量约为19 kD的鸡α干扰素.采用细胞病变抑制法在Vero-VSV*GFP系统中测定表达产物的抗病毒活性.所的表达的鸡α干扰素具有明显的抗病毒活性,活性不低于3.2×105 U/mL.利用杆状病毒载体系统成功地表达了具有抗病毒活性的鸡α干扰素的成熟蛋白,为开发廉价高效的鸡干扰素生物制剂奠定了物质基础.

为研究Cu/Zn-SOD基因在提高转基因植物抗逆性方面的作用,从一株地热芽孢杆菌（Geobacillus）中克隆得到Cu/Zn-SOD基因,以pZP211质粒为表达载体,构建了植物表达载体pZP211-Cu/ZnSOD,并通过农杆菌介导对烟草进行遗传转化。经PCR检测证明已获得转Cu/Zn-SOD基因的烟草。进而测定转基因烟草的SOD活力,结果表明Cu/Zn-SOD基因在烟草中高效表达。对转基因烟草进行耐盐性检测,证明Cu/Zn-SOD基因确实能够提高烟草对盐胁迫的耐受性。

Objective:To detect the methylation of DNA for Spodoptera litura nucleopolyhedrovirus Ⅱ(SpltNPVⅡ) was existed through the search.Method:Polymerase sequence of DNA and other three sequences in the total were chose,and detect the methylation of DNA by the method of bisulfite-modification.Result:DNA-methylation was taken place in the 14 bureaus of CG among the 246 bureaus of DNA polymerase.And 3 bureaus were taken place in the three sequences which were chose.Conclusion:DNA-methylation was existed in the SpltNPVⅡ.The methylation level of DNA-polymerase of DNA-polymerase was higher than other three sequence,however,difference had different levels.This result was important to future research about the Spodoptera litura nucleopolyhedrovirus.

Chloroplast transformation has several advantages,including high expression efficiency,high safety,stable transformation,and multiple genes in a single transformation event.Eukaryotic and prokaryotic foreign genes have been expressed successfully in the chloroplast,and more than twenty kinds of plants have been succeed in chloroplast transformation.It is noticeable that chloroplast,as a reactor,has gained an extensive application prospect.

This review overviewed mainly about the kinds of herbicide resistance genes, their main resource and the application of herbicide resistance genes. Moreover, we also discussed the discovery of the novel herbicide resistance genes, the modification of herbicide resistance gene using genetic and protein engineering, the mechanisms of herbicide resistance and new herbicide resistance transgenic crops breeding.

In current, Agrobacterium-mediated gene transferring is one of major methods used in genetic transformation of plants. This paper systematically reviewed the effect on plant transformation efficiency based on the introduction of the principle of this transformation, comparison among different kinds of Agrobacterium strains and the development to transformation vectors. In addition, the newly progresses on the Agrobacterium-mediated transformation of dicotyledonous and monocotyledons species transformation were also discussed, respectively.

Proteome analysis is becoming a powerful tool in identifying protein kinds and function.Chloroplast as an important photosynthesis organelle,its proteomics has been a hot research area,involving chloroplast total proteomics,subceller proteomics,differential proteomics,and the function of protein.This paper introduced mainly the technology of proteomics and new advances on chloroplast proteomics.

Rapid nucleic acid amplification in vitro is a revolutionary technology developed since 1983.It has been applied widely in modern agriculture,medical and food science etc..Such technology has provided a great tool for gene manipulation and functional genomics.In vitro nucleic acid amplification is usually carried out by a thermal stable enzyme,such as Taq polymerase,and requires thermal cycle equipment.The invention of isothermal amplification has improved the traditional PCR method and in some way simplified the reaction procedure.Recombinase-aid amplification(RAA) was introduced.RAA is the latest isothermal amplification technology which may be carried out under the room temperatures.The distinctive merit of RAA is mimicking in vivo T4-phage DNA amplification complex and the whole reaction is super fast and specific.Moreover,it can also be used for digital real time quantitative analysis.

Baculoviruses are obligate lethal pathogens of arthropodas.They have been highly valued and widely studied since being exploited as a expression vector in 1980s,for their excellent advantage including eukaryotic expression enviroment.In less than 30 years,great advances have achieved involving recombinant baculovirus construction and screening.Thus,the baculovirus expression vector system(BEVS)has become one of the four most popular expression system comprising bacterium,yeasts,mammalian cells and baculoviruses.The BEVS has been used in surface display,virus-like particles' production,mammalian cells' gene delivery,RNA interference,etc.The development and application of the BEVS is reviewed in this article.

Differential proteomics is an important branch subject of proteomic.Through comparative study of the protein expression profile,it reveals the progress and essence of physiological and pathological states of the cell.In recent years,differential proteomics of silkworm developed rapidly and involves extensive,and become the hot spot of silkworm research.This paper introduced the proteomics research methods and the research advancements in silkworm.

Nucleic acid amplification is a biotechnology that trace nucleic acid can be rapidly amplified in vitro.The technology has been applied widely in molecular biology,medical and forensic test etc.Such methodology has been continuously improved in its functionality and wideness of ultilization.Recombinase-aid amplification(RAA) is the most recent isothermal amplification technology,in which the classical thermal stable enzyme has been replaced by recombinase,single-stranded DNA binding(SSB) and DNA polymerase.Therefore,the RAA reaction can be carried out at optimized 37℃ or under the room temperatures without any heating assistance.The entire RAA reaction is very rapid,specific,power-saving and portable.These merits could largely facilitate RAA more applicable than PCR in the near future.

Origin recognition complex(ORC) binds to the origin of DNA replication and acts as a landing pad for the recruitment of other replication factors that form the pre-replication complex(pre-RC).Individual subunits of ORC have additional functions that are unrelated to DNA replication.Infection by B.mori nucleopolyhedrovirus(BmNPV) initially decreased and then increased the expression of BmORC at mRNA level after 36 hour,which were monitored by real-time PCR.The coordinate changes in the expression of all six BmORC mRNAs suggested that the BmORC proteins might also participate in the replication of B.mori nucleopolyhedrovirus(BmNPV).

In recently few years,people pay growing attention on the unicellular green alga Chlamydomonas reinhardtii,because it offers the potential to produce high yields of recombinant proteins more rapidly and cost lower than traditional cell culture.Being a model organism,Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation.Recombinant proteins can express in nuclear and chloroplast,but expression in the nuclear is superior to expression in the chloroplast.Recently,the successful expression of several proteins with pharmaceutical relevance has been reported from the chloroplastic genome of Chlamydomonas reinhardtii,proving capacity of Chlamydomonas reinhardtii chloroplast as a bioreactor.

In contrast to the situation of random integration of foreign genes in nuclear transformation, the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination. To establish an expression system for alien genes in rice chloroplast, the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study. Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique, and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator of psbA gene 3' sequence. Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct. Subsequently, the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained. Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome. Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome. Thus, the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome, but PPT-resistant trait for rice plants as well. It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.

A genomic library of Helicoverpa armigera multiple nucleocapsid nucleopolyhedrovirus(HearMNPV)was constructed by"partial filling-in" method,the inserted sequence showed high identity to that the 38k gene was identified and acquired,its open reading frame(ORF)has 903 base pairs which encoded 300 amino acids.It has a late gene motif TTAAG and behaves similarly to all baculovirus late gene.The amino acid sequence analysis showed that the HearMNPV 38K protein has higher identities with Alphabaculovirus and has more closer relationship with Alphabaculovirus.The prediction of 38K Protein tertiary structure showed its structure has 95% identity with that of phosphatase,and it was related with nucleocapsid assembling.

It has been reported that genes encoding antigens of bacterial and viral pathogens can be expressed in plants and are shown to induce protection antibodies. The structural protein E2 of classical swine fever virus (CSFV), which has been shown to carry critical epitopes, has been expressed in different systems. Here, we report the expression of CFSV E2 gene in tobacco chloroplasts. Mice immunized with leaf extracts elicited specific antibodies. This indicated that the expressed E2 proteins had a certain degree of immunogenicity. To our knowledge, this is the first report showing induction of protective antibody in response to classical swine fever virus (CSFV) by immunization with antigen protein E2 expressed in tobacco chloroplasts, which will open a new way to protection from, CSFV by plant chloroplasts as bioreactors.

The genomic DNA was extracted from the sheep hydatid cyst protoscolices that was isolated from Qinghai Province in China.eg95 gene was amplified using polymerase chain reaction(PCR)and cloned.The sequencing results showed the whole length of eg95 gene is 1 262bp.There are two introns and three extrons,the length of which are 70bp,306bp and 95bp respectively.So the ORF of eg95 was presumed to be 471bp and encode 157AA.The ORF was cloned by using the RT-PCR and cloned into pGEM-T Easy plasmid.The sequences were analyzed by DNAstar and show 100% similarity with the presumed sequence.The ORF was ligased into prokaryotic expression vector pGEX-5x-1.The recombinant expressed plasmid pGEX-5x-1-eg95 was expressed in procaryotic strain BL21.The specific protein strap was detected by SDS-PAGE and the mass-spectrum results indicated the expressed protein is the eg95 antigen protein.ELISA showed the special reaction and proved the right expression,thus it could provide base for further study of cystic echinococcosis.