β-carotene hydroxylase (chyb) catalyzes the conversion of β-carotene to zeaxanthin through β-cryptoxanthin in the pathway of carotenoid biosynthesis. The salt-tolerance of chyb gene in Dunaliella viridis has important research value in the aspect of plant resistance. chyb gene (GenBank accession No. JN118489) cloned in previous research was used to construct the plant expression vector pCAMBIA3301-chyb and transferred into tobacco (Nicotiana tabacum) by Agrobacterium tumefaciens LBA4404, and 16 regenerated tobaccos resisted to glufosinate ammonium were obtained. The results of PCR and RT-PCR indicated that chyb gene had been transferred successfully into 13 of them. Besides, salt tolerance of transgenic tobacco was detected through measuring plant height, leaf area, chlorophyll content, leaf relative conductivity and proline content. The result showed that plant height, leaf area and chlorophyll content were extremely significantly increased (P＜0.01) compared with the control group. The average height of transgenic plant was 37.82 cm, which was 1.37 times higher than that of the control group. Transgenic tobaccos were treated with different concentrations of NaCl. The results demonstrated that the average survival rate of transgenic tobacco was 1.58 times higher than that of control group under 100 mmol/L NaCl stress. Transgenic tobacco could grow under the treatment of 200 mmol/L NaCl, while the growth of control tobacco was inhibited. The leaf relative conductivity increased slightly, and the proline content of transgenic plants were improved extremly significantly than the control group (P＜0.01). It was concluded that chyb gene could improve salt-tolerance in transgenic tobacco plants and all the results help to lay the foundation for further exploring salt-tolerance of chyb gene.

Those antibiotics and herbicides marker genes generally used at present in plant genetic transformation might be potential treats and dangers for human being and environment, so it is urgent to search and develop new secure screening reagents instead of antibiotics and herbicides reagents. New recombinant plasmid pCAMBIA1301-xylA-Ah-Glu-P with promoter of peanut (Arachis hypogaea L.) β-1,3-glucanase gene (Ah-Glu-P) and xylose isomerase gene (xylA) from Escherichia coli was constructed successfully. pCAMBIA1301-xylA-Ah-Glu-P was then transformed to cotyledon explants of peanut variety Huayu23 by Agrobacterium mediated method using xylose as screening reagent. The frequency of regeneration seedling was 16.1% and the frequency of transformed positive plants reached to 78.4%, when the concentration of sucrose and xylose in medium was 10 and 20 g/L, respectively. The transgenic positive plants were then transplanted to field by grafting and domesticating and the survival rate of transplanted plants was up to 74.5%. The transformation method with cotyledons as explants, with xylose as screening reagent, has characteristics of simple and rapid, safe and pollution-free and higher transgenic positive frequency. So it was a safe and efficient genetic transformation method compared with other transformation methods using other explants and antibiotics screening reagent.

Plasmid molecule worked as reference material has been shown to be a good alternative in genetically modified organisms (GMOs) detection, and can promptly satisfy the regulatory requirements of GMOs. In this study, according to the transgenic information of 4 main crops including rice (Oryza sativa L.), maize (Zea mays L.), soybean (Glycine max) and rape (Brassica napus), 4 types of plasmids for GMO screening were developed and confirmed. The constructed pMD-rice contained 7 target elements including promoter of Cauliflower mosaic virus (CaMV35S), terminator of nopaline synthase (tNOS) and selective marker genes, such as biolaphos resistance (Bar), neomycin phosphotransferase (NPTⅡ) and hygromycin B phosphotransferase (Hpt), insect resistant gene of Bacillus thuringiensis (Bt) and a rice endogenous reference gene of sucrose phosphate synthase (SPS) were selected. The constructed pMD-maize was consisted of 4 target sequences including tNOS, CaMV35S, Bar and zSSⅡb as endogenous reference genes. The constructed pMD-soybean contained 5 target elements including CaMV35S, tNOS and 2 exogenous genes cowpea trypsin inhibitor gene (CpTi) and enolpyruvylshikimate phosphate synthase (EPSPS), and the Lectin precursor protein gene as endogenous reference gene. The constructed pMD-rape contained 8 target elements including CaMV35S, Figwort mosaicvirus 35S (FMV35S), nopaline synthase promoter (P-nos) and tobacco tapetum specific promoter (PTa29), tNOS, terminator-35S (T-35S) and the 7th gene of TL-DNA (g7) and high mobile group protein gene (HMG) as endogenous reference genes. In theory, all elements of the 4 plasmids covered 90% of the approved transgenic events of 4 major crops as maize, rape, soybean and rice in the world, and they covered 100% of transgenic events that had been approved in China. Then the constructed plasmids and reference materials were comparatively analyzed and all the results demonstrated that the constructed plasmids were suitable for GMO screening detection.

Bacterial blight, caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad), is the most destructive disease in anthurium (Anthurium andraeanum Linden). Appropriate reference gene selection is essential for accurate genes expression quantification about blight response by using quantitative Real-time PCR (qRT-PCR), and to elucidate the phytopathology mechanism of anthurium-bacteria interaction. In this study, 6 commonly used candidates including elongation factor1-α (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin 7(UBQ7), histidine 3 (His3), α-tubulin (TUB) and β-Actin (ACTB), were employed to identify the most reliable reference genes. The genes amplification efficiency was assessed by qRT-PCR, and expression stability in the processes of foliage and systemic infection were analyzed on both resistant and susceptible cultivars, then generated dates were evaluated comprehensively using geNorm3.5, BestKeeper0.953 and NormFinder1.0 programs. As a result, the tested genes showed different expression characters in various samples, among which, the expression of UBQ7 was much more stable in different tissues and different varieties, could be recommended as reference gene for data normalization. The result will facilitate expression analysis of anthurium genes responding to blight.

Thioredoxin (Trx) is a kind of highly conservative and low molecular weight proteins, which widely exists in plants, animals, bacteria and yeasts. It maintains the redox equilibrium of organisms, which regulates and controls biological signal transduction and other functions. This study cloned VvTrx gene from grape (Vitis vinifera) gDNA sequence (GenBank No. EU280166) of 989 bp and cDNA sequence (GenBank No. EU280164) of 561 bp and the open reading frame (ORF) was 381 bp, encoding 126 amino acids. The multiple sequence alignment and phyogenetix tree showed that the active site of VvTrx gene in Thompson Seedless (Vitis vinifera cv.) was WCIPS and belonged to the subgroup Ⅳ in Trx-h. Analysis of quantitative Real-time PCR(qRT-PCR) showed that, in different periods of the ovule development of both Thompson Seedless and Pinot Noir, the expression of VvTrx gene reached the highest level in 40 d after flowering. Besides, the difference between them was extremely significant (P＜0.001) and the expression of Thompson Seedless was about 5 times of Pinot Noir. After that, the VvTrx gene was cloned into the prokaryotic expression vector of pET-32a(+) to construct pET-32a(+)/VvTrx, and a 30 kD solubility fusion protein expressed in the Escherichia coli BL21 through isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The study showed that the VvTrx gene had a certain relation to the process of seedless grape ovules abortion, which provides the research directions to the molecular mechanism of VvTrx gene in seedless grape embryo abortion process and the breeding of seedless grape.

Leucine rich repeat containing G protein coupled receptor 8 (LGR8) plays an important role in the process of testicular descent in male mammals. LGR8 gene in half smooth tongue sole (Cynoglossus semilaevis) was cloned on the basis of the entire genome and transcriptome sequencing using RACE method. LGR8 gene contained 2 768 nucleotides, and the open reading frame (ORF) was 2 205 bp, which encoded 734 amino acids, 272 bp of 5'-UTR and 296 bp of 5'-UTR. The amino acid sequences contained 10 LRRs areas, a LDL-A regional structure and a 7tm-1 domain. The results of Real-time fluorescent quantitative PCR (qRT-PCR) showed that LGR8 mainly expressed in brains and gonads of the sexual maturity male bodies of three years old fish, which was extremely higher than that of other tissues. There was no significant difference among the most tissues in females(except the intestine and the heart).. It was noteworthy that the expression level of LGR8 in the brain and gonad of males are significantly higher than that of females (P＜0.05). The results of gonad in different periods by qRT-PCR showed that LGR8 gene began to rise at 80 d, and the expression was up to the maximum amount between 150 and 210 d, when the expression was significantly higher than that in other tissues (P＜0.05). The results suggested that LGR8 might play a significant role in gender determination and development of the gonads in half smooth tongue sole which provides certain theoretical basis for the sex determination mechanism study of half smooth tongue sole.

Miniature inverted-repeat transposable elements (MITEs) are the most abundant DNA elements identified in plants genome and involved in many important biological events. However, only few of the MITEs families are confirmed as active transposable elements, which makes it very challenging to study its mechanism. In this study, we aimed to enhance our understanding of MITEs transposition in tobacco(Nicotiana tabacum) plants. Bioinformatics analytical tools, such as MITE-Hunter, Muscle-Multiple Sequence Alignment and TIR/TSD analysis, were employed to study MITEs sequence structural features and copy numbers of tobacco genes from the NCSU database. From almost 2 million publicly available tobacco gene sequences, we were able to reveal 21 different MITEs family (named TMi 1 to TMi 21), in which, 17 of them were reported for the first time in this study and 4 of them were recently reported by other researchers. Amongst all of the newly identified MITEs family, we were particularly interested in TMi 1 family. Identification of TMi 1 element polymorphism in different tobacco strains had provided preliminary evidence of MITEs transposition activity. Moreover, we had established 2 specific biological phenotypes for transposon display assay and the results showed that TMi 1 was an active transposon element. This was the first time which an active MITEs was reported in tobacco genome. For the last 2 decades, MITEs and its transposition activities were studied mainly in rice (Oryza sativa) plant models. This study not only identified new MITEs family, but also validated that tobacco plant was a very useful tool for studying MITEs and its transposition activities. We demonstrated that tobacco genome was a transposon-rich species and potentially possess novel active MITEs elements. Such discovery provides new perspectives and improves our understanding in MITEs transposon and gene regulation. Therefore, further investigation of MITEs activities in tobacco plants is required and possibly will re-define the principal of genome evolution and genetic biodiversity formation.

Co-expression analysis is an important approach to explore the genes responsible for different traits in large biological data set. In this study, we calculated the expression abundance of genes in 10 different tissues of cucumber (Cucumis sativus L.) using RNA-Seq data and removed the genes whose maximal fragments per kilobase of exon per million fragments mapped (FPKM) values were less than 5 across 10 different tissues. Then, co-expression modules were detected according to the correlation and TO (Topological Overlap) value between genes by applying WGCNA package in R project. Finally, 1 134 modules were obtained including 16 924 genes, of which 839 modules were selected for their mean correlation coefficients more than 0.9, getting 11 844 genes in total. The great functional and morphological variation in plant tissue types arises from the differential regulation of a finite set of genomic transcripts. The relationship analysis between modules and tissue types found 323 tissue-correlated modules including 5 784 genes. These modules highly correlated with tissues (r＞0.65). Using the topGO package in R project, we identified Gene Ontology (GO) terms that appeared in modules more frequently than expected. Nine of the 10 tissues had correlated-modules highly enriched in GO biological processes respectively (Fisher's exact test, P＜0.000 1), except tendril. GO enrichment analysis (Fisher's exact test, P＜0.000 1) of tissue-specific modules showed some specific genes were related to different tissues. The overrepresented GO biological processes in tissue-specific modules was often consistent with known tissue attributes. Positional clusters were also found in the modules with size ranging from 2 to 5 genes, most of which contained 2 genes. The physical distance between clustered genes in one module on the chromosome was less than 25 kb. The clustered genes often had similar structure or function. As an example, we got 3 modules in leaf and 1 module in stem correlated to the biosynthesis pathway of cucurbitacin, and 10 genes were published in previous research. These 4 modules were enriched in the biological process of terpenoid and isoprenoid which were the precursors of cucurbitacin. We also found that module M107 collected most of genes (7/10) associated with biosynthesis pathway of cucurbitacin which were in 2 clusters, which indicated that genes having similar function were often adjacently located on the chromosome. This study identified some important co-expression modules in cucumber through transcriptome and network analysis and provided a reference for similar studies in the future.

The objective of this study was to research the expression of tumor protein p53 (Tp53) in yak (Bos grunneins) during different development period of embryo and to verify the relationship with in vitro fertilization (IVF) embryo development. In this study, we gathered the ovarian of female yak and collected cumulus-oocyte complexes (COCs) during in vitro maturation and to culture these IVF embryos. The expression of Tp53 in different periods of IVF embryos were detected by the methods of PCR, quantitative Real-time PCR and immunofluorescence at mRNA and protein levels. The results showed that Tp53 gene expressed in different development period of IVF embryos, and Tp53 in blastocysts and 2~4 cells volume was relatively low, and there was no significant difference between groups (P＞0.05); Tp53 in stage 5~8 cells and 9~16 expression quantity was relatively high, and there was no significant difference between groups (P＞0.05). The expression of Tp53 in stage 2~4 cells and blastocyst had significant difference compared with 5~8 cell embryo and 9~16 cell embryo(P＜0.05). This study suggests that Tp53 played a very important role in adjusting early embryonic development. The relationship between the expression of Tp53 and developmental block was positive correlation. Indirect immune fluorescent displayed that Tp53 mainly existed in the nucleus while a small amount existed in cytoplasm and its role was related to DNA damage.

To explore the expression of growth hormone receptor (GHR) in different tissues of yak(Bos grunneins), samples of these tissues (heart, liver, spleen, lung, kidney and testis) were collected from healthy yaks under normal physiological conditions. Quantitative Real-time PCR (qRT-PCR) was applied to analyze the expression of GHR gene in different tissues. Immunohistochemistry (IHC) was used to detect the distribution and localization of GHR protein, integrated optical density (IOD) of GHR immunoreactions were measured, and followed by an analysis for significant difference. The results of qRT-PCR showed that GHR gene had a differential expression in different tissues, comparative expression quantity of GHR gene in liver was 3.73 fold than that in heart, 4.98 fold in kidney, 5.03 fold in testis, 8.58 fold in lung and 10.21 fold in spleen, respectively. Unequal immunoreaction intensity was observed in different tissues by immunohistochemistry. Comparative analysis of IOD showed that GHR had the strongest immunoreactions in liver, and followed by the heart, kidney, testis and spleen, the weakest immunoreactions of GHR was found in lung. This experiment demonstrated that GHR had a differential expression in different tissues by studying at genetic and protein level. This work provides a theoretical information for improvement and breeding of yak.

The aim of this study is to verify whether the expression of HSP70 (heat shock protein, HSP) and apoptosis of yak (Bos grunniens) cumulus cells were regulated by IGF-1 (insulin-like growth factor, IGF-1). The yak cumulus cells were cultured in vitro supplemented with different concentrations of IGF-1 (0, 50, 100 and 200 ng/mL). The relative expression levels of HSP70 mRNA and protein in different groups were detected by Real-time quantitative PCR (qRT-PCR) and Western blot (WB), and location of HSP70 protein was detected by immunofluorescence. Furthermore, relative expression levels of Bax (B-cell lymphoma/leukemia associated x protein, Bax) and Bcl-2 (B-cell lymphoma/Leukemia-2, Bcl-2) mRNA in different groups were also evaluated by qRT-PCR, flow cytometry was used to evaluate the rate of apoptosis. The result showed that: (1) IGF-1 improved the relative levels of HSP70 mRNA and protein; these improvements were highest in the 100 ng/mL IGF-1 group, followed by 200 ng/mL and 50 ng/mL groups, the relative levels of HSP70 mRNA and protein were lowest in control group (0 ng/mL IGF-1); (2) Detection by immunofluorescence showed that HSP70 protein could be observed in yak cumulus cells cytoplasm as well as within nuclei; (3) Relative level of Bax was down-regulated by IGF-1, while Bcl-2 was up-regulated. The Bax mRNA was lowest in groups with 100 ng/mL IGF-1, where the Bcl-2 was the highest. In 200 and 50 ng/mL IGF-1 groups, the relative expression levels of Bax was decreased compared with that of in 100 ng/mL group, while relative expression levels of Bcl-2 was increased. Bax expression in the groups with IGF-1 was lower than the group without IGF-1(control group), while Bcl-2 expression was higher in groups with IGF-1 than without IGF-1; (4) IGF-1 inhibited apoptosis of yak cumulus cells, and the rate of apoptosis in cumulus cells was (6.32±0.84)% in 100 ng/mL IGF-1 group, which was lowest among these groups. In 200 and 50 ng/mL IGF-1 groups, the apoptosis rates were (13.85±2.01)% and (25.42±1.63)%, respectively, both of them were significantly higher than that in 100 ng/mL IGF-1 group (P＜0.05). Apoptosis rate of cumulus cells was (26.14±3.16)% in control group, which was lowest among these groups. The results demonstrated that expressions of HSP70, Bax and Bcl-2 on yak cumulus cells were regulated by IGF-1, and the apoptosis rate of cumulus cells was also reduced; the optimum concentration was 100 ng/mL. The results in this study provide new insights for the molecular mechanism of IGF-1 in inhibiting the apoptosis, and also provide theoretical foundation to explore the functions of IGF-1 during yak oocytes maturation and preimplantation embryo development, which will be helpful to improve the technique of yak embryo cultured in vitro.

Oviductin (Ovn) plays crucial role during fertilization and early embryonic development. The objective of this study was to clone the full open reading frame of yak (Bos grunniens) oviductin and to detect its different expression during the estrous cycle from mRNA and protein level. In this study, we cloned oviductin cDNA by RT-PCR, and analysed the expression of oviductin in oviductal ampulla and isthmus of yak in early follicular (follicles 3~6 mm), late follicular (with preponderant follicles＞6 mm), early luteal (one or more corpora hemorrhagica visible) and late luteal (one or more corpora lutea visible) by qRT-PCR (quantitative Real-time PCR) and immunohistochemistry. The oviductin cDNA of yak was successfully cloned, and the sequences had been submitted to GenBank, and accession number was KF303541, which contained the full open reading frame and determined to be 1 443 nucleotides encoding for a deduced protein of 480 amino acids. The sequence analysis revealed that oviductin cDNA sequences of yak shared very high homology with cattle and buffalo. The phylogenetic tree based on amino acid sequences of oviductin indicated that yak oviductin was closely related to its cattle counterpart, reached 99.1% with Bos taurus, followed by 93.7% with buffalo (Bubalus bubalis), 88.1% with sheep (Ovis aries), 79.8% with Macaca radiata. qRT-PCR results showed highest oviductin mRNA oviductin expression in the stage of late follicle, reduce in the stages of early luteal and late luteal. The expression of mRNA was lowest in the stages of early follicle. However, there was no difference between ampulla and isthmus. The prominent immunolabeling were observed in the late follicle and early luteal stages, which was reduced in other stages. In this study, we obtained cDNA sequence of yak oviductin containing an open reading frame, and found the highest expression in the stage of late follicle. It showed that oviductin has an important biological role in oocyte maturation and the process of ovulation. The present study provides certain theoretical references for increasing yak fertilization rate and the development of early embryos.

In order to study the sequence characteristics of heat shock 70 kD protein-12B gene (HSPA12B) and its expression in yak (Bos grunniens) early in vitro fertilization (IVF) embryos, the specific primers were designed by the conservative sequence of HSPA12B gene of the cattle (Bos taurus)，and the gene sequence could be obtained by cloning. The HSPA12B sequence was analyzed by bioinformatics. The HSPA12B in early IVF embryos were detected by the methods of quantitative Real-time PCR (qRT-PCR). The results of bioinformatics showed the coding region of the yak HSPA12B gene was 705 bp in length, encoding 234 amino acids, and the content of Alanine resduces was the highest (about 10.3%) and the content of Asparagine resduces was the lowest (about 0.9%). According to homology analysis, the similarity of HSPA12B gene in yak and cattle was up to 99.5%, showing the HSPA12B gene was highly conserved. The protein was hydrophilic and had no signal peptide, which was not a transmembrane and secreted protein. qRT-PCR results showed that the HSPA12B in yak early embryos fertilized in vitro expressed in each period, and the relative expression of HSPA12B in 2-4 cell stage embryos was 14.814 8, 4.269 9 and 40.485 8 folds respectively, compared to the 5-8 cell stage embryos, morulae and blastocysts. The difference of the relative expression of HSPA12B between 2-4 cell stage embryos and 5-8 cell stage embryos，morula and blastocysts was very significant (P＜0.01). These results showed HSPA12B played an important role in the development of embryos IVF and can inhibit cell apoptosis, cell senescence, and regulate embryonic development.

Among a significant number of described cold shock proteins, CIRP (cold-inducible RNA binding protein) has been widely researched. In order to explore the function of CIRP in the development of yak testes，and further provide datas for elucidating the regulatory mechanism by which of CIRP acts in spermatogenesis and testicular function of plateau mammalian, we investigated the dynamic expression of CIRP in yak testis during postnatal development by means of Real-time RT-PCR, Western blot and its cellular localization by the immunohistochemical SABC (strept avidin-biotin complex) method. In this study, whole testes were collected from 1-day-old, 5-month-old, 1-year-old and 3-year-old male yaks. The results showed yak CIRP open reading frame contained 642 bp, encoded for 213 amino acids and their molecular weight was estimated as 23 kD. The cDNA sequences of yak CIRP revealed significant homology of 99.8% with Bos taurus. qRT-PCR (quantitative Real-time PCR) and Western blot results showed that CIRP expressed in the testis of different age yaks in mRNA and protein levels, and increased with age. The immunostaining of CIRP was weakly expressed in the seminiferous tubules and occurred in the spermatogonium of 1-day-old yak testes. Then the immunostaining of CIRP was widely localized in the spermatogonium of 5-month-old yak testes. And in 1-year-old yak testes, primary spermatocyte was observed and positive substances were located in the nucleus of spermatogonium and primary spermatocyte. Additionally, in 3-year-old yak testes which developed well, CIRP expressed strongly in seminiferous tubules, and the positive immunostaining occurred in nucleus of spermatogonium and primary spermatocyte. Nothing was observed in the negative controls. This research suggested that CIRP might play an important role in regulating testicular development and maintaining the proliferation and differentiation of spermatogenic cells.

CD4 is a membrane-bound glycoprotein found on T cells, and also an important surface marker of helper T lymphocyte. This molecule widely involves in recognition of specific antigens in association with major histocompatibility complexⅡ(MHCⅡ) molecules and transmembrane signal transduction. Expression of CD4 is critical for cell-mediated immune response and defense mechanisms. In this study, the yak (Bos grunniens) CD4 open reading frame (ORF) was cloned and sequenced using RT-PCR, and the CD4 mRNA and protein levels of adult yak thymus, spleen, mesenteric lymph nodes and haemal nodes were detected by means of qRT-PCR (quantitative Real-time PCR), Western blots and immunohistochemical streptavidin biotin-peroxidase complex (SABC) method. The results showed that yak CD4 ORF contained 1 188 bp, encoded 396 amino acids. The cDNA sequences of yak CD4 revealed significant homology with Bos taurus. The results of qRT-PCR and Western blots showed that CD4 mRNA and protein widely expressed in lymphoid tissues. The expression levels in the thymus was higher than that in other lymphoid tissues (P＜0.01); additionally the levels of haemal nodes was similar to that of mesenteric lymph nodes, and both of them were higher than those for spleen (P＜0.01). The immunohistochemical results showed that the CD4+ cells were distributed in the cotex and medulla of yak thymus. In the spleen, many CD4+ cells were located in the periarteriolar lymphoid sheaths and red pulp. Moreover CD4+ cells in mesenteric lymph nodes and haemal nodes were mainly presented in the mantle zone and medulla cord. The results indicated the thymus was a primary 1ymphoid tissue responsible for CD4+ T lymphocytes exportation and thymic production may influence the T lymphocytes extent of secondary lymphoid tissues. Then haemal nodes might have similar functions as the mesenteric lymph nodes and spleen. This study would provide fundamental data for immunogenetics and immunobiology, and also bring a new insight into timely immunization, the pathogenesis and disease prevention.

Precise genome editing technologies not only play important roles in biological research and crop improvement but also are a potential avenue for therapy of genetic diseases. Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) has been developed as a new targeted genome editing tool since the last 2 years. Because it's flexible, efficient, cheap and easy to operate, it quickly surpass the previous technologies to become the hottest genome editing tool. Up to now, CRISPR/Cas has been applied in a wide variety of cells and organisms such as bacteria, mammalian cells, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Brachydanio rerio, Arabidopsis thaliana, Oryza sativa and so on. Furthermore, the CRISPR-Cas9 system has broad application prospects in functional genomic screens, transcriptional modulation, epigenetic control and live imaging of DNA loci. It will undoubtedly transform biological research and spur the development of biotechnology. Here, we review the biological basis of CRISPR/Cas DNA-targeting platform, with an emphasis on means to improve target specificity, provide an update of recent developments and applications, and present the challenges and future directions for this platform to provide some useful references for the later research and application of this field.