柑橘冷诱导基因及其启动子表达载体构建与瞬时表达分析

【目的】以YFP为报告基因,构建柑橘冷诱导基因及其启动子的植物表达载体。【方法】克隆获得枳冷诱导基因Ptcor8及其启动子p Ptcor8,柠檬中同源基因Clcor8及其启动子p Clcor8;双酶切含启动子的克隆载体和植物表达原始载体p1D4,连接获得含启动子的中间载体;再双酶切含冷诱导基因的克隆载体和中间载体,连接后获得重组质粒;通过冻融法将重组质粒导入农杆菌EHA105中。【结果】构建了p1D4/p Ptcor8-Ptcor8::YFP,p1D4/p Ptcor8-Clcor8::YFP和p1D4/p Clcor8-Ptcor8::YFP 3个融合表达载体,瞬时表达发现3个融合基因均可在冰糖橙叶片中表达。【结论】3个融合表达载体的成功构建为下一步转化柠檬,分析枳冷诱导基因Ptcor8及其启动子p Ptcor8的功能奠定了基础。     

Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors

Background

Virus-induced gene silencing (VIGS) is an effective technology for the analysis of gene functions in plants. Though there are many reports on virus vectors for VIGS in plants, no VIGS vectors available for Rosaceae fruit trees were reported so far. We present an effective VIGS system in apple, pear, and Japanese pear using Apple latent spherical virus (ALSV) vectors.

Results

Inoculation of ALSV vectors carrying a partial sequence of endogenous genes from apple [ribulose-1, 5-bisphosphate carboxylase small subunit (rbcS), alpha subunit of chloroplast chaperonin (CPN60a), elongation factor 1 alpha (EF-1a), or actin] to the cotyledons of seeds by a particle bombardment induced highly uniform knock-down phenotypes of each gene on the true leaves of seedlings from 2~3 weeks after inoculation. These silencing phenotypes continued for several months. Northern blot and RT-PCR analyses of leaves infected with ALSV containing a fragment of rbcS gene showed that the levels of rbcS-mRNA drastically decreased in the infected apple and pear leaves, and, in reverse, rbcS-siRNAs were generated in the infected leaves. In addition, some of apple seedlings inoculated with ALSV vector carrying a partial sequence of a TERMINAL FLOWER 1 gene of apple (MdTFL1) showed precocious flowering which is expected as a knock-down phenotype of the silencing of MdTFL1 gene.

Conclusions

The ALSV-based VIGS system developed have provides a valuable new addition to the tool box for functional genomics in apple, pear, and Japanese pear.     

Cloning,silencing, and prokaryotic expression of strawberry sucrose synthase gene FaSus1

The sucrose synthase (Sus) gene is regarded to be involved in strawberry fruit ripening; however, direct molecular and biochemical evidence is lacking. Here, the coding cDNA sequence of FaSus1 was cloned by RT-PCR and both a hairpin-mediated RNA interference pBI121 vector and a recombinant E. coli expression PET28a vector were constructed. These vectors were then used to transform strawberry fruit and for prokaryotic expression in strain BL21, respectively. The results showed that hairpin-mediated RNA silencing of FaSus1 led to a decrease in sucrose content and inhibited fruit ripening. Enzymatic activity analysis of FaSus1 showed that the reaction system contains 0.309 mg of recombinant FaSus1 protein, reaching 0.617 mg/mL. The cleavage activity of the enzyme was 0.25 U with a specific activity of 0.812 U/mg, whilst the synthetic activity of the enzyme was 0.057 U with a specific activity of 0.185 U/mg. This study provides physiological, molecular, and biochemical evidence to demonstrate that the FaSus1 has high sucrolysis activity and plays an important role in the regulation of strawberry fruit ripening.     

ModuleFinder and CoReg: alternative tools for linking gene expression modules with promoter sequences motifs to uncover gene regulation mechanisms in plants

Background  

Uncovering the key sequence elements in gene promoters that regulate the expression of plant genomes is a huge task that will require a series of complementary methods for prediction, substantial innovations in experimental validation and a much greater understanding of the role of combinatorial control in the regulation of plant gene expression.     

果实特异启动子调控薄皮甜瓜ACC合成酶cDNA反义表达载体的构建

应用反义RNA技术抑制甜瓜成熟过程中内源乙烯的合成,从而培育耐贮运品种是解决甜瓜延熟保鲜难题的可行新方法。根据GenBank中甜瓜、黄瓜ACC合成酶基因氨基酸保守序列设计引物,从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总RNA,经RT-PCR扩增得到约0.7kb的ACC合成酶cDNA片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为777bp,编码258个氨基酸;从番茄(东农706)叶片组织中提取总DNA,经PCR扩增得到约2.2kb的E8基因片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为2192bp;以pCAM2301为起始植物表达载体,pCAM-GT为中间载体,成功构建了果实特异启动子(E8)调控薄皮甜瓜ACC合成酶cDNA反义表达载体,采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。     

韧皮部特异启动子驱动密码子优化的抗菌肽D基因的植物表达载体构建

根据177个GenBank中登录的柑橘编码蛋白密码子用法的分析结果,优化并重新设计和合成了含柑橘偏爱密码子、对柑橘黄龙病有杀灭作用的柞蚕抗菌肽D基因(命名为CAPD),克隆入pUC19克隆载体并经测序验证后,获得了含新抗病基因的重组质粒pUC19-CAPD。用限制性内切酶BamHI和SacI双酶切pUC19-CAPD克隆载体和pBI121植物表达载体的质粒DNA,回收pUC19-CAPD克隆载体中的CAPD基因小片段和pBI121植物表达载体中去掉GUS报告基因的大片段,经连接、转化和鉴定后,构建了由CaMV35S组成型启动子(35SP)驱动CAPD目的基因的新植物表达载体(命名为pHZ05);用限制性内切酶BamHI和HindIII双酶切含笋瓜韧皮部特异启动子(PSP)的pUCm-PSP克隆载体和pHZ05植物表达载体的质粒DNA,分别回收pUCm-PSP克隆载体中的PSP小片段和pHZ05植物表达载体中去掉CAPD目的基因上游35SP的大片段,经连接、转化和鉴定后,构建了由PSP驱动CAPD目的基因的新植物表达载体(命名为pHZ06)。利用细胞感受态法直接将2个由不同启动子驱动的含CAPD目的基因的新重组植物表达载体分别导入根癌农杆菌LBA4404、GV3101、EHA105和发根农杆菌Ri15834等4个农杆菌菌株中,为利用农杆菌介导的遗传转化技术培育抵抗由韧皮部传导的毁灭性和检疫性病害柑橘黄龙病的新种质奠定了基础。     

Effects of epididymal P34H gene silencing on expression of P34H and activity of hyaluronidase in mouse sperm

AIM: To investigate the effects of P34H gene silencing on the expression of P34H and activity of hyaluronidase (HYD) in mouse sperm.METHODS: The recombinant plasmid series of P34H targeted short hairpin RNA (shRNA) were constructed by GV248 plasmids vector. These recombinant plasmids were transformed into DH5α competent cells, and the plasmids were taken from DNA sequencing analysis. The HEK293T cells were co-transfected with shRNA and lentiviral packaging plasmids. The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to inject into the mouse epididymis and the expression of P34H at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The location of P34H protein on the mouse spermatozoa was determined by indirect immunofluorescent staining using P34H antibody. The positive rate and activity intensity of HYD was detected by modified sodium hyaluronate-gelatin membrane. RESULTS: DNA sequencing analysis confirmed that the 3 P34H-shRNA sequences were successfully inserted into the lentiviral vectors. P34H expression in epididymis tissue was significantly decreased at both mRNA and protein levels compared with those of the non-transfected and normal control vectors (P<0.05). The GV-P34H-shRNA-1 played a significant role in reducing the percentage of P34H positive rate and the activity of HYD in mouse sperm. The silencing effect did not significantly differ between the non-transfected and normal control vectors. CONCLUSION: Silencing of P34H significantly inhibits the percentage of P34H positive rate and the activity of hyaluronidase in mouse sperm.     

A novel fluorescent pH probe for expression in plants

Background  

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants.     

Grafting and RNA transport via phloem tissue in horticultural plants

Grafting is a cultivation method that exploits a cooperative relationship between partner plants possessing different genomes. It is most commonly used for the propagation and cultivation of trees, shrubs, and fruit vegetables. In addition, as represented by florigen (flowering hormone) experiments, grafting has been utilized in the field of plant physiology to clarify the mechanism of long-distance transport by which signals arising in organs that perceive an environmental change are transmitted to response organs. Recent analytical technology has revealed that some specific RNA molecules are also transported through phloem tissue as genetic information to execute coordinated organ growth and development. Therefore, it is anticipated that the RNA transport system could be applied for the improvement of cultivars of various horticultural crops, if the mechanism were controllable by artificial means.     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.     

Transient expression of an active human interferon-beta in lettuce

Using Agrobacterium mediated transient expression method, plant bivalent expression vector pBI121 containing GUS as a report gene was transformed into lettuce (Lactuca sativa). Through designed orthogonal analysis, intact lettuce leaves infiltrated with 200 μM acetosyringone and 0.8 OD₆₀₀ bacterial suspensions under vacuum for 30 min, then co-cultured at 24 °C for 6 ds produced a maximum GUS protein of 2.5% TSP with 21.39 nmol mg⁻¹ min⁻¹ MU activity, which was 19 times of the control (1.31 nmol mg⁻¹ min⁻¹ MU). Employed these optimized conditions HuIFN-beta was expressed in lettuce leaves. Western blot and antivirus bioactivity analyses confirmed the HuIFN-beta achieved by agrobacterium infiltration had a high biological activity (3.1 × 10⁴ IU/mL). To our knowledge, it is the first detailed orthogonal optimizing study of Agrobacterium mediated transient expression and the first report on the production of the biologically active therapeutic proteins produced by Agrobacterium mediated transient expression in lettuce. In summary, transient expression by Agrobacterium vacuum infiltration can be adopted as an efficient, inexpensive and small-scaled plant expression system for therapeutic protein production.     

杜梨PbNHX1基因的克隆、表达分析及功能验证

【目的】克隆1个杜梨Na~+/H~+逆向转运蛋白基因PbNHX1,并对其序列特征、表达特点及功能进行研究。【方法】采用RT-PCR和PCR克隆PbNHX1的c DNA和DNA序列,利用生物信息学方法进行序列分析,定量PCR检测其在非生物胁迫下转录水平变化,酵母互补试验检测PbNHX1基因的离子转运能力。【结果】杜梨PbNHX1基因c DNA编码区长1 704 bp,对应基因组DNA序列长3 594 bp,由13个外显子和12个内含子组成,编码蛋白含567个氨基酸。系统进化树显示,PbNHX1位于液泡膜型Na~+/H~+逆向转运蛋白分支上,与杨树液泡膜型Na~+/H~+逆向转运蛋白Pt NHX1.3基因亲缘关系较近。正常生长条件下,PbNHX1在杜梨叶片中表达量高于根。施加200 mmol·L-1Na Cl、10%(φ)PEG6000或100μmol·L-1ABA后,PbNHX1在叶片中的转录水平持续上升;其在根中的表达量先升后降,表达高峰依次出现在处理后6、3和6 h。PbNHX1的转入可恢复Na Cl、KCl和潮霉素B对nhx1缺失酵母菌株AXT3的生长抑制,转基因酵母细胞中Na~+和K~+含量显著增加。【结论】PbNHX1具有植物NHXs基因家族的固有特征,对盐碱、渗透胁迫和ABA处理均存在转录响应,转入该基因能够提高酵母nhx1缺失突变体AXT3对盐胁迫的耐受能力,部分恢复其对阳离子的转运功能,从而促进Na~+、K~+积累。     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

The lower eudicot genus Aquilegia, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. Aquilegia is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e.g., Arabidopsis) and grass (e.g., Oryza) model species.     

Synthesization,expression, purification and activity assay of hIGF-1

AIM: To express the synthesized human insulin like growth factor I （hIGF-1） gene in E.coli with high expression level and explore the way to increase the efficiency of factor Xa cleavage. METHODS: The gene of hIGF-1 was designed and synthesized according to the preference of E.coli. A fusion protein with a recognized site of factor Xa between CBD (cellulose binding domain) and hIGF-1 was expressed and purified by cellulose affinity chromatography. MTT method was used to assay the bioactivity of CBD-IGF fusion protein. hIGF-1 was released by factor Xa. In order to improve the sensitivity of fusion protein to factor Xa, the short flexible peptide （Gly-Thr-Gly- Gly-Gly-Ser-Gly） was added before the recognized site of factor Xa. RESULTS: SDS-PAGE results indicated that the CBD-IGF fusion protein was expressed and purified . Biological assay results indicated CBD-IGF fusion protein could promote the growth of NIH3T3 cell. The short flexible peptide （Gly-Thr-Gly-Gly-Gly-Ser-Gly）, which was added before the recognized site of factor Xa, improved the sensitivity of fusion protein to factor Xa. CONCLUSION: CBD-IGF fusion protein with bioactivite are expressed and purified. The amio acid sequences changes between the site recognize of factor Xa can help to improve the cleavage efficiency of Factor Xa.     

Transgene expression for Gladiolus plants grown outdoors and in the greenhouse

Transgene expression was evaluated for Gladiolus plants transformed with either the CaMV 35S, double CaMV 35S, rolD, or Arabidopsis UBQ3 promoter controlling the uidA or bean yellow mosaic virus coat protein gene in either the sense or antisense orientation to determine differences in expression for plants grown in the greenhouse and outdoors for two years. There was more variability in GUS expression when plants were grown outdoors than in the greenhouse for two years. Four of the six transformed plant lines with the UBQ3, rolD, and CaMV 35S promoters grown outdoors showed significant differences in GUS expression from year to year as compared to two of the six lines with the UBQ3 and rolD promoters grown in the greenhouse. When grown the same year, two plant lines with the CaMV 35S and one line with the rolD promoter showed 2–16× higher levels of GUS expression outdoors than in the greenhouse, and one plant line with the UBQ3 promoter had 31× higher GUS expression in the greenhouse instead of outdoors. Three of six plant lines transformed with the bean yellow mosaic virus coat protein gene in either the sense or antisense orientation under control of the double CaMV 35S promoter showed obvious transgene expression as compared to three lines that did not show expression or negligible expression for both years when plants were grown both outdoors and in the greenhouse. This study verified long-term gene expression, rather than silencing, for Gladiolus plants when grown outdoors and in the greenhouse from year to year.     

Altered activity and expression of MMPs/TIMPs are associated with functional and structural left ventricular remodeling in hypertensive rats

AIM: To investigate the relationship between matrix metalloproteinases and tissue inhibitors of matrix metalloproteases imbalance with functional and structural left ventricular (LV) remodeling in the hypertensive rats.METHODS: 6-week-old male stroke-prone spontaneously hypertensive rats (SHR-SPs,n=40) served as the hypertensive heart disease model,and age-matched male Wistar-Kyoto (WKY) rats (n=10) were used as control.After 6 months,the rats in two groups were anesthetized for invasive hemodynamic measurement by Millar pressure-volume (P-V) conductance catheter.Then the rats were sacrificed and hearts were dissected for morphological analysis,gelatin-zymography and Western blotting analysis.RESULTS: Left ventricular (LV) hemodynamic parameters showed the systolic and diastolic dysfunction in SHR-SPs compared with that in control group (P<0.05).Collagen volume fraction,ratio of perivascular collagen area to luminal area,myocardial cross-sectional area and the medial area to luminal area ratio of the SHR-SPs were all increased remarkably (P<0.05).LV matrix metalloproteinase-2 (MMP-2) activities,MMP-2 and tissue inhibitors of matrix metalloprotease-1 (TIMP-1) protein level in SHR-SP were notably higher than those in control group (P<0.05).CONCLUSION: Chronic pressure-overload is capable of inducing imbalances of cardiac ECM and MMPs/TIMPs system,both imbalances induce LV dilation,cardiac systolic and diastolic dysfunction.     

西瓜抗病毒RNAi植物表达载体的构建

为了研究转化同一种病毒的不同基因在转基因西瓜中引发RNA沉默的效果以及利用RNA沉默培育抗多种病毒的策略,以pFGC5941为骨架质粒,分别构建了ZYMV cp、nib和Hc-Pro基因的反向重复植物表达载体pFIRZYMVCP、pFIRZYMVNIb和pFIRZYMVHc-Pro;利用重叠延伸PCR的方法,首先构建了含有CMV cp、WMV cp和ZYMV cp部分基因片段的CWZ cp三价基因,采用该三价基因构建了CWZ cp基因的反向重复植物表达载体pFIRCWZ CP。研究首次在国际上构建了西瓜转基因抗病毒RNAi植物表达载体,为探讨RNA沉默在转基因抗病毒西瓜中的应用奠定了基础。     

Inhibitory effect of antisense eukaryotic expression vectors for c-myc on rat airway smooth muscle cells

AIM: To observe the inhibitory effect of antisense eukaryotic expression vectors for c-myc on rat airway smooth muscle cells. METHODS: Antisense and sense eukaryotic expression vectors for c-myc pcDNA3-myc-antisense and pcDNA3-myc-sense were constructed. Lipofectin was used to introduce antisense and sense eukaryotic expression vectors for c-myc into rat. The inhibitory effect was assayed by MTT cell proliferation assay. Cell cycles were detected by flow cytometry technology. The expression of c-Myc was detected by immunohistochemistry. RESULTS: The results showed that antisense eukaryotic expression vector for c-myc inhibited rat airway smooth muscle cells proliferation. Rat airway smooth muscle cells were prohibited in S phase and the expression of c-Myc was decreased after antisense eukaryotic expression vectors for c-myc were transfected into cells. CONCLUSION: Antisense eukaryotic expression vectors for c-myc inhibit rat airway smooth muscle cell proliferation.