霍乱弧菌EL－Tor35A3菌株外膜蛋白主区带的纯化

选用霍乱弧菌ＥＬ－Ｔｏｒ生物型３５Ａ３菌株（血清稻叶型）以ＥＤＴＡ－溶菌酶法与超速离心法提取其外膜蛋白,并将聚丙烯酰胺凝胶电泳后一条蛋白主区带分离。ＳＤＳ－聚丙烯酰胺凝胶电泳测得该蛋白主区带的分子量为２５ｋＤ,免疫电泳显示一条线,免疫双扩试验表明,不同的霍乱弧菌菌株均有该蛋白抗原存在。     

Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

Key message

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.

Abstract

The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.     

Improved protocol for the transformation of adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues

The production of transgenic citrus plants from adult tissues is difficult because of low regeneration and transformation rates. To increase the transformation efficiency of adult citrus tissues, an improved protocol involving adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues was developed. Explants were pre-incubated in a liquid medium prior to infection by Agrobacterium tumefaciens. Plant materials were also incubated on callus-induction medium supplemented with various combinations of cytokinin (Cyt) and kanamycin (Kan). An appropriate pre-incubation of the explants increased the transformation efficiency of adult tissues. During the callus-induction period, the Cyt type and Kan concentration had the largest and smallest effects on the transformation efficiency, respectively. The most effective combination of plant growth regulator and Kan for the transformation of ‘Tarocco’ blood orange tissues was 2 mg L⁻¹ 2-isopentenyl adenine and 50 mg L⁻¹ Kan. The transformation efficiency under the optimized conditions was 11.7%. A Southern blot analysis confirmed the integration of the transgene. These results indicated that the transformation efficiency of adult citrus tissues can be enhanced by optimizing the transformation conditions.

     

Increased activity of the tandem fructose-1,6-bisphosphate aldolase,triosephosphate isomerase and fructose-1,6-bisphosphatase enzymes in <Emphasis Type="Italic">Anabaena</Emphasis> sp. strain PCC 7120 stimulates photosynthetic yield

The regulation of photosynthetic yield at the genetic level has largely focused on manipulation of the catalytic enzymes in the Calvin cycle by genetic engineering. In order to investigate the contribution of increased enzymatic activity in the Calvin cycle on photosynthetic yield, the rice fructose-1,6-bisphosphate aldolase (FBA), spinach triosephosphate isomerase (TPI) and wheat fructose-1,6-bisphosphatase (FBPase) genes were cloned in tandem and co-overexpressed in cyanobacterium Anabaena sp. strain PCC 7120 cells. The enzymatic activities of FBA, TPI and FBPase, as well as sedoheptulose-1,7-bisphosphatase (SBPase), were remarkably increased in transgenic cells relative to the wild-type. The photosynthetic yield, as reflected by photosynthetic O₂ evolution and dry cellular weight, was also markedly increased in transgenic cells versus wide-type cells. The activity of SBPase is considered the most important factor for ribulose-1,5-bisphosphate (RuBP) regeneration in the Calvin cycle, and increased activity of TPI alone in transgenic cells does not stimulate photosynthetic yield. Thus, the increased activity of FBA and FBPase, but not TPI, significantly improved photosynthetic yield in transgenic cells by stimulating SBPase activity and consequently accelerating the RuBP regeneration rate.     

Identification of novel Ssl0352 protein (NdhS), essential for efficient operation of cyclic electron transport around photosystem I, in NADPH:plastoquinone oxidoreductase (NDH-1) complexes of Synechocystis sp. PCC 6803

Cyanobacterial NADPH:plastoquinone oxidoreductase, or type I NAD(P)H dehydrogenase, or the NDH-1 complex is involved in plastoquinone reduction and cyclic electron transfer (CET) around photosystem I. CET, in turn, produces extra ATP for cell metabolism particularly under stressful conditions. Despite significant achievements in the study of cyanobacterial NDH-1 complexes during the past few years, the entire subunit composition still remains elusive. To identify missing subunits, we screened a transposon-tagged library of Synechocystis 6803 cells grown under high light. Two NDH-1-mediated CET (NDH-CET)-defective mutants were tagged in the same ssl0352 gene encoding a short unknown protein. To clarify the function of Ssl0352, the ssl0352 deletion mutant and another mutant with Ssl0352 fused to yellow fluorescent protein (YFP) and the His(6) tag were constructed. Immunoblotting, mass spectrometry, and confocal microscopy analyses revealed that the Ssl0352 protein resides in the thylakoid membrane and associates with the NDH-1L and NDH-1M complexes. We conclude that Ssl0352 is a novel subunit of cyanobacterial NDH-1 complexes and designate it NdhS. Deletion of the ssl0352 gene considerably impaired the NDH-CET activity and also retarded cell growth under high light conditions, indicating that NdhS is essential for efficient operation of NDH-CET. However, the assembly of the NDH-1L and NDH-1M complexes and their content in the cells were not affected in the mutant. NdhS contains a Src homology 3-like domain and might be involved in interaction of the NDH-1 complex with an electron donor.     

显微注射法制备遗传工程小鼠模型的研究

遗传工程小鼠模型是基因功能、人类疾病发病机制及新药研究开发的最重要的模式生物之一。在建立遗传工程小鼠模型的众多方法中,显微注射法是目前国际上公认的制备转基因及基因剔除动物模型的首选。在进行了大量显微注射工作后,简要介绍建立遗传工程小鼠模型的基本技术与方法,并对在显微操作过程中较为关键的因素进行一些分析和讨论。     

Increased activity of only an individual non-regulated enzyme fructose-1,6-bisphosphate aldolase in <Emphasis Type="Italic">Anabaena</Emphasis> sp. strain PCC 7120 stimulates photosynthetic yield

The goal of this study was to investigate the contribution of increased activity of individual non-regulated enzymes in the Calvin cycle to improve photosynthetic yield. Two non-regulated enzymes, rice fructose-1,6-bisphosphate aldolase (FBA) and spinach triosephosphate isomerase (TPI), were individually cloned and overexpressed in the cyanobacterium Anabaena sp. strain PCC 7120 cells. The enzyme activity and the photosynthetic yield, as reflected by the cell growth rate, photosynthetic oxygen evolution and dry cellular weight, were measured and compared between the wild-type and transgenic cells harboring either FBA or TPI. Though the activity of these two individual non-regulated enzymes was similarly increased in the corresponding transgenic cells, the contributions of each enzyme on the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), reflected by the levels of Rubisco large subunit, and the photosynthetic yield were different. Transgenic cells, carrying FBA, showed an evident increase in Rubisco amount and photosynthetic yield, while there was no increase in cells harboring TPI. This indicates that the contributions of non-regulated enzymes in the Calvin cycle on photosynthetic yield differed and firstly reveals that increased activity of only a single non-regulated enzyme in transgenic cells markedly improves the photosynthetic yield via stimulating the amount of Rubisco and consequently accelerating the ribulose-1,5-bisphosphate (RuBP) regeneration rate.     

Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^? allele. The promoter of both alleles contains the effector binding element (EBE_PthA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.     

Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii

Treatment with NaHSO₃ induces a 10-fold increase in H₂ photoproduction in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H₂ photoproduction in green alga. In this study, treatment with 13 mM NaHSO₃ resulted in about a 200-fold increase in H₂ production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O₂ content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO₃ treatment results in a higher maximum rate of H₂ photoproduction, greater efficiency of conversion of light energy into H₂, shorter half-time to produce the maximum accumulated H₂ levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO₃ treatment is an efficient, rapid, and economic strategy for improving photobiological H₂ production in the green alga C. reinhardtii.     

Transgenic citrus expressing synthesized <Emphasis Type="Italic">cecropin</Emphasis> B genes in the phloem exhibits decreased susceptibility to Huanglongbing

Key message

Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing.

Abstract

Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth (Antheraea pernyi), has been shown to effectively eliminate bacteria. In this study, we demonstrated that transgenic citrus (Citrus sinensis Osbeck) expressing the cecropin B gene specifically in the phloem had a decreased susceptibility to HLB. Three plant codon-optimized synthetic cecropin B genes, which were designed to secrete the cecropin B peptide into three specific sites, the extracellular space, the cytoplasm, and the endoplasmic reticulum, were constructed. Under the control of the selected phloem-specific promoter GRP1.8, these constructs were transferred into the citrus genome. All of the cecropin B genes were efficiently expressed in the phloem of transgenic plants. Over more than a year of evaluation, the transgenic lines exhibited reduced disease severity. Bacterial populations in transgenic lines were significantly lower than in the controls. Two lines, in which bacterial populations were significantly lower than in others, showed no visible symptoms. Thus, we demonstrated the potential application of the phloem-specific expression of an antimicrobial peptide gene to protect citrus plants from HLB.