Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis

Fructose-1,6-bisphosphatase（FBPase） is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends（RACE） technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region（UTR） of 92 bp, a 3′？UTR of 69 bp, and an open reading frame（ORF） of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.

A Golden2-like transcription factor, BnGLK1a, improves chloroplast development, photosynthesis, and seed weight in rapeseed

Enhancing photosynthetic efficiency is a major goal for improving crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development.In this study,we demonstrate that Golden2-like 1a(BnGLK1a)plays an important role in regulating chloroplast development and photosynthetic efficiency.Overexpressing BnGLK1a resulted in significant increases in chlorophyll content,the number of thylakoid membrane layers and photosynthetic efficiency in Brassica napus,while knocking down BnGLK1a transcript levels through RNA interference(RNAi)had the opposite effects.A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1-2(BnPYL1-2)and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit(BnCSN5A),which play essential roles in regulating chloroplast development and photosynthesis.Consistent with this,BnGLK1a-RNAi lines of B.napus display hypersensitivity to the abscisic acid(ABA)response.Importantly,overexpression of BnGLK1a resulted in a 10%increase in thousand-seed weight,whereas seeds from BnGLK1a-RNAi lines were 16%lighter than wild type.We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity.Our results not only provide insights into the mechanisms of BnGLK1a function,but also offer a potential approach for improving the productivity of Brassica species.

Os DXR interacts with Os MORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

Plant chlorophyll biosynthesis and chloroplast development are two complex processes that are regulated by exogenous and endogenous factors. In this study, we identified OsDXR, a gene encoding a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR is a chloroplast protein. Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type.Moreover, we found that the RNA editing efficiencies of ndhA-1019 and rpl2-1 were significantly reduced in the OsDXR knock-out lines. Furthermore, OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay. Finally, disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway resulted in defects in chloroplast development and the RNA editing of chloroplast genes.

PtrDJ1C,an atypical member of the DJ-1 superfamily,is essential for early chloroplast development and lignin deposition in poplar

The nuclear-encoded factors and the photosynthetic apparatus have been studied extensively during chloroplast biogenesis.However,many questions regarding these processes remain unanswered,particularly in perennial woody plants.As a model material of woody plants,poplar not only has very significant value of research,but also possesses economic and ecological properties.This study reports the Populus trichocarpa DJ-1C(PtrDJ1C)factor,encoded by a nuclear gene,and a member of the DJ-1 superfamily.PtrDJ1C knock-out with the CRISPR/Cas9 system resulted in different albino phenotypes.Chlorophyll fluorescence and immunoblot analyses showed that the levels of photosynthetic complex proteins decreased significantly.Moreover,the transcript level of plastid-encoded RNA polymerase-dependent genes and the splicing efficiency of several introns were affected in the mutant line.Furthermore,rRNA accumulation was abnormal,leading to developmental defects in chloroplasts and affecting lignin accumulation.We concluded that the PtrDJ1C protein is essential for early chloroplast development and lignin deposition in poplar.

Expression of the Thylakoid Membrane Localized PPF1 in Transgenic Arabidopsis Affects Chloroplast Development

PPF1 is a vegetative growth related gene that encodes a putative membrane protein having high homology with Arabidopsis chloroplast thylakoid protein ALB3. Immunoelectron microscopic assay showed that PPF1 was mainly localized in the thylakold membrane and was highly expressed in well-developed chloroplasts of short day (SD) grown G2 pea while having a very low abundance in chloroplasts of long day (LD) grown plants two weeks after flowering. Comparison of the leaf senescence processes in transgenic Arabidopsis and wild type plants revealed that overexpression of PPF1 delayed leaf senescence, while the depression of its Arabidopsts homologue (ALB3) with PPF1 antisense mRNA accelerated leaf senescence obviously. Ultrastructural analysis of transgenic Arabidopsis plants showed that when PPF1 was overexpressed in Arabidopsis, the chloroplasts were bigger and had much more grana and stroma thylakoid membranes than those of wild type plants. On the contrary, when PPF1 was expressed in antisense orientation to reduce the level of PPF1 homologue in Arabidopsis, the transgenic plants had smaller chloroplasts With less grana. and poorly developed thylakoid membrane systems. These results suggested that the developmental status of chloroplasts was positively correlated with the level of PPF1 or its Arabidopsts homologue, ALB3. Our results suggested that PPF1 gene might regulate plant development by controlling chloroplast development.

Protective Effects of Oral Fructose-1, 6-diphosphate on Liver Injury in Animal Models

Aim To investigate the effects of FDP on different liver injury models to explore the possibility of FDP used as an oral liver protective agent. Methods Chronic liver injury model in rats was induced by carbon tetrachloride （ CCl4 ） ; Acute liver injury model in mice was induced by aminogalactose （GAIN） or lipopolysaccharide （LPS）. Results In CCl4-induced chronic liver injury model, FDP （1 , 4 g·kg^-1·d^-1, q.d., for 10 weeks） significantly lowered ALT, AST,γ-glutamyl transpeptidase （γ-GT）, alkaline phosphatase （ALP）, and total bilirubin （T-BIL） in serum compared with vehicle; simultaneously it evidently elevated abnormal total protein （TP）, albumin （ALB） and total cholesterol （ T-CHO ） levels in serum; it also dose-dependently reduced hydroxyproline contents in hepatic tissue. 4 g·kg^-1·d^-1 of FDP apparently decreased incidence of hepatic cirrhosis, and alleviated pathological changes of liver tissue. In GaiN-induced model, 1.0 - 4. 0 g·kg^-1·d^-1 of FDP （ bid, for 3 d ） significantly lowered alanine aminotransferase （ ALT ） and aspartate aminotransferase （ AST ） levels in serum ; it also decreased liver coefficient. 4. 0 g·kg^-1·d^-1 of FDP significantly alleviated pathological changes of cell ultra-structures. In LPS-induced model, only high dose of FDP （4. 0 g·kg^-1·d^-1, bid, for 12 d） significantly decreased ALT level in serum. Conclusion This study first demonstrated the protective effect of oral FDP on chronic liver injury caused by CCl4, and confirmed its effect on acute liver injury at the same time, suggesting that Long-term oral FDP is efficacious against liver injury induced by different factors and can be used as an oral liver protective agent in clinic.

Isolation of a 1 195 bp 5′-Flanking Region of Rice Cytosolic Fructose-1,6-bisphosphatase and Analysis of Its Expression in Transgenic Rice

A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 lip 5'-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding beta-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice.

三倍体茶树‘西莲1号’叶绿体基因组特征及系统发育分析

三倍体茶树品种‘西莲1号’是桑植白茶产业发展的主推品种,与湖南现有茶树品种相比具有很强的特异性。为揭示‘西莲1号’的分类和演化地位,项目采用叶绿体(Chloroplast,cp)全基因组测序技术进行了‘西莲1号’全基因组测序,并对其所有的SSR标记进行了开发与筛选。结果表明:(1)‘西莲1号’叶绿体基因组全长为157038 bp,GC含量为37.30%,为一个四分体结构,包括1个大的单拷贝(LSC)、一个小的单拷贝(SSC)和一对反向重复序列(IR),长度分别为86612 bp、18282 bp和26072 bp。cpDNA共注释得到132个基因,其中unigenes有114个,包含80个编码基因,30个tRNA基因和4个rRNA基因。(2)共鉴定出245个简单序列重复(SSR),通过筛选有15个SSR具有较高的多态性。获得了cpDNA基因组20个氨基酸的偏好密码子,‘西莲1号’密码子偏好以A/T碱基结尾。(3)基因选择压力分析表明,与茶组其他资源相比,6个基因(accD、ndhC、petB、rpl16、rpoC1、rpoC2)可能处于正选择状态。(4)基于叶绿体基因组序列构建了基于22份茶组植物的系统发育树,聚类结果显示‘西莲1号’与其他栽培型茶树品种(Camellia sinensis var.sinensis)处于系统发育树的末端,属于进化的茶树资源类型。

Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca^(2+)-ATPase in cardiomyocytes of Adriamycin-treated rats

Objective: To observe the effects of fructose-1,6-diphosphate (FDP) on serum levels of cardiac troponin 1 (cTnl) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca2+]) and activity of sarcoplosnic Ca2+-ATPase (SRCa2+-ATPase) in Adriamycin (ADR)-treated rats. Methods: Rats were intraperitoneally injected with ADR (2.5 mg/kg every other day for 6 times) and then with different dosages of FDP (every other day for twenty-one times). Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was performed to detect serum level of cTnl. CK-MB was detected by monoclonal antibody, Myo[Ca2+] was detected by fluorescent spectrophotometry and the activity of SRCa2+-ATPase was detected by inorganic phosphate method. Results: FDP (300, 600, 1200 mg/kg) significantly reduced the serum levels of cTnl and CK-MB, while at the same time decreased calcium concentration and increased SRCa2+-ATPase activity in cardiomyocytes of ADR-treated rats (P<0.01). Conclusions: FDP might alleviate the cardiotoxic effects induced by ADR through decreasing calcium level as well as increasing SRCa2+-ATPase activity in cardiomyocytes.

CDP1, a novel component of chloroplast division site positioning system in Arabidopsis

Chloroplasts are plant-specific organelles that evolved from endosymbiotic cyanobacteria. They divide through binary fission. Selection of the chloroplast division site is pivotal for the symmetric chloroplast division. In E. coli, positioning of the division site at the midpoint of the cell is regulated by dynamic oscillation of the Min system, which includes MinC, MinD and MinE. Homologs of MinD and MinE in plants are involved in chloroplast division. The homolog of MinC still has not been identified in higher plants. However, an FtsZ-like protein, ARC3, was found to be involved in chloroplast division site positioning. Here, we report that chloroplast division site positioning 1 （AtCDP1） is a novel chloroplast division protein involved in chloroplast division site placement in Arabidopsis. AtCDP1 was discovered by screening an Arabidopsis cDNA expression library in bacteria for colonies with a cell division phenotype. AtCDP1 is exclusively expressed in young green tissues in Arabidopsis. Elongated chloroplasts with multiple division sites were observed in the loss-of-function cdpl mutant. Overexpression of AtCDP1 caused a chloroplast division phenotype too. Protein interaction assays suggested that AtCDP1 may mediate the chloroplast division site positioning through the interaction with ARC3. Overall, our results indicate that AtCDP1 is a novel component of the chloroplast division site positioning system, and the working mechanism of this system is different from that of the traditional MinCDE system in prokaryotic cells.

The Arabidopsis Spontaneous Cell Death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development, photoprotection and retrograde signalling

Carotenoids, a class of natural pigments found in all photosynthetic organisms, are involved in a variety of physiological processes, including coloration, photoprotection, biosynthesis of abscisic acid （ABA） and chloroplast biogenesis. Although carotenoid biosynthesis has been well studied biochemically, the genetic basis of the pathway is not well understood. Here, we report the characterization of two allelic Arabidopsis mutants, spontaneous cell death1-1 （spcl-1） and spc1-2. The weak allele spc1-1 mutant showed characteristics of bleached leaves, accumulation of superoxide and mosaic cell death. The strong mutant allele spc1-2 caused a complete arrest of plant growth and development shortly after germination, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that SPC1 encodes a putative ζ-carotene desaturase （ZDS） in the carotenoid biosynthesis pathway. Analysis of carotenoids revealed that several major carotenoid compounds downstream of SPC 1/ZDS were substantially reduced in spc1-1, suggesting that SPC 1 is a functional ZDS. Consistent with the downregulated expression of CAO and PORB, the chlorophyll content was decreased in spc1-1 plants. In addition, expression of Lhcb1. 1, Lhcbl. 4 and RbcS was absent in spc1-2, suggesting the possible involvement of carotenoids in the plastid-to-nucleus retrograde signaling. The spc1-1 mutant also displays an ABA-deficient phenotype that can be partially rescued by the externally supplied phytohormone. These results suggest that SPC1/ZDS is essential for biosynthesis of carotenoids and plays a crucial role in plant growth and development.

Clinical study of applying fructose-1,6-diphosphate and captopril to enhance the protective effects of cardioplegia solution on ischemic myocardium

In the present experiment,fructose-1,6-diphosphate（FDP）and captopril（Cap）wereadded to the cold potassium cardioplegia solution and the levels of malondialdehyde（MDA）,cre-atine phosphokinase MB（CPK-MB）,thromboxane B（TXB2）and 6-keto-PGF1α in plasma weremeasured during open-heart surgery.Quantitative study of myocardial ultrastructure and obser-vation of cardiac resuscitation were also undertaken.The findings suggested that FDP,especiallywhen combined with Cap could significantly strengthen the protective effects of cold potassiumcardioplegia solution on ischemic myocardium.

A New Method of Crystallization of Octahydro Trisodium Salt of Fructose- 1,6-diphosphate

In order to overcome the elementary heterogeneous nucleation whileoctahydro trisodium salt of fructose- 1, 6-diphosphate (FDPNa_3·8H_2O) is crystallized with ethanol precipitation at low temperature,a new crystallization method with alcohol precipitation combined withsalt precipitation has been presented. The ethanol-sodium ac- etatesystem for crystallization of salt of fructose-1, 6-diphosphate isbased on the mechanism of crystallization of FDPNA_3·8H_2O in theethanol-low temperature system. It is found that crystal size may becontrolled by regulating Temperature of pH value of solution in thecrystallization process, and the crystal yield increases to 95/100from 78/100 Which obtained in the ethanol-low temperature system.

白叶1号白化过程中叶绿体蛋白质组差异分析

白叶1号是一种温度敏感型白化茶树品种,叶绿体的变化是其产生阶段性白化现象的关键因素。本研究以白叶1号鲜叶叶绿体为研究对象,采用双向电泳、质谱鉴定结合生物信息学分析,研究阶段性白化过程中叶绿体蛋白的表达差异,探讨白叶1号阶段性白化现象的分子机制。结果表明,在白叶1号白化前期、白化期和复绿期叶绿体中分别识别726、748、718个蛋白质,其中差异表达的蛋白59个,质谱成功鉴定22个差异表达蛋白。生物信息学分析表明,差异表达蛋白直接或间接参与了光合作用、应激响应、核酸代谢、物质代谢和未知功能等,其中与光合作用相关的差异表达蛋白最多,占31.82%,表明阶段性白化现象可能与这些生理功能相关。通过荧光定量PCR分析发现,差异蛋白的基因表达与蛋白表达存在一定差异,这可能是由于蛋白质翻译后加工及修饰造成的。上述研究为进一步揭示白叶1号阶段性白化现象产生的分子机制奠定了理论基础。

杀雄剂SQ-1诱导小麦生理型雄性不育小花完整叶绿体差异蛋白质的鉴定

为了在蛋白质水平揭示小麦雄性不育的分子遗传机制,以小麦经杀雄剂SQ-1诱导的生理型雄性不育系,以及对应正常可育系所构建的等生理差异系为试材,应用差速离心和蔗糖密度梯度离心,首先分离出供试材料小花的完整叶绿体,制备蛋白样品后采用固相IEF/SDS-PAGE双向凝胶电泳技术对完整叶绿体蛋白质进行了分离、银染,得到了重复性较好的双向电泳图谱.PDQuest2DE软件分析识别出约150个较为清晰的蛋白质点,采用MALDI-TOF鉴定出的6个差异表达蛋白质分别是:PAP-fibrillin、ATRABB1A、底物同源结构域蛋白/RhoGAP结构域蛋白、铜锌超氧化物歧化酶(Cu/Zn-SOD)、R2R3-MYB转录因子及1个假定蛋白质.生物信息学功能分析暗示,这些蛋白直接参与了花药内激素调节、蛋白质转运、蛋白质互作、活性氧积累及花药的发育,表明SQ-1诱导的小麦生理型雄性不育其败育机理可能就与这些生理代谢过程的变异直接相关.

恶性疟原虫裂殖子表面蛋白1C末端基因导入烟草叶绿体的研究

目的将恶性疟原虫裂殖子表面蛋白1C末端msp1-42基因(3D7株)导入烟草叶绿体基因组中并进行同质化筛选,为利用叶绿体表达系统生产MSP1-42蛋白提供基础材料。方法利用烟草偏爱密码子设计克隆恶性疟原虫(3D7株)msp1-42基因的引物,从含msp1-42基因的pBluntmsp质粒中扩增出msp1-42,构建烟草叶绿体表达载体LRrrmsp。通过基因枪转化法转化烟草叶片,在500mg/L壮观霉素的选择压力下筛选抗性植株,采用PCR鉴定抗性植株的msp1-42基因及aadA基因,对鉴定阳性植株进行同质化筛选(叶片切碎、在含500mg/L壮观霉素分化培养基上分化出新的植株)3轮以上,并采用多重PCR分析其同质化情况。结果构建了恶性疟原虫msp1-42基因的叶绿体表达载体LRrrmsp。基因枪转化后获得6个转化子,转化频率为0.6个/枪。转化3～5d后,小块叶片开始增大增厚,并逐渐由绿色变为黄绿色,7～10d后黄化或白化,再经约30d的筛选培养,叶片上出现绿色小芽。PCR检测抗性植株的msp1-42及aadA基因,分别扩增出约900bp与500bp的条带,与预期相符。多重PCR分析从经过3轮同质化筛选植株的叶绿体基因中扩增出与未转基因烟草对照大小一致的弱条带,表明经过3轮同质化筛选的转基因植株仍含有未插入外源基因的叶绿体基因组。结论获得含恶性疟原虫msp1-42的烟草叶绿体表达载体,并将恶性疟原虫msp1-42基因导入烟草叶绿体基因组中,获得尚未完全同质化的转基因烟草。

小麦苗期对非生物胁迫的响应及叶绿体果糖-1,6-二磷酸醛缩酶基因的表达分析

叶绿体果糖-1,6-二磷酸醛缩酶(Chloroplast Fructose-1,6-bisphosphate aldolase,CpFBA)参与叶绿体(质体)的碳、氮代谢,在光合作用中具有重要功能,并能对植物的非生物胁迫产生积极响应。为了深入研究小麦CpFBA的生物学功能和对外界逆境的响应模式,以矮变1号、返白系、中国春、西农928、西农2000和晋麦47等六个不同生理特性的小麦品种(系)为材料,分析其在低温、NaCl、PEG模拟干旱和ABA处理下最大叶长和主根根长受到的影响,并采用半定量RT-PCR的方法研究小麦幼苗中CpFBA基因表达模式的变化。结果表明,各种外界非生物胁迫均不同程度的抑制了小麦幼苗叶片和根的生长,其中低温和ABA处理的抑制最为显著。在各种处理条件下,小麦幼苗CpFBA均有明显的上调表达,但不同品种间存在差异,表达模式的改变与品种的生理特性之间表现出一定的相关性。

水分胁迫对小麦叶绿体果糖——1.6—双磷酸酯酶活性的影响

在PEG溶液处理所引起的水分胁迫下,小麦幼苗叶片的相对含水量、净光合速率、蒸腾速率、气孔导度、细胞间隙二氧化碳浓度降低。在轻度水分胁迫下,叶片叶绿体果糖—1.6—双磷酸酯酶(E.C.3.1.3.11)活性没有受影响,而严重水分胁迫可使此酶活性显著降低,这是此时叶片净光合速率下降的原因之一。叶片轻微失水时,其内淀粉含量有一定上升,但继续失水则含量下降。水分胁迫使小麦叶片蔗糖含量降低。

香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基克隆与生物信息学分析

通过PCR技术成功克隆1个香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长序列,并登录GeneBank,登陆号为FJ973633。利用生物信息学方法,分析香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因序列,对其推导的氨基酸的理化性质、亲水性/疏水性、跨膜结构、导肽、二级结构进行预测,并对香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的氨基酸做进化发育分析,为进一步了解香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基在香蕉光合吸收中的作用奠定基础。

烟草NtFtsZ2-1基因参与叶绿体分裂和扩大的调节(英文)

叶绿体虽然是植物细胞内一种极其重要的细胞器,但其分裂的分子机制尚不很清楚。已经证明FtsZ 蛋白作为真核细胞分裂装置的一个关键成分,参与叶绿体的分裂过程。烟草的 FtsZ 基因属于 2 个不同的家族,在对 NtFtsZ1 家族成员研究的基础上,用正义和反义表达技术研究了 NtFtsZ2 家族成员NtFtsZ2-1 基因在转基因烟草中的功能。显微分析结果表明NtFtsZ2-1基因的表达水平异常增强或减弱都会严重干扰叶绿体的正常分裂过程,导致叶绿体在形态和数目上的异常(体积明显增大,数目显著减少),而单个叶肉细胞中叶绿体的总表面积在正反义转基因烟草和野生型烟草之间保持了相对稳定,没有发生明显的变化。同时还证明NtFtsZ2-1基因表达的变化对叶绿素含量和叶绿体的光合作用能力没有直接的影响。据此我们认为NtFtsZ2-1基因参与叶绿体的分裂和体积的扩大,其表达水平的波动会改变植物中叶绿体的数目和大小,而且在叶绿体的数目与体积之间可能存在一种补偿机制,保证叶绿体能最大限度地吸收光能,从而使光合作用得以正常进行。