Rubisco副合基因的构建及其在E.coli中的表达

将编码番茄核酮糖－１,５－二磷酸羧化酶／加氧酶小亚基转运肽的一段ＤＮＡ序列与菠菜Ｒｕｂｉｓｃｏ大亚基的编码区连接,构建了一个Ｒｕｂｉｓｃｏ融合基因。限制性内切酶图谱和ＤＮＡ序列分析证明副合部位的核苷酸序列符合构建前的三联密码子框架。将Ｒｕｂｉｓｃｏ融合基因转入Ｅ．ｃｏｌｉ,用ＩＰＴＧ进行诱导表达。利用蛋白质印迹技术检测到诱导产物的存在。     

水稻和烟草核酮糖1，5－二磷酸羧化酶／加氧酶大小亚基之间的分子杂交

利用固定化Ｒｕｂｉｓｃｏ大小亚基解离重组技术,进行水稻和烟草Ｒｕｂｉｓｃｏ大小亚基之间的分子杂交,实验表明,无论同源或异源的小亚基重组到固定化的大亚基上去后,其羧化酶活性没有明显的变化,但对加氧酶活性却有明显的影响。当水稻Ｒｕｂｉｓｃｏ的大亚基同烟草小亚基杂交重组后,其加氧酶活性同固定化水稻Ｒｕｂｉｓｃｏ相比有明显的增高,因而其羧化／氧化比值下降,并且接近于对照的固定化烟草Ｒｕｂｉｓｃｏ。反之,当烟草Ｒｕｂｉｓｃｏ的大亚基与水稻小亚基杂交重组后,其加氧酶活性同固定化烟草Ｒｕｂｉｓｃｏ相比有明显降低,因而其羧化／氧化比值升高,并接近于对照的固定化水稻Ｒｕｂｉｓｃｏ。由此推测,高等植物Ｒｕｂｉｓｃｏ的小亚基对酶的羧化／氧化比值有一定的影响。     

小麦返白系返白期间Rubisco变化研究

以小麦返白系和对照矮变１号为材料,选用返白系三个特殊的时期,返白初期、全白期、复绿初期,对其叶片可溶性蛋白进行了Ｎａｔｉｖｅ－ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ的研究。结果表明：随着叶片白化,核酮糖１．５－二磷酸羧化酶／加氧酶（Ｒｕｂｉｓｃｏ）全酶谱带逐渐变小,全白叶全酶谱带消失,随着复绿全酶谱带又出现,并逐渐恢复。而且Ｒｕｂｉｓｃｏ大、小亚基（ＬＳ、ＳＳ）谱带减少幅度差异很大,大亚基减少远远大于小亚基。     

低温锻炼对水稻幼苗叶片中Rubisco的影响

低温锻炼能提高水稻幼苗的抗冷力,低温锻炼虽不能明显提高Ｒｕｂｉｓｃｏ活性,却提高了冷却条件下Ｒｕｂｉｓｃｏ的稳定性和增强了胁迫后正常生长条件下其活性的恢复能力。分别用火箭免疫电泳分析Ｒｕｂｉｓｃｏ蛋白和ＳＤ－ＳＰＡＧＥ分析大,小亚基量表明：低温锻炼未提高Ｒｕｂｎｉｓｃｏ蛋白的合成能力,但增加了大,小亚基的合成量。     

Rubisco活化酶的研究进展

Ｒｕｂｉｓｃｏ活化酶是最近发现的一种该编码的叶绿体蛋白,它在叶绿体内具有激活光合碳同化限速酶Ｒｕｂｉｓｃｏ的功能,该酶能在生理水平ＲｕＢＰ,ＣＯ２浓度（１０μｍｏｌ／Ｌ）下使Ｒｕｂｉｓｃｏ达到最大的活化程度,Ｒｕｂｉｓｃｏ活化酶的研究揭示了长期以来未能解决的Ｒｕｂｉｓｃｏ在体内活化的机理,Ｒｕｂｉｓｃｏ活化酶能解除磷酸糖对Ｒｕｂｉｓｃｏ活性的抑制作用,它的活化活性需要有ＡＴＰ的存在,同时它有ＡＴＰ     

影响水稻光合日变化的酶和相关因素的分析

水稻叶片的最大光合速率出现在上午１０：００时,Ｒｕｂｉｓｃｏ初始活力也在此时达到最大,然后逐渐降低,下午１４：００时略上升后又下降．Ｒｕｂｉｓｃｏ初始活力与光合速率之间极显著相关,相关系数为 ０．９４７４．运用相关性分析、回归分析、通径分析,对晴（有时有云）天气水稻叶片的光合速度、光合关键酶及有关因素的日变化进行综合评估,结果发现中午光合下降主要来自气孔限制,同时Ｒｕｂｉｓｃｏ活性也下降;Ｒｕｂｉｓｃｏ活性是影响光合日变化的又一重要生化因子．体内Ｒｕｂｉｓｃｏ活性受Ｒｕｂｉｓｃｏ活化酶的调节．     

Rubisco的装配一种研究监护分子（molecular chaperone）作用的模型系统

Ｒｕｂｉｓｃｏ的装配──一种研究监护分子（ｍｏｌｅｃｕｌａｒｃｈａｐｅｒｏｎｅ）作用的模型系统李立人（中国科学院上海植物生理研究所,上海２０００３２）关键词Ｒｕｂｉｓｃｏ,装配１．引言核酮糖１,５－二磷酸羧化酶／加氧酶（简称Ｒｕｂｉｓｃｏ,Ｅ．Ｃ．４...     

水稻Rubisco小亚基前体cDNA的克隆及其产物向豌豆叶绿体的运输

用ＲＴ－ＰＣＲ方法克隆了完整的水稻Ｒｕｂｉｓｃｏ小亚基前体ｃＤＮＡ基因,经耦联的体外转录和翻译系统合成了带同位素标记的小亚基前体蛋白,然后与新制备的豌豆完整叶绿体共保温,进行蛋白质的跨膜运输研究显示：异源的水稻Ｒｕｂｉｓｃｏ小亚基前体能穿膜运输入豌豆叶绿体。     

水稻叶片在自然衰老过程中1,5—二磷酸核酮糖羧化酶／加氧酶的降解

１,５－二磷酸核酮糖羧化酶／加氧酶（Ｒｕｂｉｓｃｏ）是光合碳同化的关键酶,研究其降解机理对合理调控水稻生长后期光合衰退具有重要意义。前人用人为诱导植物衰老的方法,研究了Ｒｕｂｉｓｃｏ的降解机理,认为该酶降解之前,必需发生亚基间的交联聚合和向类囊体膜转移,这样在结构和空间上有利于水解酶的作用。我们用自然衰老叶片进行研究的结果表明：Ｒｕｂｉｓｃｏ在降解过程中其比活基本保持恒定,意味着未发生酶的失活,     

水稻和烟草核酮糖1,5—二磷酸羧化酶／加氧酶大小亚基之间的分子杂交

利用固定化Ｒｕｂｉｓｃｏ大小亚基解离重组技术,进行水稻和烟草Ｒｕｂｉｓｃｏ大小亚基之间的分子杂交,实验表明,无论同源或异源的小亚基重组到固定化的大亚基上去后,其羧化酶活性没有明显的变化,但对加氧酶活性却有明显的影响。当水稻Ｒｕｂｉｓｃｏ的大亚基同烟草小亚基杂交重组后,其加氧酶活性同固定化水稻Ｒｕｂｉｓｃｏ相比有明显的增高,因而其羧化／氧化比值下降,并且接近于对照的固定化烟草Ｒｕｂｉｓｃｏ。反之,当     

Cloning and Structure Analysis of rbcS Gene from Wild Soybean

About 1 kb fragment of rbcS (ribulose 1, 5-bisphosphate carboxylase small subunit) gene in wild soybean (Glycine soja, Ji 50017) was amplified from total DNA by PCR assay. Sequence analysis of the fragment indicated that 1089 bp sequenced included the whole coding region for Rubisco small snbunit. The rbcS gene in wild soybean encoded a precursor composed of a transit peptide of 55 amino acids and a mature protein of 123 amino acids. There were two introns found in the rbcS gene as other dicotyledonous species previously sequenced. Comparison of DNA sequences showed high degree homology of rbcS genes between wild soybean and cultivated soybean (Glycine max var. wayne). Some changes of amino acids emerged from the diverse nucleotides did not affect the function of the small subunit. These results may contribute some basic data in molecular biology to study the origin and evolution of soybean.     

Acyl carrier protein-derived sequence encoded by the chloroplast genome in the marine diatom Cylindrotheca sp. strain N1.

The chloroplast genome of chromophytic and rhodophytic algae differs from the plastid genome of plants and green algae in that it encodes the gene for the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Hybridization studies indicated that there was a second region of chloroplast DNA from the marine diatom Cylindrotheca sp. strain N1 that strongly hybridized to a previously isolated Cylindrotheca fragment that contained the rbcS gene and flanking sequences. Subsequent determination of the oligonucleotide sequence of this second chloroplast DNA fragment, however, indicated that hybridization was due to identical sequences 3' to the previously cloned Cylindrotheca chloroplast rbcL rbcS genes. Sequences derived from the 5' end of the second chloroplast DNA fragment contained a short open reading frame of 80 amino acids which was found to be highly homologous to bacterial acyl carrier protein and nuclear-encoded acyl carrier protein from plants. Amino acid residues in the environment of Ser-36 of the Escherichia coli protein, which is bound to a 4'-phosphopantetheine moiety, are virtually identical in the Cylindrotheca deduced sequence and all other sources of this protein. Unlike plant acyl carrier-deduced amino acid sequences, there was no leader peptide sequence found for the presumptive Cylindrotheca protein, consistent with the location of this DNA fragment on the chloroplast genome of this organism. DNA encoding the putative acyl carrier protein gene and rbcS thus represent two genes that are chloroplast-encoded in the chromophytic marine diatom Cylindrotheca, a significant departure from the organization of such genes in plants.     

叶绿体超氧化物歧化酶（ChlSOD）基因的构建及序列分析

构建叶绿体超氧化物歧化酶基因（ChlSOD),采用RT-PCR方法分离豌豆RUBP羧化酶小亚基导肽基因（TP）,定向克隆至pUC19测序,定向克隆烟草MnSOD成熟蛋白基因（SODm)至pUC19;采用平粘端连接法将二者在pUC19中构成嵌合基因ChlSOD,并对此基因进行序列分析,序列分析表明：TPcDNATP,12bp的Linker及615bp SODm。TP与ChlSOD基因的序列分析与国外报道序列完全吻合。     

Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes.

Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB. rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits. rbcL-rbcS was the same as that reported previously (A. M. Viale, H. Kobayashi, T. Takabe, and T. Akazawa, FEBS Lett. 192:283-288, 1985). A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced. We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites. rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively. Edman degradation analysis of the subunits of RuBisCO isolated from C. vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium. The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively. Among hetero-oligomeric RuBisCOs, the C. vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%). A similar situation has been observed for the C. vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits.     

Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin)

Abstract The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin. A PCR-amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fim A, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.     

Cloning, sequence, and expression of the Drosophila cAMP-dependent protein kinase catalytic subunit gene

Genomic DNA containing the protein coding region for Drosophila cAMP-dependent protein kinase catalytic subunit has been cloned and sequenced. The probe used to detect and isolate the gene fragment was constructed from two partially complementary synthetic oligonucleotides and contains 60 base pairs that encode (using Drosophila codon preferences) amino acids 195-214 of the beef heart catalytic subunit. In reduced stringency hybridization conditions, the probe recognizes two target sites in fly genomic DNA with 85% homology. One of these sites is in the cAMP-dependent protein kinase catalytic subunit gene, which was isolated as a 3959-base pair HindIII fragment. This fragment contains all of the protein coding portion, 900 base pairs upstream of the initiator ATG, and 2000 base pairs downstream of the termination codon (TAG). The coding portion of the gene contains no introns and yields a protein of 352 amino acids. There is a 2-amino acid insertion near the N terminus of the fly protein relative to the beef and mouse enzymes. Of the remaining 350 amino acids, 273 are invariant in the three species. A probe derived from the coding sequence of the HindIII clone hybridizes strongly to a 5100-base poly(A)+ RNA and weakly to 4100- and 3400-base poly(A)+ RNAs expressed in adult flies. A 2100-base pair EcoRI genomic fragment containing the second site recognized by the 60-base pair probe has also been cloned. DNA sequence analysis demonstrates that this fragment is part of the cGMP-dependent protein kinase gene or a close homolog. The catalytic subunit gene and the cGMP-dependent protein kinase gene have been located in regions 30C and 21D, respectively, of chromosome 2.     

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.     

The amino acid sequence of the A2B1a subunit of glycinin

The amino acid sequences of the acidic and basic components of the A2B1a subunit of glycinin, the major seed reserve protein of the soybean (Glycine max L. Merr.), were determined. They contain 278 and 180 amino acids, respectively, and have molecular weights of 31,600 +/- 100 and 19,900 +/- 100. The molecular weight of the acidic component is considerably less than that estimated by sodium dodecyl sulfate-gel electrophoresis (37,000). Sequence heterogeneity was detected at several positions scattered throughout the primary structures of both components, indicating that the preparation sequenced was composed of several nearly identical polypeptides. These data, in conjunction with a recently determined nucleotide sequence of the 3'-terminal two-thirds of the analogous glycinin subunit gene, illustrate the complexity of the gene family responsible for synthesis of glycinin subunits.