小麦体细胞无性系Glu-1基因突变体的遗传分析

对 Glu-1基因的Glu-Al和Glu-Bl位点都发生突变的1个小麦体细胞无性系后代的高分子量谷蛋白亚基组成进行的分析结果,进一步证实了体细胞无性系发生了基因突变, 且发现同源染色体的等位基因易发生相同突变,突变体多是纯合基因型。认为这是体细胞无性系变异稳定快的遗传根源。 Abstract:Evidence for specific gene mutation has been obtained in our previous research by analyzing high-molecular-weight glutenin subunits(HMW-GS)controlled by Glu-1 gene in wheat somaclons.On the basis of the result,the present study detected in the offspring of a HMW-GS somaclone the mutants at both Glu-A1 and Glu-B1 loci.The occurrence of gene mutation in wheat somaclones was further proved.The results showed that a allele on homologous chromosomes often mutated identically.Genotype of this kind mutation migh be pure.Thie is considered to be genetic reason for quickly stabilizing of somaclonal variation.     

小麦新品种(系)Glu-1位点等位基因变异研究

应用SDS-PAGE技术分析了40份小麦新品种(系)的高分子量麦谷蛋白亚基等位基因变异。在Glu-1位点共检测到10种变异类型,其中Glu-Al位点有3种类型：Null、1、26 ,Glu-B1位点有5种类型：7 8、7 9、14 15、7、17 18,Glu-D1位点有2种类型：2 12、5 10;Null(54．3％)、7 8(51．4％)和2 12(62．9％)分别是Glu-Al、Glu-B1和Glu-D1位点上的主要亚基变异类型。另外,在2份材料的Glu-B1和Glu-D1位点各检测到1个新的亚基,分别命名为1By8．1和1Dx5^ 。Glu-1位点的Nei‘s遗传变异指数平均为0,5648,Glu-B1的遗传多样性最高,Glu-D1最低。供试小麦材料Glu-1位点的HMW-GS组合共有17种类型,以(Null,7 8,2 12)组合为主要类型,占31．4％;有9种亚基组合类型分别只在1份材料中出现,占26.1％。结果表明,这些小麦新品种(系)存在着丰富的亚基组合类型。     

Genetic Diversity of Gli-1，Gli-2 and Glu-1 Alleles Among Chinese Endemic Wheats

Genetic diversity at Gli-1, Gli-2 and Glu-1 loci was investigated in 32 accessions of Chinese endemic wheat by using acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulfate (SDS)-PAGE. There were 8 gliadin and 3 high-molecular-weight (HMW)-glutenin patterns in 14 Yunnan hulled wheat ( Triticum aestivum ssp. yunnanese King) accessions, 9 gliadin and 4 HMW-glutenin patterns in 9 Tibetan weedrace ( T. aestivum ssp. tibetanum Shao ) accessions, and 9 gliadin and 5 HMW-glutenin patterns in 9 Xinjiang rice wheat ( T. petropavlovskyi Udacz. et Migusch.) accessions. One accession (i.e. Daomai 2) carried new subunits 2.1+10.1 encoded by Glu-D1 . Among the three Chinese endemic wheat groups, a total of 10, 14 and 11 alleles at Gli-1 locus; 11, 14 and 12 alleles at Gli-2 locus; and 5, 6 and 8 alleles at Glu-1 locus were identified, respectively. Among Yunnan hulled wheat, Tibetan weedrace and Xinjiang rice wheat, the Nei's genetic variation indexes were 0.3798, 0.5625 and 0.5693, respectively. These results suggested that Tibetan weedrace and Xinjiang rice wheat had higher genetic diversity than Yunnan hulled wheat.     

中国特有小麦Gli-1、Gli-2和Glu-1位点的遗传多样性(英文)

运用APAGE和SDS_PAGE方法 ,研究了 32份中国特有小麦Gli_1、Gli_2和Glu_1位点的遗传多样性。在 1 4份云南铁壳麦 (Triticumaestivumssp .yunnaneseKing)中 ,共出现 8种醇溶蛋白带型和 3种高分子谷蛋白带型。在 9份西藏半野生小麦 (T .aestivumssp .tibetanumShao )中 ,发现 9种醇溶蛋白带型和 4种高分子谷蛋白带型。在 9份新疆稻麦 (T .petropavlovskyiUdacz.etMigusch .)中 ,观察到 9种醇溶蛋白带型和 5种高分子谷蛋白带型 ,其中 1份新疆稻麦 (稻麦 2 )具有Glu_D1编码的新亚基 2 .1 1 0 .1。在这 3种中国特有小麦群体中 ,Gli_1位点分别检测出 1 0、1 4和1 1个等位基因 ;Gli_2位点各具有 1 1、1 4和 1 2个等位基因 ;Glu_1位点也分别出现 5、6和 8个等位基因。云南铁壳麦、西藏半野生小麦和新疆稻麦群体内的Nei’s遗传变异系数分别为 0 .3798、0 .56 2 5和 0 .56 93。这些结果说明 ,与云南铁壳麦相比 ,西藏半野生小麦和新疆稻麦群体内的遗传变异相对较大。     

普通小麦F_1杂种Glu-1基因表达过程中的共显性,基因组互作和剂量效应

普通小麦F_1杂种Glu-1基因表达过程中的共显性,基因组互作和剂量效应@潘幸来$山西农业科学院棉花研究所!运城044000小麦;;基因表达;;基因组     

四川小麦地方品种Gli-1、Gli-2和Glu-1位点的遗传多样性(英文)

运用APAGE和SDS_PAGE方法 ,研究了 89个四川小麦 (TriticumaestivumL .)地方品种Gli_1、Gli_2和Glu_1位点的遗传多样性。在这些地方品种中 ,总共发现 32种醇溶蛋白带型和 3种高分子谷蛋白带型。在Gli_1、Gli_2和Glu_1位点上 ,分别检测出 14、15和 5个等位基因。在每一个位点上 ,出现频率最高的等位基因分别为Gli_A1a(89% ) ,Gli_B1h (46 % ) ,Gli_D1a (6 5 % ) ,Gli_A2a (6 4% ) ,Gli_B2j (45 % ) ,Gli_D2a (48% ) ,Glu_A1c (99% ) ,Glu_B1b (99% )和Glu_D1a (10 0 % )。四川小麦地方品种的Nei’s遗传变异系数平均为 0 .370 6 ,变幅为 0到 0 .70 87;其中Gli_B2位点的遗传多样性最高 ,而Glu_D1位点最低。同时 ,Gli位点的遗传多样性高于Glu_1位点的遗传多样性 ,但又低于现代品种Gli位点的遗传多样性。这些结果说明四川地方小麦品种的遗传基础狭窄。在研究中 ,“成都光头”与“中国春”的醇溶蛋白和高分子谷蛋白的带型完全一致 ,进一步证实“中国春”是“成都光头”的一个选系。     

Mg2+ ligands (Glu-384, Glu-429) of β-galactosidase from Lactococcus lactis ssp. lactis 7962

The secondary and the tertiary structures of -galactosidase from Lactococcus lactis ssp. lactis 7962 were designed by Nnpredict and Sybyl Version 6.3. Structural modeling of -galactosidase has shown that Glu-384 and Glu-429 are ligands for Mg²⁺ and Mg²⁺ is required for maximum activity. To confirm this prediction, we generated seven site specific mutants: Glu-384-Gln; Glu-384-Val; His-386-Phe; Asn-428-Asp; Glu-429-Gln; 384Gln-429Gln and 384Val-429Gln. The -galactosidases substituted at Glu-384 or Glu-429 had < 1% of the activity of the native enzyme with ONPG as substrate. The substitution of Glu-384 or Glu-429, which removed only one of the coordinating ligand for Mg²⁺, was still affected by Mg²⁺, but the mutants 384Gln-429Gln or 384Val-429Gln, which had been modified both Mg²⁺-binding sites, were not affected by Mg²⁺. Thus, Glu-384 and Glu-429 are probably ligands of Mg²⁺ and the three dimensional disposition of Mg²⁺ and its neighborhood interactions (Glu-384, Glu-429, Asp-428 or His-386) are important in the maintenance of –galactosidase activity.     

Is the glutamate residue Glu-373 the proton acceptor of the excitatory amino acid carrier 1?

Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na(+)/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na(+) binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na(+)/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.     

Glu-333 of Nicastrin Directly Participates in ��-Secretase Activity

γ-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for γ-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in γ-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in γ-secretase activity. By normalizing to the active pool of γ-secretase with two separate methods, we establish that γ-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by γ-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because γ-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in γ-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.The brains of Alzheimer disease patients are characterized by dense neuritic plaques that consist of the insoluble β-amyloid peptide (Aβ)² and neurons containing neurofibrillary tangles of the Tau protein (1, 2). The Aβ peptide is produced via the sequential proteolysis of APP by β- and γ-secretase (3). γ-secretase is a multisubunit complex consisting of at least four proteins: presenilin (PS), NCT, APH-1, and PEN-2, all of which are necessary and sufficient for activity (4–9). The formation of the γ-secretase complex is tightly controlled, with an ordered assembly of subunits coupled to spatial restriction (10). It is believed that the last step of the complicated γ-secretase maturation and activation process involves in cis endoproteolysis of the PS holoprotein (11–13). It is this form of γ-secretase with PS in its N- and C-terminal fragments (NTF and CTF, respectively) that represents the fully mature, proteolytically active enzyme.γ-Secretase is a unique protease that cleaves within the lipid bilayer a large number of Type 1 single transmembrane-spanning proteins that vary widely in their sequence and size (14–16). In a previous report, we demonstrated that NCT functions as a substrate receptor for γ-secretase (4). In that report, we showed that NCT recruits substrates that have had their large extracellular domains first removed by an upstream protease in a process termed “ectodomain shedding.” This process generates a new, short extracellular stub with a free N terminus, which is required for proteolysis by γ-secretase. We also established that Glu-333 of NCT participates in activity within the larger context of the DYIGS and peptidase-like (DAP) domain, which shares distant homology to amino- and carboxypeptidases. A recent study by Chávez-Gutiérrez et al. (17) confirmed that mutations at the equivalent rodent residue impair γ-secretase. However, the authors attributed the reduction in activity to a role for Glu-333 in γ-secretase maturation but not directly in activity per se. Although a role for NCT and Glu-333 in γ-secretase assembly and maturation is consistent with our early work (4, 18, 19), the authors'' conclusion that mature γ-secretase complexes containing the Glu-333 mutant NCT are fully active presents a challenge to the model that NCT is a receptor for γ-secretase substrates in mature, active enzyme. Although PS-NTF or -CTF alone is an adequate measure of active γ-secretase complexes, Chávez-Gutiérrez et al. (17) measured specific activity by normalizing γ-secretase products to the sum of PS1-CTF and PEN-2 presumably due to the levels of PS-NTF/CTF by themselves being at the detection limit of Western blotting with electrochemiluminescence (ECL). Such an approach has caveats, as normalizing to the sum of PS1 and PEN-2 does not represent a measurement of the intrinsic activity per single, active enzyme; rather, this mode of normalization instead skews the data to minimize the effects of the mutations, especially when compounded with the unreliability of ECL measurement at the detection limit of Western blotting. Indeed, normalizing to the amount of mature, active γ-secretase in a rigorous, quantitative manner would be necessary to accurately compare the intrinsic activities of wild-type and mutant enzymes.In this study we used two γ-secretase reconstitution methods, including one that bypasses endoproteolysis and two separate normalization approaches to demonstrate that γ-secretase complexes containing NCT-E333A are indeed intrinsically less active than wild-type NCT. We show that this mutant is deficient in its ability to directly bind to γ-secretase substrates. Moreover, we confirm our observations with a second γ-secretase substrate, C83, which is itself the physiological product of α-secretase cleavage of APP. We also examine a series of substrate truncation mutants and find that γ-secretase can cleave substrates that lack the entire extracellular domain, provided that such substrates also contain a free N-terminal amine. Taken together, we conclude that Glu-333 participates directly in activity after γ-secretase complex maturation. Finally, we put forth a model wherein the dual role of Glu-333 in γ-secretase maturation and substrate recognition could be explained in the context of NCT being a substrate receptor. In this model Glu-333 partakes in the recruitment of not only the ectodomain-shed Type 1 membrane proteins but also of the intracellular loop of PS for its endoproteolysis, a hallmark event of γ-secretase maturation and activation.     

The detection of a de novo allele of the Glu-1Dx gene in wheat–rye hybrid offspring

Key message

This study provides a link between a de novo gene and novel phenotype in wheat–rye hybrids that can be used as a model for induced de novo genetic variation.

Abstract

Wide hybridization can produce de novo DNA variation that may cause novel phenotypes. However, there is still a lack of specific links between changed genes and novel phenotypes in wide hybrids. The well-studied high-molecular-weight glutenin subunit (HMW-GS) genes in tribe Triticeae provide a useful model for addressing this issue. In this study, we investigated the feasibility of a wheat–rye hybridization method for inducing de novo phenotypes using the Glu-1Dx2.2 subunit as an example. We developed three hexaploid wheat lines with normal fertility and a Glu-1Dx2.2 variant, named Glu-1Dx2.2 ^v , derived from three F₁ hybrids. The wild-type Glu-1Dx2.2 has two direct repeats of 295 bp length separated by an intervening 101 bp in its central repetitive region. In the mutant Glu-1Dx2.2 ^v , one copy of the repeats and the intervening sequence were deleted, probably through homology-dependent illegitimate recombination (IR). This study provides a direct link between a de novo allele and novel phenotype. Our results indicate that the wheat–rye method may be a useful tool to induce de novo genetic variations that broaden the genetic diversity for wheat improvement.     

~(60)Co-γ射线诱发皖麦50 Glu-1A亚基等位基因突变体差异蛋白质组学研究

研究了皖麦50及~(60)Co-γ射线诱发的Glu-1A突变体的比较蛋白质组学,结果表明,突变体蛋白质含量增加,2-D电泳图上有11个蛋白质斑点表达差异显著,其中3个蛋白点上调,8个蛋白点下调。经MALDI-TOF/TOF质谱鉴定,差异蛋白点涉及44个蛋白质。通过GO分析,参与生物过程的差异蛋白中,18个蛋白质参与代谢过程,18个蛋白质参与细胞过程,8个蛋白质参与刺激响应。参与分子功能差异蛋白中,18个蛋白质参与催化活性,10个蛋白质参与链结,7个肽段为分子功能调解剂,9个肽段为未知蛋白。参与细胞成分的差异蛋白中,12个蛋白质为胞外区蛋白,12个蛋白质为细胞,9个蛋白质为细胞器,2个蛋白质为膜,9个蛋白质为未知蛋白。KEGG通路注解表明,差异蛋白质中,4个蛋白质参与淀粉和蔗糖代谢,3个蛋白质参与氨基糖和核苷酸糖代谢,1个参与丙酮循环,1个参与半胱氨酸和甲硫氨酸代谢,1个参与丙酮酸代谢,1个参与乙醛酸和二羧酸代谢,1个参与光合生物碳固定,1个参与碳代谢信息通路。     

Glu-44 in the Amino-terminal α-Helix of Yeast Vacuolar ATPase E Subunit (Vma4p) Has a Role for VoV1 Assembly

The proton (H⁺) pumping vacuolar-type ATPase (V-ATPase) is a rotary enzyme that plays a pivotal role in forming intracellular acidic compartments in eukaryotic cells. In Saccharomyces cerevisiae, the membrane extrinsic catalytic V₁ and the transmembrane proton-pumping V_o complexes have been shown to reversibly dissociate upon removal of glucose from the medium. However, the basis of this disassembly is largely unknown. In the earlier study, we have found that the amino-terminal α-helical domain between Lys-33 and Lys-83 of yeast E subunit (Vma4p) in the peripheral stalk of the V₁ complex has a role in glucose-dependent V_oV₁ assembly. Results of alanine-scanning mutagenesis within the domain revealed that the Vma4p Glu-44 is a key residue in V_oV₁ disassembly. Biochemical analysis on Vma4p Glu-44 to Ala, Asn, Asp, and Gln substitutions indicated that Glu-44 has a role in V-ATPase catalysis. These results suggest that Glu-44 is one of the key functional residues for subunit interaction in the V-ATPase stalk complex that allows both efficient rotation catalysis and assembly.     

Protein and DNA sequence analysis of a ‘private’ genetic variant: albumin Ortonovo (Glu-505 → Lys)

Albumin Ortonovo is a slow moving variant of human serum albumin which has been found only in people coming from the small villages of Ortonovo and Nicola (Liguria, Italy) and reaches polymorphic frequency (≥1%) in the poorly admixed population group living in that area. This is the first report of a ‘private’ varint detected in a Caucasin population. It probably originated as a mutation in a founder individual many generations ago. Isoelectric focusing analysis of CNBr fragments from the purified variant localized the mutation in fragment CNBr (residues 447–548). This fragment was isolated on a preparative scale by reversed-phase HPLC and subjected to V8 proteinase digestion. Sequence analysis of the abnormal V8 peptide revealed that the variant arises from a previously unreported substitution at position 505 where glutamic acid has been replaced by lysine. The protein data were confirmed by DNA sequence analysis which indicated a single nucleotide change of $\text{G}\text{AA}\text{→}\text{A}\text{AA}$ in the corresponding codon of the structural gene. Since the amino acid substitution found in albumin Ortonovo accords with its electrophoretic mobility on cellulose acetate, residue 505 is probably exposed to the solvent. The clustering of the mutations in the intersubdomain connection linking subdomains IIIA and IIIB (residues 492–511) accords with the fact that this region lies on the molecular surface and is accessible to solvent.     

Basic carboxyl groups of hemoglobin S: Influence of oxy-deoxy conformation on the chemical reactivity of Glu-43(β)

The -carboxyl groups of Glu-43() and Glu-22() of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide atp H 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence ofN-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22() and Glu-43() with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43() with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43() (i.e., chemical reactivity) as a function of thepH in the region of 5.5–7.5, the apparentpKa of its -carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43() of HbS atpH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the -carboxyl group and could be one of the crucial preludes to the polymerization process.     

Development of a PCR-based DNA marker for <Emphasis Type="Italic">Glu-1By</Emphasis> alleles in the old Hungarian Bánkúti wheat

Based on sequence alignment, phylogenetic, and dotplot analyses, primers were designed in order to distinguish the wheat high-molecular-weight glutenin subunit alleles By8 and By9. The primers were tested on 26 lines of Bánkúti wheat, an old Hungarian variety, and a number of other varieties. Consistency was observed between their known By protein subunit and the obtained DNA marker. Comparison of the B subunit content and the By alleles of the Bánkúti lines was also in agreement with the previous prediction that the By8 and By9 subunits are linked to the Bx7 and Bx7? subunits, which are responsible for dough quality in Bánkúti wheat, respectively. Thus, the developed molecular marker would be appropriate for marker assisted selection of the dough quality trait in the introgressive breeding of Bánkúti lines into modern cultivars.     

Site-directed mutagenesis of Glu-297 from the α-polypeptide of Phaseolus vulgaris glutamine synthetase alters kinetic and structural properties and confers resistance to L-methionine sulfoximine

In this paper we examine the functionality of Glu-297 from the -polypeptide of Phaseolus vulgaris glutamine synthetase (EC 6.3.1.2). For this purpose, the gln cDNA was recombinantly expressed in Escherichia coli, and site-directed mutants constructed, in which this residue was replaced by alanine. The level of glutamine synthetase transferase catalytic activity in the mutant strain was 70-fold lower while biosynthetic activity remained practically unaffected. Kinetic parameters for both enzyme activities were not greatly altered except for the K_m for ammonium in biosynthetic activity, which increased 100-fold. A similar result was reported when mutagenizing Glu-327 from E. coli glutamine synthetase, a residue shown to be present at the active site. This suggests that the Glu residue mutated in the higher-plant enzyme could develop a similar catalytic role to that of bacteria. Another characteristic feature of the mutant protein was its higher resistance to inhibition of the biosynthetic activity by L-methionine sulfoximine, a typical inhibitor of glutamine synthetase. In addition, we show that immunoreactivity of the glutamine synthetase mutant protein, both under native and denaturing conditions, is similar to the wild type, indicating that no deep conformational changes were produced as a consequence of the introduced mutation. However, structural changes in the active site can be predicted from alterations detected in the behaviour of the mutant protein towards affinity chromatography on 2,5-ADP-Sepharose, as compared to the wild type. Nevertheless, complementation of an E. coli glnA mutation indicated that the E297A mutant enzyme was physiologically functional.     

微生物发酵生产十一烷1，1 1二羧酸的研究

从热带假丝酵母(Candiada tropicalis)T25—14经过紫外线和亚硝酸的多次诱变,获得4株产十一烷l,11二羧酸(DC₁₃)较多的突变株,其中最优的NP-159株以20％(V／V)正十三烷(nC₁₃)为碳源摇瓶发酵4天,DC₁₃达80g／L左右。在16L罐上,以30％(V／V)nC₁₃发酵6天,DC₁₃高达139g／L,回收残烃后,对nC₁₃的转化率为80％以上。后处理收率为78．9％,DC₁₃的纯度为95．3％。     

IBVS1基因表达载体PG-S1的构建

以含有鸡传染性支气管炎病毒S1基因的载体P^IBV-Z和含有CMV启动子的栽体质粒P^EGFR-C1为材料,构建了可表达IBV S1基因的表达载体P^G-S1,经酶切、电泳和PCR检测结果证实,构建的载体符合目的要求,可以作为表达载体用于鸡的抗病育种。     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。