Characterization of a Novel 1Ay Gene and Its Expression Protein in Triticum urartu

High molecular weight glutenin subunit （HMW-GS） plays an important role in determining dough property and breadmaking quality, and the exploration of novel genes for HMW-GS will be crucial for quality improvement program. A gene coding the y type HMW-GS at Glu-A1 locus in Triticum urartu （AA, 2n=2×= 14） with an electrophoretic mobility similar to that of 1Dyl2, was cloned, sequenced, and heterologously expressed. This novel active lay gene FJ404595 was confirmed by structure analyses of nucleotide and deduced amino acid sequences combining with phylogenetic analysis. The open reading frame （ORF） of this gene was 1 830 bp, encoded a protein of 608 amino acid residues containing 46 hexapeptides and 14 nonapeptides, which was mostly similar to the lAy gene AM183223 at a high identity of 99.62% with the two substitutions of both leucine/proline and valine/glutamate, obviously different from the lAy gene EU984504 with 587 residues containing 44 hexapeptides and 13 nonapeptides in T. urartu. The amino acid （leucine） at 446 differed from that （proline） of all the eight compared active lAy subunits. The predicted secondary protein structure implied that this lAy subunit might also have positive impact on flour processing quality.     

Cloning and Characterization of Full Length cDNA of a CC-NBS-LRR Resistance Gene in Sweetpotato

Conserved domain such as nucleotide binding site （NBS） was found in several cloned plant disease resistance genes. Based on the NBS domain, resistance gene analogues （RGAs） have been isolated. A full-length cDNA, SPR1 was obtained by rapid amplification of cDNA ends （RACE） method. Sequence analysis indicated that the length of SPR1 was 3 066 bp, including a complete open reading frame of 2 667 bp encoding SPR1 protein of 888 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has a typical structure of nonT1R-NBS-LRR genes, with NB-ARC, CC, and LRR domains. The SPR1-related sequences belonged to multicopy gene family in sweetpotato genome according to the result of Southern blotting. Semi-quantitative RT-PCR analysis showed SPR1 expressed in all tested tissues. The cloning of putative resistance gene from sweetpotato provides a basis for studying the structure and function of sweetpotato disease-resistance relating genes and disease resistant genetic breeding in sweetpotato. The gene has been submitted to the GenBank database, and the accession number is EF428453.     

Cloning and Analysis of NBS-LRR Type Resistance Gene Analogues in Sweet Potato (Ipomoea batatas)

The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically designed degenerate primers. After cloning and sequencing,20 NBS-LRR type of disease-resistance gene analogue (RGAs) in sweet potato were observed. The deduced amino acid sequence of DNA fragment contains the conserved motifs of NBS-LRR type RGAs,such as P-loop,Kinase-2α,Kinase-3α and GLPL domain. The 20 RGAs could be sorted into two subclasses,namely TIR-NBS-LRR type and non-TIR-NBS-LRR type. Compared with the known resistance genes including N,L6 and M,the percentages of homologous amino acid sequence in 10 TIR-NBS-LRR range between 21%-44%. While other 10 non-TIR-NBS-LRR assume 15%-46% homology with the known resistance genes (Prf,RPM1,RPS2,etc.). Consequently the RGAs may further be used as molecular marker for screening the candidate disease-resistance genes in sweet potato.     

Cloning and Characterization of a PGIP-Encoding Gene from Solanum torvum

Verticillium wilt is a severe disease in eggplant caused by Verticillium dahliae.Polygalacturonase-inhibiting proteins （PGIPs） have been shown to be involved in preventing the invasion of fungus including V.dahliae.Cloning genes encoding PGIPs is quite valuable for plant resistance breeding to Verticillium wilt.In this study,a cDNA encoding the polygalacturonase-inhibiting protein was isolated from Solanum torvum by RT-PCR and RACE,designated StPGIP （accession no.FJ943498）.The cDNA sequence of StPGIP was 1 097 bp long and contained an open reading frame of 990 bp.The predicted amino acid sequence of the gene consisted of 329 amino acids and had conserved LRRs.The StPGIP protein had a high identity with PGIPs from other species.Analysis of StPGIP expression at the mRNA level by RT-PCR showed that the gene was expressed in all organs and could be induced to increase expression by V.erticillium dahliae infection.     

棉铃虫蛋白酶体β5基因Habeta5的克隆与序列分析(英文)

[Objective] Using molecular biotechnology to clone the proteasome β5 gene from cotton bollworm (Helicoverpa armigera), this research aimed to provide basis for further research on the function of proteasome β5 gene in cotton bollworm. [Method] Total RNA was extracted from midgut of cotton bollworm. The full length cDNA of Habeta5 gene was cloned by using rapid amplification of cDNA ends (RACE) technology, then sequence analysis was carried out. [Result] The full length cDNA sequence was successfully cloned and isolated, named as Habeta5. It was 947 bp in length, contained an ORF (843 bp) and encoded 280 amino acid residues, with the predicted mass of 30.87 kD and isoelectric point(pI) of 9.60. In the deduced amino acid sequence, a proteasome β5 subunit domain lies between 74th to 261st amino acid residues. It has more than 62% identity to other insects such as Drosophila melanogaster. The proteasome β5 subunit conservative regions were very similar with each other. Molecular evolution by Neighbor Joining method indicated that Habeta5 was homologous with other proteasome β5 subunit of species. [Conclusion] Sequence alignment shows that the cloned fragment is a proteasome β5 subunit gene (GenBank accession number: FJ358434).     

Construction and Characterization of a cDNA Library from the Pulp of Coconut （Cocos nucifera L.）

To investigate the gene expression profile of endosperm development, a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages. The constructed cDNA library incorporated approximately 1 × 10^7 clones in total, and the size of the insertion fragments ranged from 800 to 2 000 bp. Sequencing results of 100 randomly picked clones showed that the recombination rate was 96%. In subsequent sequence analysis, 41 clones （41%） were homologous to known function proteins, and 23 clones showed high amino acid identity （more than 80%） with the corresponding genes of different plants. Semi-quantitative RT-PCR indicated that oleosin and globulin genes are pulpspecific expression, and have differential expression level in different developmental stage. Clone 29, recognized as homologous to KIAA1239 protein （Homo sapiens）, was observed to occur nine times, indicating that this gene may be over-expressed during the endosperm development stage. However, the homologous protein was found only in mammals, and the detailed function is still unknown. Elucidation of the functional characterization of these genes will be carried out immediately.     

Identification and Functional Analysis on Abiotic Stress Response of SoybeanCl-Channel Gene GmCLCnt

The Cl^- homeostasis was known as the major mechanism of soybean to achieve NaCl tolerance, but studies on the role of chloride channel under abiotic stress were relatively few. We cloned a putative CLC-type chloride channel gene GmCLCnt from soybean via RACE and it was predicted to encode a protein of 783 amino acids with 9 possible transmembrane domains and 2 tandem CBS domains. Real-time RT-PCR analysis showed that the GmCLCnt gene was expressed in all tissues of soybean but enriched in leaves and its expression was induced by NaCl, polyethylene glycol （PEG）, coldness and ABA treatments. The Arabidopsis seedlings overexpressing GmCLCnt were more tolerant to higher concentration of NaCl than those of wild type. The results suggested that the GmCLCnt may be a CLC-type chloride channel and play an important role in salt tolerance.     

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz.et Migusch)

A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dyl0.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dyl0.1 from seed.     

西藏普通小麦地方品种特有高分子量谷蛋白亚基组合“Tibetan Dx5*+Tibetan Dy10”中Tibetan Dy10亚基基因测序与分析

 【目的】鉴定在西藏小麦地方品种中发现的特有高分子量谷蛋白亚基组合“Tibetan Dx5*+Tibetan Dy10”中的Tibetan Dy10亚基是否与普通小麦Dy10亚基为同一亚基。【方法】利用SDS-PAGE分析和Tibetan Dy10亚基基因的克隆和测序。【结果】表明Tibetan Dy10亚基与普通小麦中Dy10亚基广泛存在的Dx5+Dy10组合形式中的Dy10亚基的分子序列非常相似，但分别在2个六肽中的1个氨基酸部位发生替换，第335位的甘氨酸（G）和第451位的谷氨酰氨（Q）在Tibetan Dy10 中均被替换为精氨酸（R）。【结论】Tibetan Dy10与普通小麦中常见的Dy10亚基基因的DNA序列存在微小差异，属于Dy10位点的一个新变异。     

小麦地方品种半截芒Glu-B1位点HMW-GS基因的克隆及序列分析

高分子量谷蛋白亚基(HMW-GS)与小麦的品质特性紧密相关,挖掘小麦中HMW-GS新基因,对小麦品质改良具有重要意义。以小麦地方品种半截芒为材料,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)分析其HMW-GS组成,并设计特异性引物,利用PCR技术克隆其Glu-B1位点x型和y型亚基基因。序列分析表明,2个基因具有完整编码框,长度分别为2 367bp和2 106bp(GenBank登录号分别为KJ579439和KJ579440),编码789个氨基酸和702个氨基酸的蛋白,被命名为1Bx14*和1By15*。同源性搜索结果显示2条氨基酸序列与典型的HMW-GS有较高的同源性,且与普通小麦亚基1Bx14和1By15的同源性均为95%。系统进化树分析表明,1Bx14*和1By15*分别与1Bx14和1By15的遗传距离较近,聚类到同一分支上。     

Composition and Content of High-Molecular-Weight Glutenin Subunits and Their Effects on Wheat Quality

Sedimentation values, flour glutenin macropolymer (GMP) contents, composition and contents of high-molecular-weight (HMW) glutenin subunits (GS) of 233 flour samples were determined. Our data indicated that subunit 1 occurred more frequently at Glu-A1, subunit pair 7 + 8 at Glu- B1 and 2 + 12 at Glu-D1. The significant relationships between Glu-1 quality score and total HMW glutenin content, sedimentation value and GMP content suggested that the composition of HMW-GS affects wheat quality strongly. Moreover,the total content of HMW-GS was correlated with certain quality parameters more significantly. Relationship between subunit 5 + 10 content and breadmaking quality was better than others, but 2 + 12, 7 + 8, 7 + 9 and 4 + 12 also correlated with certain quality parameters significantly. The contents of total HMW-glutenin, x-type subunits and y-type subunits related with sedimentation value, flour GMP content, and Glu-1 quality score more strongly than that of individual subunit or subunit pair. The flour GMP content, with excellent correlation to sedimentation value, total contents of HMW glutenin, x- and y-type subunits and many other quality parameters, could be an ideal indicator of breadmaking quality at earlier generations for breeding purpose for its simple procedure and small scale.     

节节麦高分子量谷蛋白Dx5~t+Dy12~t亚基组合中Dy12~t亚基的序列测定与分析

为了认识节节麦5t+12t亚基品质表现突出的分子基础,利用SDS-PAGE分析研究了该亚基组合中12t亚基的电泳迁移率并克隆和测序了该亚基基因。结果表明,该12t亚基在SDS-PAGE上的电泳迁移率与普通小麦中的Dy12具有一致的电泳迁移率,但与Dy10的电泳迁移率差异明显。而氨基酸序列比较结果显示,12t亚基的分子序列与Dy10的相似程度非常高,二者仅存在4个氨基酸的替换,而它与Dy12的分子序列存在较大的差异,不但包括12个氨基酸的替换,同时包括2个六肽氨基酸和另外4个氨基酸部位的插入和缺失变化。本研究结果表明,节节麦高分子量谷蛋白Dx5t+Dy12t亚基赋予小麦优良加工品质的主要原因可能与12t亚基与小麦Dy10非常相似,导致这一亚基组合更倾向与Dx5+Dy10有一定关系。     

毛竹PePsbS1基因的分子特征及其原核表达

PsbS蛋白在植物非光化学淬灭(NPQ)中发挥着重要作用。采用同源比对方法从毛竹(Phyllostachys edulis)全长cDNA文库中得到1个PsbS同源基因序列(FP091683),命名为PePsbS1。该基因全长1 069 bp,具有多种光应答元件和参与光应答的顺式作用元件。PePsbS1的开放阅读框为807 bp,编码一个268 aa的蛋白。蛋白结构分析表明,该蛋白由转导肽(53 aa)和成熟蛋白(215 aa)组成,成熟蛋白包含1个叶绿素a/b结合蛋白功能域、4个跨膜区;疏水性分析表明该蛋白组成氨基酸以疏水性氨基酸为主,占42.5%;Blastp分析发现该蛋白与玉米的一致性最高,达80.3%。构建含有PePsbS1编码成熟蛋白序列的原核表达载体pET23a-PePsbS1-mature,转化大肠杆菌,经IPTG诱导表达后进行SDS-PAGE电泳分析。结果表明,分离纯化获得目的蛋白的分子量约为28 kD,与预测PePsbS1编码成熟蛋白的大小相符。这将有助于对竹子PsbS蛋白结构与功能的深入研究。     

一种节节麦高分子量麦谷蛋白与小麦同源蛋白氨基酸序列的比较

对节节麦的一种y类高分子量麦谷蛋白基因进行了序列测定和比较。结果显示,该亚基与小麦A、B和D和节节麦D染色体编码的y型亚基的全蛋白氨基酸残基数目均不一致。6个y型基因的信号肽、N-端和C-端氨基酸残基数目均一致,但有个别氨基酸的替换。而重复区氨基酸残基数目均不一致,主要由于重复单元六肽和九肽的数目不等。在目前已知的D基因组编码的y型亚基中,Dy13t的重复区是最短的,它比Dy10少4个九肽,比Dy12少4个九肽,2个六肽,比Dy12t少4个九肽。由N-Calign序列所作的聚类分析将这6个y型亚基分为3支,A、B和D基因组编码的亚基各占一支。来源于D基因组的4个基因的一支,又可将来源于节节麦和小麦D基因组的2个基因分别聚类在一起。     

Effect of high-molecular-weight glutenin subunit Dy10 on wheat dough properties and end-use quality

High-molecular-weight glutenin subunits (HMW-GSs) are the most critical grain storage proteins that determine the unique processing qualities of wheat. Although it is a part of the superior HMW-GS pair (Dx5+Dy10), the contribution of the Dy10 subunit to wheat processing quality remains unclear. In this study, we elucidated the effect of Dy10 on wheat processing quality by generating and analyzing a deletion mutant (with the Dy10-null allele), and by elucidating the changes to wheat flour following the incorporation of purified Dy10. The Dy10-null allele was transcribed normally, but the Dy10 subunit was lacking. These findings implied that the Dy10-null allele reduced the glutenin:gliadin ratio and negatively affected dough strength (i.e., Zeleny sedimentation value, gluten index, and dough development and stability times) and the bread-making quality; however, it positively affected the biscuit-making quality. The incorporation of various amounts of purified Dy10 into wheat flour had a detrimental effect on biscuit-making quality. The results of this study demonstrate that the Dy10 subunit is essential for maintaining wheat dough strength. Furthermore, the Dy10-null allele may be exploited by soft wheat breeding programs.     

Comparative Analysis of Hina Gene Sequences in Wild （Hordeum spontaneum） and Cultivated （H. vulgare） Barleys

The Hina gene is one of the two known Hin genes for hardness, and its RNA expression is correlated with grain hardness and dry matter digestibility variation. In this study, only one clone of Hina gene was obtained from one barley accession. A total of 121 Hina gene sequences were isolated from 121 wild barley {Hordeum spontaneum) accessions in Israel, Iran, and Turkey, and then their molecular characteristics were compared with 97 Hina gene sequences from 74 cultivated barley (H. vulgäre) lines in Europe and 23 landrace (H. vulgäre) with global distribution and other 26 Hina gene sequences from cultivated barleys (H. vulgäre) with unknown global distribution. C/s-acting regulatory element (CARE) searching revealed that there were different types of regulatory element for the Hina gene in wild and landrace/cultivated barleys. There were six consistent cw-acting binding sites in wild and landrace/cultivated barleys, whereas 8 to 16 inconsistent TATA-boxes were observed. In addition, three special elements (E2Fb, Spl, and boxS) were only observed in wild barley, while one (AT 1 -motif) was only found in landrace/cultivated barley. Forty-four deduced amino acid sequences of HINA from wild and landrace/cultivated barleys were obtained by deleting repetitive amino acid sequences, and they were clustered into two groups on the basis of Neighbor-Joining analysis. However, there was no obvious difference in the amino acid sequences of HINA between wild and landrace/cultivated barleys. Comparing to protein secondary structure of wheat PINA, it was indicated that HINA also existed a signal peptide. In addition, HINA was a hydrophilic protein on the basis of the protein properties and composition.     

七彩神仙鱼Vasa基因cDNA的克隆及表达分析

源自DEAD-box家族的Vasa基因是生殖细胞的分子标记之一。本研究克隆了七彩神仙鱼（Symphysodon haraldi） Vasa基因的全长序列,并进行了不同组织的表达分析。结果表明：七彩神仙鱼Vasa 基因cDNA序列共2 370 bp,其中5''UTR占123 bp,3''UTR为279 bp,ORF编码655个氨基酸,长1 968 bp。氨基酸序列同源性分析表明七彩神仙鱼的Vasa基因与尼罗罗非鱼（Oreochromis niloticus）的同源性最高。半定量分析表明,Vasa基因在成熟七彩神仙鱼的性腺中特异表达,在其它组织中无表达信号。qRT-PCR结果显示,Vasa在七彩神仙鱼早期胚胎发育阶段及出膜后50日内均有表达。在受精卵至囊胚期阶段,Vasa基因表达持续增加,在囊胚期至孵化期阶段,表达持续降低。在仔鱼阶段,Vasa分别在25日龄和40日龄出现了最低和最高表达量。繁殖前后比较发现,繁殖前的精巢组织中Vasa表达量比繁殖后的表达量低,而卵巢恰好相反。本研究结果可为研究七彩神仙鱼的性分化、生殖细胞分子标记及其发育提供参考。