Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene

[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification;B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained;CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine.

Identification and molecular characterization of Cathepsin L gene and its expression analysis during early ontogenetic development of kuruma shrimp Marsupenaeusjaponicus

Cathepsin L gene is a member of the cysteine proteinase gene group. In this study Cathepsin L gene was isolated from Kuruma shrimp Marsupenaeusjaponicus （Mj-Cathepsin L） and the full-length DNA sequence was 1 963 bp. Mj-Cathepsin L protein showed high homologies with other Cathepsin L proteins documented in vertebrates, mollusks and other crustaceans. Expression analysis of Mj-Cathepsin L gene in different tissues revealed that it was predominant in hepatopancreas. During early ontogenetic development stages Mj-Cathepsin L showed a development-regulated expression, and the Mj-Cathepsin L showed a molting stage-regulated expression during the five molting stages, inferring its role in the ontogenic development of M.japonicus. Two kinds of forms of Mj- Cathepsin L protein： pro-Cathepsin L and Cathepsin L were measured in hepatopancreas, stomach and intestine by Western Blotting.

Effects of Lipoxygenase Null Genes of Soybean in Controlling Beany-flavor of Soymilk and Soyflour

The flavor of the soymilk and soyflour obtained from the lipoxygenase mutant isolines was concentrated by simultaneous distillation and extraction (SDE), and its constituents were identified by gas chro-matography (GC) and gas chromatography-mass spectrometry. Results showed that the same 24 flavor constituents were isolated in both soymilk and soyflour, and most of them were aldehydes and alcohols. Lox2 was most responsible for the production of the volatile and beany-flavor components, and Lox1 less responsible. Lox3 was least responsible and can reduce the yield of hexanal. Either Lx1 or Lx2 could significantly reduce the volatile and beany-flavor, and Lx3 could significantly increase the yield of hexanal. Primary and secondary interactions existed among the null mutant genes, and the major effects and interactions could be affected by processing conditions. The isoline with triple lipoxygenase null genes yielded the least volatile and beany-flavor components, and the isoline without the lipoxygenase gene Lx3 produced the greatest amount of the volatile and beany-flavor components. The amounts of volatile and beany-flavor components produced by the other isolines were between that of the isoline with triple lipoxygenase null genes and the isoline without lipoxygenase gene Lx3. According to the correlation analysis, the hexanal amount could be used as an index in evaluating the importance of lipoxygenase isozymes in the yield of beany-flavor compounds, and the effects of the different types of lipoxygenase null mutants in controlling beany-flavor compounds. The cultivars with triple lipoxygenase null genes will be a quality raw material for soy food processing.

Phenotypic Characterization and QTL/Gene Identification for Internode Number and Length Related Traits in Maize

Internode number and length are the foundation to constitute plant height, ear height and the above-ground spatial structure of maize plant. In this study, segregating populations were constructed between EHel with extremely low ear height and B73. Through the SNP-based genotyping and phenotypic characterization, 13 QTL distributed on the chromosomes (Chrs) of Chr1, Chr2, Chr5-Chr8 were detected for four traits of internode no. above ear (INa), average internode length above ear (ILaa), internode no. below ear (INb), and average internode length below ear (ILab). Phenotypic variation explained (PVE) by a single QTL ranged from 6.82% (qILab2-2) to 12.99% (qILaa5). Zm00001d016823 within the physical region of qILaa5, the major QTL for ILaa with the largest PVE was determined as the candidate through the genomic annotation and sequence alignment between EHel and B73. Product of Zm00001d016823 was annotated as a WEB family protein homogenous to At1g75720. qRT-PCR assay showed that Zm00001d016823 highly expressed within the tissue of internode, exhibiting statistically higher expression levels among internodes of IN4 to IN7 in EHel than those in B73 (P Zm00001d016823 might provide novel insight into molecular mechanism beyond phytohormones controlling internode development in maize.

STRUCTURE OF THE MITOCHONDRIAL URFA6L GENE AND tRNA^(Lys) GENE FROM CARP

The carp mitochondrial URFA6L gene consists of 165 base pairs. The overall structural organization of the gene is very similar to that of the Xenopus URFA6L gene. Their nucleotide sequences exhibit 68% homology. The carp URFA6L gene encodes a protein of 54 amino acids. The amino acid composition of the protein is unusual because almost half of the residues consist of 5 hydrophobic amino acids(proline, tryptophan, leueine, isoleueine and tyrosine). A comparison between the amino acid sequences of 5 vertebrate URFA6L proteins and the yeast ATPase8 showed that they have weak but very important common structural features, suggesting that the vertebrate URFA6L proteins may function asATPase8. The nucleotide sequence of the lysine tRNA gene from carp has been determined and represented in cloverleaf secondary structure. Similar to amphibian and mammalian mitochondrial tRNA^(Lys) genes, the carp mitochondrial tRNA^(Tys) gene also has some unusual structural features as compared with its cytoplasmic counterpart. A comparison between the nucleotide sequences of the tRNA^(Lys) gene from 7 vertebrates showed that the most conservative portions are the anticodon loop, nucleotides 8 and 9, the variable loop, the anticodon stem and the aminoacyl stem. The least conservative portions are the D-loop and the T-loop. These structural features may show that the mitochoudrial tRNA^(Lys) has a different interaction with mitochondrial ribosome.

Progress of CACNA1S Gene and Hypokalemic Periodic Paralysis

CACNA1 S gene is the gene encoding L-type calcium channel αa-subunit. CACNA1 S gene mutations can cause hypokalemic periodic pa- ralysis （HOKPP）. The related research speculated that CACNA1 S gene was the candidate genes which affect meat quality traits. In the present ar- ticle, the biological characteristics of CACNA1 S gene, structure, genetic diseases and the research development were respectively reviewed so as to provide a reference for further research.

辣椒EIL基因家族的鉴定及其在盐碱胁迫下的表达分析

EIL(Ethylene-insensitive 3-like)基因在参与植物乙烯信号通路的转导和生长发育过程中发挥着重要作用。为了解辣椒EIL基因家族成员信息,利用生物信息学手段对辣椒EIL基因家族成员的理化性质、蛋白质结构、系统进化、基因结构、保守域、启动子顺式作用元件及CaEILs基因在不同组织部位和不同非生物胁迫条件下的表达模式进行分析。同时使用实时荧光定量PCR(qRT-PCR)对辣椒叶片中CaEILs在盐碱胁迫下的表达模式进行研究。结果表明,辣椒9个CaEILs分布在6条染色体上,氨基酸数目、蛋白质理论分子质量和脂肪系数分别介于209~677、23.77~76.07 ku和63.10~87.75,且主要为酸性、亲水的不稳定性核蛋白。系统进化分析显示,CaEILs被分成了4个亚家组,9个CaEILs在根、茎、叶、花蕾、花中具有不同程度的表达,而低温、高温、高盐、干旱胁迫下均能诱导CaEILs的表达,其对以上非生物胁迫均具有不同程度的响应。此外,利用qRT-PCR对盐碱胁迫下辣椒叶片中CaEILs进行了检测,结果发现,随着处理时间的延长,CaEIL1、CaEIL2、CaEIL4、CaEIL5、CaEIL8的表达量整体呈上升趋势,而CaEIL3、CaEIL6、CaEIL7、CaEIL9的表达量整体呈下降趋势。

Expression of ACC Oxidase Gene from Sugarcane Induced by Hormones and Environmental Force

In the present study, a full-length cDNA encoding 1-aminocyclopropane-1-carboxylic acid oxidase gene has been cloned from sugarcane （named GZ-ACO）. Two primers were designed for coding the ORF in the full-length cDNA of GZ-ACO gene from sugarcane. PCR amplification was performed with sugarcane DNA template, and a fragment of 1 104 bp （GZ34） was obtained. GZ34 was labeled with [α-32P] dCTP as the probe and used for hybridization after cloning and sequencing. Southern blotting analysis indicated that there were at least three other sequences, which weakly hybridized with the GZ34. Northern analysis showed that GZ34 was strongly induced by treatment with IAA, BA, ethephon, LiC1 and cold stress, respectively. As a contrast, the mRNA for ACO gene was at lower levels for both the light-grown and dark-grown plants without additional treatment. There were two transcripts in the dark-grown plants and three transcripts in the treatments with IAA, BA and cold stress, but there was only one transcript in ethephon treatment. It showed that GZ-ACO might be a gene connected with ethylene formation and take part in response to the induction of plant hormone and environmental stress.

Effects of the Fhb1 gene on Fusarium head blight resistance and agronomic traits of winter wheat

The gene Fhb1 has been used in many countries to improve wheat Fusarium head blight(FHB) resistance. To make better use of this gene in the Yellow-Huai River Valleys Winter Wheat Zone(YHWZ), the most important wheat-producing region of China, it is desirable to elucidate its effects on FHB resistance and agronomic traits in different genetic backgrounds. Based on a diagnostic marker for Fhb1, six BC2 populations were developed by crossing dwarf-male-sterile(DMS)-Zhoumai 16 to three Fhb1 donors(Ningmai 9, Ningmai 13, and Jianyang 84) and backcrossing to Zhoumai 16 and Zhoumai16’s derivative cultivars(Lunxuan 136 and Lunxuan 13) using marker-assisted backcross breeding. The progenies were assessed for FHB resistance and major agronomic traits.The Fhb1 alleles were identified using the gene-specific molecular marker. The plants with the Fhb1-resistant genotype(Fhb1-R) in these populations showed significantly fewer infected spikelets than those with the Fhb1-susceptible genotype(Fhb1-S). When Lunxuan 136 was used as the recurrent parent, Fhb1-R plants showed significantly fewer infected spikelets per spike than Fhb1-R plants produced using Lunxuan 13 as the recurrent parent, indicating that the genetic backgrounds of Fhb1 influence the expression of FHB resistance. Fhb1-R plants from the DMS-Zhoumai 16/Ningmai 9//Zhoumai 16/3/Lunxuan 136 population showed the highest FHB resistance among the six populations and a significantly higher level of FHB resistance than the moderately susceptible control Huaimai 20. No significant phenotypic differences between Fhb1-R and Fhb1-S plants were observed for the eight agronomic traits investigated. These results suggest that it is feasible to improve FHB resistance of winter wheat withoutreducing yield potential by introgressing Fhb1 resistance allele into FHB-susceptible cultivars in the YHWZ.

甘蓝型油菜REVEILLE家族鉴定及诱导表达分析

【目的】REVEILLE家族基因具有重要生物学功能,而在甘蓝型油菜(Brassica napus L.)中该家族基因研究较少,深入认识甘蓝型油菜中RVE家族基因功能及其与非生物胁迫的关系。【方法】利用生物信息学方法,在甘蓝型油菜、白菜(Brassica rapa)和甘蓝(B.oleracea)中分别鉴定到33、16和16个RVE基因,并对其系统进化、染色体定位、基因结构和理化性质以及启动子顺式元件进行分析。【结果】所有的RVE蛋白被分为两个亚家族。33个BnaRVE分别定位在18条A亚基因组的染色体和15条C亚基因组的染色体上。大部分成员属于较稳定的疏水蛋白;多数Ka/Ks值小于1,说明该家族受到强烈的纯化选择作用。基因结构变异较大,内含子数目在4-11之间。共线性分析结果表明,甘蓝型油菜和拟南芥、白菜、甘蓝存在大量的同源基因。大部分甘蓝型油菜RVE家族成员在子叶和叶都有表达且表达量最多。BnaRVE启动子上含有大量与激素和生物逆境相关的元件,通过定量PCR分析,4个BnaRVE在ABA与MeJA诱导和低温胁迫下的表达量显著上调。【结论】在甘蓝型油菜基因组中鉴定出33个BnaRVE家族成员。不同基因在不同发育时期和不同组织中表现出不同的表达模式。不同基因对各种胁迫存在响应,且表达模式不一。RVE家族成员对ABA、MeJA和冷胁迫具有正向响应功能。

Relationship between differ- ential gene expression pat- terns in functional leaves of maize (Zea mays L.) at milk filling stage and heterosis using cDNA-AFLP

To understand the molecular mechanism of maize heterosis, differential gene expression patterns in the functional leaves of 35 maize hybrids relative to their parents involving 10 elite inbreds at milk filling stage were analyzed by using cDNA-AFLP. The correlation analyses of various differential expression patterns with the performance and heterosis of main maize agronomic traits were evaluated. The main results were as follows: For uniparental specific expression, significant positive correlations were detected with the performance of seed weight per ear and 100-seed weight at 0.01 and 0.05 probability levels respectively. For biparental specific expression, significant negative correla-tions were detected with the performance of ear diameter and seed weight per ear at 0.01 probability level. For uni-parental specific expression, significant positive correlations were detected with the heterosis of ear diameter and seed weight per ear at 0.01 and 0.05 probability levels respectively. For biparental specific expression, significant negative cor-relation was detected with the heterosis of ear diameter at 0.05 probability level. However, for F1-specific expression, for fragments detected only in one parent and F1, and for fragments detected only in two parents or only in F1, no sig-nificant correlation was detected with the performance or heterosis of all agronomic traits surveyed.

Gene Expression of Fas,Soluble Fas and Fas-Ligand in Thyroid Tissues and Thyrocytes from Patients with Graves′ Disease

ObjectiveTo investigate Fas,soluble Fas(sFas)and Fas ligand(Fas L)gene expression in thyroid tissues and thyrocytes from patients with Graves disease(GD)and to find the interrelationship between apoptosis and pathogenesis of GD. MethodsThyroid tissues were obtained from 7 GD patients and 3 healthy subjects who died accidentally. Thyrocytes were cultured in Eagle′s medium. Total RNA was isolated from thyroid tissues and cultured thyrocytes. The cDNA was prepared by reverse transcription and amplified for Fas,sFas and Fas L by polymerase chain reaction(PCR). ResultsFas and sFas mRNA were detected in all samples from both GD and normal thyroid tissues and thyrocytes,but Fas L mRNA was only found in GD thyroid tissues and thyrocytes. Semi quantitative analysis showed that when compared with those of normal controls,the Fas and sFas mRNA levels were markedly increased in GD thyroid tissues(P<0.01),whereas in GD thyrocytes only the sFas mRNA levels was significantly elevated(P<0.01). ConclusionGene expression of Fas,sFas and Fas L showed abnormality in both thyroid tissues and thyrocytes from GD. The increased production of sFas might be involved in the hyperplasia of thyroid gland.

The Value of Different GA Insensitive Rht Dwarfing Genes in Winter Wheat Breeding

The value of different dwarfing genes in winter wheat breeding was studied using 6 near-isogenic lines carrying different Rht dwarfing genes over three years experiment.Results showed that both the Rht1 and Rht2 semi-dwarfing genes had significantlypositive effects on kernel number and grain weight per spike, and had significantlynegative effects on 1000-grain weight comparing to the tall line(rht) and the Rht3 line.The Rht3 dwarfing gene had a significantly negative effect on kernel number per spike,and had positive effect on 1000-grain weight. The combination of the Rht2 and Rht3 geneshowed significantly negative effect on yield components. All of these 5 dwarfing orsemidwarfing genotypes mentioned above had a significantly negative effect on plantheight and no significant effect on the area of flag leaf, spikelets per spike and spikelength.

Seedling and adult plant resistance to leaf rust in 46 Chinese bread wheat landraces and 39 wheat lines with known Lr genes

Wheat leaf rust,caused by Puccinia triticina(Pt),is an important foliar disease that has an important influence on wheat yield.The most economic,safe and effective way to control the disease is growing resistant cultivars.In the present study,a total of 46 wheat landraces and 34 wheat lines with known Lr(leaf rust resistance)genes were inoculated with 16Pt pathotypes for postulating seedling resistance gene(s)in the greenhouse.These cultivars and five wheat differential lines with adult plant resistance(APR)genes(Lr12,Lr22b,Lr34,Lr35 and Lr37)were also evaluated for identification of slow rusting resistance in the field trials in Baoding,Hebei Province of China in the 2014–2015 and 2015–2016 cropping seasons.Furthermore,10 functional molecular markers closely linked to 10 known Lr genes were used to detect all the wheat genotypes.Results showed that most of the landraces were susceptible to most of the Pt pathotypes at seedling stage.Nonetheless,Lr1 was detected only in Hongtangliangmai.The field experimental test of the two environments showed that 38 landraces showed slow rusting resistance.Seven cultivars possessed Lr34 but none of the landraces contained Lr37 and Lr46.Lr genes namely,Lr9,Lr19,Lr24,Lr28,Lr29,Lr47,Lr51 and Lr53 were effective at the whole plant stage.Lr18,Lr36 and Lr45 had lost resistance to part of pathotypes at the seedling stage but showed high resistance at the adult plant stage.Lr34 as a slowing rusting gene showed good resistance in the field.Four race-specific APR genes Lr12,Lr13,Lr35 and Lr37 conferred good resistance in the field experiments.Seven race-specific genes,Lr2b,Lr2c,Lr11,Lr16,Lr26,Lr33 and LrB had lost resistance.The 38 landraces showed slow rusting resistance to wheat leaf rust can be used as resistance resources for wheat resistance breeding in China.

Cloning and Characterization of a Salt Tolerance-Associated Gene Encoding Trehalose-6-Phosphate Synthase in Sweetpotato

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes： trehalose-6-phosphate synthase（TPS） and trehalose-6-phosphate phosphatase（TPP）. In the present study, a TPS gene, named IbTPS, was first isolated from sweetpotato（Ipomoea batatas（L.） Lam.） cv. Lushu 3 by rapid amplification of cDNA ends（RACE）. The open reading frame（ORF） contained 2 580 nucleotides encoding 859 amino acids with a molecular weight of 97.433 kDa and an isoelectric point（pI） of 5.7. The deduced amino acid sequence showed high identities with TPS of other plants. Real-time quantitative PCR analysis revealed that the expression level of IbTPS gene was significantly higher in stems of Lushu 3 than in its leaves and roots. Subcellular localization analysis in onion epidermal cells indicated that IbTPS gene was located in the nucleus. Transgenic tobacco（cv. Wisconsin 38） plants over-expressing IbTPS gene exhibited significantly higher salt tolerance compared with the control plant. Trehalose and proline content was found to be significantly more accumulated in transgenic tobacco plants than in the wild-type and several stress tolerance related genes were up-regulated. These results suggest that IbTPS gene may enhance salt tolerance of plants by increasing the amount of treahalose and proline and regulating the expression of stress tolerance related genes.

Molecular Cloning and Functional Characterization of a Salt Tolerance-Associated Gene IbNFU1 from Sweetpotato

Iron-sulfur cluster biosynthesis involving the nitrogen fixation（Nif） proteins has been proposed as a general mechanism acting in various organisms.NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses.Based on the EST sequence selected from salt-stressed suppression subtractive hybridization（SSH） cDNA library constructed with a salt-tolerant mutant LM79,a NFU gene,termed IbNFU1,was cloned from sweetpotato（Ipomoea batatas（L.） Lam.） via rapid amplification of cDNA ends（RACE）.The cDNA sequence of 1 117 bp contained an 846 bp open reading frame encoding a 281 amino acids polypeptide with a molecular weight of 30.5 kDa and an isoelectric point（pI） of 5.12.IbNFU1 gene contained a conserved Cys-X-X-Cys motif in C-terminal of the iron-sulfur cluster domain.The deduced amino acid sequence had 66.08 to 71.99% sequence identity to NFU genes reported in Arabidopsis thaliana,Eucalyptus grandis and Vitis vinifera.Real-time quantitative PCR analysis revealed that the expression level of IbNFU1 gene was significantly higher in the roots of the mutant LM79 compared to the wild-type Lizixiang.Transgenic tobacco（cv.Wisconsin 38） plants expressing IbNFU1 gene exhibited significantly higher salt tolerance compared to the untransformed control plants.It is proposed that IbNFU1 gene has an important function for salt tolerance of plants.

Cloning of TaCYP707A1 Gene that Encodes ABA 8′-Hydroxylase in Common Wheat (Triticum aestivum L.)

The plant hormone abscisic acid （ABA） regulates many important physiological and developmental processes in plants. The objective of this study was to clone the ABA 8′-hydroxylase gene in common wheat. In the present study, we used the eDNA sequence of barley HvCYP707A1 gene （GenBank accession no. AB239299） as a probe for BLAST search against the common wheat （Triticum aestivum L.） EST database in GenBank. All wheat ESTs sharing high similarity with the reference gene were subjected to contig assembly. Primers were designed based on the constructed contigs to clone the wheat CYP707A1 gene, designated as TaCYP707A1. The genomic DNA sequence of TaCYPTO7A1 gene comprised five exons and four introns, with a size of 2225 bp. The corresponding cDNA sequence of TaCYP707A1 was 1737 bp, containing an open reading frame （ORF） of 1431 bp, a 42-bp 5′-untranslated region （UTR） and a 264-bp 3′UTR, with 94.9% of identical sequences to HvCYP707A1 gene （AB239299）. The neighbor joining tree indicated that the deduced amino acid sequences of TaCYP707A1 gene was highly similar to those of barley and rice. The TaCYP707A1 gene was located on chromosome 6BL using a set of Chinese Spring nullisomic-tetrasomic lines and ditelosomic line 6BS. These results will be of high importance in understanding of molecular mechanism of ABA catabolism.

Transferring a Gene Expression Cassette Lacking the Vector Backbone Sequences of the 1Ax1 High Molecular Weight Glutenin Subunit into Two Chinese Hexaploid Wheat Genotypes

1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of Amp^R gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest （GOI） and the selectable marker （MG）, bombardment pressure and genotypes are vital for the expression of a transformed GEC.

Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants

In contrast to the situation of random integration of foreign genes in nuclear transformation, the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination. To establish an expression system for alien genes in rice chloroplast, the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study. Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique, and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator ofpsbA gene 3 sequence. Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct. Subsequently, the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained. Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome. Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome. Thus, the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome, but PPT-resistant trait for rice plants as well. It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.

TaARR1, a cytokinin response regulator gene in Triticum aestivum, is essential in plant N starvation tolerance via regulating the N acquisition and N assimilation

Plant N starvation response is closely associated with the N signaling components that involve transduction of the low-N cues. In this study, we functionally characterized Ta ARR1, a cytokinin(CK) response regulator gene in Triticum aestivum, in mediating the N starvation adaptation in plants. Ta ARR1 harbors two conserved domains specified by plant ARR family members;subcellular localization analysis indicated its target onto nucleus after endoplasmic reticulum assortment. Ta ARR1 displayed modified expression upon the N starvation stressor, showing upregulated expression in roots and leaves over a 27-h N starvation treatment and whose induced transcripts were gradually recovered along with progression of the N recovery treatment. The tobacco lines overexpressing Ta ARR1 displayed improved low-N stress tolerance, displaying enlarged phenotype, increased biomass and N accumulation, and enhanced glutamine synthetase(GS) activities compared with wild type(WT) following the N starvation treatment. Expression analysis on genes encoding the nitrate transporter(NRT) and GS proteins in Nicotiana tabacum revealed that Nt NRT2.2 and Nt GS3 are upregulated in expression in the N-deprived transgenic lines, whose expression patterns were contrasted to other above family genes that were unaltered on transcripts between the transgenic lines and WT. Transgene analysis validated the function of Nt NRT2.2 and Nt GS3 in regulating N accumulation, GS activity, growth traits, and N use efficiency in plants. These results suggested the internal connection between the Ta ARR1-mediated N starvation tolerance and the modified transcription of distinct N acquisitionand assimilation-associated genes. Our investigation together indicates that Ta ARR1 is essential in plant N starvation adaptation due to the gene function in transcriptionally regulating distinct NRT and GS genes that affect plant N uptake and assimilation under the N starvation condition.