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[Objectives]The paper aimed to provide a basis for studying the function of 14-3-3 protein and breeding of Jerusalem artichoke, we identified the 14-3-3 gene family of Jerusalem artichoke and analyzed their expression patterns in response to high temperature, cold, salt and drought stresses. [Methods]Cloning and bioinformatics analysis were used to study gene properties, and RNA-seq data analysis and RT-qPCR were used to study gene response patterns to abiotic stress. [Results]Ten members of the 14-3-3 gene family, namely HtGRF1-HtGRF10(GenBank accession No.OP132618-OP132627), were cloned from Jerusalem artichoke. According to the evolutionary relationship, these genes were divided into two subfamilies, HtGRF1-HtGRF7 belonged to the non-ε group, and HtGRF8-HtGRF10 belonged to the ε group, which usually formed homologous or heterodimers. The expression level of HtGRF2/3/6/9 in sprouts, roots, stems and leaves was higher than those of other genes. The expression level of HtGRF3/5/9 was higher in stolon and tuber formation stages than that in the tuber rapid intamescentia and maturity stages during the tuber development. Comprehensive analysis of HtGRF in response to abiotic stress in roots and leaves showed that HtGRF6 expression level decreased under high temperature, salt and drought stresses, while HtGRF2/3/7/8/9 expression level decreased under salt stress, but increased under drought stress. HtGRF1 expression level increased under cold stress, HtGRF4/5 expression level decreased under drought stress, and HtGRF10 expression level did not change significantly. [Conclusions]The 14-3-3 protein family in Jerusalem artichoke was highly conserved and encoded by multiple genes, and played an important role in the growth and development and in coping with complex environments. [ABSTRACT FROM AUTHOR]

[Objectives]The purpose of this experiment was to explore the differential expression of neuromedin B（NMB）and its receptors（neuromedin B receptor, NMBR;gastrin releasing peptide receptor, GRPR;bombesin receptor subtype 3,BRS3）in goat testis at different development stages, which provided reference for analyzing the mechanism of mammalian testicular development and functional regulation. [Methods]Twelve male goats（3-and 9-months-old, six individuals per age group）with clear pedigree and good body conditions were selected for castration and collected the testicular tissue. Based on gene cloning and sequence analysis of NMB and its receptors, immunohistochemistry was used to detect the location of NMB and its receptors in testicular tissue, and RT-qPCR and Western blot were used to detect the expression of NMB and its receptors in goat testis at different development stages. [Results]NMB and its receptors gene had the most recent evolutionary relationship with sheep and cattle. The nucleotide sequence of the coding region of NMB and its receptors gene had more than 96% homology with cattle and sheep, and structural analysis showed that NMBR,GRPR and BRS3 belonged to seven transmembrane receptors. NMB and its receptors were widely distributed in testicular tissues of 3-months-old and 9-months-old goats, and a strong staining intensity in leydig cells, myoid cells and sperm cells. In addition, compared with the 3-months-old goats, the mRNA and protein expression levels of NMB and GRPR in testis of 9-months-old goats significantly decreased（P＜0.05）,while the mRNA and protein expression levels of NMBR and BRS3 in testis of 9-months-old goats significantly increased（P＜0.05）. [Conclusions]NMB and its receptors may be involved in the regulation of goat testicular development. [ABSTRACT FROM AUTHOR]

[Objectives]This research aimed to explore the function of SmICE1（Inducer of CBF expression 1）gene in cold response and anthocyanin biosynthesis in eggplant. [Methods]Using photosensitive eggplant variety ‘Lanshanhexian’as material, the full-length sequence of SmICE1 was homology-based cloned and bioinformatics analysis was performed. The seedlings with four leaves treated at 4 ℃ were used to analyze the expression level of SmICE1 gene. To study the subcellular localization of SmICE1,the PHB-SmICE1-YFP vector was constructed and transformed into the tobacco leaves via Agrobacterium-mediated transient transformation. SmICE1 gene was overexpressed in Arabidopsis thaliana using the Agrobacterium-mediated floral dip method. Then, the cold resistance, physiological indexes and the expression levels of AtCBF（CRT binding factor）genes were analyzed in transgenic plants. The Agrobacterium containing the PHB-SmICE1-YFP and SmCBFpro-LUC was transiently co-transformed into the tobacco cells to perform the dual luciferase（Dual-LUC）assay. In order to carry out the yeast two-hybrid（Y2 H）assay, the yeast bait vector SmYABBY-pGBKT7 and prey vector SmICE1-pGADT7 were constructed and transformed into yeast strain AH109. The plant expression vector SmYABBY-nYFP and SmICE1-cYFP were constructed, and the Agrobacterium was injected into tobacco cells to carry out bimolecular fluorescence complementation（BiFC）assay. [Results]SmICE1 gene was cloned, and it contained 1 524 bp open reading frame encoding 507 amino acids with a predicted molecular weight of 54.75×10³ and a theoretical isoelectric point of 5.6. Then, it was found that the SmICE1 had the high similarity to SlICE1 in Solanum lycopersicum by multiple sequence alignment and phylogenetic tree analysis. Quantitative real-time PCR indicated that low temperature could induce the expression of SmICE1 gene. SmICE1 protein was localized in the nucleus according to the subcellular localization analysis. The SmICE1 protein had strong transactivation activity, and Dual-LUC assay indicated that SmICE1 could enhance the expression level of SmCBF genes. Ectopic overexpression of SmICE1 enhanced the freezing tolerance of transgenic Arabidopsis. After low temperature treatment, the proline content and the expression levels of AtCBF genes were higher in transgenic plants than in wild type（WT）plants, and the transgenic plants had lower electrolyte leakage than WT. The Y2 H and BiFC assays indicated that SmICE1 could interact with SmYABBY. [Conclusions]SmICE1 could induce the expression of SmCBF genes and increase the cold-resistance of transgenic plants, furthermore the interaction between SmICE1 and SmYABBY might be involved in anthocyanin accumulation. [ABSTRACT FROM AUTHOR]

[Objectives]This article aimded to clone the ERA1（ENHANCED RESPONSE TO ABA) gene of Chrysanthemum morifolium,and analyze its expression pattern preliminarily, so as to lay the foundation for exploring its role in regulating the flowering of chrysanthemums. [Methods]The gene encoding farnesyltransferase β subunit in chrysanthemum was screened from the transcriptome database of the summer chrysanthemum ‘Yuuka’,and designated as CmERA1. The total RNA from the leaves of wild-type C morifolium Jinba’was extracted, and the full-length cDNA of CmERA1 was cloned using the total cDNA of‘Jinba’leaves as a template. Bioinformatics methods were applied to analyze the genetic relationship of CmERA1 and characteristics of its encoded proteins, and yeast one-hybrid system was applied to analyze transcriptional activity of CmERA1. Real-time fluorescent quantitative PC (RT-qPCR) was used to analyze their expression levels in different tissues and under different photoperiods. Green fluorescent protein GFP was applied as a label to determine subcellular location of CmERA1. [Results]CmERA1 gene had 1 356 bp open reading frame, encoding 451 amino acids. Its molecular mass was 50.0×103,and its theoretical pI was 5.12. Hylogenetic analysis showed that it had the closest relationship with ERA1 in Artemisia annua. The secondary structure analysis of CmERA1 showed that the percentage of the alpha helix was the biggest. The analysis of transcriptional activation activity showed that CmERA1 protein had no transcriptional activation activity. Subcellular localization analysis showed that CmERA1 was localized in the nucleus. The analysis of the promoter of CmERA1 showed that it contained elements related to the response to the meristem expression, light, ABA and SA. The RT-qPCR showed that the expression level was the highest in leaves during vegetative growth, the lowest in roots, the highest in roots during reproductive growth, and the lowest in ray florets. CmERA1 changed rhythmically in response to photoperiod. [Conclusions]The analysis of expression pattern showed that CmERA1 responded to photoperiod changes. [ABSTRACT FROM AUTHOR]

[Objectives] Cloning and analyzing the function of rice leaf fusion gene LF1 (Leaf Fusion 1) were intended to lay the foundation for elucidating the molecular mechanism of rice leaf development. [Methods] lf1 was a leaf fusion mutant obtained by tissue culture of rice variety‘02428’. The rice leaf fusion mutant lf1 and indica rice variety‘N22’were used to construct a genetic segregation population, and finely map lf1. The candidate genes of LF1 were determined by resequencing analysis and verified by knocking out experiment, and the expression pattern of LF1 and the protein subcellular location were further analyzed. [Results]lf1 was primarily located in the region between molecular markers InDel6-6 and RM30 on chromosome 6 using 10 mutant individuals and 10 normal individuals selected from lf 1/N22 F2 population. Further, lf1 was finely mapped in the range of about 3.2 Mb between the molecular markers InDel6-6 and RM6818 by 256 extreme individuals and increasing the density of molecular markers. Compared with wild type‘02428’,nine genes showed sequence differences in lf1 at the fine mapping interval. Among of them, six genes were encoding transposons, one was annotated as expressing protein gene, and another one was a NAC transcription factor gene Os06 g0344900. A 33 bp deletion in the first exon of Os06 g0344900 resulted in a deletion of 11 amino acids in lf1 compared with the wild type‘02428’. Then, Os06 g0344900 was identified as the candidate gene for LF1. Using CRISPR/Cas9 technology, four different type knockout lines of Os06 g0344900 were obtained. Similarly with lf1 mutant, all knockout lines displayed leaf fused phenotype. These results indicated that Os06 g0344900 was the target gene of LF1. Further, qRT-PCR analysis showed that LF1 were constitutively expressed in various tissues, and relatively higher in the young spikes. The subcellular location showed that LF1 was located in the nucleus. [Conclusions]The leaf fusion phenotype of rice mutant lf1 is caused by the mutation of Os06 g0344900 on chromosome 6. The cloning and preliminary functional analysis of this gene lay the foundation for elucidating the molecular regulation mechanism of rice leaf development. [ABSTRACT FROM AUTHOR]

[Objectives] Halloween genes are involved in the biosynthesis of ecdysone. The ecdysone plays an important role in regulation of the development and reproduction in insects. The stem rice borer, Chilo suppressalis, is one of the most destructive pests in rice plant. It is very important to understanding the characterizations and expression profiles of Halloween genes of C.suppressalis,which is helpful for uncovering the growth and development regulation mechanism in C.suppressalis. [Methods]The complete transcript sequence of Cyp302a1 and Cyp315a1,two members of Halloween genes in C.suppressalis were cloned by using reverse transcriptase polymerase chain reaction（RT-PCR), and rapid amplification of cDNA end (RACE ), methods. The conserved structures of the sequences were analyzed by multiple alignments. The molecular phylogenetic tree was constructed by MEGA 7.0 software. The expression profiles of Halloween genes at different developmental stages and in different tissues were detected by real-time quantitative PCR（RT-qPCR）. [Results]The Halloween gene members, CsCyp302 a1 and CsCyp315 a1,were cloned from C.suppressalis. Each gene contained its own five conserved domains, WxxxR（Helix-C）,GxE/DTT/S（Helix-I）,ExxR（Helix-K）,PxxFxPE/DRF（PERF motif）,PFxxGxRxCxG/A（heme-binding domain）. Both CsCyp302 a1 and CsCyp315 a1 clustered into a clade with their own family members. There were differences in the expression levels of each gene in different developmental stages and tissues. The expression patterns had differences between each gene. The relative expression level of Cyp307 a1 in C.suppressalis was the highest and that of Cyp315 a1 was the lowest. Besides, Cyp307 a1 was highly expressed in the head, the expression level of Cyp306a1 was higher in the midgut, Cyp302a1 and Cyp315a1 were highly expressed in the head and integument, and the expression level of Cyp314a1 was higher in the fat body and integument. [Conclusions]The sequence characterizations of CsCyp302a1 and CsCyp315a1 and the temporal and spatial expression profiles of five Halloween genes of C.suppressalis were determined. [ABSTRACT FROM AUTHOR]

[Objectives] This paper aimed to clone the CmCDKL9 gene of Chrysanthemum morifolium 'Jinba', and analyze its expression pattern preliminarily, so as to lay the foundation for exploring its function in regulating flowering. [Methods] The CmSTKI/ CDKL9 encoding serine/threonine protein kinase was screened from the summer chrysanthemum variety' Yunka' transcriptome data base and named as CmCDKL9. The full-length cDNA of this gene was cloned by using wild-type C. morifolium Jinha cDNA as a template. The function and characteristics of the protein were analyzed and predicted by bioinformatics methods. The expression of the gene under different photo-period process was analyzed by real-time quantitative PCR. [Results] The CmCDKL9 gene cDNA had a full length of 1 680 bp, including an open reading frame of 1 671 bp and encoding 556 amino acids. The relative molecular mass was 62.3x10³,and the theoretical pl was 9.60. Phylogenetic free analysis showed that CmCDKL9 had the same origin as other species such as Arabidopsis thaliana, and it was closely related to the homologous genes of CDK19 in Artemisia annua. Subcellular localization indicated that CmCDKL9 was located in the nucleus and cell membrane. Promoter element analysis showed that the promoter of CmCDKL9 gene contained G-box, AE-box and other cis-elements in response to light signals. Fluorescence quantitative PCR analysis showed that the CmCDKL9 gene was expressed in the roots, stems, old leaves and young leaves flower bands of the C. morifolium 'Jinba' during the vegetative growth period and flower bud differentiation period. During the vegetative growth period, the expression level was the highest in the young leaves and the lowest in the old leaves, and during the flower bud differentiation period, it was the highest in the flower buds while the lowest in the root. Different photo-periods treatments showed that CmCDKI9 responded to photo period treatment, and the expression level showed a diurnal trend under long-day conditions. [Conclusions] We cloned the CCDKL9 gene, and the expression pattern analysis showed that it responded to photoperiod clunges. [ABSTRACT FROM AUTHOR]

[Objectives] This study tries to seek a new molecular approach substitute by cloning the acetylcholinesterase genes(ace)from the Sesamia inferens and analyzing their polymorphism and resistance related mutations. It provides a new way to establish high-throughput insecticide screening and suitable insecticide resistance molecular detection methods.[Methods] Acetylcholinesterase genes were cloned by polymerase chain reaction(PCR)and rapid amplification of cDNA ends(RACE),and confirmed by their molecular characteristics and phylogenetic tree analysis. The polymorphism frequencies were analyzed for the key gene ace1 among different geographical populations. The corresponding resistance levels to triazophos were also tested,and finally the resistance mutations were pursued.[Results] Two full-length cDNA sequences from S.inferens were cloned,characterized,and designated as Sinace1 and Sinace2(GenBank accession No.:MK641586 and MK641585),respectively. They both shared all the conserved structural features of the ace family,and the phylogenetic tree analysis indicated that they had the closest relationship with the corresponding ace genes from lepidopeterians. Polymorphism analysis of the 97 Sinace1 sequences showed 107 nucleotide polymorphism sites and 21 amino acid mutations all with varied frequencies among the four geographical populations. No resistant mutations previously reported in different ace genes were found. Four of the geographical populations were tested and showed that their sensitivities to triazophos varied within 5 times. Comparison analysis found no amino acid mutations with their occurrences consistent with triazophos resistance among the geographical populations.[Conclusions] No detectable resistance mutations have happened in Sinace1 of S.inferens. However,the two ace genes and the polymorphism firstly reported in this work should lay a foundation for the study of the resistance mechanism for organophosphate resistance,and technique development for selecting efficient insecticides by enzyme activity inhibition. [ABSTRACT FROM AUTHOR]

Rice is the most important food crop in China. Rice seed vigor is a complex quantitative trait controlled by multiple genes and it is of great significance to improve rice seed vigor for high yield, quality and efficiency of production under direct seedling rice. This paper reviewed the main research advances including evaluation index, gene identification and cloning, molecular regulation mechanism relate to rice seed vigor in recent years. Based on the molecular markers and map-based cloning, genome-wide association study(GWAS), high throughput sequencing, and omics techonology, more than twenty-three key genes related to seed vigor have been cloned, including transcription factor genes, metabolism-related genes of plant hormone ABA and GA. Through the balance of ABA and GA metabolism, it regulates maturing of embryos, seed dormancy and germination, seedling growth. It will be very helpful to improve genetics of rice seed vigor by molecular design breeding technology in the future. [ABSTRACT FROM AUTHOR]

[Ｏｂｊｅｃｔｉｖｅｓ]Ｇｉｖｅｎ ｔｈｅ ｇｒｅａｔ ｉｍｐｏｒｔａｎｃｅ ｏｆ ｆｅｒｔｉｌｉｔｙ ｉｎ ｒｉｃｅ ｐｏｐｕｌａｔｉｏｎꎬｗｅ ｔｒｙ ｔｏ ｓｔｕｄｙ ｔｈｅ ｓｔｅｒｉｌｅ ｍｕｔａｎｔ ｆｍｓ(ｆｅｍａｌｅ ａｎｄ ｍａｌｅ ｓｔｅｒｉｌｉｔｙ)ｃｌｅａｒｌｙ ｓｏ ｔｈａｔ ｍｕｃｈ ｍｏｒｅ ｇｅｎｅｓ ａｎｄ ｆｉｎｅｒ ｇｅｎｅ ｒｅｇｕｌａｔｏｒｙ ｎｅｔｗｏｒｋ ａｂｏｕｔ ｒｉｃｅ ｓｔｅｒｉｌｉｔｙ ｃａｎ ｂｅ ｆｏｕｎｄ. [Ｍｅｔｈｏｄｓ] Ｃｙｔｏｌｏｇｉｃａｌ ｏｂｓｅｒｖａｔｉｏｎ ｗａｓ ｍａｄｅ ｏｎ ｔｈｅ ｓｔａｂｌｅ ｍｕｔａｎｔ ｆｍｓꎬ ｗｈｉｃｈ ｗａｓ ｏｂｔａｉｎｅｄ ｂｙ ｅｔｈｙｌ ｍｅｔｈａｎｅｓｕｌｆｏｎａｔｅ( ＥＭＳ)￣ｉｎｄｕｃｅｄ ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｏｆ ｊａｐｏｎｉｃａ ｒｉｃｅ ｖａｒｉｅｔｙ‘Ｎｉｎｇｊｉｎｇ ４’. Ｂｅｓｉｄｅｓꎬｗｅ ｃｒｏｓｓｅｄ ｔｈｅ ｓｔｅｒｉｌｅ ｍｕｔａｎｔ ｆｍｓ ｗｉｔｈ‘Ｎ２２’ｔｏ ｃｏｎｓｔｒｕｃｔ ａ Ｆ２ ｐｏｐｕｌａｔｉｏｎ ｆｏｒ ｇｅｎｅｔｉｃ ａｎａｌｙｓｉｓ ａｎｄ ｇｅｎｅ ｍａｐｐｉｎｇ. [Ｒｅｓｕｌｔｓ]Ｂｏｔｈ ｔｈｅ ｍａｌｅ ａｎｄ ｆｅｍａｌｅ ｇａｍｅｔｅｓ ｏｆ ｔｈｅ ｆｍｓ ｍｕｔａｎｔ ｗｅｒｅ ｓｔｅｒｉｌｅ. Ｔｈｒｏｕｇｈ ｔｈｅ ｔｒａｎｓｖｅｒｓｅ ｓｅｃｔｉｏｎꎬｌａｓｅｒ ｓｃａｎｎｉｎｇ ｃｏｎｆｏｃａｌ ｍｉｃｒｏｓｃｏｐｙ ａｎｄ ｍａｇｅｎｔａ ｓｔａｉｎꎬｗｅ ｏｂｓｅｒｖｅｄ ｔｈｅ ａｂｓｅｎｃｅ ｏｆ ｔｈｅ ｔａｐｅｔｕｍ ａｎｄ ｔｈｅ ｍｉｄｄｌｅ ｌａｙｅｒ ｏｆ ｔｈｅ ａｎｔｈｅｒ ｃｅｌｌ ａｎｄ ｔｈｅ ｄｅｖｅｌｏｐｍｅｎｔａｌ ａｒｒｅｓｔ ｏｆ ｔｈｅ ｐｏｌｌｅｎ ｍｏｔｈｅｒ ｃｅｌｌ. Ｍｏｒｅｏｖｅｒꎬｉｎ ｔｈｅ ｅｍｂｒｙｏ ｓａｃ ｏｆ ｔｈｅ ｍｕｔａｎｔꎬｔｈｅ ｎｕｍｂｅｒ ｏｆ ｅａｒｌｙ ｓｐｏｒｏｃｙｔｅ ｉｎｃｒｅａｓｅｄꎬａｎｄ ｔｈｅ ｍａｔｕｒｅ ｅｍｂｒｙｏ ｓａｃ ｃｏｍｐｌｅｔｅｌｙ ｄｅｇｅｎｅｒａｔｅｄ ｏｒ ｄｉｓｐｌａｙｅｄ ｍｏｒｅ ｔｈａｎ ８ ｎｕｃｌｅｕｓꎬｃａｕｓｉｎｇ ｔｈｅ ｆｅｍａｌｅ ｇａｍｅｔｅｓ ｔｏ ｂｅ ｓｔｅｒｉｌｅ. Ｇｅｎｅｔｉｃ ａｎａｌｙｓｉｓ ｉｎｄｉｃａｔｅｄ ｔｈａｔ ｔｈｅ ｓｔｅｒｉｌｉｔｙ ｔｒａｉｔ ｏｆ ｍｕｔａｎｔ ｆｍｓ ｗａｓ ｃｏｎｔｒｏｌｌｅｄ ｂｙ ａ ｒｅｃｅｓｓｉｖｅ ｎｕｃｌｅａｒ ｇｅｎｅ ａｎｄ ｆｉｎａｌｌｙ ｔｈｅ ｌｏｃｕｓ ｗａｓ ｍａｐｐｅｄ ｔｏ ａ ８１８ ｋｂ ｒｅｇｉｏｎ ｂｅｔｗｅｅｎ ｔｈｅ ｍａｒｋｅｒｓ Ｔａ￣４ ａｎｄ Ｔａ￣７ ｏｎ ｔｈｅ ｌｏｎｇ ａｒｍ ｏｆ ｒｉｃｅ ｃｈｒｏｍｏｓｏｍｅ １２. Ｔｈｅｒｅ ｗｅｒｅ １３ ＯＲＦ(ｏｐｅｎ ｒｅａｄｉｎｇ ｆｒａｍｅ)ｉｎ ｔｈｉｓ ｒｅｇｉｏｎ. Ｂａｓｅｄ ｏｎ ｔｈｅ ｇｅｎｏｍｅ ｓｅｑｕｅｎｃｉｎｇ ｒｅｓｕｌｔꎬａ ｎｕｃｌｅｏｔｉｄｅ ｓｕｂｓｔｉｔｕｔｉｏｎ ｗａｓ ｆｏｕｎｄ ｏｎ ｔｈｅ ｇｅｎｅ Ｏｓ１２ｇ０４７２５００ꎬｗｈｉｃｈ ｔｕｒｎｅｄ ｔｒｙｐ￣ ｔｏｐｈａｎ ｉｎｔｏ ａｎ ｅｎｄ ｃｏｄｏｎꎬｒｅｓｕｌｔｉｎｇ ｉｎ ｔｈｅ ｐｒｅｍａｔｕｒｅ ｔｅｒｍｉｎａｔｉｏｎ ｏｆ ｔｒａｎｓｌａｔｉｏｎ. Ｔｈｅ ｅｘｐｒｅｓｓｉｏｎ ｏｆ ＦＭＳ ｗａｓ ｂｏｔｈ ｈｉｇｈｅｒ ａｎｄ ｓｐｅｃｉｆｉｃ ｉｎ ａｎｔｈｅｒꎬａｎｄ ｔｈｅｎ ｉｎ ｔｈｅ ｅｍｂｒｙｏ ｓａｃ. [Ｃｏｎｃｌｕｓｉｏｎｓ]Ｔｈｅ ｆｅｒｔｉｌｉｔｙ ｏｆ ｆｍｓ ｍｕｔａｎｔ ｉｓ ｃｏｎｔｒｏｌｌｅｄ ｂｙ ａ ｒｅｃｅｓｓｉｖｅ ｎｕｃｌｅａｒ ｇｅｎｅ(Ｏｓ１２ｇ０４７２５００) ｗｈｉｃｈ ａｆｆｅｃｔｓ ｔｈｅ ｄｅｖｅｌｏｐｍｅｎｔ ｏｆ ｒｉｃｅ ｐｏｌｌｅｎ ａｎｄ ｅｍｂｒｙｏ ｓａｃ. [ABSTRACT FROM AUTHOR]

[Objectives] Nicotinic acetylcholine receptor(nAChR) plays an important role in excitatory transmission at cholinergic synapse in insect central nerve systems and is also the target of neonicotinoid insecticides. The Colorado potato beetle(CPB), Leptinotarsa decemlineata, is an important worldwide quarantine defoliator. The ten nAChR subunit genes in L.decemlineata had been obtained from previous reports, namely Ldα1-Ldα4, Ldα6-Ldα10 and Ldβ1. The aim of the study is to further improve the composition and expression profile of the nAChR subunits of L.decemlineata. [Methods] The nAChR α subunit gene was cloned by polymerase chain reaction(PCR) and rapid amplification of cDNA ends(RACE) based on bioinformatic analysis of L.decemlineata genome dataset. The cloned gene was subjected to phylogenetic relationship analysis by constructing phylogenetic tree. Quantitative real-time PCR was applied to the investigation of the expression levels of different expression profiles of the Ldα5 in different development stages or body parts of adults and under imidacloprid and thiacloprid treatments. [Results] In this study, the full-length cDNA of a new nAChR α subunit gene from L.decemlineata was cloned. It was named as Ldα5(GenBank accession:MF197919). The quantitative real-time PCR [Results] showed Ldα5 had the highest expression level in 1st instar larvae and heads of adults respectively. In the 4th instar larvae exposed to the insecticides tested, imidacloprid significantly decreased the expression level of Ldα5 and thiamethoxam had no effects, whereas in the adults, the two insecticides led to the up-regulation of Ldα5 significantly. [Conclusions] The findings may provide basis for further study on the function of nAChR subunits and the molecular mechanisms of target resistance to neonicotinoids of L.decemlineata. [ABSTRACT FROM AUTHOR]

[Objectives] Aquaporin, a member of the major intrinsic protein superfamily, functions mainly for transporting water and small molecule compounds across cell membrane, and the aim of this study was to analyze the function of the aquaporin and its possible contribution to insecticide metabolism in an important agricultural pest Laodelphax striatellus. [Methods] In this study, aquaporin genes in L. striatellus were cloned based on the transcriptome data and with PCR and RACE cloning techniques. The cloned genes were subjected to phylogenetic relationship analysis by constructing phylogenetic tree with previously reported insect aquaporin genes, tests of the expression level in the susceptible and resistant strains by real-time quantitative PCR, and functional verification for their transporting capability by combinant expression in vitro. [Results] Six full length aquaporin genes were cloned from L. striatellus and designated as LsAQP1, LsAQP2, LsAQP3, LsAQP4, LsAQP5 and LsAQP6, respectively. Among them, LsAQP6 was found expressed with higher level in all three resistant strains tested as compared to the susceptible strain. Furthermore, functional expression showed that LsAQP6 had strong transport capacity for water and significant transport capacity for methomyl, but not for glycerol and bisultap. [Conclusions] Thus, it is speculated that LsAQP6 plays a significant role in transporting water and some small molecule insecticides in L. striatellus. [ABSTRACT FROM AUTHOR]

[Objectives]The aim is to identify rice dwarf mutant htr, localize the target gene and provide new dwarf gene resources for rice breeding. [Methods]Sections of the stem were prepared for cytological observation. The dwarf mutant htr was crossed with ‘N22’ and ‘9311’ to construct F2 populations for genetic analysis and gene mapping, respectively. We selected the recessive extreme individuals to localize the gene HTR. [Results]htr was a stably inherited dwarf mutant. The plant height, panicle length, 1 000-grain weight, and tiller number in mature plants all decreased compared with wild type, while the leaf width and culm diameter were increased and the leaf colour of htr was much darker green than wild type. By observing sections of the second internodes at the mature stage, we found in the longitudinal direction of the stem, the cells failed to elongate normally and became small, but the cell number increased. Genetic analysis showed that the dwarf trait of mutant htr was controlled by a pair of recessive gene. The HTR locus was mapped to a region between the markers ttr-1 and ttr-2 on chromosome 5, which was further narrowed down to a 144 kb region. Sequence analysis revealed that the coding region of the AP2-like ethylene-responsive transcription factor had a single nucleotide substitution, resulting in a change in the conserved amino acid sequence in the htr mutant. Subcellular localization assay showed that HTR was located in the nucleus, and the HTR was constitutively expressed, but it had the highest expression in the stem. [Conclusions]The phenotype of htr was controlled by a single recessive gene, which encodes the AP2-like ethylene-responsive transcription factor. The cloning of HTR gene could provide some theoretical reference for cultivating thick-walled and lodging resistant new rice varieties. [ABSTRACT FROM AUTHOR]

[Objectives] Cinnamoyl coenzyme A reductase(CCR) is a key enzyme in lignin biosynthesis. The aim of this study is to investigate the synthesis mechanism of pomegranate lignin and cultivate new varieties of soft-seed pomegranate by cloning and characterization of pomegranate CCR gene. [Methods] The full length of PgCCR was obtained from the seeds of pomegranate variety ' Hongyushizi' by the method of RACE and RT-PCR. The expression levels of PgCCR in different cultivars, different tissues and seeds of different developmental stages were analyzed by qRT-PCR. [Results] The lull-length cDNA sequence was 1017 bp, encoded a deduced protein of 338 amino acids, and contained the conserved sequence KNWYCYGK, which was considered to be the catalytic site of CCR protein. Phylogenetic tree analysis showed that PgCCR shared the same origin with other species, and had the closest relationship with Vitis vinifera VvCCR. PgCCR was detected in three cultivars of pomegranate, and the expression in 'Hongyushizi' was higher, while lower in 'Huiliruanzi' and 'Tunisiruanzi'. PgCCR expression was detected in leaves, flowers and stems, and the expression in stems was highest, while the lowest in leaves. PgCCR expression was detected in 'Hongyushizi' seeds of different periods. The relative expression ol PgCCR gradually increased from 20-80 d, reached a peak at 80 d, and then declined from 80 d. [Conclusions] The pomegranate CCR gene was cloned and its expression level was correlated with the synthesis and content of lignin. [ABSTRACT FROM AUTHOR]

[Objectives]Ascorbate peroxidase(APX)removes excess plant peroxides by using an electron donor from ascorbic acid,which is an essential mechanism in response to abiotic stress. The aim of the paper is to study the response of DcAPX gene in the process of resisitance to abiotic stress. [Methods]The DcAPX gene was cloned from carrot variety‘Kurodagosun’. Expression profiles under high and low temperature,drought,and salinity treatments were performed to further understand the roles of the gene in response to abiotic stress in carrot. The gene was submitted to GenBank and the accession number is KR364573. [Results]Sequence analysis indicated that the length of DcAPX gene was 753 bp,which encoded 250 amino acids. It is predicted that the relative molecular mass of its protein was 27.76×103,and pI was 5.65. Amino acid sequence comparison showed that the evolutionary relationship of DcAPX from carrot was close to Pimpinella brachycarpa,which both belong to the Apiaceae family. Structure analysis showed that DcAPX was made up of 38.40% alpha helix,10.40% extended strand,and 51.20% random coil. Analysis on three-dimension structure implied that the enzyme was composed of 10 alpha helixes and 4 beta sheets. Quantitative real-time PCR analysis demonstrated that carrot DcAPX gene was tissue-specific,and had the highest expression level in the leaf. [Conclusions]DcAPX gene was significantly induced by abiotic stress,and the gene may play important roles in resistance against abiotic stress. [ABSTRACT FROM AUTHOR]

[Objectives]We had a preliminary study on the function of CmMYB59 and explained its response to the cold stress. [Methods]In this study,a novel MYB and its alternative splicing transcript were isolated from Chrysanthemum morifolium‘Jinba’by RT-PCR and RACE methods. The expression of CmMYB59 in different tissues and cold treatment conditions was studied by qRT-PCR. Furthermore,its transcriptional activation and subcellular localization were analyzed by the yeast one-hybrid assay and transient expression in onion epidermal cells respectively. [Results]The full length of this gene was 904 bp,containing an open reading frame of 768 bp,encoding 255 amino acids,with relative molecular mass of 61.99×103. The multiple alignment of the protein sequences showed the protein had a typical R2R3-MYB domain and was most closed to AtMYB59. Thus,the gene was named CmMYB59. qRT-PCR showed that the expression of CmMYB59 was the highest in roots,higher in stems and leaves,the lowest in shoot tip and floral organ,and it was responsive to the cold stress. The subcellular localization indicated that the protein was located in nucleus. The yeast one-hybrid assay showed that the CmMYB59 protein had a transcriptional activation function. [Conclusions]It is speculated that CmMYB59 might be related to cell division,participating in the development process of root,and being associated with cold stress. [ABSTRACT FROM AUTHOR]

In the current study, Squaliobarbus crriculus MyoG gene fragments were amplified by RT-PCR, 3'RACE and 5' RACE methods. A full-length of 1 628 bp MyoG cDNA sequence in S.crriculus was assembled, which includes a complete ORF encoding a 274 amino acids peptide with a typical bHLH structure in the protein. Sequence analysis indicated that the S. crriculus MyoG gene exhibited low similarity of nucleotide and amino acid with other fishes, and the highest one of nucleotide similarity and amino acid similarity is Cyprinus carpio, which was 78.42% and 74.09%, respectively. A phylogenetic tree of 13 species was constructed using the Neighbor-Joining method by MAGE 4.2, and the phylogenetic relationship of the 13 species was revealed. It indicated that the S.crriculus MyoG gene was highly conserved in evolution. MyoG gene was detected in all eight examined tissues by semi-quantitative RT-PCR, MyoG mRNA expressed highest in muscle, followed in kidney, heart and brain, weakly expressed in gill, and traced in spleen, implying that the fish MyoG gene would play an important role in muscle development. [ABSTRACT FROM AUTHOR]

CELI was the first mismatch endonuclease, which was identified from the celery(Apium graveolens L. ). CELI is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. This study measured the different varieties of celery CELI gene expressions under abiotic stress, in order to further study the function and application of endonuclease. The cDNAs encoding the CELI were cloned from two celery variaties of ' Liuhehuangxinqin' and 'Ventura', respectively. The sequences were analysed and used for quantitative real-time PCR to detecte the expression profiles under different abiotic stress treatments. CELI sequences of various types of plants in this study were from the NCBI database. The amino acid composition and physicochemical properties were analysed by the Expasy related software. MEGA 5 test and editor of the evolutionary tree by DNAMAN 6.0 was used to generate report graphics. The results showed that the lengths of CELI genes were 891 bp, encoding 296 amino acids. There were 20 different nucleotide sites, and 3 amino acid residues were changed between the two celery cultivars. There was highly nucleotide identity between the CELI genes from the two celery cultivars. Multiple sequence alignment display showed 77.42% similarity. Analysis of amino acid composition and physicochemical properties showed that the predicted molecular weights of the CELI were 33.88.103 and 33.92.103 ,and the pI values were 6. 51 and 6. 37,respectively. Phylogenetic analysis illuminated that the CELI of celery was close to Petroselinum crispum,an Apiaceae plant. The CELI of Apiaceae plant was close to the plant of Compositae. The expression profiles of the CELI genes of organizations in two-month-old mature plants were detected by quantitative real-time PCR. The expression of each tissue descended as flowers, leaves, roots, stems in 'Liuhehuangxinqin', but stems, flowers, roots, leaves in'Ventura'. Quantitative real-time PCR analysis showed that the CELI genes were tissue-specific expressed in the two celery cultivars. The expression profiles of the CELI genes exposed to various treatments(4 ?,38 ?,0.2 mol·L-1 NaCl and 200 g·L-1 PEG) for 2 h were detected by quantitative real-time PCR. The gene expression levels were significantly different in the two celery cultivars, and the level significantly decreased after treated by 200 g·L-1 PEG. Conclusions: Under the treatments of high temperature,low temperature, drought, high salt, the celery plants of 'Ventura' resistance performance is better. In this study, through analysis of gene expression after these abiotic stress treatments, it was found that the expression level of CELI gene induced in 'Liuhehuangxinqin' was greater than in 'Ventura'. [ABSTRACT FROM AUTHOR]

To obtain the arginine kinase gene( AK) from Haemonchus contortus, total RNA was extracted from the adult worms, followed by RT-PCR, using a pair of primers( P1:5'CGCGGATCCTTCACCATGTCTGTTCCTCCG-3' P2:5'CCCAAGCTTCCCT- TCAAGCCTTCTTCTCCA-3')targeting the AK gene of H. contortus. Then the RT-PCR product was purified and cloned into pMD19-T vector, and the recombinants were identified by PCR and enzyme digestion. The sequence of this gene was compared with the sequences of AK gene available in GenBank, and the characters were analyzed by DNAStar. By subcloning the ORF of arginine kinase into the vector pET32a(+),the prokaryotic expression plasmid pET32/ AK was constructed. The recombinants were transformed into Escherichia coli BL21 and induced by isopropyl-B-D-thiogalactopyranoside( IPTG) for 5 hours, after which, the bacterial cells were collected by centrifugation, followed by sonication. The supernatant and inclusion bodies were collected and analyzed by SDS-PAGE. The recombinant AK was purified with protein purification kit, refolded by utilizing diluted urea as solvent step by step, and analyzed by Western blot. To assay the enzyme activity of the recombinant arginine kinase, the standard curve was established with standard phosphorus solution( potassium dihydrogen phosphate,0. 439 3 g, dissolved with double distilled water, diluted to 1 000 mL) and phosphate determination reagent(1% ( NH4 )6 Mo7 024.4H2 0,1 mol.L-1 H2 SO4 and 5% FeSO4.7H2 O). The reaction mixture consisting of 100 mmol.L-1 Tris,10 mmol.L-1 L-arginine,10 mmol.L-1 β-mercaptoethanol, 2 mmol.L-1 ATP-Na, and 5 mmol.L-1 magnesium acetate was allowed to incubate at certain temperature for 10 min, after 10.L 6 μg.μL-1 enzyme(the recombinant AK) was added. To stop the reaction, the solution was placed in boiling water for 1 min and plunged into an ice bath for another minute, after which,500 μL phosphate determination reagent was added into the above-mentioned solution at 30°C, and the absorbance was measured at 660 nm. The activity of arginine kinase was calculated according to the contents of phosphorus. Results: A gene fragment of 1 104 bp was produced by RT-PCR. It was founded that the gene was 89% similar with the gene of H. contortus AK available in GenBank. SDS-PAGE analysis showed that the recombinant protein was expressed with the relative molecular weight of 60. 3×103 . Result of Western blot showed that the recombinant protein could be recognized by the serum from the goat infected with H. contortus. Analysis of the activity of the recombinant protein indicated that the optimum reaction temperature was 30°Cand the optimum pH was 7. 5 for this enzyme. Conclusion: Arginine kinase gene of H. contortus was cloned and expressed. The recombinant protein showed the antigenicity and some activities of arginine kinase. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors(nAChRs) play an important role in excitatory synaptic transmission in insects and are also one kind of the most important insecticide targets. Colorado potato beetle(CPB, Leptinotarsa decemlineata) is a serious defoliator of potato and often causes great yield loss. Cloning and investigation of the expression patterns of the nAChRα genes from L. decemlineata may provide important basis for understanding of the function of nAChR subunits and neonicotinoid resistance in L. decemlineata. The gene fragments encoding nAChR subunit proteins were isolated by bioinformatic analysis of L. decemlineata transcriptome dataset and polymerase chain reaction(PCR)amplification. Full-length cDNAs were further obtained by rapid amplification of cDNA ends(RACE). Sequence analysis was conducted using BLAST programs on the National Center for Biotechnology Information(NCBI). Phylogenetic analysis was carried out using ClustalW program and neighbor joining method in MEGA 5. 0 program. Quantitative real-time PCR was employed to investigate the expression levels of the three-subunit genes at various developmental stages(1 to 4 instar larvae, pupa and adult) and different body parts in adults(head, thorax and abdomen). Results:1)Three subunit gene fragments encoding nAChRα4, α7 and α9 subunit proteins were cloned from L. decemlineata and full-length cDNAs of these genes were further obtained, correspondingly designated as Ldα4, Ldα7 and Ldα9 respectively. The complete cDNA sequence of Ldα4,Ldα7 and Ldα9 contained 1 607 bp,2 342 bp,1 227 bp, and encoded a protein of 348, 529 and 341 amino acids respectively. 2) Sequence analysis showed that these genes possessed the typical structure characteristics of nAChR gene family and shared high similarities with other insect nAChR α subunit proteins, especially with those from the coleopteran insect, Tribolium castaneum(87%,89% and 47% for Ldα4, Ldα7, Ldα9, respectively). The analysis of phylogenetic tree indicated that Ldα4, Ldα7 and Ldα9 were clustered into the same class with those from T. castaneum respectively, confirming the close evolutionary relationship between L. decemlineata and T. castaneum. 3) All the three genes,Ldα4,Ldα7 and Ldα9,exhibited the higher expression levels in the head and thorax compared with those in abdomen for adults (P<0.05). These genes also showed different expression levels among different developmental stages. Ldα4 had the highest expression levels in adult and pupa stages,while lowest levels in larvae. However,no significant difference was found between the pupa and larval stages. For Ldα7,the highest level of transcription was observed in the 3rd instar larvae,followed with the 1st instars larvae,and the lowest level was in the 2nd and 4th instar larvae,pupae and adults. For Ldα9,expression level in pupae was higher than those in the adults and larvae(P<0.05). Three full-length cDNAs encoding nAChR αsubunit proteins were obtained from L. decemlineata,named Ldα4,Ldα7 and Ldα9,respectively. The expression levels of each gene in larva,pupa and adult stages were different,suggesting that the nAChR subunit genes may play specific roles in different developmental stages of L. decemlineata. [ABSTRACT FROM AUTHOR]

Two novel RDR6 genes were identified in soybean by homologous clones, named as GmRDR6a and GmRDR6b. Sequences of the two genes were analyzed by bioinformatics softwares, and genes expression of different tissues and their reponse to biotic and abiotic stresses were performed using real-time PCR. The results showed that GmRDR6a gene was located on chromosome 6 of soybean genome, and the length of it was 4 389 bp, inclulding an intron of 744 bp. The ORF of GmRDR6a was 3 615 bp and encoded 1 204 amino acids(297. 5⨰103 )with an isoelectric point of 4. 86. GmRDR6b gene was located on chromosome 4 of soybean genome and the length of it was 4 002 bp, contained an intron of 387 bp. The ORF of GmRDR6b was 3 615 bp and encoded 1 204 amino acids(296. 89⨰103 ) with an isoelectric point of 4. 86. The consereved motifs " DLDGD" of RDRs gene family were identified in GmRDR6a and GmRDR6b; The expression of the two novel genes were identified in all tissues of soybean,and showed the higest expression level in flower of soybean;Real-time PCR analysis revealed that the mRNA levels of these two genes in the resistant material' Kefeng No. 1' were significant higher than the susuceptible material' Nannong 1138-2' after soybean mosaic virus( SMV) treatment. For abiotic stress,GmRDR6a showed high expression level in roots, stems and leaves under the treatments of salinity and drought, and showed the highest expression level in roots under salt stress. There was an early response after ABA treatment. There was no significant expression level difference of GmRDR6a gene under chilling stress. GmRDR6b showed higher expression level under salt stress and ABA treatment and there was an early response under chilling stress. There was no significant expression level difference of GmRDR6b gene under drought stress. [ABSTRACT FROM AUTHOR]

The full-length cDNA of Δ 6 fatty acyl desaturases (FAD6) of Coilia nasus, which was involved in the biosynthesis of highly unsaturated fatty acids ( HUFA), was cloned by RT-PCR ( reverse transcription polymerase chain reaction) and RACE ( rapid- amplification of cDNA ends) methods. The results revealed that the FAD6 cDNA was 2 032 bp,including an ORF( open reading frame) of 1 338 bp specified a protein of 445 amino acids, which contained three histidine-rich regions and two putative transmembrane domains. The deduced amino acid sequence shared 75% - 79% identity with that in Muraenesox cinereus and Rachycentron canadum, and low similarity of freshwater fish. Actually, it was consistent with the results of phylogenetic analysis. The tissue-specific expressions of FAD6 mRNAs were detected in C. nasus. A high expression was observed in the brain and intestine, while relatively high expression was encountered in muscle and liver,and a weak expression in the heart, kidney and gill. [ABSTRACT FROM AUTHOR]