Intron Retention Fine-Tunes the Resistance of the Rice Mutant pls4 to Rice Sheath Blight(Rhizotonia solani AG I.1a)

OsPLS4 encodes aβ-ketoacyl carrier protein reductase(KAR).The role of OsPLS4 in rice sheath blight(Rhizoctonia solani)remains unclear.Our preliminary studies showed that premature leaf senescence mutants(pls4)were highly susceptive to sheath blight in the early stage of rice development.To explore the role of this gene in the development of rice sheath blight,the transcriptome profiles of the rice pls4 mutant and wild type were compared by RNA-seq.The results revealed 2,569 differentially expressed genes(DEGs).The down-regulated genes were significantly enriched in the defense response-related biological processes.These down-regulated genes included the chitinase genes and WRKY genes,which were significantly changed in pls4 mutants.Furthermore,467 genes induced significant alternative splicing(AS)events.Among them,intron retention(IR)affected gene expression levels and functions of the vitamin B6(VB6)metabolism pathway related to sheath blight.This result suggests that IR plays an important role in the sheath blight resistance of mutant pls4.Together,these results indicate that pls4 could be involved in the biological process of sheath blight via DEGs and the fine-tuning of IR.The present study provides a molecular basis for further investigation of the resistance of rice to sheath blight.

猪H-FABP基因intron3全序列测定

[目的]为将H-FABP基因应用于猪育种过程中的标记辅助选择提供基础资料。[方法]根据GenBank数据库上公开发表的相关的猪H-FABP基因序列设计特异性扩增引物,对H-FABP基因内含子3的PCR产物纯化后直接进行测序。[结果]成功扩增出猪H-FABP基因intron 3的全序列,全长为1 350 bp,已向GenBank数据库提交,检索号为DQ 002993。[结论]该研究为确定影响肌内脂肪沉积的主效基因奠定了理论基础。

牦牛和普通牛DRB1*Intron1-exon2序列变异分析

为揭示牦牛抗逆性及抗病育种积累更多的分子遗传学资料,通过检测DRB1基因在牦牛和普通牛群体中的变异,分析该基因检测区域遗传参数。以甘南牦牛、青海牦牛、天祝白牦牛、大通牦牛和普通牛为研究对象。应用PCR-SSCP方法检测BoLA-DRB1基因第1内含子及第2外显子部分序列多态性。DRB1基因第1内含子区检测到4处SNPs及1处插入/缺失突变,第2外显子区检测到17处SNPs,两区域均表现为高度多态;单倍型连锁分析发现21种intron 1-exon 2单倍型组型且存在单倍型连锁不平衡现象,A-A1、A-B1、B-A1和B-B1单倍型在牦牛和普通牛中频率较高;聚类分析表明,牦牛DRB1基因第2外显子区碱基序列与普通牛及山羊的同源性最高,系统进化情况与它们亲缘关系远近一致。牦牛和普通牛BoLA-DRB1基因第1内含子及第2外显子多态性丰富,可作为牦牛和普通牛BoLADRB1的遗传标记。

慢性肾衰竭继发性甲旁亢患者中CaSR基因intron5多态性的研究

为研究中国江苏汉族人中钙敏感受体 (calcium-sensingreceptor, CaSR)基因单核苷酸多态性与慢性肾衰竭(chronicrenalfailure, CRF)继发性甲旁亢(secondaryhyperparathyroidism, SHPT)的关系.为此采集江苏省122例慢性肾衰竭继发性甲旁亢(CRF SHPT)患者及 25例正常人血液样本,通过PCR-RFLP的方法分析CaSR基因内含子 5(intron5 )BseRI多态性;以标准方法测定血清钙、血清磷、血清全段甲状旁腺素 (intactPTH,iPTH)3项生理指标;对检测的结果进行统计分析.结果发现,CRF-SHPT患者intron5多态性频率与正常人没有明显差异;汉族人intron5多态性频率与其它种族有显著差异;TT基因型患者的Ca2+、iPTH的血清浓度显著低于CC基因型的浓度.结论:CaSR基因intron5多态性频率具有种族差异性;intron5多态性与Ca2+、iPTH的血清水平密切相关,影响CRF SHPT病情的严重程度.

基于CDS..join特征域的Exon/Intron数据库的构建

基因进化的研究和重构通常是在序列水平上进行的,包括比对它们的基因序列或蛋白序列.而对基因外显子/内含子结构的分析能够提供更多有价值的信息,比如绘制更为可靠的系统发生图谱,或更精确地阐明内含子的进化.为此,本文设计了相应的Perl脚本程序来提取、比较和搜索基因说明文档中CDS..join特征域的Exon/Intron结构.通过该方法,可构建相关物种的Exon/Intron数据库(EID),其主要内容包括内含子的相位,Exon或Intron的数量和大小,剪接位点的模式以及选择性剪接(Alternative splicing,AS)的相关信息.

Relationship Between the First Base of the Donor Splice Site of Waxy Gene Intron 1 and Amylose Content in Yunnan Indigenous Rice Varieties

There exists a single nucleotide polymorphism, G or T, at the first base of the donor splice site of waxy gene intron 1 in rice. In order to study the relationship between the first base of the donor splice site of waxy gene intron 1 and amylose content in rice, the one-step PCR method was used to determine whether it is G or T in 220 Yunnan indigenous rice varieties from 14 districts, 55 towns/counties of Yunnan Province, and 101 varieties of which were validated by the PCR-Acc I method. According to the G/T polymorphism, 164 rice varieties showed GG-genotype, while the other 56 fell into TT- genotype, accounting for 74.5% and 25.5% of all the test varieties, respectively. When all the rice varieties were divided into indica and japonica subspecies, it was found that 80.5% of indica rice and 67.0% of japonica rice belonged to GG-genotype. The rice varieties with GG-genotype had significantly higher amylose content （18.95% on average） than those with TT- genotype （all below 16%）, but 33 rice varieties with GG-genotype still had low amylose content ranging from 3.91% to 15.93%, and most of them came from the Dai minority area in the Southwest of Yunnan Province. However, there was no significant difference in the mean amylose content of the same GG or TT genotypes between indica and japonica rice, suggesting that different genetic backgrounds, indica or japonica, had no effect on amylose content. The coefficient of correlation between the genotype and amylose content was 0.733 （P〈0.01）.

The Sequence Variations of Intron-3 of the α-Amylase Gene in Adzuki Bean

This study describes variation of intron-3 of α-amylase gene from 156 breeds of adzuki beans using SSCP(single-strand conformation polymorphism)analysis. Based on α-amylase gene structure and sequence, A pair of PCR primers, F (CCTACATTCTAACACACCCT) and R (GCATATTGTGCCAGTACAAT) were designed to amplify intron-3 fragments of α-amylase gene. 14 variant types were detected, including 13, 9, 10, 4 variant types in the wild, weed, locally cultivated and modern brought-up adzuki beans respectively, 9, 8, 7 variant types of the wild adzuki beans from Japan, China and Korea respectively, and some other variant types in the local adzuki beans from China and Bhutan. 60% of subjects of cultivated races were found to be EE type in the experiment. In addition, sequence analysis of intron-3 of α-amylase gene from 8 variant types reveals the evolution process of various variant types in adzuki beans.

Specific intronic p53 mutation in esophageal squamous cell carcinoma in Southern Thailand

AIM:To investigate p53 mutations in esophageal cancer in a high-risk population,and correlate them with smoking,alcohol consumption and betel chewing.METHODS:One hundred and sixty-five tumor samples of esophageal squamous cell carcinoma(ESCC) obtained from a university hospital in Songkhla province,Southern Thailand were investigated for p53 mutations in exons 5-8,using polymerase chain reaction-single strand conformation polymorphism analysis,followed by direct sequencing.A polymerase chain reactionrestriction fragment length polymorphism(RFLP) assay was additionally used to confirm possible germline mutation in intron 6.A history of risk habits was obtained by interviews.The association between risk habits and mutation frequency was evaluated using the χ 2 test.RESULTS:The studied specimens were from 139 male and 26 female patients with ESCC,treated at Songklanagarind Hospital.Most of the patients were smokers(86.7%) and alcohol consumers(72.73%),and 38.3% were betel chewers.Forty-three mutations of the p53 gene were detected in 25.5%(42/165) of tumor samples.Mutations were most commonly found in exon 5(25.6%) and exon 8(25.6%).Mutations in the hot-spot codon 248 were found in four cases(9.3% of all mutations).G:C→C:G(30.23%),G:C→A:T(27.90%) and G:C →T:A(16.28%) were the prevalent spectra of mutations.Unexpectedly,among 10 intronic mutations,eight cases harbored a similar mutation:G→C substitution in intron 6(nucleotide 12759,GenBank NC_000017).These were additionally confirmed by the RFLP technique.Similar mutations were also detected in their matched blood samples using RFLP and direct sequencing,which suggested germline mutations.There was no significant correlation between risk habits and p53 mutation frequency.CONCLUSION:A proportion of Thai ESCC patients harbored specific intronic p53 mutations,which might be germline mutations.Further studies are needed to explore this novel finding.

Intron-Targeted Intron-Exon Splice Conjunction (IT-ISJ) Marker and Its Application in Construction of Upland Cotton Linkage Map

To develop a new DNA maker, which could be used in genetic diversity analysis and genetic map construction in plants, IT-ISJ （intron targeted intron-exon splice junction） primer combinations, which were designed according to the intronexon splice junction conserved sequences, were used to construct cotton genetic linkage map in the present study. 49 out of 704 IT-ISJ primer combinations showed polymorphism between upland cotton high quality cultivar Yumian 1 and multiple dominant gene line T586, and the polymorphic primer combinations accounted for 7.0% of total primer combinations. 49 IT-ISJ primer combinations were used to genotype 270 F2：7 recombinant inbred lines developed from （Yumian 1 × T586） F2, and 58 IT-ISJ loci were obtained. 58 IT-ISJ, together with 150 SSR and 8 morphological loci, were used to conduct linkage analysis, and a linkage map including 22 linkage groups and 113 loci （49 IT-ISJ, 62 SSR, and 2 morphological loci） was constructed. The linkage map covered 714.5 cM with an average interval of 6.3 cM between two markers, accounting for 16.1% of cotton genome. The present study demonstrated that the polymorphism of IT-ISJ marker is high, and it could be effectively applied in plant genetic map construction.

Utilizing modified ubi1 introns to enhance exogenous gene expression in maize(Zea mays L.) and rice(Oryza sativa L.)

supported by the National Natural Science Fund of China （30970231）;the Genetically Modified Organisms Breeding Major Project of China （2014ZX08003001）

Identification,development,and application of cross-species intron-spanning markers in lentil(Lens culinaris Medik.)

Lentil(Lens culinaris Medik) is one of the most important food legumes in the world. The use in lentil of molecular marker-assisted breeding is limited, owing to the low availability of polymorphic markers. In the present study, we developed a set of polymorphic intron-spanning markers(ISMs) using a cross-species mapping approach. In this approach, putative unique transcripts(PUTs) of L. culinaris were mapped onto the Medicago truncatula genome, exploiting its closeness with the lentil genome. Spliced alignment of the PUTs resulted in a total of 25,717 alignments, allowing the development of 1703 ISMs. From these, a subset of 105 ISMs were synthesized and validated with a 51% amplification success rate in 32 lentil genotypes. Of these ISMs, 40(74%) were polymorphic and generated 2–11 alleles per locus in a genetically diverse panel of 32 lentil genotypes including wild species.This set of polymorphic ISMs along with their functional annotation data will be useful in lentil breeding.

Constructing Competitive Reverse Transcription Polymerize Chain Reaction Inter-Reference of PC mRNA by Intron Approach

Inter-reference of competitive reverse transcription polymerase chain reaction (RT-PCR)was constructed by intron method to detect the change of PC mRNA level in the pathway ofcarbohydrate metabolism. The experiment based on the principle that 81bp intron sequencewas deleted in PC mRNA compared with PC DNA sequence. The 466bp competitive DNA templaterecombinant plasmid of PC mRNA was successfully built by a pair of primer and was clonedonce, PC DNA and PC mRNA could be inter-referred each other. The intron approach used inthe experiment has broken through the traditional method of constructing competitivetemplate.

Investigation on the Interface between Exons and Introns in the Genomic DNA of Arabidopsis thaliana in the Phase Space

In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot products of P vector and three weight vectors were the feature coordinates for the exons and introns in 3-dimensional phase space.The expression for the interface between the exons and the introns in the genomic DNA of Arabidopsis thaliana in 3-dimensional phase space was established,which could be used to distinguish the exons and the introns in the genomic DNA of Arabidopsis thaliana with an accuracy higher than85%in 3-dimensional phase space.

Voltage-Driven Adaptive Spintronic Neuron for Energy-Efficient Neuromorphic Computing

A spintronics neuron device based on voltage-induced strain is proposed.The stochastic switching behavior,which can mimic the firing behavior of neurons,is obtained by using two voltage signals to control the in-plane magnetization of a free layer of magneto-tunneling junction.One voltage signal is used as the input,and the other voltage signal can be used to tune the activation function(Sigmoid-like) of spin neurons.Therefore,this voltage-driven tunable spin neuron does not necessarily use energy-inefficient Oersted fields and spin-polarized current.Moreover,a voltage-control reading operation is presented,which can achieve the transition of activation function from Sigmoid-like to Re LU-like.A three-layer artificial neural network based on the voltage-driven spin neurons is constructed to recognize the handwritten digits from the MNIST dataset.For the MNIST handwritten dataset,the design achieves 97.75% recognition accuracy.The present results indicate that the voltage-driven adaptive spintronic neuron has the potential to realize energy-efficient well-adapted neuromorphic computing.

Increment of hFIX expression with endogenous intron 1 in vitro

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.

Analysis and prediction of exon, intron, intergenic region and splice sites for A. thaliana and C. elegans genomes

Although a great deal of research has been undertaken in the area of the annotation of gene structure, predictive techniques are still not fully developed. In this paper, based on the characteristics of base composition of sequences and conservative of nucleotides at exon/intron splicing site, a least increment of diversity al-gorithm (LIDA) is developed for studying and predicting three kinds of coding exons, introns and intergenic regions. At first, by selecting the 64 trinucleotides composition and 120 position parameters of the four bases as informational parameters, coding exon, intron and intergenic sequence are predicted. The results show that overall predicted accuracies are 91.1% and 88.4%, respectively for A. thaliana and C. ele-gans genome. Subsequently, based on the po-sition frequencies of four kinds of bases in regions near intron/coding exon boundary, initia-tion and termination site of translation, 12 position parameters are selected as diversity source. And three kinds of the coding exons are predicted by use of the LIDA. The predicted successful rates are higher than 80%. These results can be used in sequence annotation.

Novel intron 2 polymorphism in the melanophilin gene is in Hardy-Weinberg equilibrium and is not associated with coat color in goats

Pigmentation plays important adaptation and physiological efficiency roles in animals. In the sequence of a 648 bp fragment representing intron 1, exon 2, and part of intron 2 of the MLPH mammalian pigmentation gene, we identified a novel g.469C> G mutation in intron 2, and genotyped it in 266 Nigerian goats using PCR-RFLP analysis. The C allele had frequencies of 0.9625, 0.9804 and 0.97405 in West African Dwarf (WAD),Sahel(SH) and Red Sokoto (RS) breeds, respectively. The G allele was the highest in WAD (0.0375), followed by RS (0.02595), and then SH (0.0196). Overall low FIS and FST and high Nm values demonstrate little differentiation within and among the goat breeds at this intronic locus. This g.469C> G polymorphism in MLPH gene is the first in any goat breed and also first in Nigerian goats. Our results suggest that this intronic SNP locus is maintained at Hardy-Weinberg equilibrium (P < 0.05) and the lack of association of this SNP with coat color may indicate its neutrality in goats.

Evolutionarily conserved features of the retained intron in alternative transcripts of the <i>nxf1</i>(nuclear export factor) genes in different organisms

One of the features of intron-containing genes of the nxf (nuclear export factor) family in different organisms is the presence of an evolutionarily conserved exon-intron block: exon 110nt-intron-exon 37nt. The intron in this evolutionarily conserved block, which we call a “cassette” intron, can be excised or retained in alternative transcripts of nxf1. It corresponds to intron 10 -11 inthe genes that are orthologous to nxf1 in vertebrates, and intron 5 -6 inthe genes that are orthologous to nxf1 in Drosophilidae. The alignment of sequences of cassette introns in nxf1 genes in vertebrates has revealed four evolutionarily conserved sequences: 1)5’flanking sequence, 2) a region containing СТЕ (constitutive transport element), 3) third conserved sequence, and 4)3’flanking sequence. Introns 5-6 of nxf1 in Drosophilidae have no similar conserved sequences. The results of sequence alignment demonstrate a similarity between cassette introns of nxf1 in Drosophilidae in two poly(A) sequences. The prevalence of Dm nxf1 transcripts containing cassette intron 5-6 under completely spliced transcripts in the heads of adult Drosophila melanogaster suggests a functional importance of transcripts that contain a retained intron. Evolutionary conservation, which in Drosophilidae is evident in the presence of poly(A) sequences in cassette introns of the nxf1 genes, is an adaptive feature: the poly(A) sequences are capable of mimicking the 3’-end of transcripts, promote transport from the nucleus to the cytoplasm, or are involved in NMD control. The ability to form characteristic secondary structures is a common feature of nxf1 cassette introns.

Sequencing of Intron 3 of Porcine Heart Fatty Acid-Binding Protein Gene

[ Objective] The aim of this paper is to provide the basic data for marker-assisted selection of pig breeding using porcine heart fatty acid- binding protein （H-FABP） gene. [Method] According to the related sequences of porcine H-FABP gene released in GenBank, specific primers were designed to amplify the intron 3 of porcine H-FABP gene. [ Result] The intron 3 of porcine H-FABP gene was amplified successfully. Its whole sequence was 1 350 bp in length and had been submitted to GenBank （Accession no. ： DQ 002993）. [Condusion] The study lays a theoretical foundation for determination of the major genes affecting intramuscular fat deposition.

FDA顾委建议批准用Intron A治疗慢性乙肝

一个医学顾问委员会一致建议美国食品药物管理局(FDA)批准Schering Crop.(Madison,NJ)的干扰素-α2a产品Intron A用于治疗慢性乙型肝炎。该药已获准在美国治疗丙型肝炎、卡波氏肉瘤、阴疣及发状细胞白血病。在临床试验中,以乙肝病毒标记的减少量为指标,测出Intron A的有效率为42％。乙型肝炎目前尚未有批准的治疗药物。仅美国就有1百万患者。急性乙肝患者中有5％—10％发展到顽固状态。