为了探究转录调节因子LIM同源盒蛋白8基因(LIM homebox protein 8, LHX8)内含子5多态性与松辽黑猪繁殖性状的关联性,试验选取120头经产松辽黑猪母猪为研究对象,采用Sanger直接测序法检测LHX8基因第5内含子的单核苷酸多态性(SNP)位点,使...为了探究转录调节因子LIM同源盒蛋白8基因(LIM homebox protein 8, LHX8)内含子5多态性与松辽黑猪繁殖性状的关联性,试验选取120头经产松辽黑猪母猪为研究对象,采用Sanger直接测序法检测LHX8基因第5内含子的单核苷酸多态性(SNP)位点,使用SPSS 22.0分析LHX8基因内含子5的SNP与松辽黑猪繁殖性状的关联性。结果表明,松辽黑猪LHX8基因内含子5存在T/C突变;共检测到TT、TC和CC共3种基因型,T、C两种等位基因,优势等位基因型为CC,优势等位基因为C。χ^(2)适合性检验表明,该SNP在松辽黑猪群体中处于Hardy-Weinberg平衡状态,为中度多态(0.25<PIC<0.5)。关联分析结果表明,CC基因型个体断奶仔猪数显著多于TC基因型(P<0.05)。TC基因型个体初生重极显著高于TT基因型与CC基因型(P<0.01),CC基因型显著高于TT基因型(P<0.05)。说明松辽黑猪LHX8基因第5内含子T30C是潜在的与繁殖性状相关的遗传位点。但该突变位点能否作为松辽黑猪繁殖性状的遗传标记,需要进行更大规模的群体验证。展开更多
Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes a...Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition.3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.展开更多
Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned ...Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals.展开更多
目的研究肺表面活性蛋白B（surfactant protein B，SP-B）内含子5基因多态性与新生儿支气管肺发育不良（bronchopulmonary dysplasia，BPD）的相关性，从基因水平探讨BPD的发病机制。方法收集2016年11月至2017年11月在内蒙古医科大学附...目的研究肺表面活性蛋白B（surfactant protein B，SP-B）内含子5基因多态性与新生儿支气管肺发育不良（bronchopulmonary dysplasia，BPD）的相关性，从基因水平探讨BPD的发病机制。方法收集2016年11月至2017年11月在内蒙古医科大学附属医院NICU住院治疗的BPD患儿为BPD组（n＝50），其中蒙古族13例，汉族37例，选择同时期内、同群体中未发生BPD的50例新生儿为对照组，其中蒙古族15例，汉族35例。利用PCR技术检测SP-B基因内含子5多态性及其基因型、等位基因分布。结果不论蒙古族或汉族，SP-B内含子5基因型均可检出3种基因型：即野生型、插入型及缺失型。汉族BPD组此3种基因型频率分别为73.0%（27/37）、10.8%（4/37）和16.2%（6/37），等位基因频率分别为77.0%（57/74）、9.5%（7/74）和13.5%（10/74）；对照组上述3种基因型频率分别为82.9%（29/35）、11.4%（4/35）和5.7%（2/35），等位基因频率分别为85.7%（60/70）、8.6%（6/70）和5.7%（4/70）。蒙古族BPD组上述3种基因型频率分别为53.8%（7/13）、15.4%（2/13）和30.8%（4/13），等位基因频率分别为61.5%（16/26）、15.4%（4/26）和23.1%（6/26）；对照组上述3种基因型频率分别为53.3%（8/15）、26.7%（4/15）和20.0%（3/15），等位基因频率分别为66.7%（20/30）、20.0%（6/30）和13.3%（4/30）。汉族BPD组与汉族对照组、蒙古族BPD组与蒙古族对照组相比较，SP-B内含子5等位基因及基因型频率差异均无统计学意义（P均〉0.05）。结论未发现SP-B内含子5多态性与内蒙古地区汉族及蒙古族新生儿BPD的发生有明显相关性。展开更多
Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regu...Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.展开更多
基金Supported by the National Natural Science Foundation of China(31470107)the National Key Basic Research Program of China(2014CB138302)the China Agriculture Research System(CARS24)
文摘Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition.3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.
文摘Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals.
文摘目的研究肺表面活性蛋白B（surfactant protein B，SP-B）内含子5基因多态性与新生儿支气管肺发育不良（bronchopulmonary dysplasia，BPD）的相关性，从基因水平探讨BPD的发病机制。方法收集2016年11月至2017年11月在内蒙古医科大学附属医院NICU住院治疗的BPD患儿为BPD组（n＝50），其中蒙古族13例，汉族37例，选择同时期内、同群体中未发生BPD的50例新生儿为对照组，其中蒙古族15例，汉族35例。利用PCR技术检测SP-B基因内含子5多态性及其基因型、等位基因分布。结果不论蒙古族或汉族，SP-B内含子5基因型均可检出3种基因型：即野生型、插入型及缺失型。汉族BPD组此3种基因型频率分别为73.0%（27/37）、10.8%（4/37）和16.2%（6/37），等位基因频率分别为77.0%（57/74）、9.5%（7/74）和13.5%（10/74）；对照组上述3种基因型频率分别为82.9%（29/35）、11.4%（4/35）和5.7%（2/35），等位基因频率分别为85.7%（60/70）、8.6%（6/70）和5.7%（4/70）。蒙古族BPD组上述3种基因型频率分别为53.8%（7/13）、15.4%（2/13）和30.8%（4/13），等位基因频率分别为61.5%（16/26）、15.4%（4/26）和23.1%（6/26）；对照组上述3种基因型频率分别为53.3%（8/15）、26.7%（4/15）和20.0%（3/15），等位基因频率分别为66.7%（20/30）、20.0%（6/30）和13.3%（4/30）。汉族BPD组与汉族对照组、蒙古族BPD组与蒙古族对照组相比较，SP-B内含子5等位基因及基因型频率差异均无统计学意义（P均〉0.05）。结论未发现SP-B内含子5多态性与内蒙古地区汉族及蒙古族新生儿BPD的发生有明显相关性。
基金supported by grants from the National Key Research and Development Program of China (2016YFD0101804)the National Natural Science Foundation of China (31788103 and 31420103912)as well as the Chinese Academy of Sciences (QYZDY-SSWSMC030 and GJHZ1602)
文摘Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.
