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Verticillium wilt is a severe disease in eggplant caused by Verticillium dahliae.Polygalacturonase-inhibiting proteins （PGIPs） have been shown to be involved in preventing the invasion of fungus including V.dahliae.Cloning genes encoding PGIPs is quite valuable for plant resistance breeding to Verticillium wilt.In this study,a cDNA encoding the polygalacturonase-inhibiting protein was isolated from Solanum torvum by RT-PCR and RACE,designated StPGIP （accession no.FJ943498）.The cDNA sequence of StPGIP was 1 097 bp long and contained an open reading frame of 990 bp.The predicted amino acid sequence of the gene consisted of 329 amino acids and had conserved LRRs.The StPGIP protein had a high identity with PGIPs from other species.Analysis of StPGIP expression at the mRNA level by RT-PCR showed that the gene was expressed in all organs and could be induced to increase expression by V.erticillium dahliae infection. 相似文献
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A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds. 相似文献
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Conserved domain such as nucleotide binding site （NBS） was found in several cloned plant disease resistance genes. Based on the NBS domain, resistance gene analogues （RGAs） have been isolated. A full-length cDNA, SPR1 was obtained by rapid amplification of cDNA ends （RACE） method. Sequence analysis indicated that the length of SPR1 was 3 066 bp, including a complete open reading frame of 2 667 bp encoding SPR1 protein of 888 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has a typical structure of nonT1R-NBS-LRR genes, with NB-ARC, CC, and LRR domains. The SPR1-related sequences belonged to multicopy gene family in sweetpotato genome according to the result of Southern blotting. Semi-quantitative RT-PCR analysis showed SPR1 expressed in all tested tissues. The cloning of putative resistance gene from sweetpotato provides a basis for studying the structure and function of sweetpotato disease-resistance relating genes and disease resistant genetic breeding in sweetpotato. The gene has been submitted to the GenBank database, and the accession number is EF428453. 相似文献
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野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析(英文) 总被引：7，自引：3，他引：4
[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion. 相似文献
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CHEN Bo WANG Tai-xia WANG Han-zhong LI Yun-chang YAN Xiao-hong WANG Li-jun WEIWen-hui 《中国农业科学(英文版)》2010,9(4):488-496
AINTEGUMENTA （ANT） gene has been proved to have a novel function on controlling the organ size and the seed weight in Arabidopsis thaliana, the research on its orthologous gene in Brassica napus will be of potential interest to elucidate the molecular mechanism of rapeseed development and increase the rapeseed output. A SMART cDNA library of the flower bud from B. napus cultivar Zhongshuang 9 was constructed, and the full-length cDNAs of two homologous BnaANT candidate genes were cloned by PCR in B. napus, namely BnaX.ANT.a （GenBank accession no. DQ211969） and BnaX.ANT.b （accession no. DQ211970）. They shared 87.1 and 83.4% amino acid identity with ANT of A. thaliana, respectively. There is 87.4% amino acid identity between BnaX.ANT.a and BnaX.ANT.b. By quantitative real-time PCR, the obviously differential expression levels of the two BnaANT candidate genes were detected in the 28 DAF seeds of the big and small grains B. napus lines, the expression abundance of BnaANT in the 28 DAF seed of 9311 was over three times compared to that of 9260, which indicates that these two BnaANT genes are also likely related to the floral organ size and the seed weight in B. napus. 相似文献
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黄瓜细胞质型谷氨酰胺合成酶基因(GS1)的克隆及其在低氮条件下的表达 总被引：1，自引：1，他引：0
【目的】分离和克隆黄瓜谷氨酰胺合成酶GS1基因,分析其序列特征,了解其在低氮条件下的表达情况。【方法】依据黄瓜基因组数据库中Csa015274基因编码区全序列,应用引物设计软件Primer Premier 5.0设计引物,从黄瓜叶片cDNA中克隆该基因,用生物信息学方法对获得的cDNA序列及推定氨基酸序列进行分析鉴定,并用实时荧光定量PCR法研究GS1基因在不同氮素浓度下的表达变化。【结果】分离到GS1基因,GenBank登录号为JQ277263。该基因长1 071 bp,编码356个氨基酸,与甜瓜(Cucumis melo L.)GS1基因同源性高达97%。该基因编码的蛋白是1个不稳定的疏水蛋白,无跨膜结构,无信号肽,存在蛋白激酶C磷酸化位点,酪蛋白激酶Ⅱ磷酸化位点,N-十四酰化位点,酪氨酸激酶磷酸化位点等活性位点。GS1基因表达模式分析显示,在低氮条件下,该基因下调表达,随着氮素浓度的增高GS1基因的表达量增加。在高浓度的氮素水平下,该基因的表达同样受到抑制。【结论】成功从黄瓜叶片中分离克隆到GS1基因,该基因具有已知物种GS1基因的特征,可用于该基因的功能研究。 相似文献
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大白菜果胶甲酯酶基因BrPME1的克隆及特征分析 总被引：4，自引：0，他引：4
【目的】克隆大白菜果胶甲酯酶基因,为进一步探讨果胶代谢在大白菜育性调控中的分子机制提供帮助。【方法】利用cDNA-AFLP技术分析大白菜核雄性不育两用系‘AB02’可育株(msms)和不育株(Msms)花蕾的基因表达谱,在可育株混合花蕾cDNA中扩增出1条特异条带TDF-24,通过RACE技术扩增该基因的cDNA全长序列,采用生物信息学软件分析所克隆基因的编码蛋白特性,利用荧光定量PCR技术分析基因时空表达模式。【结果】该基因编码大白菜果胶甲酯酶(EC 3.1.1.11),被命名为BrPME1(GenBank登录号：HM185497)。BrPME1全长cDNA序列为1 290 bp,编码1个包含363个氨基酸的前体蛋白。BrPME1蛋白N末端1—23位为信号肽,成熟蛋白含有保守的果胶甲酯酶酶活结构域,而不含酶活抑制位点。预测BrPME1蛋白含有10个磷酸化位点、1个酰胺化位点和6个N端豆蔻酰基化位点。BrPME1在两用系不育株花蕾中表达量很低,在可育株的大花蕾以及成熟花药中高水平表达。【结论】BrPME1是一个受大白菜细胞核雄性不育基因抑制的果胶甲酯酶基因家族成员。 相似文献
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该研究运用RT-PCR技术,首次从大熊猫Ailuropoda melanoleuca的肌肉组织总RNA中成功克隆了AP2S1基因的编码序列,并对其进行了全面分析.结果表明:大熊猫AP2S1基因编码序列克隆片段全长为512bp,开放阅读框(ORF)为429bp,编码142个氨基酸的蛋白质,该蛋白的相对分子质量为1.7×104,等电点pI为5.82,含有4种类型共5个功能位点,即1个蛋白激酶C磷酸化位点、2个酪蛋白激酶Ⅱ磷酸化位点、1个N-酰基化位点和1个网格蛋白调节素小肽链标签;且该蛋白在哺乳动物网状细胞中的半寿期(half-life)大于30h,不稳定指数(in-stability index)为26.99,属于稳定蛋白.进一步分析发现,大熊猫AP2S1基因与已报道的人、苏门答腊猩猩、牛、褐家鼠和小家鼠5个哺乳动物物种的编码序列及其编码的氨基酸序列具有很高的相似性,表明该基因及其编码蛋白在物种漫长的进化过程中极其保守. 相似文献
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[目的]分离和鉴定扩展莫尼茨绦虫(Moniezia expansa)电压门控钙通道β亚基(cavB)基因,预测蛋白二级结构和B细胞抗原表位,从而探讨Cavβ在寄生虫病治疗中所起的作用.[方法]构建扩展莫尼茨绦虫成虫cDNA文库,随机挑取重组阳性克隆进行测序,对部分序列进行引物步移法测序,获取全长cDNA序列;采用生物信息学方法对其对应的蛋白进行二级结构和B细胞抗原表位的预测.[结果]获得扩展莫尼茨绦虫Cavβ基因,全长946 bp,编码180个氨基酸,理论分子质量为19.788 8 kD,等电点为5.06,属于Ca_channel_B超家族.二级结构以无规则卷曲为主,结构预测存在4个可能的B细胞抗原表位.[结论]研究对于扩展莫尼茨绦虫Cavβ基因的获得和B细胞抗原表位的预测,为该基因功能的试验性鉴定工作奠定了基础. 相似文献
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COP1 E3连接酶是一个光形态建成的抑制子和光调控植物发育的分子开关。对山核桃Carya cathayensis花芽454测序获得CcCOP1 E3连接酶的片段, 通过cDNA末端快速扩增技术(RACE), 分别获得该基因的全长, 大小为2 331 bp, 它由2个特殊的结构域组成即环形锌指结合域和WD-40重复序列, 其编码的蛋白质有较强的亲水性, 在氨基端主要是亲水性氨基酸, 而羧基端主要是疏水性氨基酸。CcCOP1 E3连接酶与毛果杨Populus trichocarpa等的COP1 E3连接酶同源基因相似度较高, 总体高达77.80%。实时荧光定量聚合酶链式反应(real-timePCR)结果显示:CcCOP1 E3连接酶的表达贯穿于在山核桃雌雄花的发育过程, CcCOP1 E3连接酶在山核桃的茎、叶、果实、花芽中均有表达, 但在花芽中表达量最高, 3月中旬表达量最高, 在5月中旬雄花表达量相对较高。CcCOP1 E3连接酶与山核桃雌雄花分化有关。 相似文献
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COP1 E3连接酶是一个光形态建成的抑制子和光调控植物发育的分子开关.对山核桃Carya cathayensis花芽454测序获得CcCOP1 E3连接酶的片段,通过cDNA末端快速扩增技术（RACE）,分别获得该基因的全长,大小为2 331 bp,它由2个特殊的结构域组成即环形锌指结合域和WD-40重复序列,其编码的蛋白质有较强的亲水性,在氨基端主要是亲水性氨基酸,而羧基端主要是疏水性氨基酸.CeCOP1 E3连接酶与毛果杨Populus trichocarpa等的COP1 E3连接酶同源基因相似度较高,总体高达77.80％.实时荧光定量聚合酶链式反应（real-timePCR）结果显示：CcCOP1 E3连接酶的表达贯穿于在山核桃雌雄花的发育过程,CcCOP1 E3连接酶在山核桃的茎、叶、果实、花芽中均有表达,但在花芽中表达量最高,3月中旬表达量最高,在5月中旬雄花表达量相对较高.CcCOP1 E3连接酶与山核桃雌雄花分化有关. 相似文献
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利用RACE方法,在山核桃嫁接过程中扩增出山核桃水通道蛋白同源基因CcPIP。应用生物信息学软件进行分析,预测该序列编码294个氨基酸,具有6个跨膜区,有MIP家族信号序列和高等植物PIP高度保守序列。通过NCBI同源性比较分析表明,该基因与葡萄、菜豆等物种的水通道蛋白基因同源性达到99%。荧光定量RT-PCR分析表明,CcPIP基因在芽中的表达量最低,其次是雄花序,果实,幼叶和茎,而在根中表达量最高。在山核桃嫁接前后,CcPIP基因在接穗中表达趋势和在砧木中的表达一致。CcPIP基因的表达量在山核桃嫁接前砧木和接穗中都有强烈表达,但在嫁接后3 d急剧下降,分别在砧木和接穗中下降了5倍和2倍。在随后的11 d里,CcPIP基因的表达量在砧木和接穗中开始上升,至嫁接后14 d基因的转录水平比嫁接后3 d分别增加了7倍和4倍。该CcPIP基因参与山核桃嫁接成活的水分运输过程中起到基因表达调控的作用。 相似文献
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百子莲CONSTANS同源基因的克隆及表达分析 总被引：1，自引：1，他引：0
根据前期百子莲转录组测序分析的结果,获得了1 个与光周期调控开花途径关键基因CONSTANS(CO)同源
性较高的核心片段。采用cDNA 末端快速扩增(RACE) 方法得到了百子莲CO 基因cDNA 全长序列,命名为
ApCOL,GenBank 登录号为KF683287。序列分析表明,百子莲ApCOL 基因cDNA 全长1 648 bp,5非编码区(5UTR)
和3非编码区(3UTR)分别为126 和355 bp,开放阅读框(ORF,127 ~ 1 293 bp)1 167 bp,编码388 个氨基酸。
ApCOL 具有CO 蛋白典型的结构域:氨基末端有2 个B-box 结构域,羧基末端有1 个CCT 保守结构域。氨基酸同源
性比对发现,ApCOL 与葡萄(XP_002274384.2)、可可(EOX99181.1)和大豆(NP_001241023.1)等植物的CO 蛋白有
很高的相似性,同源性均在50%以上。系统进化树分析表明,ApCOL 与小麦CO(EMS51329.1)聚类关系最近。实
时荧光定量qRT-PCR 结果表明:叶片中ApCOL 的表达量在花芽分化3 个时期均高于花芽中的表达量,且叶片中的
最高表达量和花芽中最低表达量均出现在花芽诱导期;在整个初花期内,各器官中ApCOL 表达量由高到低依次是
花梗、叶片、幼果、子房、花瓣、茎和花葶,花梗中表达量分别为叶片和茎中的2.68 和114.3 倍;不同光周期处理的叶
片中ApCOL 基因表达模式相近,均在暗处理时期呈现高表达。说明该基因的表达具有明显的时空差异性和生物钟
调节特性,推测ApCOL 在百子莲成花过程中以及花发育过程中发挥着一定的作用。 相似文献
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花分生组织特征基因LEAFY在植物花芽分化与成花诱导途径中起着重要的调控作用。通过RACE方法从香草兰中克隆Vp LFY基因的全长c DNA序列。结果显示,该基因包含一个1 493 bp的开放阅读框,编码491个氨基酸残基。经蛋白质序列同源比对分析结果发现,Vp LFY属于FLO-LFY家族。组织特异性研究结果表明,Vp LFY基因在香草兰组织中属于组成型表达。荧光定量分析结果表明,Vp LFY基因分别在香草兰纯花芽和混合花芽的不同发育阶段中有着较强的表达。 相似文献
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[目的]克隆茶树甲羟戊酸激酶基因(CsMVK),分析其生物学信息及在不同组织及乌龙茶做青过程中的表达特性,为研究茶叶加工过程中香气形成机制提供参考.[方法]RT-PCR扩增CsMVK基因,应用生物信息学软件对其编码蛋白(CsMVK)的氨基酸序列进行预测分析,通过实时荧光定量PCR(qPCR)分析CsMVK在茶树不同组织及乌龙茶做青过程中的表达特性.[结果]克隆获得的CsMVK基因(GenBank登录号MF668187)全长1455 bp,开放阅读框(ORF)长度为1164 bp,编码387个氨基酸,蛋白质分子量41.18 kD,理论等电点(pI)5.88.CsMVK蛋白具有GHMP_kinases_N domain和GHMP_kinases_C domain 2个功能结构域,定位于细胞质中,不存在跨膜结构和信号肽.系统发育进化树分析结果显示,CsMVK与三七、常春藤的MVK聚为一类,表明茶树与三七、常春藤的亲缘关系最近.qPCR检测结果显示,CsMVK基因在果实中的表达量显著高于其他组织(P<0.05,下同),在不同组织中的表达量排序为:果实>根>叶>茎>花;在乌龙茶做青过程中,从鲜叶到晒青叶,CsMVK基因的表达量上升,晒青到一摇过程中表达量下降,经过3次摇青,其表达量持续升高并在杀青前达最大值,与其他做青阶段差异显著.[结论]CsMVK基因表达与茶叶香气品质形成有关. 相似文献