利用交错延伸剪接PCR技术扩增油菜花粉育性基因MS2Bnap全长cDNA序列

交错延伸剪接PCR是一种用于分子进化研究的DNA改组技术。本实验利用其原理,将已获得的油菜花粉育性MS2Bnap有部分序列重叠的三段PCR产物作为模板进行扩增,获得了花粉育性基因MS2Bnap的全长cDNA序列。     

采用RACE技术获得全长人新基因MAGE-D1

 c DNA末端快速扩增 (RACE)技术是快速获得新基因 5′和 3′端未知序列的有效手段 .鉴于新报导的人肿瘤基因 MAGE家族成员之一 MAGE- D1的 c DNA序列不完全 ,从而拟用 RACE技术获得 MAGE- D1的全长 c DNA序列 .结果显示 ,MAGE- D1的 c DNA全长为 2 80 0个碱基 ,编码778个氨基酸 .同时对 RACE技术应用时的一些关键问题进行了讨论 .     

甘蓝型油菜BnCOP1基因编码区全长cDNA的克隆与功能研究

通过分析拟南芥、豌豆、番茄和水稻的COP1 (constitutively photomorphogenic 1) 的cDNA序列, 运用RT-PCR和改进的基因组步行 (genome walking) 技术相结合的方法, 首次从甘蓝型油菜中克隆到油菜 BnCOP1编码区cDNA的全长序列, 其全长2 034 bp, 编码677个氨基酸. 同源 性分析表明, 其编码的氨基酸序列与拟南芥的同源性高达94%. 对BnCOP1编 码序列（cDNA）演绎出的氨基酸序列分析表明, 其编码的蛋白包含有N端的 环形锌指结合域(ring finger zinc binding domain, RING)、中间的卷曲 螺旋形结构域(coiled-coil domain, coiled-coil ), 7个C端的WD-40重复 序列(WD-40 repeats, WD-40)的功能域. 半定量RT-PCR和实时荧光定量PCR 分析该基因在油菜中的表达模式,结果显示, BnCOP1在甘蓝型油菜的各个组 织器官中均有表达,其中在花中的表达明显高于在根、叶、茎、果荚及子叶 和胚轴中,暗示该蛋白可能与开花途径相关. 过表达BnCOP1的转基因拟南芥 植株在高度、主茎的直径和叶片大小上都呈现出比野生型弱小的表型, 表 明BnCOP1抑制了拟南芥光形态建成从而影响了植物的生长发育.     

Analysis of MS2Bnap genomic DNA homologous to MS2 gene from Arabidopsis thaliana in two dominant digenic male sterile accessions of oilseed rape (Brassica napus L.)

PCR technique was employed to isolate gene homologous to the MS2Bnap (X99922.1) from two rapeseed (Brassica napus L.) dominant digenic male sterile lines, namely 220A (male sterile) and 220B (male fertile), 6A (male sterile) and 6C (male fertile). The isolated 2,581 bp sequences from 220A (named 220A-gDNA, GenBank accession number AY288778), 220B (220B-gDNA, AY257490), 6A (6A-gDNA, DQ060318) and 6C (6C-gDNA, DQ060319) all contained six introns. Forty-one single nucleotide polymorphism (SNP) sites were detected by alignment of these four sequences, seven of them dispersed in the exon regions. Two SNPs (1247, 1656) were detected between 220A-gDNA and 220B-gDNA, and the one at nucleotide 1247 of 220A-gDNA with A replaced by C was a missense mutation, which may be the putative male sterility site in 220A. All eight SNPs identified between 6A-gDNA and 6C-gDNA were located in the third intron, so the proteins encoded by them are the same. The one SNP between 6A-/6C-gDNA and 220A-/220B-gDNA at nucleotide 2474 of 220A-/220B-gDNA with C replaced by G was a missense mutation. Mutation site of BNMS2PROT (CAA68190.1) encoded by MS2Bnap in 220A(254) and 6A/6C(584) is different, which indicated dominant digenic male sterile line 220AB and 6CA have some difference in the molecular level. Comparison of structure of MS2Bnap in B. napus with that of MS2 in Arabidopsis thaliana revealed that the similarity of exons between these two genes is higher than that of introns.     

Sequence of an oleocin cDNA from Brassica napus

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 pb and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3 untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.     

油菜叶绿体基因ndhB的序列

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野生茄子（Solanum torvum）抗黄萎病相关基因Sto Vel的克隆与分析

采用RT—PCR和RAcE技术从野生茄子中扩增克隆到一个抗黄萎病相关基因,命名为StoVel,其cDNA全长3400bp,含有3153bp的完整开放阅读框,编码1051个氨基酸,该基因编码的蛋白序列与刚果野茄、类番茄和番茄Vel编码的氨基酸序列同源性分别为82％、81％和80％,且有很高的功能区段保守性。将该cDNA全长序列提交Gen Bank,登陆号为DQ020574。半定量PCR表明该基因为组成型表达,在根中表达最多,叶中最少。