兴安落叶松肌动蛋白基因的分离与序列分析

以兴安落叶松(Larix gmelinii)实生苗为材料,提取总RNA和基因组DNA;利用兼并PCR及RACE技术克隆肌动蛋白基因(Lg-act).序列分析的结果显示,该基因cDNA全长1662 bp,编码377个氨基酸;基因组DNA长度约为2564bp,包含4个内含子,5个外显子.所克隆的Lg-act cDNA序列与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性均在77％以上,氨基酸序列的相似性高达93％以上.根据高等植物肌动蛋白相似性构建的系统树显示,兴安落叶松肌动蛋白与挪威云杉肌动蛋白之间的亲缘关系较为密切,在进化中分化时间最为接近.     

甘薯叶绿体rbcL基因的克隆与序列分析

根据烟草、水稻和菠菜叶绿体的全基因组序列设计引物,以甘薯的叶绿体基因组DNA为模板,PCR扩增包舍甘暑叶绿体rbcL完整基因(GenBank登录号为AY942199)在内的一段序列.序列分析表明:此片段的全长为1 627 bp,包括1 443bp的编码区序列在内.推测编码480个氨基酸,同时构建了此片段的限制性酶切图谱.相似性比较显示,此基因编码区序列与烟草、菠菜、小麦、水稻、玉米、矮牵牛、紫花苜蓿、拟南芥、莨菪、葡萄以及甜菜的rbcL基因核苷酸的同源性为85%～98%,氨基酸的同源性为92%～95%.     

甜菜ATP合酶β亚基基因αtpB的克隆、序列分析及进化

αtpB基因编码ATP合酶β亚基,是光合作用中的重要基因。ATP合酶是生物体内能量代谢的关键酶,参与氧化磷酸化和光舍磷酸化反应。利用植物叶绿体基因组在进化过程中高度保守的特点,根据已知植物烟草、水稻和菠菜等的叶绿体基因组全序列,设计并合成了一对引物,以甜菜叶绿体DNA为模板,PCR扩增得到包含αtpB完整基因（GenBank登录号为DQ067451）在内的一段序列,测序与序列分析表明：该克隆片段全长2293bp,其中包括有1497bp的编码区序列,推测编码498个氨基酸。同源性比较,该克隆基因与烟草、菠菜、油菜、水稻αtpB基因的核苷酸序列同源性分别为90.92％、95．79％、87．71％和86．37％,推测的氨基酸序列同源性分别为94．58％、97．19％、92．17％和91．97％。同时,建立了几种植物的氨基酸序列系统进化树。     

甜菜ATP合酶β亚基基因atpB的克隆、序列分析及进化

atpB基因编码ATP合酶β亚基,是光合作用中的重要基因。ATP合酶是生物体内能量代谢的关键酶,参与氧化磷酸化和光合磷酸化反应。利用植物叶绿体基因组在进化过程中高度保守的特点,根据已知植物烟草、水稻和菠菜等的叶绿体基因组全序列,设计并合成了一对引物,以甜菜叶绿体DNA为模板,PCR扩增得到包含atpB完整基因(GenBank登录号为DQ067451)在内的一段序列,测序与序列分析表明:该克隆片段全长2 293 bp,其中包括有1 497 bp的编码区序列,推测编码498个氨基酸。同源性比较,该克隆基因与烟草、菠菜、油菜、水稻atpB基因的核苷酸序列同源性分别为90.92%、95.79%、87.71%和86.37%,推测的氨基酸序列同源性分别为94.58%、97.19%、92.17%和91.97%。同时,建立了几种植物的氨基酸序列系统进化树。     

甘薯质体中的乙酰辅酶A羧化酶亚基基因accD的克隆与序列分析

依据烟草质体全基因组序列设计引物,以甘薯质体基因组DNA为模板,PCR扩增包含质体accD基因完整编码区在内的一段序列（GenBank登录号为GQ395771）。序列分析表明：该片段全长为2209bp,包括1548bp的nccD基因编码序列,推测编码515个氨基酸的蛋白质,该蛋白序列具有异质型β-CT中保守的锌指结构和C末端5个基元。同时绘制了该DNA片段的限制性酶切图谱。相似性比较显示,甘薯accD基因与大豆、马铃薯、拟南芥、人参、莴苣、葡萄、海岛棉、甘蓝、辣椒、菠菜、番茄和烟草的accD基因核苷酸相似性为72％-87％,氨基酸相似性为58％-83％。     

杨树叶绿体3′rps12基因,rps7基因和ndhB基因片段的克隆及序列分析

通过烟草叶绿体相关序列设计引物 ,从杨树的叶绿体基因组中克隆出 2个相邻的DNA片段 .经分析发现 ,这 2个DNA片段包含核糖体蛋白 3′rps12、rps7基因和NADH脱氢酶第二亚基ndhB基因片段 .利用DNAMAN等软件 ,将扩增到的杨树叶绿体DNA片段与烟草、拟南芥、玉米和黑松的相关序列进行比较 ,证实所扩增的片段具有较高的保守性 ,尤其是在这 3个基因的编码区 ,同源性均在 90 %以上 .插入或缺失常发生在基因间隔区 ,同源性在 80 %左右 ;对其编码区所推导的氨基酸序列进行了比较 ,同源性均在 92 %以上 .首次克隆了杨树叶绿体的部分DNA序列 ,详细报道并分析了杨树 3′rps12、rps7基因和ndhB基因片段及其边界序列信息 .所报告的基因序列均已登录GenBank .     

水牛SRY基因的克隆及序列分析

根据荷斯坦牛SRY基因设计一对引物,采用聚合酶链式反应（PCR）技术,以中国沼泽型水牛（Swamp Buffalo）基因组DNA为模板,扩增得到SRY（Sex Deterimation region of Y chromosome）全序列约2005bp,其中1-504bp为5’启动子区,1196-2005bp为3’侧翼序列,在505-1195bp为SRY的外显子,编码229个氨基酸。在SRY HMG box区域设计探针,用地高辛标记后分别与雄性、雌性水牛基因组DNA进行Southern 杂交,结果显示该段序列只在雄性DNA样本中有杂交信号,证明SRY基因为雄性特异。BLAST比对结果显示与牛属动物SRY基因的同源性为96％,其中SRY基因HMG box区域同源性高达99%,说明SRY基因具有高度的进化保守性     

甜菜ATP合酶β亚基基因atpB的克隆、序列分析及进化

atpB基因编码ATP合酶β亚基,是光合作用中的重要基因。ATP合酶是生物体内能量代谢的关键酶,参与氧化磷酸化和光合磷酸化反应。利用植物叶绿体基因组在进化过程中高度保守的特点,根据已知植物烟草、水稻和菠菜等的叶绿体基因组全序列,设计并合成了一对引物,以甜菜叶绿体DNA为模板,PCR扩增得到包含atpB 完整基因（GenBank登录号为 DQ067451）在内的一段序列,测序与序列分析表明：该克隆片段全长2 293 bp,其中包括有1 497 bp的编码区序列,推测编码498个氨基酸。同源性比较,该克隆基因与烟草、菠菜、油菜、水稻atpB基因的核苷酸序列同源性分别为90.92%、95.79%、87.71%和86.37%,推测的氨基酸序列同源性分别为94.58%、97.19%、92.17%和91.97%。同时,建立了几种植物的氨基酸序列系统进化树。     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT—PCR和PCR—RACE技术,从菊科植物甘菊（Dendranthema lavandulifolium）中克隆到2个甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase,BADH）基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97％,推导的氨基酸序列的同源性为98％。与已发表的其它植物BADH基因氨基酸序列的同源性在64％以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽（VTLELGGKSP）以及与酶功能有关的半胱氨酸残基（C）。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT—PCR—Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

湖北海棠MhPR1a基因的克隆与表达特性分析

以水杨酸诱导的湖北海棠[ Malus hupehensis (Pamp.) Rehd.]全长cDNA文库和基因组DNA为模板,克隆其PR1a基因(MhPR1a)的全编码区序列,并对该序列进行生物信息学分析;在此基础上利用荧光定量RT-PCR技术对湖北海棠根、茎和叶中该基因的表达特性及经过10μmol·L-1ABA、4℃低温处理及苹果蚜虫(Aphis citricola van der Goot)侵染后叶中该基因的表达特性进行了测定.结果表明:克隆获得的MhPR1a基因全长518 bp,最大开放阅读框为492 bp,编码162个氨基酸残基;编码的蛋白质为酸性蛋白,其相对分子质量为16 960,等电点pI 5.46;其基因组DNA序列与cDNA序列完全一致,说明MhPR1a基因内部没有内含子.湖北海棠MhPR1a基因与苹果(M.domestic Borkh.)和沙梨[Pyrus pyrifolia( Burm.f.)Nakai] PR1基因的cDNA序列及其编码的氨基酸序列同源性均较高,其中cDNA序列的同源性均为97％,氨基酸序列的同源性分别为95％和97％;系统树也显示MhPR1a基因编码的氨基酸序列与苹果和沙梨的亲缘关系最近,聚为一类.MhPR1a基因编码的氨基酸序列具有SCP保守结构域,含有1个信号肽和6个保守的半胱氨酸残基.在湖北海棠的叶、茎和根中MhPR1a基因均能表达,在根中的表达量最高.10 μmol·L-1ABA和4℃低温处理48 h后均可诱导MhPR1a基因的表达,且相对表达量明显高于对照(处理0h);苹果蚜虫也可诱导MhPR1a基因的表达,说明MhPR1a基因在湖北海棠抵抗植食昆虫和低温胁迫的过程中可能发挥着重要作用.     

An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation.

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.     

青藏高原东南部山丹nrDNA ITS和cpDNA petB/petD 序列的分子进化特点

采用改良CTAB法提取青藏高原东南部山丹25个居群所有个体的基因组DNA,选取核基因ITS和叶绿体petB/petD区域进行PCR扩增、纯化和测序。对所有序列对位排列,其中ITS序列总长696bp,变异位点有4处,共产生7种单倍型,变异位点百分率为0.72%,（G+C）含量60.4%;petB/petD序列总长616bp,仅1处变异位点和2种单倍型,变异位点百分率为0.16%,（G+C）含量34.6%。表明山丹中,petB/petD区域较ITS序列保守,变异速率较慢。对ITS序列单倍型进行失配分布和中性检验分析发现,山丹现有分布范围可能经历了近期居群小范围扩张,AMOVA分析发现山丹居群的遗传变异主要存在于居群内,N_ST >G_ST（P>0.01）,表明山丹的遗传变异有着不显著的谱系地理结构。因此,山丹ITS序列适合该种的谱系地理学研究。     

Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii

We have mapped and sequenced the petA (cytf), petB (cytb6) and petD (subunit IV) genes on the chloroplast genome of Chlamydomonas reinhardtii. At variance with the pet genes in higher plant chloroplasts, the petB and petD genes are continuous, not adjacent and not located next to the psbB gene. The corresponding polypeptide sequences are highly conserved when compared with their counterparts from other sources but have a few features specific of algal cytb6/f complexes. In particular the transit sequence of cytf displays unique characteristics when compared with those previously described for cytf in higher plants.     

5'' to 3'' exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways

Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5' and 3' untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5'-->3' and 3'-->5' exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions -20, +25, +165 or +25/+165 relative to the mature petD 5' end, mRNAs accumulate with 5' ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5' end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5'-->3' exoribonucleolytic activity. In the case of the -20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5' end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5' ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) -20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.     

Localization of the genes coding for the 51 kDa PSII chlorophyll apoprotein,apocytochrome b6, the 65–70 kDa PSI chlorophyll apoproteins and the 44 kDa PSII chlorophyll apoprotein in pea chloroplast DNA

Summary DNA probes isolated from previously mapped spinach genes were used to locate 5 genes on pea ctDNA by heterologous hybridization. The genes mapped include psbC, psaA, psaB, psbB, and petB. PsbB and petB mapped to a 6.7 kbp XbaI DNA fragment adjacent to the petD gene. Northern probes from within the DNA which codes for psbB and petD hybridized to 6 RNAs ranging from 1.2 to 5.6 kbp. The psaA and psaB genes, which code for 65–70 kDa proteins of Photosystem I, were mapped to a 7.5 kbp. XbaI DNA fragment. A 5.8 kbp RNA is transcribed from the region which contains the psaA and psaB genes suggesting that these genes are co-transcribed. Finally the psbC gene which codes for a 44 kDa chlorophyll-protein of Photosystem II was mapped to a 12.3 kbp PstI DNA fragment. The pea psbC open reading frame overlaps the psbD coding sequence and this gene pair is within 3 kbp of the psaA-psaB genes. Overall, the organization of the 3 gene clusters analyzed in peas is similar to that reported for spinach.