多枝赖草DNA导入小麦引起重要农艺性状变化及相应的分子证据

多枝赖草DNA导入小麦引起重要农艺性状变化及相应的分子证据@李维琪$中国科学院新疆化学研究所!乌鲁木齐830000小麦;;多枝赖草DNA;;农艺性状     

星星草脱氢抗坏血酸还原酶（PtDHAR）cDNA的克隆及表达分析

脱氢抗坏血酸还原酶是抗坏血酸代谢循环中的关键酶,在多种植物中与抗胁迫相关。为了获得抗盐碱植物星星草中该基因序列,利用RACE技术,从星星草中克隆出脱氢抗坏血酸还原酶基因（PtDHAR）的cDNA全长序列,其GenBank登录号为HM125046。PtDHAR cDNA核苷酸序列长度为987bp,开放阅读框为639bp,编码213个氨基酸。该基因编码的氨基酸序列与水稻、小麦等禾本科作物具有很高的同源性。Northern杂交分析表明,该基因在盐碱胁迫下表达量显著升高。     

多枝赖草基因组可转化人工染色体（TAC）文库的构建和鉴定

为了克隆和转化多枝赖草（Leymus multicaulis）耐黄矮病、耐蚜虫、耐干旱、耐盐碱等抗逆性基因,利用脉冲电泳分离纯化2Mb以上核DNA,酶切后回收10～200kbDNA片段按大小分5组与TAC载体连接后电转化导入细菌DH10B,在卡那霉素和蔗糖选择压下均有阳性克隆．用两种TAC载体pYLTAC17和pYLTAC747H／sacB分别构建了文库I和II,约16．5和23．6万个克隆,估计覆盖3～5倍多枝赖草基因组大小．文库以混合克隆形式保存在12×2块深孔96孔板中,每孔1．2mL菌液中含300～600个克隆,40多万个克隆转存到3块384孔板,留24个孔存叶绿体和线粒体DNA探针菌液,可用于后续文库鉴定．此外,从文库I和II分别挑取2501和2890个单克隆存于14块384孔板中．17块384孔板都用GeneTAC^TMG3复制了2份,点高密度杂交膜6张,以多枝赖草谷胱甘肽还原酶基因5＇RACE,GR和3＇RACE＋GR为探针初步筛选到19个阳性克隆,两文库为多枝赖草抗性基因的克隆、物理图谱的构建、功能验证等基因组有关研究奠定了基础。     

小麦—大赖草易位系的RFLP分析

利用辐射、花药培养及杀配子基因效应已创制出一系列小麦-大赖草易位系.为在其中找出可能的纯合易位系、明确易位所涉及的相关染色体以及易位断裂点的确切位置,利用了已被定位于小麦7个部分同源群染色体长、短两臂上的67个探针进行了RFLP分析,结果鉴定出3个纯合的易位系:T1BL*7Lr#1S、T4BS*4BL-7Lr#1S和T6AL*7Lr#1S.其中,易位系T1BL*7Lr#1S和T6AL*7Lr#1S中染色体7Lr#1的断裂点位于标记MWG808和标记ABG476.1之间,而1B和6A染色体上的断裂点都在着丝粒附近.易位系T4BS*4BL-7Lr#1S中染色体7L#1的断裂点位于标记BCD349和标记CDO595之间,4B染色体断裂点则位于标记CDO541和标记PSR164之间的长臂上.     

赖草属5个种的核型与进化

报道了国产赖草属５个种的核型,即大赖草,２ｎ＝４ｘ＝２８＝２４ｍ（２ＳＡＴ）＋４ｓｍ（２ＳＡＴ）;粗穗赖草２ｎ＝４ｘ＝２８＝２２ｍ（２ＳＡＴ）＋４ｓｍ＋２ｓｔ（２ＳＡＴ）;若羌赖草,２ｎ＝４ｘ＝２８＝２０ｍ（４ＳＡＴ）＋６ｓｍ＋２ｓｔ（２ＳＡＴ）;羊草,２ｎ＝４ｘ＝２８＝２２ｍ（４ＳＡＴ）＋２ｓｍ＋４ｓｔ（４ＳＡＴ）;窄颖赖草,２ｎ＝４ｘ＝２８＝２２ｍ（２ＳＡＴ）＋４ｓｍ（２ＳＡＴ）＋２ｓｔ（２Ｓ     

大鼠4.5S RNAs的cDNA克隆与序列分析

利用ＲＴ－ＰＣＲ方法,首次从大鼠肝脏细胞总ＲＮＡ中扩增出４．５Ｓ ＲＮＡｓ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,经酶切电泳鉴定,然后测序。与报道的小鼠和仓鼠４．５Ｓ ＲＮＡｓ序列进行了比较研究,并对该分子的结构特点进行了初步分析。     

小麦与赖草远缘杂交的受精和胚胎发育

对小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）和赖草（Ｌｅｙｍ ｕｓｓｅｃａｌｉｎｕｓＴｚｒｅｌ．）杂交的受精和胚胎发育进行了观察．赖草花粉在小麦柱头上萌发良好,花粉管可顺利长入花柱和胚囊,在检查过的３１９个小麦子房中,６２个（１９．４４％ ）发生了双受精,产生了胚和胚乳,但胚乳的发育往往落后于胚的发育;４９个（１５．３６％ ）发生了单卵受精,只产生胚而无胚乳;７个（２．１９％ ）发生了单极核受精,只产生胚乳而无胚．小麦×赖草虽然总受精率可高达３６．９９％ ,然而由于胚乳的缺乏或发育不完全,致使最后结实率很低．从１５０朵授过赖草花粉的小麦颖花中,只得到１粒种子．表明利用胚培养技术对杂种胚进行早期离体培养,可望提高杂种植株的获得率     

切断根茎对根茎禾草沙鞭的赖草克隆生长的影响

为了研究半干旱内陆沙化生境中植物克隆整合对克隆植物基株扩展能力和对克隆植物株定居逆境能力的贡献,在内蒙古鄂尔多斯高原毛乌素沙地对根茎禾草沙鞭和赖草,进行了以切断根茎为处理的野外实验。实验结果表明,切断根茎处理使根茎禾草沙鞭基株的幼小部分（观测单元）在实验期间根茎数量增量,地上枝数量增量,地上枝总长度增量,主根茎节数增量,根茎节总数增量,主根茎长度增量和根茎总长度增量显著地减少。然而,切断根茎处理对     

Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.     

植物谷胱甘肽还原酶的生物学特性及功能

谷胱甘肽还原酶(glutathione reductase,GR: EC 1.6.4.2)是植物体内一种重要的抗氧化酶类,其主要的生理功能是将氧化型谷胱甘肽(oxidaized glutathione disulfide,GSSG)还原成还原型谷胱甘肽(reduced glutathione,GSH),从而为活性氧（reactive oxygen species,ROS）的清除提供还原力,保护植物免受伤害.文中主要从Gr基因及其氨基酸序列的比较等方面分析了该酶的生物学特性;又对植物逆境响应,酶基因的缺失等方面的研究进行综述,阐释了GR酶在植物体内的作用原理、在逆境胁迫中抗逆表达调控途径及其作用机制;并对已有的研究成果进行总结分析,探讨了GR酶可能的起源及系统进化过程,为今后该酶的研究提供理论参考.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

Cloning,Nucleotide Sequence,and Disruption of Streptococcus mutans Glutathione Reductase Gene (gor)

We cloned and sequenced the glutathione reductase gene (gor) of an oxygen-tolerant Streptococcus mutans, and constructed a gor-disruption mutant by homologous recombination. The gor gene consisted of 1,350 bp, coding for a protein of 450 amino acid residues. The deduced amino acid sequence of the S. mutans gor gene product showed extensive similarity with those of glutathione reductases from prokaryotes and eukaryotes. Although the mutant could grow aerobically, it showed no growth in the presence of 2 mM diamide, a thiol-specific oxidant. In contrast, growth of the wild-type strain was not significantly inhibited by 2 mM diamide, and glutathione reductase activity was increased 2.2-fold under these conditions. In addition, the level of glutathione reductase activity in the wild-type strain was increased 3.6-fold upon exposure to air, and the elevated level of the enzyme was retained throughout the aerobic growth. Thus, glutathione reductase may be important in protection of S. mutans against oxidative stress.     

RT-PCR克隆甜菜硝酸还原酶cDNA全长序列及分析

根据GenBank中已公布的甜菜（Beta vulgaris）硝酸还原酶（nitrate reductase）基因序列（gb∣ABW05098.1∣）,设计引物,以50 mmol·L^-1 KNO₃溶液处理的甜菜幼苗为材料,从总RNA中通过RT-PCR分离得到一个硝酸还原酶基因,其cDNA长2 760 bp,包含了完整的基因编码序列,与已公布的硝酸还原酶基因序列相似性达99%。Southern杂交分析表明,硝酸还原酶基因在甜菜基因组中可能以两个拷贝或低拷贝形式存在。根据其编码的氨基酸序列,利用生物信息学预测了其亚细胞定位和蛋白质的三级结构。     

谷胱甘肽还原酶和超氧化物歧化酶在玉米幼苗热激诱导的交叉适应中的作用

玉米幼苗经过热激处理后,在随后的高温、低温、干旱和盐胁迫环境中,其存活率明显高于未热激的幼苗,表明热激能提高植物的抗热性、抗冷性、抗旱性和抗盐性,证实了玉米幼苗交叉适应现象的存在;热激还可提高谷胱甘肽还原酶(GR)和超氧化物歧化酶(SOD)的活性,且在上述4种胁迫过程中GR、SOD活性水平与玉米幼苗的存活率呈正相关,表明GR和SOD参与玉米幼苗交叉适应的形成.     

巴西橡胶树的脂酰辅酶A还原酶cDNA克隆及其序列分析

从巴西橡胶树差减cDNA文库中筛选到一个与脂酰辅酶A还原酶同源性较高的基因片段,根据该基因片段序列信息,设计特异引物,采用RACE进行差异片段的5’和3’端的扩增,获得长度为1365bp的cDNA克隆R28（GenBank登陆号：AY461413）。序列分析表明,该基因包含1149bp的开放阅读框,5＇-UTR为96bp,3＇-UTR为128bp,编码382个氨基酸,推测其蛋白质的分子量为43．5kDa,等电点为8．97,有一个跨膜螺旋N（187至215位氨基酸）和1个由17个氨基酸组成的信号肽（1至17位氨基酸）。R28含有脂酰辅酶A还原酶的保守（NADP结合蛋白保守区）,推测该基因是一个脂酰辅酶A还原酶基因。     

羊草OEE1基因的克隆及盐胁迫下的表达

从羊草(Leymus chinensis )叶片cDNA文库中克隆得到可能编码33 kD的光系统Ⅱ(PSⅡ)外周蛋白(oxygen-evolving enhancer protein1,OEE1)全长cDNA(GenBank登录号为EF583851),命名为LcOEE1.序列分析结果表明,该cDNA全长1 107 bp,5′非编码区为32 bp,3′非编码区为71 bp,编码区长987 bp,编码328个氨基酸.BALSTp比对发现,该基因氨基酸序列与已报道的小麦和水稻中的OEE1序列具有95%和94%的相似性.聚类分析表明,该基因与小麦和水稻的亲缘关系较近,与拟南芥和菠菜OEE1基因的亲缘关系较远.Northern杂交结果表明,在200 mmol/L的NaCl处理7 d的幼叶中,OEE1 mRNA的表达量明显高于未处理的对照,说明羊草中OEEl基因受盐诱导.     

羊草光合作用相关基因的克隆与分析

在构建了羊草叶片cDNA文库的基础上,利用M13载体通用引物筛选其亚文库,挑选阳性克隆进行测序,将测序结果在NCBI基因库中进行比对,得到一个Rubisco大亚基基因全长序列和Rubisco小亚基基因部分序列,并对其核苷酸及其编码的氨基酸序列进行分析。结果显示,Rubisco大亚基基因长度为1 796 bp,与禾本科大麦、小麦、野雀麦、粗山羊草、旱麦草、异形花草、黑麦等的核苷酸序列同源性达98%以上;羊草的Rubisco小亚基基因部分序列含有一个开放阅读框,其长度为186 bp,编码61个氨基酸,与禾本科的小麦、大麦、燕麦、黑麦以及扁穗雀麦Rubisco小亚基基因氨基酸序列的同源性分别为93%、93%、91%、91%、92%。羊草Rubisco基因的克隆与分析有利于进一步研究其光合作用效率。