通过农杆菌介导法获得耐盐转甜菜碱醛脱氢酶基因白三叶草

通过农杆菌介导法将耐盐植物山菠菜甜菜碱醛脱氢酶(Betaine Aldehyde Dehydrogenase,BADH)基因成功地转化了白三叶草。转基因植株在经过48h 1％NaCl胁迫后相对电导率为20％左右,而非转基因植株高达40％,表明转基因植株细胞膜在盐胁迫下受到的伤害较非转基因的轻,并且转基因植株能够在含有0．5％NaCl的水培养中正常生长两周以上,而非转基因植株则呈现不正常生长。     

棉花Trihelix转录因子GhGT29基因的克隆及功能分析

Trihelix转录因子在植物抵御各种逆境胁迫中扮演重要作用,克隆棉花Trihelix转录因子基因并分析其表达特性和功能,为最终利用转基因手段改良棉花抗逆性奠定基础。本文依据生物信息学分析,采用RT-PCR方法从陆地棉中克隆了一个Trihelix转录因子基因,命名为GhGT29(GenBank登录号：JQ013097)。该基因最大开放阅读框(ORF)为1092 bp,编码363个氨基酸,预测分子量为40.9 kDa,等电点为5.45。SMART蛋白结构预测发现,该蛋白含有1个Trihelix家族典型的SANT结构域。系统进化树分析表明,GhGT29属于Trihelix转录因子SH4亚家族,与拟南芥AtSH4-like1、AtSH4-like2亲缘关系最近。实时荧光定量PCR结果表明,GhGT29受高盐、干旱、低温胁迫和ABA诱导表达;GhGT29在陆地棉的根、茎、叶、花、开花后当天胚珠以及开花后12 d(12 DPA)纤维中均有表达,其中在花中表达量最高,在茎中表达量最低。利用拟南芥原生质体系统进行分析,结果显示GhGT29主要定位于细胞核中,并且具有转录激活活性。以上结果表明GhGT29基因可能参与棉花逆境信号通路中对抗逆功能基因表达的调控。     

水稻盐胁迫应答cDNA的克隆、表达和染色体定位

从150mmol／L盐胁迫3h和没有胁迫的耐盐水稻(Oryza Sativa L.)品种特三矮2号叶片中提取mRNA,构建cDNA文库。通过示差筛选,得到3个盐胁迫应答cDNA克隆Ts1、Ts2和Ts3。Northern分析表明,3h的盐胁迫可使Ts1、Ts2和Ts3的转录水平明显上升;3～24h期间,Ts1和Ts2的转录水平继续上升,而Ts3的转录水平则下降。序列分析表明,这3个cDNA克隆与已知功能的基因没有同源性。利用特三矮2号的耐盐亲本ZYQ8和粳稻JXl7组合构建了DH群体和分子标记连锁图谱,将Ts1、Ts2和Ts3分别定位在第l、3和7染色体上。值得注意的是,Ts1、Ts3和Ts3与用同一群体定位的主效和微效耐盐QTL位于同一或相邻区域。     

Identification of salt-stress responsive genes in rice (Oryza sativa L.) by cDNA array

To identify salt stress-responsive genes, we constructed a cDNA library with the salt-tolerant rice cultivar, Lansheng. About 15000 plasmids were extracted and dotted on filters with Biomeck 2000 HDRT system or by hand. Thirty genes were identified to display altered expression levels responding to 150 mmol/L NaCl. Among them eighteen genes were up-regulated and the remainders down-regulated. Twenty-seven genes have their homologous genes in Gen-Bank Databases. The expression of twelve genes was studied by Northern analysis. Based on the functions, these genes can be classified into five categories, including photosynthesis-related gene, transport-related gene, metabolism-related gene, stress- or resistance-related gene and the others with various functions. The results showed that salt stress influenced many aspects of rice growth. Some of these genes may play important roles in plant salt tolerance.     

甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板,通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上,得到重组质粒ｐＧＢＹ５６。序列分析结果表明,内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ,其中包含３个开放阅读框架,分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比,其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上,构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明,２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后,可特异地表达２５ｋＤ蛋白     

Isolation and characterization of five rice telomere-associated sequences

Ｔｅｌｏｍｅｒｅｉｓｔｈｅｅｓｓｅｎｔｉａｌｇｅｎｅｔｉｃｌｏｃｕｓａｔｔｈｅｅｎｄｓｏｆａｌｌｅｕｋａｒｙｏｔｉｃｃｈｒｏｍｏｓｏｍｅｓ.ＴｈｅｙｗｅｒｅｐｒｏｐｏｓｅｄｔｏｃａｐｃｈｒｏｍｏｓｏｍｅｓｐｒｅｖｅｎｔｉｎｇｔｈｅｅｎｄｔｏｅｎｄｆｕｓｉｏｎｓｂｅｔｗｅｅｎｂｒｏｋｅｎｅｎｄｓａｎｄｃｏｎｔｉｎｕａｌｔｅｒｍｉｎａｌＤＮＡｌｏｓｓｄｕｒｉｎｇｒｅｐｌｉｃａｔｉｏｎ.Ｔｈｅｙａｌｓｏｈａｖｅｉｎｆｌｕｅｎｃｅｓｏｎｍｅｍｂｒａｎｅｃｈｒｏｍｏｓｏｍｅｉｎｔｅｒａｃｔｉｏｎａｎｄｔｈｅ…     

一个种子特异的大豆AP2类转录因子参与调控种子萌发

植物种子的发育和萌发受严格的时空调控,其中转录因子起着重要的作用．本研究发现了一条在发育过程中的大豆种子里特异表达的表达序列标签（expressed sequence tag,EST）．通过末端快速扩增技术（rapid amplification of cDNA end,RACE）得到该基因的全长序列并命名为GmSGR．序列分析表明该基因属于AP2／ERF类转录因子家族,其AP2结构域与拟南芥（Arabidopsis thaliana）中的AP2／ERF基因家族DREB亚家族中A-3组的AtABI4的AP2结构域同源性极高．在酵母系统中,未检测到GmSGR具有转录激活活性．将该基因在拟南芥中过量表达,在高浓度的脱落酸（abscisicacid,ABA）及葡萄糖的条件下,转基因植物种子的萌发率均高于对照组;而在高浓度的NaCl条件下,转基因植物种子的萌发率低于对照组．在转基因植物的幼苗中,AtEm6和AtRD29B的表达较野生型幼苗高．这些结果表明GmSGR可能通过调控AtEm6和AtRD29B的表达,从而导致转基因种子对ABA敏感度降低,对盐胁迫更敏感．     

Identification of salt-stress responsive genes in rice (Oryza sativa L.) by cDNA array

To identify salt stress-responsive genes, we constructed a cDNA library with the salttolerant rice cultivar, Lansheng. About 15000 plasmids were extracted and dotted on filters with Biomeck 2000 HDRT system or by hand. Thirty genes were identified to display altered expression levels responding to 150 mmol/L NaCl. Among them eighteen genes were up-regulated and the remainders downregulated. Twenty-seven genes have their homologous genes in GenBank Databases. The expression of twelve genes was studied by Northern analysis. Based on the functions, these genes can be classified into five categories, including photosynthesis-related gene, transportrelated gene, metabolismrelated gene, stress-or resistancerelated gene and the others with various functions. The results showed that salt stress influenced many aspects of rice growth. Some of these genes may play important roles in plant salt tolerance.     

一种无桥的水平板型凝胶电泳

凝胶电泳是研究核酸、蛋白质等生物大分子的一项重要技术,也是DNA的限制性核酸内切酶切割片段的分析、分离、纯化的一种不可缺少的方法。虽然垂直(管柱型及板型)凝胶电泳早已被广泛应用,但它不能用低浓度琼脂糖凝胶分离大分子DNA。水平板型凝胶     

应用微卫星标记进行大豆种质多样性和遗传变异性分析

微卫星标记又称ＳＳＲ标记是近年来发展的一种新型的分子标记可有效地进行基因型鉴定,系统谱分析并可估算材料间的遗传距离。用５对ＳＳＲ引物对１５份大豆材料进行扩增,共得到２１条多态性条带。每个ＳＳＲ座位的等侠基因数目为３～＾个,基因多样性范围为０．４３７～０．６６８,对这些材料进行了遗传距离分析。家系分析表明微卫星ＤＮＡ经过多世代的九分裂的后产生了突变,在ＲＩＬ Ｆ８代中有的个体在个别ＳＳＲ等基因的大小