小粒野生稻STK类抗病基因同源序列的克隆与分析

根据丝氨酸/苏氨酸蛋白激酶类(serine-threonine kinase,STK)抗病基因结构中保守结构域,设计引物,以小粒野生稻基因组DNA为模板,扩增获得10条STK类抗病基因同源序列.同源性分析表明其都具有STK保守结构域,与已克隆的STK类抗病基因有不同程度的相似性,为进一步克隆小粒野生稻中的STK类抗病基因提供依据.     

小麦NBS-LRR类抗病基因同源序列的分离与鉴定

根据已知植物抗病基因的保守区域设计引物,从抗锈病小麦品种西农88基因组DNA扩增出3条与植物抗病基因同源的序列,分别为WRGA1、WRGA2和WRGA14。这三条同源片段均含有典型的NBS-LRR类抗病基因所拥有的保守性结构域Kinase-2a、Kinase-3a和疏水结构域(HD)．它们与部分已知NBS-LRR类抗病基因的氨基酸序列同源性为46．0%-9．9%,三个片段间在氨基酸水平上的同源性为80．7%-56．8%。Northern杂交表明WRGA1在小麦中受水杨酸正调控,属诱导型表达。     

三浅裂野牵牛NBS类抗病基因同源序列的克隆与分析

NBS类植物抗病基因保守结构域的克隆为利用简并引物扩增抗病基因同源序列提供了可能.根据抗病基因Gro1-4、Gpa2、N等的P-loop和GLPL保守结构域设计简并引物,分离甘薯近缘野生种三浅裂野牵牛NBS类型抗病基因同源序列,共获得6条相关序列,核苷酸序列的相似性为48%~97%,推测氨基酸序列的相似性在25.2%~95.1%之间.系统进化分析表明,6条三浅裂野牵牛RGA序列可分为2个不同的类群:TIR-NBS和non-TIR-NBS.三浅裂野牵牛RGA序列与源自甘薯的RGA序列有很高的相似性,这在一定程度上反映了三浅裂野牵牛与甘薯之间的亲缘关系.分离的6条RGA序列分别命名为ItRGA1~ItRGA6,GenBank登录号分别为DQ849027~DQ849032.     

云南悬钩子蔷薇NBS LRR类抗病基因同源克隆与分析

为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBS LRR序列中的保守区域设计简并引物,利用RT PCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBS LRR保守结构域的抗病基因同源序列(RGAs),分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。     

云南悬钩子蔷薇NBS-LRR类抗病基因同源克隆与分析

为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBSLRR序列中的保守区域设计简并引物,利用RTPCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBSLRR保守结构域的抗病基因同源序列（RGAs）,分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。     

小麦NBS类抗病基因同源cDNA序列的克隆与特征分析

根据已克隆植物抗病(R)基因NBS保守结构域设计简并引物,采用RT-PCR和cDNA末端快速扩增技术(RACE),在小麦抗叶锈病近等基因系材料TcLr19中进行抗病同源基因cDNA全长的扩增。获得了1个通读的NBS类抗病同源基因S11A11cDNA序列,该序列全长2923bp,编码878个氨基酸序列。生物信息学分析结果表明,该片段含有NB-ARC保守结构域和多个LRR结构域。聚类分析表明,S11A11编码的蛋白与小麦抗叶锈病基因Lr1编码的蛋白亲缘关系较近,而与Lr10亲缘关系较远。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr19小麦中成功获得了抗病基因同源序列,为最终克隆小麦抗叶锈病目的基因奠定了基础。     

野生稻NBS类抗病基因同源序列的分离与序列分析

为了挖掘野生稻中的抗病资源,根据已克隆的植物抗病基因核苷酸结合位点序列中的保守结构域设计3对简并引物,从疣粒、药用、高秆、宽叶和斑点野生稻基因组DNA中分离出13条NBS类抗病基因类似物,其中11条具有连续的ORF,具有NBS类R基因的保守基元P-loop、kinas-2、kinas-3a和GLPL。在NCBI上进行同源性搜索发现,其中12条RGAs的核苷酸序列与水稻已知的NBS类R基因具有66%～94%的同源性,与其他植物已知R基因具有67%～84%的同源性;其对应的氨基酸序列与水稻已知的NBS类R基因具有43%～93%的同源性,与其他植物已知R基因具有37%～79%的同源性。另外1条的核苷酸序列与水稻假定的NBS类R基因具有76%的同源性,其氨基酸序列与水稻假定的NBS类R基因具有74%的同源性。根据序列分析结果设计6对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,RN1BD5、RN1BD10、RN1GG2和RN1YY6均能表达,说明这些片段可能是功能性抗病基因的部分序列;而RN1KY9和RN1GG5没有表达,可能是假基因。     

小麦抗赤霉病品种NBS同源序列克隆与分析

根据二穗短柄草NBS-LRR类基因的保守序列设计同源引物,以小麦抗赤霉病品种苏麦3号、宁7840和望水白基因组DNA为模板,通过PCR扩增,得到43条序列,其中4条为非编码序列或结构域不完整;39条与植物抗病基因同源,其中的7条内部存在终止密码子,可能是假基因,经过比对分析,其余32条具有连续的开放阅读框和保守结构域,推导的氨基酸序列均具有Kinase-1a、Kinase-2和Kinase-3a及GLPL区等几个保守区,在GenBank中均能找到与之高度同源的其他物种的核酸序列,并且Kinase-2的最后一个氨基酸均为色氨酸(W),属于non-TIR类NBS基因。32条序列可分为4大类,它们之间核苷酸同源性为64%-98%,编码氨基酸同源性为22%-98%。根据序列分析随机设计5对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,7-1、s-3、s-4和w-2均能表达,说明这些片段可能是功能性抗病基因的部分序列;7-13不表达,再次证明属于假基因。32条序列在之前未被报道过,这些RGA可以作为筛选赤霉病功能性抗病基因的候选序列。     

巴西橡胶Pto类抗病同源序列的克隆与系统发育重建（英文）

番茄Pto基因是一类可以编码丝氨酸/苏氨酸激酶(STK)序列的广谱抗性候选基因,其序列克隆与鉴定为深入了解番茄的抗病机制奠定了基础。在该研究中,一对依据Pto基因的保守序列设计的简并引物被用来扩增巴西橡胶中Pto基因抗病同源序列,扩增得到了一个约550 bp的基因片段,其随后被克隆并测序。序列分析发现,其中的7个抗病同源序列与Pto基因高度同源(BLASTX E value3e-53),所以其被认为是Pto基因抗病同源序列(Pto-RGCs)。通过巴西橡胶的Pto-RGCs多序列比对表明,这些序列包含了多个STKs保守的次级结构域。此外,系统发育分析也表明,巴西橡胶的Pto-RGCs属于Pto基因同源的R基因。该研究结果中Pto-RGCs可为巴西橡胶抗病的发展提供一个有效的基因资源。     

甜瓜抗霜霉病基因同源序列克隆与分析

采用RT—PCR扩增的方法,从高抗霜霉病甜瓜品种‘日本安农二号’中克隆到约3kb的cDNA片段（命名为MRGH-D,该基因是一个连续的通读编码框,编码1007个氨基酸。推测的蛋白质分子量为113．7kDa,等电点为7．88,蛋白质预测无跨膜区。根据推测的氨基酸序列,该基因属于TIR—NBS—LRR类抗病基因,具有TIR-NBS—LRR类抗病基因所有的保守结构域。核苷酸序列和氨基酸序列同源性分析结果显示,MRGH-J与甜瓜抗病基因的同源序列MRGHl2及抗霜霉病相关基因mp-19均具有高达99％的同源性,推测该基因可能在甜瓜抗霜霉病中起作用。     

Characterization and linkage mapping of R-gene analogous DNA sequences in pea (Pisum sativum L.)

Pea (Pisum sativum L.) sequences that are analogous to the conserved nucleotide binding site (NBS) domain found in a number of plant disease resistance genes (R-genes) were cloned. Using redundant oligonucleotide primers and the polymerase chain reaction (PCR), we amplified nine pea sequences and characterised their sequences. The pea R-gene analog (RGA)- deduced amino acid sequences demonstrated significant sequence similarity with known R-gene sequences lodged in public databases. The genomic locations of eight of the pea RGAs were determined by linkage mapping. The eight RGAs identified ten loci that mapped to six linkage groups. In addition, the genomic organization of the RGAs was inferred. Both single-copy and multicopy sequence families were present among the RGAs, and the multicopy families occurred most often as tightly linked clusters of related sequences. Intraspecific copy number variability was observed in three of the RGA sequence families, suggesting that these sequence families are evolving rapidly. The genomic locations of the pea RGAs were compared with the locations of known pea R-genes and sym genes involved in the pea-rhizobia symbiosis. Two pea RGAs mapped in the genomic region containing a pea R-gene, Fw, and four pea RGAs mapped in regions of the genome containing sym genes. Received: 4 August 1999 / Accepted: 11 November 1999     

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some con-served motifs. In the present work,a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences,21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group,A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced,and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2-10 blotted bands. In addition,since three non-TIR-NBS-RGAs have the same hybridization pattern,we conjecture that these three RGAs form a cluster distribution in the genome.     

Starch-synthase III family encodes a tandem of three starch-binding domains

The starch-synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII-specific domain (SSIII-SD) with three internal repeats and a C-terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch-binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved.     

Origin, diversity and evolution of NBS-type disease-resistance gene homologues in coffee trees (Coffea L.)

The majority of plant disease-resistance genes (R-genes) isolated so far encode a predicted nucleotide-binding site (NBS) domain. NBS domains related to R-genes show a highly conserved backbone of amino acid motifs, which makes it possible to isolate resistance gene analogues (RGAs) by PCR with degenerate primers. Multiple combinations of primers with low degeneracy, designed from two conserved motifs in the NBS regions of R-genes of various plants, were used on genomic DNA from coffee trees, an important perennial tropical crop. Nine distinct classes of RGAs of the NBS-like type, representing a highly diverse sample, were isolated from Coffea arabica and C. canephora species. The analysis of one coffee RGA family suggested point mutations as the primary source of diversity. With one exception, coffee RGA families appeared to be closely related in sequence to at least one cloned R-gene. In addition, deduced amino acid sequences of coffee RGAs were identified that showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)-type R-genes. The high degree of similarity between particular coffee RGAs and R-genes isolated from other angiosperm species, such as Arabidopsis, tomato and rice, indicates an ancestral relationship and the existence of common ancestors. The data obtained from coffee species suggests that the evolution of NBS-encoding sequences involves the gradual accumulation of mutations and slow rates of divergence within distinct R-gene families, rather than being a rapid process. Functional inferences drawn from the suggested pattern of evolution of NBS-type R-genes is also discussed.     

植物抗病基因同源序列及其在抗病基因克隆与定位中的应用

近10年来已有20多个植物抗病基因被克隆,测序,这些抗病基因所编码的蛋白中大多含有核苷酸结合位点,富含亮氨酸重复序列,蛋白激酶,亮氨酸拉链结构,跨膜结构域,Toll白介素－1区域等保守结构域。利用这些保守结构域合成PCR引物,已扩增出大量的植物抗病基因同源序列（RGA）。对RGA与抗病基因的关系进行了分析,讨论了RGA在研究抗病基因进化中的作用,指出RGA在抗病基因定位和转基因中具有重要意义。     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHARdehydroascorbatereductase基因的cDNA与DNA全长序列,命名为BoDHAR。并利用双链接头介导PCR的染色体步行技术(genomewalking)克隆了其上游644bp的5′端序列。所获的BoDHAR基因全长1486bp,存在两个内含子,DNA编码区序列633bp,编码210个氨基酸;序列分析表明BoDHAR与同源基因AT1G19570.1cDNA序列有82.3%的一致性,推导的氨基酸序列有79.6%的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明BoDHAR在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。     

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group, A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced, and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2–10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.     

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group, A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced, and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2–10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.