【作者】 张晓峰；

【导师】 薛庆中；

【作者基本信息】 浙江大学 ， 作物遗传育种， 2004， 博士

【摘要】 1．水稻全基因组NBS-LRR编码基因分析 采用BLAST(Basic Local Alignment Search Tool)和HMM(Hidden Markov Model)分析，对水稻籼亚种9311全基因组47077个预测基因进行搜索比对，鉴定出549个类似NBS-LRR的抗性基因，其中具有完整的NBS区域和富亮氨酸重复区域的共258个(占47％)。序列结构分析表明，有169个基因的N端存在由20～50个氨基酸组成的CC结构。其中12个水稻CNL基因N端含有两个CC结构。水稻NBS-LRR基因以及拟南芥的CNL形成的2条共有序列(consensus sequence)。相对保守区域主要分布在5个主要motif(P-loop、kinase2、RNBS-B、GLPL以及MHD)位置。我们发现水稻两个基因家族在NBS上游至N端域内中均存在相对保守的基序(ELRELAYDAEDVIDEFXYX)，该基序在大约90％的基因序列中保守程度很高，而在拟南芥CNL基因序列中并不存在。绝大部分NBS区域约为300(305.6±49.6)个氨基酸，但也搜索到NBS区域长达1048个氨基酸的基因。新发现的motif9、motif7、motif10、motif1、motif8、motif6等也有很强的保守性。域中每个氨基酸的保守程度也不尽相同。在P-loop(GMGGLGKTT)中除第二和第五位氨基酸的保守性较差外，其余氨基酸保守性均很强，仅发生个别位置氨基酸变异。而GLPL的第二位氨基酸及MHD的第一位氨基酸保守性相对较差。LRR在整个NBS至C末端序列中均有分布，但多数位于MHD下游大约80个氨基酸左右。NBS-LRR基因中包含LRRs的数目为2～33个，平均为10.5个。水稻NBS-LRR基因中共鉴定出12个主要的富亮氨酸重复LRRs，长度在10～24氨基酸之间，这些LRRs序列中大多以两个亮氨酸L之间间隔2个其它氨基酸居多，即LXXLXXL这种重复形式较多。我们还发现一小部分基因中含有特殊的结构，其中8个NBS-LRR基因中存在两个完整的NBS区域。大约85％基因中发现存在内含子，他们广泛分布于整个基因序列的各个位置。常见的内含子的剪切位置位于激酶2(Kinase2)之前，约占40％左右，但内含子的剪切位点同样显示多样性。将258个NBS-LRR基因的NBS区域，即从P-loop第一个氨基酸G开始前大约10个氨基酸至MHD domain之后大约30个氨基酸左右的基因蛋白序列用来形成系统亲缘树。当取bootstrap值大于75％作为分组依据时，这些基因共形成88组。玉米的RP1和拟南芥的RPM1基因在亲缘树呈现独立分枝，而其它的7个已知功能的NBS-LRR基因水稻的NBS基因形成不同的分枝，说明这些基因同水稻基因之间在序列上存在相当的保守性。四个TIR基因构成的分枝中居然还包含一个拟南芥的CNL基因家族成员At 1 933560，暗示这些基因起源于共同的祖先。2.水稻和拟南芥NBS一LRR基因家族同义密码子使用偏好的比较 应用多重变量分析软件CodonW对全基因组的水稻和拟南芥NBS一LRR基因家族的同义密码子进行分析，分别鉴定出25种和16种最优密码子，而且这些最优密码子完全不一致。两个NBS一LRR基因家族中所鉴定的最优密码子与其全基因组的最优密码子一致，说明该基因家族的密码子使用特点受物种自身的影响很大。而且水稻和拟南芥的各NBS一LRR基因家族成员间密码子使用上发生偏向的程度不尽相同。同一物种内基因家族对密码子的使用也存在一定变异性。密码子发生强烈偏向使用的是以G或C结尾的密码子，而拟南芥中则是以A或T结尾的密码子。密码子中位于第一、三位的G+C含量明显高于第二位的G+C的含量。最适密码子使用频率Fop(Frequeney of optimum eodons)与基因的G+C含量，第三位密码子的G十C百分含量GC3s、以及物种特定的密码子适合性因子CAI呈现极显著相关。3.釉稻全基因组非NBS一LRR抗性基因的结构及其分布特点 从釉稻9311全基因组精细图中采用同源比对结合蛋白结构域分析的方法，鉴定出276个非NBS一LRR抗性基因，大约占水稻基因组全部基因的0.6%。在各类基因家族中鉴定出10一12个相对保守的序列区域。对全部水稻抗性基因(534)在染色体上的分布进行统计表明，260个抗性基因组成88个基因簇，其中异质基因簇巧个，其它为单身。本研究剖析了水稻基因组中抗性基因的类别、分布及结构等特点，有助于研究基因的功能和进化，为进一步克隆新的抗性基因提供重要信息和依据。更多还原

【Abstract】 1. Genome-wide analysis of NBS-LRR-encoding genes in indica rice(Oryza sativa L.)By BLAST(Basic Local Alignment Search Tool) and HMM(Hidden Markov Model) analysis, we screen 549 genes encoding resistance proteins, similar to the NBS-LRR(nucleotide binding site and leucine-rich repeats), from total 47077 predictive genes in indica rice cv9311genome. 258 (47%) genes contain the full NBS region (from P-loop to MHD motif) and LRR domain in their sequences, and 157 (61%)genes own one CC motif, and 12 genes had two CC respectively, which consist of 20-50 amino acid residues in the upstream of NBS domain. The major conserved regions gather on NBS region, such as five main motifs P-loop, kinase2, RNBS-B, GLPL and MHD, in the two consensus sequences covered in NBS-LRR gene of rice and CNL of Arabidopsis. As high a conservative sequence as 90% in N terminus, which is named as motifl (ELRELAYDAEDVIDEFxYx), can be detected in both the CNL and n-CNL gene families of rice, but cannot be in CNL family of Arabidopsis. Most of NBS domains of rice genes span -300 amino acids with an extreme type of 1048 amino acids. In NBS region, we identify numerous conserved motifs, some of which showed high conservation, such as motif9 motif7, motif10 motif1 motif8 motif6, except five major motifs mentioned above.The majority amino acid residue in each motifs shows a considerable degree of conservation, except the second and the fifth that in P-loop (GMGGLGKTT). The second in GLPL and first amino acid residue in MHD also showed lower conservation. LRR distributed in all the C terminal sequences, but most were located at -80 amino acids after the MHD domain. The LRR domain ranges from 2 to 33 with a mean of 10.5 LRRs. By MEME, we identified 12 major patterns of LRRs with 10-24 amino acids residues, of which most LRR motifs shared the L(XX)n pattern, that is two leucines (L) were separated by random amino acid. We also found several special domains in few genes, eight NBS-LRR genes of them having two full NBS domains. Introns scatter in about 85% NBS-LRR gene sequences of rice, and show their diversity in splicing sites, about 40% of them being at the beginning of the kinase2. By analyzing sequence diversity and relationships in NBS domains (i.e. from the 10 amino acids N terminal to first Gly in the P-loop motif to ~30 amino acids after MHD motif)of NBS-LRR genes between rice and other species, we have constructed two phylogenetic trees based on the genes encoding protein sequences of about 30 amino acids. The trees are composed of 88 groups at any values when bootstrap used to define the groups exceeds 75%. Expect Rpl of maize and RPMl oi Arabidopsis results single clade respectively, seven known rice NBS-LRR genes dispersed on the different clades in the trees, indicating that higher sequence similarity between these genes. What interesting was that in cluster formed by four TIR-NBS-LRR genes included Atlg33560, a gene of CNL family member of Arabidopsis, suggesting that they might be arise from the same ancient. This may be generated by biology to detect the presence of pathogens and induce corresponding recognition and defense responses in the long evolution.2. Comparison of synonymous codon usage of NBS-LRR gene family in rice and ArabidopsisWe investigated variation in codon usage of NBS-LRR gene in completely sequenced genomes of rice and Arabidopsis families both within and between genomes using a universal multivariate analysis program-CodonW. In 59 synonymous codons we detected, 25 optimal codons of rice and 16 optimal codons of Arabidopsis were identified. NBS-LRR genes of rice had a specific codon usage bias that differed from which of Arabidopsis. The patterns of synonymous codon usage of two NBS-LRR families had a tendency with their whole genome, which reflected their specific behavior within the genome. The each member of two NBS-LRR gene families shared different bias in codon usage and also presented differences within each NBS-LRR genes. The most strongly biased codons of rice NBS-LRR genes were mainl更多还原