【作者】 杨明挚；

【导师】 陈善娜； 黄兴奇；

【作者基本信息】 云南大学 ， 植物学， 2004， 博士

【摘要】 云南野生稻是我省乃至全世界珍贵的野生植物资源,是改良栽培稻,可供人类长期持续发掘利用的重要基因宝库。本论文立足云南野生稻及其他野生稻资源,研究野生稻中功能已知或未知的植物抗病基因。从云南不同野生稻及其他来源的野生稻中分离克隆了一些抗病基因、抗病相关基因或抗病相关基因片段。对所获得的基因或基因片段的结构、功能及其在野生稻或抗病基因本身的分子进化等方面展开了一系列相对系统的研究。先后从云南元江普通野生稻中克隆了一个完整的抗稻瘟病Pi-ta 基因和一个Pi-b 基因,并从不同种或类型的云南野生稻以及其他野生稻中克隆了抗稻瘟病Pi-ta、Pi-b 以及抗白叶枯病Xa21、Xa1 基因的部分序列,从云南不同野生稻中获得了大量的R-片段,通过分析这些R-片段,克隆了一个新的丝氨酸/苏氨酸蛋白激酶类抗病侯选基因。应用相关软件,对所克隆的的抗病基因或抗病侯选基因推导产物的二级结构与功能间的关系进行了探讨。依据所克隆的基因序列间的同源性分析,初步建立了一个云南野生稻及其他野生稻与栽培稻间的分子进化模型。同时构建了三个云南野生稻叶片的cDNA文库,其中一个云南元江普通野生稻自然状态下营养生长期叶片的cDNA 文库及两个由稻瘟病和白叶枯病病菌诱导下的疣粒野生稻和药用野生稻的cDNA 文库。在cDNA 文库构建过程中,建立了一种高效且简便快速的检测cDNA 文库中插入片段大小范围的方法。为进一步从云南野生稻中发掘有用的抗病基因奠定了良好基础。 第一部分:云南野生稻中抗病基因同源片段的克隆及研究从野生稻中大规模的分离克隆抗病基因相关片段(R-片段)尚未见报道。利用简并引物我们从云南不同野生稻中获得了60 多个可以编码完整氨基酸序列的R-片段,其中30 多个为NBS-LRR 类片段,15 个丝氨酸/苏氨酸蛋白激酶类(STK)片段。此外还从小粒野生稻(Oryza minuta)、江西东乡普通野生稻(Oryza rufipogon)及长雄蕊野生稻(Oryza longistaminata)中获得了20 多个NBS-LRR类R-片段。并有选择性的在基因库中登录了20 个R-片段序列。对这些片段的序列分析发现,大多数R 片段与基因库中水稻及其他植物中获得的R 片段或基因更多还原

【Abstract】 gene pool that can continually provide us valuable genes for the modification of cultivation rice. This paper focuses primarily on the Yunnan wild rice by cloning and sequence of some disease resistance genes which functions were know or not, or disease-resistance relative genes or gene fragments by polymerase chain reaction (PCR) from different species and types of Yunnan wild rice and other wild rice species. Two complete rice blast genes from Yunnan Yuanjiang O. rufipogon were cloned. One was Pi-ta. The other was Pi-b. A lot of R gene analogues were also cloned. And by analysis of the resistance gene analogues, one new STK type of candidate gene was found and cloned from Yunnan erect type of O. rufipogon. Structure, function analysis and molecular evolution research on wild rice and the genes themselves have carried out on all the genes or fragments we cloned. At the meantime, One cDNA library at normal condition of Yuanjing type of O. rufipogon, and two cDNA libraries of O. officinalis and O. meyeriana after inducing with rice blast and rice bacterial blight pathogens, have been constructed for further study. All these researches have provided us a good foundation for the further cloning and function research of valuable genes from Yunnan wild rice. Part 1: Cloning and sequence of disease-resistance gene analogues from Yunnan wild rice. Massive cloning and sequence of disease resistance gene analogues from wild rice have not been reported yet. We have got more than 60 disease resistance gene analogues by using degenerate primers, including 47 of NBS-LRR type and 15 of Ser/thr protein kinase (STK) type. And we have also got more than 20 of NBS-LRR type disease resistance gene analogues from Dongxiang type of O. rufipogon(from Jiangxi province), O. longistaminata(Africa) and O. minuta(from Hainan province). Most of the fragments have very low homology with other cloned disease resistance analogues and genes in gene bank, and belong to new members of R-gene family. Some of the STK analogues were got by reverse transcription PCR(RT-PCR) method and must come from genes that can be transcribed. All these cloned disease resistance gene analogues can use as probes for cloning newly disease resistance genes from wild rice. And also provide us many clues for studying the molecular evolution of the disease resistance genes in wild rice. Part 2: The cloning, sequence and structure analysis on the rice blast resistance gene: Pi-ta. By using primers that were designed according to the reported rice blast resistance gene(Pi-ta) sequence of O. sativa, we cloned the DNA sequences of the Pi-ta gene extron I homologs from different type of Yunnan wild rice (including Yuanjiang type of O. rufipogon, erect type of O. rufipogon, red awn type of O. rufipogon) and Dongxiang type of O. rufipogon, O. longistaminata. All these cloned Pi-ta homologs from different wild rice have very high in homology with that of O. sativa. They have only 2~5 nucleotide acids differences among the cloned nucleotide sequences. But these differences can also provide us some clues for the study on the evolution of these wild rice and the gene itself. The sequences comparison indicated that the erect type of O. rufipogon has the closest relationship with that of the O. sativa. Erect type of O. rufipogon may be the direct ancestor of O. sativa. Further analysis indicated that the O. longistaminata from Africa may be the common ancestor of Yunnan different type of O. rufipogon and Dongxiang type of O.rufipogon. The red awn type of O.rufipogon may also be one of the ancestors of O.sativa, because of a higher homology with O. logistaminata in Pi-ta gene extron I. The Yuanjiang type of O.rufipogon is lies in a relatively independent affinity among the concerned wild rice. The complete reading frame of Pi-ta gene in Yuanjiang type of O. rufipogon has been cloned and sequenced. There were only 7 nucleotide acid differences compared with that of O. sative. And they had lead 5 amino acid changes. There is a serine instead of a alanine at position 906 in C-terminal of Pi-ta gene deduced amino acid sequence from Yuanjiang type of O. rufipogon. According to the prevail study results, the Pi-ta gene from Yuanjiang type of O.rufipogon belongs to a Pi-ta-gene alleles, that can not cause resistance response with rice blast fungus, Magnaporthe grisea containing avirulence gene AvrPita. This different single amino acid does cause a major difference in predicted secondary structures among Pi-ta gene deduced amino acid sequences from Yuanjiang type of O. rufipogon and O.sativa, respectively. Other changed amino acids had caused little difference in secondary structure of Pi-ta protein. Yuanjiang type of O. rufipogon more sensitive to rice blast pathogen than any other type of O. rufipogon may because it containing a Pi-ta-not a Pi-ta+ gene. And the difference in disease-resistance response of Pi-ta-and Pi-ta+ may because of the structure difference in Pi-ta protein that caused by the single amino acid. Part 3: The study of Pi-b gene in Yunnan wild rice. PCR results indicated that P-ib gene (another cloned rice blast gene), exist in all the rice materials that we have studied. We have cloned the first extron DNA sequences from O. meyeriana, O. officinalis, Yuanjiang type of O. rufipogon, erect type of O.rufipogon, white awn type of O.rufipogon, Dongxiang type of O.rufipogon and O. longistaminata, and the extron II DNA sequence of Yuanjiang type of O.rufipogon, red awn type of O.rufipogon , erect type of O.rufipogon and O. longistaminata. These sequences provided us more information on the evolution study of wild rice and the gene itself. Compared with the Pi-b gene extron I sequences, there is a 87bp deletion in that of Yuanjiang type of O. rufipogon. Furthermore, among the Yuanjiang type of O. rufipogon deletion range, the erect type of O. rufipogon, white awn type of O.rufipogon and O. sativa has one code (GCT) deletion at the same position compared with that of O. officinalis. Judging from this result, Yuanjiang type of O.rufipogon can’t be the ancestor of O. sativa. And indicated that the erect type of O.rufipogon, red awn type of O. rufipogon and O.sativa have closer evolution relationship. Interestedly there are three single nucleotide acids insertion among a very short sequence range in O. longistaminata Pi-b gene extron I compared with that of O. officinalis. In extron II of Pi-b gene, O.longistaminata has 100% homology with that of red awn type of O. rufipogon. There has a code deletio at the same position in the sequence of the erect type of O.rufipogon and Yuanjiang type of O. rufipogon. And the homology between the erect type of O.rufipogon and O.sativa is also very high. The complete reading frame of Pi-b gene from Yuanjiang Type of O.rufipogon was also cloned. Part4: Study on Xa21 and Xa1 rice bacterial blight genes in Yunnan wild rice. The rice blight resistance gene Xa21 are exist in all types of O. rufipogon including Yunnan erect type of O.rufipogon, red awn type of O. rufipogon, white awn type of O. rufipogon, Yuanjiang type of O.rufipogon, and O.longistaminata. but we can not detect this gene in different varities of O. sativa. Here we cloned partial DNA sequences from different Yunnan wild rice and compared them with the reported sequences of Xa21 gene initially from O.longistaminata on homology and structure analysis. Most of the Yunnan wild rice have stronger resistance to rice bacterial blight, so there exist big opportunities to find valuable Xa21 gene alleles with new resistance properties in Yunnan wild rice. In Yunnan wild rice as well as other wild rice species, the other rice blight resistance gene Xa1 has also been studied. Xa1 gene homologs were not only exist in diffrernt type of O.rufipogon but also in O.munita. O.officinalis and O.longistaminata. Part 5: Construction and testing of cDNA libraries containing importantfunction genes of Yunnan wild rice. One in normal condition cDNA library of leaf of Yuanjiang type of O.rufipogon, and two pathogen induced cDNA libraries of Yunnan O.officinalis and O.meyeriana respectively, had been constructed by using the cDNA library construction kit from CLONTECH. All the constructed cDNA libraries have reach the demand for further use. The construction of these libraries has used a newly technology of “Clontech’s”. That insured all the inserted fragments were the complete mRNA products, every clone is containing a complete gene. Furthermore, we had set up a rapid and easy method to test the insertion fragment size of cDNA library by PCR by using the common sequences in two terminals of every insertion. Higher density agrose gel electrophonesis with DNA markers can test the size range of insertion fragments of the cDNA library. This method can be used in difference analysis of different cDNA libraries, by little modification. Part 6: Cloning, structure and function analysis of a new serine-threonine protein kinase gene (BEPK) in Yunnan wild rice. A broad existing STK like analogues in different wild rice was found in analysis of the STK analogues from wild rice. We called this gene in which this STK type fragment belongs to as: “broad existing serine/thrine protein kinase gene (BEPK). According to the homolgous sequence of BEPK fragments from different species of wild rice, specific primers have been designed. We can get this BEPK fragments from all the materials that we concerned. RT-PCR studies found that this gene is constitutive expression in different wild rice and O.sativa. we have sequenced all the BEPK partial fragments from different species of wild rice and cultivation rice (hexi NO.35). We used one BEPK fragment as bait, searching homologous sequences in gene bank and found a cDNA clone’s partial sequence has almost 100% homology with the bait sequence. So, BEPK gene must belong to this cDNA clone. The cDNA clone containing BEPK gene encode a putative 419 amino acids cytoplasmic protein, belongs to a serine/threonine protein kinase. Further searching in gene bank found BEPK gene belongs to a BAC clone that from chromesome 9 of O. sativa (cultivar: Japonica group). Comparison of the cDNA clone and the BAC clone, BEPK gene has two extrons with a 114bp intron within the reading frame. The BEPK gene has 43 more than 10% leucine among the deduced amino acid sequence. Nevertherless, BEPK gene not contain LRR domain. BEPK gene is a newly member of serine/threonine receptor kinase super family that have not find any genes has more than 50% homology with it. The secondary structure and primary function analysis of this gene has also been done by using the software of DNASIS and DNA-star.更多还原