【作者】 谭明谱；

【导师】 张端品； 林兴华；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2004， 博士

【摘要】 由Xanthomonas oryzae pv．oryzae引起的白叶枯病严重危害水稻，可是国内一直集中利用Xa3和Xa4两个抗性基因，因而利用新的抗性基因改良现有品种的抗性日趋重要。 云南地方粳稻扎昌龙（Zhachanglong，ZCL）对分别来自中国、菲律宾和日本的12个水稻白叶枯病菌表现抗性，对PXO61表现出的成株期抗性受Xa22（t）控制。已从明恢63中克隆的Xa26与Xa3可能是相同的基因，ZCL中含有Xa26相同的基因。 （1） 目标基因Xa22（t）的精细定位 构建了7680个单株的由珍珠矮和扎昌龙配制的F₂群体，用两个SSR标记RM144和RM224筛选PXO61接种后的极端感病单株得到29个极端感病重组单株，目标基因Xa22（t）与RM144和RM224分别有20个和9个极端感病重组。利用这些重组单株分析Xa22（t）区域的标记，将Xa22（t）定位在明恢63-BAC克隆M3H8的亚克隆3／7A10和R1506之间，和这两个标记分别有2个和1个重组，同时得到三个共分离的标记M3H8-1、Sub11和X4-88。由于3／7A10是M3H8的亚克隆，并且R1506能和M3H8杂交，因而Xa22（t）定位在M3H8上。 利用Xa26的序列检索得到日本晴序列克隆AC116367，分析这个克隆发现抗病基因有关的序列信息很丰富，而且AC116367在目标基因Xa22（t）区域，因而将AC116367作为目标基因Xa22（t）的电子定位克隆。 （2） 确定候选基因及验证功能 对M3H8的两个片段的序列进行酶切位点分析，Spel可以将包含Xa26的19-kb片段切下来，NheI可以将8-kb的LRR片段切下来,AccI酶切片段可以覆盖12-kb的LRR-Kinase。于是根据M3H8的序列，采用等位酶切法回收ZCL对应大小的片段区域，克隆到合适载体上用特异引物筛选，再将得到的阳性克隆的外源片段转接到pCAMBIA1301，最后转化到感病品种台北309中来验证功能。 转化ZCL中含有Xa26．的19-kb片段得到10株抗性植株，证明了ZCL中的Xa26有功能。对于包含NBS结构的80112-2、LRR结构的8-kb片段和LRR-Kinase结构的12-kb片段的转化验证，每个片段的转基因植株数量都在100株左右。 得到一个ZCL-BAC克隆80112，其12-kb片段与R1506杂交，而且具有NBS结构。定位克隆M3H8也有NBS结构，且NBS在ZCL中表达，于是将12-kb的包含NBS结构的片段转化到台北309中，得到1株抗性植株，说明ZCL中具NBS结构的基因可能有功能。 （3） Xa22（t）区域相关片段的RT-PCR表达分析 对于Xa26内的两对引物RkbLZR和Rkb469一2145，在三个抗病材料zCI林政22（t）)、waso Aikoku3(从孕。，携带Xa匀和IRBB4(xa为中组成型表达，而在ZZA中没有表达，这可能与Xa26表现的抗病功能有关。 利用一个激酶结构RKORFI内的几对引物作RT-PCR表达分析，RKORFI在ZCL、WA3和IRBB4中组成型表达，在ZCL和WA3中的表达相同，而在ZCL与IRBB4中的表达有差异，Xa22（t）区域可能具有激酶结构的基因zCL祝K。 NBS在ZCL和IRBB4中组成型表达，而在WA3中没有表达，说明NBS.LRR与Xa3（可能为受体激酶结构）无关。由电子定位克隆日本晴的ACll6367越过约500一kb的转座子富集区后，NBS.LRR结构的保守序列很多，而且M3HS也扩增出NBS结构，Xa22（t）区域可能有NBS-LRR结构的基因zCL价旧S。RT-PCR表达分析表明Xa22（t）区域有Xa26、zCL.RK和ZCL小旧S等不同结构的基因;结合几个白叶枯病抗性基因在Xa22（t）区域的定位结果，zCL中的Xa22（t）与Xa4和XaZ‘紧密连锁，Xa22（t）与Xa4连锁更紧密。 （4）目标基因区域进化 许多抗性有关的基因在目标基因区域紧密连锁，Xa22（t）区域进化有亚麻抗锈病基因L模式，其中的一个基因XaZ石在IRBB3、明恢63、日本晴（Nipponbare，Nip）和9311中都有等位基因。Xa22（t）区域的进化也有亚麻抗锈病基因M模式，明恢63中在XaZ石的附近还有3个家族成员（Sun etal.，2004），日本晴中在XaZ石的等位基因NIPS一26H附近也有8个同源的受体激酶结构。聚类分析xa22（t）区域的抗病基因同源序列表明，paralogs的同源性较低，而orthologS间的同源性相对较高，进化采用Birth一and一Death模式。 对目标基因Xa22（t）的克隆，可以开发更好用的PCR标记作分子标记辅助选择育种，更好地研究ZCL的广谱抗病特性，探索目标基因区域的进化机制。更多还原

【Abstract】 Bacterial blight, caused by Xanthomonas oryzae pv. Oryzae, seriously reduces rice production world-wide. Two bacterial blight resistance genes, Xa3 and Xa4, were concentratively used for many years in China. It is very necessary to utilize new bacterial blight resistance genes to improve the resistance of target varieties. Xa22(t), identified from Zhachanglong(ZCL), conferred high levels of adult resistance to all 12 bacterial blight strains tested. Xa26, cloned from Minghui63-BAC clone named M3H8, had identical alleles in IRBB3 and ZCL, furthermore Xa3 and Xa26 may be the same gene and ZCL contained Xa26.(1) Fine mapping of Xa22(f)p2 population of 7680 plants derived from the cross of Zhenzhu’ai and Zhachanglong was screened for recombination events by RM144 and RM224. In all, 29 susceptible recombinants plants were identified by RM144 and RM224 after inoculation with PXO61 at the booting stage. Among the 29 susceptible recombinants plants, 20 were derived from recombination events between Xa22(i) and RM144 and 9 between Xa22(i) and RM224. Two recombination events were identified between Xa22(i) and 3/7A10 (one subclone of M3H8), and one recombinant was identified between Xa22(t) and R1506. In addition, both 3/7A10 and R1506 hybridized to BAG clone M3H8, this indicated that the Xa22(t) locus resided on this 100-kb BAC clone, M3H8.One Nipponbare sequence clone named AC116367 was identified by Xa26 sequence. Further analysis showed that many resistance gene analogs. In combination with the map position of AC116367 in Xa22(i) region, Nipponbare AC116367 was regarded as the electronic map clone of Xa22(i).(2) Candidates ofXa22(t)Restriction enzyme maps of two fragments in M3H8 were analyzed and equipotential digestion was used to recover the corresponding fragments from ZCL. A fragment of 19-kb digested by Spel contained Xa26 and a fragment of 8-kb digested by Nhel recovered LRR, while 12-kb fragment digested byAccl contained LRR and Kinase. After digestion, fragments of the expected size were recovered and verified by specific primers. Then the expected fragments were cloned into intermediate vector and confirmed by PCR further. Finally, the target fragment was transferred to pCAMBIA1301 for transformation. Now, 10 transformants ofXa26 and one transformant of LRR containing fragment showed resistance to PXO61. For each transformation mentioned, about 100transformants of each fragment grew up.One ZCL-BAC clone named 80112 was identified and a 12-kb fragment of it hybridized with R1506, and the 12-kb fragment proved containing NBS. Further analysis showed that M3H8 contained NBS and NBS was expressed in ZCLby RT-PCR with NBS amplification. Finally, one transformant of this 12-kb fragment conferred resistance to PXO61.(3) RT-PCR expression analysisThe RT-PCR expression of Xa26, LRR, Kinase and RKORF1 was assayed in three resistant varieties ZCL (Xa22(i)\ Wase Aikoku 3 (WA3, Xa3) and IRBB4 (Xa4).Primers in Xa26 and RKORF1 were used to carry out RT-PCR analysis. Xa26 was constitutively expressed in above three resistant varieties, while no expression was detected in ZZA. RKORF1 identified the same result as Xa26.NBS was constitutively expressed in ZCL and IRBB4, while not in WA3, which contained Xa3. This indicated that NBS-LRR was possibly related to Xa4 and ZCL-NBS, while not to Xa3. From the expression analysis, several genes of different structure such as Xa26, ZCL-RK and ZCL-NBS located in Xa22(i) region. Xa22(t) might tightly link to alleles ofXa4 andXa26, andXa4 might be the closer one.(4) Evolution of Xa22(t) regionMany genes of different structure tightly linked in Xa22(i) region. Two typical evolutionary model of resistance genes were found in evolution of Xa22(t) region. One model was similar to flax L model, where many alleles of Xa26 were found in IRBB3, Minghui63, Nipponbare and 9311 and they might be orthologs in evolution. Another model was similar to flax M model for many members were adjacent to Xa26 alleles in Minghui63 and Nipponbare. In general, the identity between orhtolo更多还原