小麦外源抗黄矮病基因相关片段的克隆和鉴定

【作者】 许景升；

【导师】 沈银柱； 张增艳；

【作者基本信息】 河北师范大学 ， 细胞生物学， 2003， 硕士

【摘要】 根据已克隆植物抗病基因编码蛋白质的保守结构设计引物，利用基于同源序列的候选基因法，扩增中间偃麦草、抗黄矮病易位系及感病材料，得到一条与抗性共分离的特异带，约538bp、将此特异标记带转化为SCAR（sequence characterized amplified region）标记，命名为TirgaZ1，用于筛选HW642基因组TAC文库，得到4个阳性克隆T1，T2，T3，T4，对阳性克隆进行了限制性内切酶分析，发现四个阳性克隆分为两类：T1，T2，T3为一类，T4为另一类。将阳性克隆T1和T4的酶切产物转膜，以RGA基因片段TirgaZ1做探针进行Southern杂交，各酶切产物均有明显的不同杂交带，并且同一酶切产物杂交带中包含1-2条相同带和1-3条相异带，即这2个克隆可能部分重叠、位于1个contig的两侧。对T1进行序列分析，表明T1含有抗病基因保守结构域，说明这2个克隆为含有NBS类R基因片段TirgaZ1的候选抗病基因克隆。 以抗病中间偃麦草、HW642和感病小麦亲本的基因组DNA做探针，对阳性克隆T1和T4的酶切产物进行Southern杂交分析，由杂交图谱可知，XhoI，EcoRV，EcoRI，PstI酶切产物与中间偃麦草、HW642的基因组DNA产生了小麦不存在的特异杂交带，进一步说明阳性克隆T1和T4是来源于中间偃麦草7XL的侯选抗黄矮病基因克隆。 根据已克隆植物抗病基因编码蛋白质的保守结构设计引物，利用抗病基因类似物多态性技术（resistance gene analog polymorphism，RGAP），研究与Bdv₂相关的RGAP标记和RGA基因片段。用187对引物组合，对具Bdv₂的不同抗病材料和不具Bdv₂感病材料进行分析，结果表明有2对引物组合cf9F／PtoKin2UIN（Tgp-1）、PtoF／XLRRinv2（Tgp-2）能稳定地扩增出与Bdv₂相关的抗病特异带，大小分别为350bp和210bp左右，说明这2个标记Tgp-1₃₅₀和Tgp-2₂₁₀是Bdv2相关的RGAP标记。用F₂群体进行的连锁分析结果表明这2河北师范大学硕士研究生学位论文个RGAP标记与丑动心共分离。从聚丙烯酞胺凝胶上回收特异扩增产物，二次扩增后克隆、侧序.序列分析表明，T纷13，和T争入l。是某些基因ORF的一部分，是新的基因片段。根据它们序列，重新设计引物，成功地将RGAP标记T争13，转换为特异PCR标记sC一gPI，FZ群体分析结果表明sc一gPI也与及扣2共分离。更多还原

【Abstract】 Primers were designed according to the conserved sequences of cloned resistance genes in plants. Using the method of candidate genes based on homologous sequences, the primers were used to screen the Th. Intermedium, wheat-77z. Intermedium and the susceptible wheat materials. A specifically amplified band, which was co-segregated with BYDV resistance, was obtained. It was about 538bp and converted to a SCAR （sequence characterized amplified region） marker, named as TirgaZ1. The marker was used to screen HW642 genomic TAC library and resulted in four positive clones, which were Tl, T2, T3 and T4.Restriction enzyme digestion analysis showed that the four clones were divided into two kinds, one of which included Tl, T2. T3 and the other included T4. Then the hybridization analysis was performed after the digestion products of Tl and T4 were transferred to a nylon membrane, with the RGA fragment TirgaZl as a probe. Obviously different hybridized bands were shown on the digestion products by different enzymes. And the same digestion products also demonstrated 1-2 identical bands and 1-3 different bands. This indicates that the two clones are perhaps partly overlapped and are on the two sides of a contig. Sequencing analysis showed that Tl contained a conserved domain of resistance genes. Therefore, the two clones might be resistance gene candidates for R gene fragment TirgaZl containing NBS-like sequence.Using the DNA of BYDV-resistant Th. Intermedium, HW642 and susceptible wheat parents as probes, Southern hybridizations were carried out on the digestion products of Tl and T4. Specifically hybridized bands with Th. Intermedium and HW642 but not with wheat parents were shown on the digestion products by Xhol, EcoRV, oRI and Pstl. The results further demonstrate that Tl and T4 are BYDV-resistance gene candidates derived from 7XL of Th. Intermedium.To screen the Bdv₍3-rrelated RGAP markers and RGA fragments, degenerated primers were designed according to the conserved sequence of cloned resistance genes in plants and used in the method of resistance gene analog polymorphism （RGAP）. After the first screening with the wheat-Th. intermedium translocation line HW642 containing a BYDV-resistance gene Bdv₂ and the susceptible recurrent wheat parents Zhong8601 and further analysis using different resistance materials with Bdv₂: and susceptible materials without Bdv₂. only 2 out of 187 primer combinations designated as cf9F/PtoKin2IN （Tgp-1） and PtoF/XLRRinv2 （Tgp-2）. 350bp and 210bp respectively, could stably amplify two specific band related to resistance gene Bdv₂. This proved the validation of Tgp-1 and Tgp-2 as RGAP markers for Bdv₂. The linkage analysis using the F₂ progeny also shows that the two markers are co-segregated with Bdv₂. The specifically amplified bands were excised from the gels of SDS-PAGE for a second PCR and the products were cloned and sequenced. The sequencing results indicate that Tgp-hso and Tgp-2₂₁₀ are parts of the ORF of certain genes and they are new gene fragments. Based on the two sequences, new primers were designed and Tgp-I₃₅₀ was successfully converted to PCR marker SC-gpl. which was also proved to be co-segregated with Bdv₂ in F₂ progeny analysis.更多还原