梨S基因cDNA芯片杂交条件优化及对梨品种S基因鉴定
发布时间：2018-08-27 12:16
【摘要】：梨属于蔷薇科苹果亚科梨属植物,是典型的配子体自交不亲和性植物(gametophytic self-incompatibility,GSI),其自交不亲和性由单一基因位点的S-RNase复等位基因(S基因)所控制。在梨栽培中必须配以不同的S基因型授粉品种才能保证授粉座果的顺利进行。梨品种S-RNase基因的鉴定可以为梨栽培过程中授粉品种的准确配置和育种过程中亲本的合理选择提供理论依据。我国梨品种S基因型复杂,不管是地方栽培品种还是野生型个体都蕴含着丰富的S基因资源。为完善梨S基因鉴定的技术体系,加快梨品种S基因资源挖掘与梨品种S基因型鉴定的进程,本文制备了梨S-RNase基因cDNA芯片并对芯片系统检测条件进行了优化,利用S-RNase基因cDNA芯片并行检测的特点快速准确鉴定了部分梨品种S基因型、挖掘新的S基因资源并对新挖掘的S基因资源进行分离鉴定和分子进化分析。主要研究结果如下:(1)S基因cDNA芯片制备。根据存在于东方梨品种中已知的S1-S10、S12-S13、S15-S22、S25-S32和S34-S52的序列特征及结构特点,以各S等位基因包含HV区的PF-PR1 mRNA序列作为cDNA探针,研制了 S基因cDNA检测芯片;实现了利用S基因芯片检测系统快速检测梨品种S基因的准确性和可靠性。(2)S基因cDNA芯片杂交体系优化。获得芯片最佳杂交条件为:最佳杂交温度42℃,最适杂交时间8～9 h,最适PCR纯化产物终浓度为200 ng μl-1,洗涤条件:2× SSC + 0.1%SDS、O.1× SSC + 0.1%SDS、O.1× SSC 溶液依次避光洗涤 3 min,蒸馏水冲洗2～3次(每次不超过10s),玻片甩干机离心机干燥玻片。(3)50个梨品种S基因型鉴定。利用S基因cDNA芯片优化杂交体系完成了对50份不同种质资源梨品种(主要是砂梨品种)S基因鉴定,确定了各品种的S基因型。优化后的S基因芯片检测系统能成功鉴定出砂梨品种所含有已知的S基因资源,同时根据芯片杂交信号可推测品种中新S基因的存在。(4)分离和鉴定了梨7个新S等位基因。对芯片系统鉴定只含有1个杂交信号、无杂交信号的品种,辅以酶切和DNA测序,从砂梨品种中分离获得了PpS53(P.Pyrifolia S53)、PpS54、PpS55、PpS56、PpS57、PpS58、PpS59 等新 S等位基因的序列,均已登录到GenBank数据库。(5)7个新S基因资源的进化分析。利用砂梨品种S基因和蔷薇科其它植物的S基因DNA序列研究了砂梨S基因的序列多态性和分子进化关系,确定7个新S基因间及与蔷薇科其它植物的S基因间的进化关系。(6)完善了梨S基因鉴定的技术体系。通过梨S基因芯片检测系统的建立,完善了梨S基因鉴定和S基因资源研究的技术体系,为梨丰产栽培和新品种的选育奠定了科学基础。
[Abstract]:Pear is a typical gametophyte self-incompatibility plant (gametophytic self-incompatibility,GSI), which is controlled by S-RNase complex allele (S gene) of single locus. Different S genotype pollination varieties must be used in pear cultivation in order to ensure the success of pollination. The identification of S-RNase gene in pear varieties can provide theoretical basis for the accurate allocation of pollinated varieties and the reasonable selection of parents in the course of pear breeding. The S genotypes of pear varieties in China are complex, both local cultivars and wild individuals contain abundant S gene resources. In order to perfect the technical system of pear S gene identification and accelerate the process of resource mining of pear variety S gene and identification of pear variety S genotype, the S-RNase gene cDNA chip of pear was prepared and the detection conditions of the chip system were optimized. Based on the parallel detection of S-RNase gene cDNA chip, the S-genotypes of some pear varieties were quickly and accurately identified, new S-gene resources were mined, and the newly excavated S-gene resources were isolated and identified, and the molecular evolution analysis was carried out. The main results are as follows: (1) S gene cDNA chip preparation. According to the sequence characteristics and structural characteristics of S1-S10 S12-S13 S15-S22 S25-S32 and S34-S52, the S gene cDNA detection chip was developed using the PF-PR1 mRNA sequence of each S allele including HV region as cDNA probe. The accuracy and reliability of rapid detection of S gene by using S gene chip detection system were realized. (2) the cDNA chip hybridization system of S gene was optimized. The optimum hybridization conditions were obtained as follows: the optimum hybridization temperature was 42 鈩，
本文编号：2207250
[Abstract]:Pear is a typical gametophyte self-incompatibility plant (gametophytic self-incompatibility,GSI), which is controlled by S-RNase complex allele (S gene) of single locus. Different S genotype pollination varieties must be used in pear cultivation in order to ensure the success of pollination. The identification of S-RNase gene in pear varieties can provide theoretical basis for the accurate allocation of pollinated varieties and the reasonable selection of parents in the course of pear breeding. The S genotypes of pear varieties in China are complex, both local cultivars and wild individuals contain abundant S gene resources. In order to perfect the technical system of pear S gene identification and accelerate the process of resource mining of pear variety S gene and identification of pear variety S genotype, the S-RNase gene cDNA chip of pear was prepared and the detection conditions of the chip system were optimized. Based on the parallel detection of S-RNase gene cDNA chip, the S-genotypes of some pear varieties were quickly and accurately identified, new S-gene resources were mined, and the newly excavated S-gene resources were isolated and identified, and the molecular evolution analysis was carried out. The main results are as follows: (1) S gene cDNA chip preparation. According to the sequence characteristics and structural characteristics of S1-S10 S12-S13 S15-S22 S25-S32 and S34-S52, the S gene cDNA detection chip was developed using the PF-PR1 mRNA sequence of each S allele including HV region as cDNA probe. The accuracy and reliability of rapid detection of S gene by using S gene chip detection system were realized. (2) the cDNA chip hybridization system of S gene was optimized. The optimum hybridization conditions were obtained as follows: the optimum hybridization temperature was 42 鈩，
本文编号：2207250
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