【作者】 林兴娥；

【导师】 胡彦民； 汤继华；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2008， 硕士

【摘要】 本研究对玉米温敏自交不亲和材料(TGSI)HE97的生态学机制、遗传机理进行了研究,并对亲和相关基因进行了克隆与功能分析。利用分期播种和田间叶龄调查的方法,对HE97的亲和性转换机制进行了研究,结果表明,HE97亲和性转换的敏感时期是雄穗小花分化期(雌穗生长锥伸长期),主要受日最低温度的影响,属于温敏型自交不亲和,其亲和性转换的温度区间为20-24℃。当最低温度低于20℃时表现不亲和,最高温度高于24℃时表现亲和,与日照时数不存在显著的相关关系。通过对HE97与郑58的F2群体进行QTLs分析,在第二染色体上检测到一个影响自交不亲和的主效QTL,贡献率为21.37%,位于SSR标记umc1635和nc133之间;该基因在F2:3群体中能同时检测到,贡献率为16.8%。经典遗传学分析与数量性状基因定位结果均表明HE97的自交不亲和特性由1对隐性基因控制,命名为tgsi1,并通过BSA分池的方法将该基因定位在第二染色体上,与SSR标记nc131的遗传距离为2.4cM,与bnlg1633之间的遗传距离为2.4cM。在本课题研究的基础上,从HE97亲和花丝与不亲和花丝的正向差减文库中挑选了一个与水稻CIPKs高度同源的玉米类CIPKs基因,利用3’RACE技术和生物信息学方法对该基因进行了克隆(在GenBank上的登陆号是EU424058)。该候选基因cDNA为1918bp,基因组DNA为2520bp,包含一个1332bp的开放阅读框,编码443个氨基酸,功能域分析表明靠近N端有一个丝氨酸/苏氨酸蛋白激酶催化结构域和一个保守的NAF结构域,BLAST分析发现,该基因与水稻、黑三角叶杨、高粱等生物体的CIPKs基因有很高的相似性。建立了SYBR Green法实时荧光定量RT-PCR表达分析系统,研究了该基因在不同植物组织间表达量差异,结果发现,玉米候选基因类CIPKs基因在根、茎、叶、花丝、花粉中均有表达,其中在不亲和花粉中表达量最高,在亲和花丝中的表达量高于不亲和花丝中的表达量,与SSH结论一致。由于类CIPKs基因在不亲和花粉中高表达量直接诱导了不亲和花粉中Ca2+浓度的增加,可以阻止花粉在柱头的萌发或抑制花粉管的生长,由此该基因在温敏自交不亲和特性发生过程中可能起到了第二信号因子的作用。更多还原

【Abstract】 In this study, the ecological and genetic mechanism of a novel ecological self-incompatibility (SI) line, HE97, in maize was studied, and a self-compatible related gene was cloned and analysed.For validating the main environmental factors that affecting the characteristics of SI, the inbred line was identified in the field through sowing in different stages, and the leaf number was investigated in the field. The results showed that the sensitive period of self-compatible conversion for the ecological SI line was the floret differentiation of staminate inflorescence, and it was mainly affected by daily minimum temperature. The result proved that the ecological SI line, HE97, belonged to the thermo-sensitive genic self-incompatibility (TGSI) line. The temperature boundary of self-compatible conversion was 20-24°C. When the daily minimum temperature was below 20°C in the floret differentiation of staminate inflorescence, it exhibited complete self-incompatibility; whereas plants exhibited self-compatibility when the daily maximum temperature was higher 24°C, and the SI has not significant correlation with sunlight.A set of F2 and the corresponding F2:3 populations, which derived from two inbred line, HE97 and Z58, were evaluated for two years to dissect the inheritance of the TGSI line. There was one main QTL was detected with the seed setting rate of the F2 populations as the input data when using the comfindance interval mapping method, accounting for 21.37% self-incompatibility genotypic variance. This QTL was localized to chromosome 2 between the SSR markers umc1635 and nc133. This QTL was also detected in the F2:3 population, accounting for 16.8% self-incompatibility genotypic variance. Classical genetic analyses and QTL mapping results all revealed that the self-incompatibility of HE97 was governed by a single main recessive gene, named tgsi1. The tgsi1 gene was mapped to chromosome 2 between SSR markers nc131 and bnlg1633 using bulked segregation analysis (BSA) method, with a distance of 2.40 cM from nc131 and 2.44 cM from bnlg1633.A CIPKs-like gene, which has homologous to calcineurin B-like protein-interacting protein kinases (CIPKs) in Oryza sativa L., was cloned by rapid amplification of cDNA ends (RACE) and bioformatics(GenBank accession number: EU 424058). The full-length cDNA of the candidate gene was 1918bp and contained an 1332 bp open reading frame, which encoded 443aa, and the deduced amino acid sequence contained an acidic Ser/Thr rich region toward its N-terminus and a conserved NAF domain. The results of Blastn and Blanstx all showed that it had highly homologous to Oryza sativa, Populus trichocarpa and Persea americana. From the phylogenetic tree, we know the CIPKs-like gene in maize has tightly homologous to Oryza sativa.The Real time RT-PCR method was used to analyze the expression of the CIPKs-like gene in various tissues of the TGSI line between self-incompatible and self-compatible stages. The result showed that the gene had high expression quantity in self-incompatible pollen, and there was little difference between self-compatible silk and self-incompatible silk. The candidate gene CIPKs-like which had high expression quantity in the self-incompatible pollen of the TGSI line, urged the Ca2+ concentration in the self-incompatible pollen and prevented pollen germination in the silk. These results demonstrated that the CIPKs-like gene played an important role in regulation of the characteristics of the TGSI line in maize.更多还原