【作者】 万平；

【导师】 刘大钧；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2000， 博士

【摘要】 一、小麦矮秆基因Rht3和赤霉酸反应不敏感基因Gai3的分子标记及其与α-淀粉酶的表达研究 小麦的Rht3是来源于中国西藏大拇指矮小麦中对赤霉酸反应不敏感的显性矮秆基因。Rht3等位基因被认为具有对F₁代籽粒产量呈超显性，使成熟籽粒α-淀粉酶活性水平降低等效应，因而在杂交小麦育种中受到重视。Rht3与α-淀粉酶的表达、GA信号转导途径，以及抗穗发芽和改善面粉加工品质密切相关。因此，研究和克隆Rht3基因具有重要理论意义和应用价值。Rht3的遗传方式已较清楚，但长期以来有关Rht3与Gai3基因是否为两个独立的基因问题仍有争议。本研究利用Rht3供体经与其隐性等位载体品种（扬麦3号与苏麦3号）回交22次的近等基因系与轮回亲本杂种F₁自交产生的BC₂₂F₂较大分离群体，从经典遗传学到分子遗传学水平，系统地研究了Rht3与Gai3的遗传关系及它们对α-淀粉酶的表达和穗发芽的影响等问题。并筛选出与Rht3、Gai3紧密连锁的RAPD和RFLP标记。为Rht3基因的有效利用提供了许多新的信息，也为其图位克隆奠定了基础。 研究表明Rht3与Gai3应为极紧密连锁的两个基因，在两个近等基因系分离群体中Rht3与Gai3间的重组值分别为0.045±0.010和0.04040±0.011。Rht3、Gai3通过抑制α-淀粉酶基因的表达，降低α-淀粉酶活性，从而提高了抗穗发芽能力。在重组类型中Rht3表现出对α-淀粉酶表达和抗穗发芽的更大影响。等电聚焦同功酶分析结果显示Rht3、Gai3主要对位于第6部分同源群长臂的α-Amyl基因的表达起作用，但群体α-Amyl同功酶谱带较为复杂，可能Rht3、Gai3调控的α-Amyl的表达机制较复杂。RAPD分析共筛选了310个随机引物，其中UBC389、OPV-06和S1060三个引物在高矮亲本间能稳定地扩增出多态带，但群体验证结果仅S1060₁₉₀₀和S1060₂₀₀₀扩增片段与Rht3、Gai3连锁，遗传距离分别为16.7cM和29.5cM。RFLP分析选用了53个第4部分同源群短臂探针，其中Xpsr584、XksuF8和Xcdo38三个探针在高矮亲本间揭示多态性，接连锁分析结果，Xpsr584与Rht3、Gai3共分离。二、利用RFLP分于标记确走导人小壹的羹观草（k kwi）染色休邱分同回群归回扭究 从小麦亲缘物种向普通小麦转移优良基因，并在分子水平上进行有效地检测，对于进一步利用小麦亲缘物种中所蕴囤的丰宫基因资源具有十分重要的理论与实际意义。yLP可以准确地确定导入外源染色体及染色体片段的大小、位置和所涉及的外源染色体与普通小麦染色体的部分同源关系，为外源优良基因的利用提供极有用的信息。本研究选用来自小麦7个部分同源群的26个DNA探针对45个小麦－鹅观草衍生后代株系及鹅观草、中国春和扬麦5号进行yLP分析。结果表明大部分探针在鹅观草基因组有3条以上的杂交带，在中国春．扬麦5号与鹅观草之间的多态性水平较高。 16个小麦一鹅观草异附加、代换或可能的易位系中，所涉及鹅观草染色体分别属子第1、3、5、6、7部分同源群。小麦一鹅观草异染色体系中导入的成对鹅观草染色体能够较稳定地遗传给后代。K139、K141、K214、KZ18、KZ19、K224 H体附加系所添加的鹅观草染色体属第 1部分同源群，但 KZI4、KZIS所添加的鹅观草染色体与 KZIg、K224所添加的鹅观草染色体来自鹅观草不同的染色体组。K147端体添加系涉及第！部分同源群鹅观草染色体长臂，而K139、K141和 K147所涉及的鹅观草染色体分别来自鹅观草不同的染色体组。鹅观草 U染色体与小麦第1部分同源群有同源关系。属第1部分同源群的鹅观草染色体尤其是它的长臂与赤霉病抗性有关．研究中还观察到鹅观草第！部分同源群与第6部分同源群染色体之间的可能重排。另外，K203添加的两条鹅观草染色体分别与第！和6部分同源群同源。K166中导人的鹅观草染色体涉及第5部分同源群。短臂。K177（Zn—41，20II－I）中，所渗入的鹅观草染色质涉及第5（SL）、6（6S）、7（SL）部分同源群。本研究结果同时也证明了鹅观草 S、H和 Y三个染色体组间的部分同源关系。更多还原

【Abstract】 Molecular markers linked to Rht3 and Gai3 genes that affect the expression of a-amylase The wheat dwarf gene Rht3 derived from Torn Thumb, a Tibetan wheat of China, is a dominant gene with the insensitivity to gibberellic acid. The alleles of Rht3 show overdominance for grain yield of hybrid F1 and depress of the ~-amylase activity in ripened grains. So it抯 possible utilization in hybrid wheat breeding is of great concern It抯 found that Rht3 is also closely related with the x-amylase activity, GA transduction, resistance to preharvest sprouting and processing quality of wheat. The irnpoitance of studies on molecular mapping and isolation of Rht3 is obvious The hereditary mode of Rht3 has been clarified, but whether Rht3 and Gai3 are two independent genes still remains as a problem to be answered In this dissertation systematic studies on the genetic relationship between Rht3 and Gai3 and how they affect the ci-amylase activity were made using large BC22F2 populations derived from two set of dwarf isogenic lines including Yangmai3 and Suirnai3 as well as their prototypes The dissertatior~ also present RAPD and RFLP markers linked to Rht3 and Gai3 which could be of use for the applications and map-based cloning of Rht3 The results of this paper revealed that Rht3 and Gai3 are tightly linked to each other with the recombination rate of 0 045眥) 010 and 0 140? 011 in the two segregating populations, respectively Rht3 and Gai3 could depress the gene expression of i-arnylase, reduce its activity and hence enhance the resistance to pre-harvest sprouting Higher effects of Rht3 compare to Gai3 on ni-amylase activity and resistance to pre-harvest sprouting were discovered from data of the recombinants. The results of isoelectric focusing isoenzyme (IEF) of a-amylase verified that Rht3 and Gai3 mainly inhibit the expression of a-Amy 1 gene located on the long arms of hornoeologous group 6 of wheat. The results also expressed that a-Amy 1 isoenzyme electrophesis patterns within the populations are very complicated which probably show the cornplex expression mechanism of a-Amy 1 regulated by Rht3 and Gai3. In the RAPD analysis, out of 310 random primers (lObp) screened, only 3 primers e.g. UBC3 89, OPV-0 6 and 51060 revealed polymorphisms in NIL, and fragments SIO6OI9a6 and S10602000 amplified by primer S1060 were shown as to be linked to Rht3 and Gai3 with a genetic distance 16.7cM and 29.5cM. respectively. In RFLP analysis, 53 probes specific for short arms of homoeologous group 4 were screened, and Xpsr584, XksuF8 and Xcdo3S showed polymorphism between the bilLs. The linkage analysis showed that Xpsr5S4 co-segregated with Rht3 and Gai3 genes. Homoeologous grouping of R. kanwji chromosomes introduced in wheat via RFLP analysis Twenty six DNA probes located on 7 homoeolgous groups of wheat were screened to reveal the RFLP between 45 wheat-R.kamoji derivatives, R.kamoji, Chinese Spring and Yairnai5. The results showed that most probes could produce more than 3 hybridized bands showing distinct polymorphisms between Chinese Spring, Yangmai 5 and 1? .kamoji. The introduced R.kamoji chromosomes in the 16 wheat-R.kamo,i chromosome lines including addtions, substitutions or possible translocations were grouped into homoeologous group 1, 3, 5, 6 and 7. Alien chromosome pairs could be readily transmitted into the descendants. Of the 16 alien chromosome lines, the added chromosomes in K139, K141, K214, K218, K219 and K224 disomic ad更多还原