【作者】 马翎健；

【导师】 何蓓如；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2002， 博士

【摘要】 本研究采用在7个不同生态点的多点试验，对光周期敏感小麦雄性不育系A31的雄性育性变异进行了研究：采用分期播种试验和室内人工控光试验对A31的育性转换机制和光周期敏感阶段进行了研究；通过A31花粉发育过程中酶活性研究，对A31雄性败育的生理机制进行了探讨；运用RAPD技术对小麦光敏雄性不育基因进行了分子标记，并对小麦RAPD扩增体系进行了优化。获得的主要研究结果如下： 1．在所选择的云南昆明、元谋、陕西杨凌、青海互助、河北石家庄、甘肃武威、黑龙江依安七个生态点的试验结果表明，A31表现明显的育性变异。试验点的海拔与A31育性并无明显相关关系，而A31育性有明显的随纬度增加降低的趋势。拔节至抽穗期的光周期是影响A31雄性育性的主要因素，光周期越长，A31育性越低，当平均日长达到一定值(约为14.5h左右)时，A31表现为完全雄性不育。拔节至抽播期平均气温与A31育性并无明显相关关系，但在日长相近条件下，温度对A31育性也有一定的效应，高温有使A31的育性恢复提高的趋势。但当日长大于一定值时(≥15h)，不论气温多高，A31仍能保持稳定的雄性不育。对比分析可以推断出A31的育性转换临界日长大约为14.5h。 2．通过A31在杨凌的分期播种试验和室内人工控光试验反应出与多点试验一致结果：随日照日数的增加，A31自交结实率有明显下降趋势；在A31的育性变化中，光照起着主导作用，但在光照长度没有达到一定值时，温度对结实也有影响，高温具有消减长日效应、促进结实的作用。 3．A31的雄性败育形式具有多样性。心皮化现象是A31雄蕊败育的一种表现形式，而且心皮化现象也与一定的日长有关，但心皮化率与A31的育性并无明显相关关系。因此，影响A31雄蕊心皮化的机制可能与A31雄性不育机制并不相同。三 光周期敏感小麦雄性不育系 A3育性转换机制研究及光敏基因分子标记 4．通过对A31花药发育过程中的不同时期长光照分阶段处理试验，表明长光照分阶段处理对 A3的育性影响差异很大，说明 A3的雄性育性对光周期的敏感性存在着时期上的差异。进一步研究表明，在 A3幼穗分化的小穗原基分化期、小花原基分化期、药隔期和四分体期这4个时期中，小花原基分化期对光周期的反应最敏感，药隔期次之，小穗原基分化期和四分体期作用很小。A3的育性转换是这几个时期长日照效应的累加所致，但其中起关键作用的是小花原基分化期和药隔期，这两个时期内如果能满足 A3长日照的要求（31 sh），就可使 A3发生育性转换，表现完全不育。 5．对光周期敏感小麦雄性不育系 A3 的倒 H叶和花药在花粉发育不同时期的 且SOD、CAT活性比较研究表明，SOD、CAT的酶活性在H核期之前与其核供体普通小”麦差异很小，二核期以后 A3的 SOD、CAT的酶活性急剧下降，到三核期显著低于其核供体普通小麦；A3花粉败育的关键时期是二核期至三核期；花粉败育的根本原因之一可能是异质系核质关系不协调、酶代谢失调，导致SOD和CAT活性下降、活性氧积累、膜系统损伤。 6．以光敏雄性不育系A3及基恢复系1376为材料，对影响RAPD扩增的因素进行了比较研究，确定了模板、MgZ＼dNTPs、引物和Tap0NA聚合酶的适宜浓度和最佳的循环次数，实验结果表明在25ul反应体系中使用20～40ng的模板DNA，1．5～2．ommol／L的呢＋，0．3～0．4nunol／L的dNTPs，0．15～0 20umol／L随机引物，1．0～1．5单位Taa0NA聚合酶；反应条件为94℃预变性smin，然后进行94C45秒，36”45秒，72”90秒，45个循环后，再在72OC延伸10分钟，小麦RAPD扩增效果较好。 7．通过对光敏雄性不育系A31与恢复系1376杂交所得的F。分离群体育性的研究分析表明，F。分离群体共582株的平均结实率为42．16％，变异范围为0～86．67％。经卡方测验表明，Fi群体中完全不育个体与可育个体的比值符合3：1的分离比例，所以，A3光敏育性可能是由一对基因控制的。 8．本实验将A3与恢复系1376杂交所得的F。单株按育性分群，建立可育DNA池和不育DNA池，分别以其为模板进行RAPD分析，用10十ase随机引物进行多态性＼引物筛选。从147试引物中筛选出2个引物在不育群体和可育群体间扩增出多态性产物。经过进一步对 F。分离群体的单株检测，其中的一个引物（Sll 06）扩增出的分子量为400hP左右的特异条带得到重复验证，证明了这种多态性的可靠性，该多态性扩增产物是与小麦A31光敏雄性不育基因连锁的，可以作为A31光敏不育基因的连锁标记，但距离多远尚不清楚，有待于以后进一步研究。更多还原

【Abstract】 Through out the multi-area experiments in 7 different ecological areas, the male fertility variation of photoperiod-sensitive male sterile line wheat (A31) was studied. The fertile change mechanism and photoperiod sensitive stages of A31 were also studied by adopting sowing by stages and indoor artificial light control experiments, Meanwhile, the male sterility physiological mechanism of A31 was probed through the research of enzyme activity during pollen growing periods. The RAPD techniques were used for molecular marker of wheat Photo -sensitive male sterile genes and RAPD amplification system was optimized. The main results showed that:1. The results of experiments carried out in 7 ecological areas in Kunming, Yuanmou of Yunnan, Yangling of Shaanxi, Huzhu of Qinghai ,Shijiazhuang of Hebai ,Wuwei of Gansu and Yi’an of Heilongjiang showed that A31 had obvious fertility variation. The fertility of A31 did not have obvious correlation with the elevation of the experimental areas, but it dropped obviously with the raise of latitude of the areas .The photo period of jointing to earring stage was the main cause of male fertility of A31, the longer of the photoperiod, the lower of the fertility and it would be sterile completely when the average day -length reached a stable numerical value (about 14.5h ). The average air temperature of jointing to earring stage did not have obvious correlation with the fertility of A31, but it had effect to same extent when the day -length was near each other; high temperature could make the fertility resumed But no matter how high the temperature was, A31 could maintain stable male sterility when the day -length was higher than a stable numerical value (about 15 h ). So the critical day-length of fertile changes of A31 could be deduced by contrasting and analysis, it was about 14.5 hours.2.The sowing by stages and indoor artificial light control experiments carried out in Yangling showed the identical results with the multi-area experiments: the self breedfructification ratio of A31 dropped obviously with the sunshine day-length increasing, The length of illumination had the main effect on the fertile changes of A31 ,but temperature could also influence the fructification if the illumination-length did not reach a stable numerical value. High temperature could dispel long sunshine effects and accelerate fructification.3.The male sterility forms of A31 had diversity. The carpellody phenomenon was one form of stamen fertility, and it is also related to the day-length, but carpellody ratio hadn’t obvious correlation with the fertility of A31.So it suggested the effect mechanism of stamen carpellody might be different with that of male sterility.4.The experiment of long sunshine treatment by stages in different periods during the anther growing was carried out. The result showed that the effect of long sunshine by stages treatments to fertility of A31 had great difference, that means the photoperiod sensitivity of A31 was different in different period, Further study showed that: in four spike differentiation periods period of floret primordial differentiation was the most sensitive to the photoperiod anther-partition period took the second place and there was little effect in spikelet primordial differentiation period and tetrad phase. The fertile changes of A31 was the result of accumulation of long sunshine effects during in these periods, and the key periods were floret primordial differentiation period and anther-partition period. If the demand of long sunshine could be satisfied in these two periods ,the fertility of A31 would change and show complete sterility.5. photo-period sensitive made sterile lines wheat (A31) were selected to study the activities of superoxide Dismutase (SOD) and Catalase (CAT) in functional leaves and anthers. The results showed that SOD and CAT activities of A31 were little different with its nuclear donor common wheat before the period of dinucleate stage, but they would drop sharply after the dinucleate stage and would be remarkably更多还原