【作者】 黄细松；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2006， 硕士

【摘要】 白菜（Brassica rapa ssp.chinensis）类蔬菜绝大部分属于冬性一年生蔬菜作物,需要经历一个低温阶段才能顺利完成发育转变。在低温阶段,感温先期抽薹是生产上一个迫切需要解决的问题。培育晚抽薹品种是解决这一问题的根本途径。菜心是白菜类蔬菜的一个变种,对低温要求不严格,在华南地区可以周年生产,是代表极端早抽薹的一类白菜类作物。 本研究以冬性类型‘上海青’普通白菜和无冬性类型‘四九’菜心杂交的F₂代为材料,根据基于性状的分析法,利用F₂群体抽薹开花分布的两个极端构建极端早抽薹开花池和极端晚抽薹开花池,利用AFLP分子标记技术,筛选二者基因组的差异,以期获得与熟性或抽薹性状有关的分子标记。同时对人工春化条件下耐抽薹材料‘上海青’白菜花芽形态分化进行观察,运用同源基因克隆法,克隆拟南芥的重要开花时间基因的正向同源基因,比较其编码序列和内含子序列的异同,为白菜抗抽薹育种,研究抽薹开花相关基因的功能打下基础。主要结果如下: （1） 对F₂代极端早抽薹开花池和极端晚抽薹开花池进行BSA-AFLP分析,获得5条早花池或晚花池特异条带,其中差异条带E2-AAG/M8-CTT₃₁₇和E6-ACG/M6-CTC₂₆₆仅在极端晚抽薹开花池出现;而E3-ACA/M1-CAA₂₉₅、E8-AGG/M3-CAG₂₇₀、E8-AGG/M4-CAT₁₇₄仅在极端早开花池出现,分别将E3-ACA/M1-CAA₂₉₅、ES-AGG/M3-CAG₂₇₀和E6-ACG/M6-CTC₂₆₆转化为SCAR标记和CAPS标记。用3个标记对F₂代分离群体的转化结果表明,SCAR标记E3M1SCAR₂₇₁和E8M3SCAR₁₅₁推测与一个控制早开花的QTL连锁,分别位于该QTL的两侧,遗传距离分别为28.5cM和21.3cM。 （2） 分别用‘上海青’进行人工低温春化处理,观察花芽形态分化进程。结果表明低温处理20d,‘上海青’已开始从营养生长向生殖生长过渡。 （3） ‘四九’菜心与‘上海青’的BrcFLC的编码序列几乎完全一致,‘四九’菜心与‘上海青’的BrcFLC的第一个内含子序列差异也很小。 （4） 低温处理20d时,BrcFLC的表达已经非常弱,此时茎端形态为已开始向生殖生长过渡,BrcFCA和BrcFT随着低温处理时间的延长表达有增强趋势;BrcFCA与BrcFLC的关系不明显;BrcFT的表达模式与BrcFLC相反。更多还原

【Abstract】 Brassica rapa ssp. chinensis var. communis and B. rapa ssp. chinensis var. parachinensis is the extreme representative of flowering habit, and the former is a winter biennial, and need a prolonged period of cold time, a process named vernalization to induce flowering, the latter, however, is a summer annual dose not need vernalization to induce flowering. In cultivation practice, premature bolting is serious problem that not only cause a great of loss in production, but also reduce the commercial value. So bolting-resistance is an important breeding goal.In the present study, a F₂ segregating population derived a cross between a flowering Chinese cabbage cultivar ’Sijiu’ （B. rapa ssp. chinensis var. parachinensis cv. Sijiu） and Chinese cabbage-pak-choi cultivar ’Shanghai-qing’ （B. rapa ssp. chinensis var. communis cv. Shanghai-qing） was developed for the the study of genetic control of flowering time.A trait-based analysis methodology, bulked segregant analysis （BSA）was adopted to make up two bulks that were composed of individuals of extreme early flowering and extreme late flowering .Bulked segregant analysis in combination of AFLP techoneques was used to screen the two bulks for differential bands that may be candidate marker associated with early flowering habit or late flowering habit. On the other hand, ’Shanghai-qing’ was artificially vernalized for the study on the morphological differentiation of floral bud in Chinese cabbage-pak-choi, meanwhile RNA was extrated from the SAM of ’Shanghai-qing’ pak-choi at different time point of vernalization treatment, and from the leaf of non-vernalized ’Shanghai-qing’ and ’Sijiu’ that was used for the cloning of vernalization-related gene by PCR method and for the expression analysis of them by RT-PCR in the different differentiation stage of bud .The main result was listed as follows:（1） Sevral specific AFLP bands between two bulks were obtained by BSA-AFLP analysis.Among them, E2-AAG/M8-CTT317 and E6- ACG/M6-CTC₂₆₆ is specific to the extreme late flowering bulks and parent ’Shanghai-qing’;and E3-ACA/M1-CAA₂₉₅, E8-AGG/M3-CAG₂₇₀, E8-AGG/M4-CAT₁₇₄ is specific to the extreme early flowering bulks and parent ’Sijiu’. Then, three of them was converted to SCAR or CAPS marker, and were named E3MlSCAR₂₇₁, E8M3SCAR₁₅₁ and E6M6CAPS_238/168. The segregant analysis in F₂ segregating population show E3MlSCAR₂₇₁ and E8M3SCAR₁₅₁ is linked to a putative flowering time QTL, flanking it on both side .being away from it 21.3 cM and 28.5 cM.（2） ’Shanghai-qing’ was artificially vernalized for the study on the morphological differentiation offloral bud in Chinese cabbage-pak-choi, the results show treatment for 20 d at 4"C is sufficient for the transition from vegetative growth to reproductive development.（3） The nucleotide sequence of intron 1 of ’Shanghai-qing’ and ’Sijiu’ show no substantial difference. RT-PCR analysis of the expression of flowering time-related genes BrcFLCJircFCA and BrcFT shows that the expression level of BrcFLC is reduced gradually and was at very low level after 20 days of vernalization treatment ,and was undetectable after 30 days of vernalization treatment, which is consistent with the fact that treatment for 20 d at 4*C is sufficient for the transition from vegetative growth to reproductive developmentThe expression of BrcFT was progressively enhanced, which show an inverse expression pattern compared with that of BrcFLC. The expression level of BrcFCA was only slightly elevated with time.更多还原