【作者】 李强；

【导师】 朱月林； 张凤兰；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2015， 硕士

【摘要】 大白菜作物属十字花科芸薹属作物,通常被认为是再生率低和难以遗传转化的作物。提高离体再生频率,建立高效离体再生体系是利用基因工程改良大白菜作物的关键。植株再生涉及到胚性愈伤组织产生、体细胞胚发生和不定芽/根生长等过程,其中胚性愈伤组织产生是关键的起始环节,而LBD是促进胚性愈伤组织发生的关键转录因子。本研究以37份白菜高代自交系为材料,参照前人的不定芽再生体系并稍作优化,对不同材料的不定芽再生频率进行统计,获得7个再生频率高的优良自交系;继而选用再生频率差异大的材料,通过荧光定量PCR测定大白菜愈伤组织发生过程中LBD基因的表达模式,初步分析了LB 基因表达对外植体愈伤组织的产生和再生频率的影响。主要结果如下:1.选用白菜类(大白菜和小白菜)作物子叶再生体系进行芽再生频率统计,结果表明不同品系材料的不定芽再生率差异巨大,再生率最低为0%,最高可达到67.2%。2.选用再生率为2.4%的21#(来源于杂一代品种’刑都优丰’)和55.7%的29#(源于杂一代品种’夏宝’)自交系为材料,利用荧光定量PCR在愈伤培养的0d、7 d、14 d、21 d四个时间点检测白菜LBD基因的表达。结果表明,白菜外植体在诱导产生愈伤组织的过程中,LBD基因四个时间点的表达量呈现先上升后下降的趋势;再生频率不同的材料其LBD基因的表达量也不同。3.选用4份再生率有明显差异的自交系材料,进一步验证了上述LBD基因在愈伤组织发生过程的表达规律。并且研究表明外源激素能显著诱导白菜LBD基因表达量的增高,有效促进材料愈伤组织的形成。在建立的子叶再生体系的基础上,本研究成功建立大白菜子叶培养农杆菌的遗传转化体系。利用本研究组基于全基因组关联分析GWAS(Genome-Wide association study)和传统QTL(Quantitative trait locus)遗传定位,在A02染色体上定位了 一个与白菜抽薹开花密切相关的候选基因—BrVIN3(Bra006824)。为验证Bra006824功能,本研究利用上述筛选获得的再生频率高的自交系材料,采用农杆菌侵染转化法将目的基因Bra006824转入相应植株中;利用相同的农杆菌采用浸花法将基因导入拟南芥并进行表型调查分析,结果显示:1.在再生过程中,外植体再生芽对潮霉素的最高耐受浓度是3 mg/L;利用该浓度进行抗性芽筛选,初步获得5株经过PCR验证的转基因植株。2.利用农杆菌浸花法将抽薹相关基因Bra006824导入拟南芥,结果表明Bra006824基因的异位高表达可以促进植株提早进入抽薹开花阶段。更多还原

【Abstract】 Chinese cabbage is one of cruciferous Brassica crops,which was considered the regeneration rate is low and difficult to apply in genetic engineering.Improving the regeneration frequency and establishing efficient regeneration system in vitro are necessary foundation for genetic engineering of Chinese cabbage.Callus formation,embryogenesis and adventitious bud development are major steps of plant regeneration,in which callus formation is a key step.In the study,37 inbred lines of Chinese cabbages and 1 commercial cultivar were tested to evaluate their shoot regeneration frequency.Then,selected seven materials with significant different regeneration frequency were used to further study.Here,we focused on the function of Brassica LBD genes in callus formation and plant regeneration by analyzing.The results preliminary suggested that LBD genes are involved in callus formation and may impact shoots regeneration frequency.Main results are as follows:1.Optimization of shoot regeneration system as the basic condition was used for regeneration rate analysis,the results showed that different inbred lines had different regeneration rate,the rate ranged from the lowest 0%to the highest 67.2%.2.21#inbred line which was derived from ’Xin Du You Feng’,with a regeneration rate of 2.4%,and 29#inbred line which was derived from ’Xia Bao’,with a regeneration rate of 55.7%,were used for function study of LBD.Expressions of LBD genes were recorded by real-time quantitative PCR in the course of 0 d,7 d,14 d,and 21 d after shoot induction.The results showed that LBD impression first increased and then decrease in the process of regeneration.The expresson of LBD genes were also different within different materials of regeneration frequency.3.The expression pattern of LBD genes were further proved by 4 materials with different regeneration rates.Moreover,LBD were induced by exogenous auxin,a hormone which could effectively promote the formation of callus.Based on the cotyledon regeneration system,the genetic transformation system of the Agrobacterium-mediated for the Chinese cabbage was successfully established in vitro.A QTL for flowering time was detected on A02 chromosome based on SLAF sequencing and genome-wide association analysis,which was consistent with the QTL determined by traditional genetic mapping.In order to understand the function of Bra006824,we selected few high regeneration frequency inbred lines.The Agrobacterium-mediated transformation method was taken to transform Bra006824 gene into the plant genome.The results showed that:1.In the screening process of regeneration,the suitable concentration of hygromycin for screening resistant buds was 3 mg/L;At least 5 positive transgenic Chinese cabbage plants were obtained,which was further confirmed by PCR analysis of candidate Bra006824.2.Bra006824 gene closely related to Chinese cabbage bolting was transformed into Arabidopsis thaliana by Agrobacterium floral dip.High ectopic expression of Bra006824 gene promotes early bolting in Arabidopsis.更多还原