【作者】 段艳欣；

【导师】 郭文武； 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2006， 博士

【摘要】 柑橘是世界上最重要的常绿果树之一，其种植面积和总产量均居水果之首。长期以来，运用传统的育种手段进行柑橘遗传改良受到珠心胚干扰、性器官败育、育种周期长以及遗传上高度杂合等因素的影响，导致育种进程缓慢。转基因技术自1983年应用到植物育种以来，发展迅猛，已经成为基因功能鉴定不可缺少的手段，也是实现植物目标性状改良的一种快速和直接的途径。植物童期长短受开花基因调控，将开花调节基因转入植物有望改变童期。本研究以拟南芥花分生组织特异基因LEAFY(LFY)、APETALA1(AP1)为目标基因，以胚性愈伤组织和实生苗上胚轴分别为外植体，通过根癌农杆菌介导法进行柑橘遗传转化研究，采用GUS组织化学染色和PCR、Southern blotting等技术对转基因材料进行鉴定，采用real-time PCR技术进行基因表达分析；对不同基因型愈伤组织转化率存在差异的原因进行了探讨：对API、LFY基因与内源开花相关基因间的互作进行了分析，并对APl促进早花的机理进行初步研究；主要结果如下：1．柑橘基因型、愈伤组织褐化影响转化率。对15种不同基因型柑橘胚性愈伤组织的转化特性进行研究的结果表明，a)在筛选过程中，抗性愈伤组织多从褐化组织中再生；b)不同基因型愈伤组织褐化程度不同，其中宽皮橘类及其杂种类基因型愈伤组织的褐化最为严重，山金柑和葡萄柚次之，澳洲指橘和伏令夏橙与金柑体细胞杂种的愈伤组织不褐化，甜橙类愈伤组织的褐化表现为褐化、轻微褐化和不褐化；c)褐化严重的柑橘基因型转化率较高，而不褐化类型转化率较低，表明愈伤组织的褐化程度与转化率有关：d)愈伤组织的褐化与其本身的总多酚含量有关，总多酚含量高的易表现褐化，而总多酚含量受基因型的影响。综上所述，推测在根癌农杆菌介导的柑橘胚性愈伤组织的遗传转化中，基因型通过愈伤组织的褐化间接影响转化率。而柑橘愈伤组织转化pBIN-mGFP5的GFP瞬时表达结果，表明总多酚含量高的宽皮橘类及其杂种类基因型的转化率显著高于总多酚含量低的柑橘基因型，与前面结果相一致。2．在前人研究基础上，进一步优化了柑橘愈伤组织转化再生体系，建立了5步筛选再生法。基于该体系，获得19种基因型的转基因细胞系245个，其中6种基因型再生，获得120个转基因芽系，有34个转基因株系移栽成活，主要表现在，1)冰糖橙胚性愈伤组织转化：a)获得转AP1基因的转化细胞系7个，胚状体再生率高达88％：再生芽经GUS染色、PCR分析鉴定为阳性，Southern杂交结果表明所检测的4个转化细胞系中AP1基因均以单拷贝插入；再生芽生根困难，通过试管嫁接获得了完整植株，但嫁接苗在生长后期表现落叶、芽枯、死亡：b)获得转LFY基因的转化细胞系2个，其中一个获得再生植株，经GUS染色、PCR分析鉴定为阳性，Southern杂交结果表明LFY基因以3个拷贝插入；再生植株初期生长健壮，移栽后表现株形矮小，分枝多，叶片小，整株成丛状。以上结果表明AP1、LFY对植株生长有较大影响。2)椪柑胚性愈伤组织转化：a)获得转LFY基因的转化细胞系9个，其中4个细胞系获得再生植株，有3个细胞系经PCR检测为阳性，Southern杂交结果表明外源基因以单拷贝插入；b)获得转AP1基因的转化细胞系113个，其中再生细胞系61个，移栽植株104棵，源自29个株系；移栽株系经GUS染色、PCR分析，表明有93％为阳性，经Southern杂交表明外源基因以1-2个拷贝插入；转基因植株腋生组织生长旺盛，多有2-4个分枝／株，与未转化对照形态差异较大；real-time RT-PCR结果表明AP1基因在芽中表达量最高，其次是根，而在愈伤组织与胚状体中表达量较低，内源开花基因CiAP1、CiLFY、CiFT、CiTFL受APl基因影响发生上调或下调表达。3)其他基因型愈伤组织转AP1基因获得转化植株的有，伏令夏橙、Succari甜橙、改良橙与尾张杂种、茶枝柑。3．以实生苗上胚轴为试材进行转化，获得3种基因型的GUS阳性株系42个，其中21个株系移栽成活，18个株系经PCR鉴定为阳性。一株转AP1基因的金柑表现早花性状。Real-time RT-PCR结果表明转基因(LFY、AP1)对内源开花相关基因的表达有较大影响。以上结果主要表现在，1)金柑实生苗上胚轴转化AP1基因：从1021个外植体中获得GUS阳性芽19个，平均转化率为1.86％；从移栽的8株GUS阳性植株中，有6株经PCR鉴定为阳性，其中3株(J3、J10、J17)进行了Southern杂交鉴定，表明外源基因以1-5个拷贝插入。转基因植株生长正常，但平均分枝数／株大于未转化对照。转基因植株(J3)在移栽11个月后开花，而正常植株需3年左右才可开花，表明金柑中异位表达AP1基因可以促进早花。Real-time RT-PCR结果表明AP1基因表达量与其插入的拷贝数成正相关，而与开花早晚无关；AP1基因通过促进CiLFY与CiFT的表达，抑制CiTFL的表达促进开花。2)冰糖橙上胚轴切段转化LFY基因：最终转化率为8.88％；获得1株转AP1基因和12株转LFY基因的GUS阳性植株；转化植株生长正常，与未转化对照无明显差异；PCR分析表明，除一株转化LFY基因的植株为阴性外，其余均为阳性：转LFY基因植株(B1)经Southern杂交鉴定，表明外源基因以单拷贝形式插入；real-time RT-PCR结果表明LFY基因在不同转基因株系中表达量不同，根中表达量略低于芽；内源开花基因CiAP1、CiLFY、CiFT在不同转化株系中分别表现上调或下调表达，CiTFL在所有转化株系中均表现下调表达，表明LFY基因影响内源开花相关基因的表达。3)山金柑转化AP1基因：从50个外植体中获得8个抗性芽，其中3个经GUS染色鉴定为转化子，但再生芽弱小，未能获得植株。4．本文还对如何提高根癌农杆菌介导的柑橘转化效率，T-DNA插入整合的拷贝数，转基因植株群体建立的必要性，以及开花基因在植物童期控制方面的应用潜力进行了探讨。更多还原

【Abstract】 Citrus is one of the most important ever green fruit crops in the world, which ranks the first in both acreage and yield. The conventional breeding program in citrus has long been hampered by such problems as polyembryony, apomixis, long juvenility, high heterozygosity and arthenogenesis. Transgenic technology, first applied to plant breeding in 1983, has developed rapidly, and now is indispensable for gene function analysis and plant genetic modification. The juvenile length is regulated by flowering genes, and ectopic expression of these genes in plants is helpful for altering the transition to flower. In this study, the Arabidopsis flowering identity genes LEAFY (LFY) and APETALA1 (AP1) were introduced into citrus embryogenic calluses or epicotyl segments by Agrobacterium tumefaciens, aiming at achieving transgenic citrus plants with shortened juvenile phase. Histochemical GUS assay, PCR and Southern blotting analysis were used to confirm the integration of transgenes. Real-time RT-PCR was used to analyze the expression of AP1 or LFY and endogenous flowering genes. Studies focused on the causation of diversities of transformation efficiency among different geotypes, the effect of AP1 or LFY on expression of endogenous flowering related genes, and the mechanism of early-flowering by ectopic expression of AP1. The main results are as follows:1. Citrus genotype and callus browning significantly influenced transformation efficiency. The transformation potentials of embryogenic calluses from 15 citrus genotypes were assessed. The results showed that, a) most resistant calluses regenerated from browning ones; b) genotypes were divided into four groups according to their browning degrees. Severe browning was observed in mandarins and its hybrids, browning in grapefruit and kumquat, and browning, weak or no browning in different sweet oranges, and no browning in microcitrus and the hybrid of Valencia and ’Meiwa’ kumquat; c) transformation efficiency was higher in severe browning genotypes and lower in non-browning ones, which indicated that callus browning was related to transformation; d) the browning degree of calluses positively correlated with the total phenolic content which was genotype-dependent, while had nothing with PPO activity. To sum up, callus browning contributed to the effect of genotype on transformation efficiency, in Agrobacterium-mediated transformation of citrus calluses. The high frequency of transient GFP expression in genotype with high content phenolics also confirmed the conclusion above, when embryogenic calluses were transformed with pBIN-mGFP5.2. The Agrobacterium-mediated transformation of citrus embryogenic calluses was opitimized and five-step regeneration protocol was established, on the basis of previous researches. A total of 245 transgenic callus lines were achieved from 19 genotypes, of which 120 lines from 6 genotypes were regenerated into shoots and 34 lines were successfully transferred to the greenhouse. These included the following results.1) Transformation of Bingtang sweet orange (C. sinensis Osbeck.): a) Seven AP1 transgenic lines were acheived, and the conversion rate from embryo to shoot was 88％. Shoot-tip graft was adopted to regenerate whole plant due to the difficulty in rooting for shoots. Regenerated shoots were confirmed as transformants by GUS, PCR analysis. Single copy of AP1 integrated into the citrus genome, as certificated by Southern blotting analysis. Grafted plants grew rapidly at the initial stages, but later tended to be defoliating and welting, and finally died, while non-transformed plants grew well. b) Two LFY transgenic lines were obtained, of which only one regenerated into plant. Transgenic plant had a strong growth at the beginning, but later grew slowly with small leaves and muti-branches, compared to non-transformed controls. Transformants were confirmed by GUS and PCR analysis, and integrated 3 copies of NPTII certificated by Southern blotting analysis. The results of abnormal growth in transgenic plants indicated that the integration of transgenes (LFY or AP1) could affect the plant growth.2) Transformation of Ponkan(C. reticulata Blanco.): a) Nine LFY transgenic cell lines were achieved, four of which regenerated into plants. PCR analysis confirmed the integration of transgenes in 3 lines, and one line was further certificated by Southern blotting. b) One hundred and thirteen AP1 trangenic cell lines were achieved, 61 lines of which regenerated into shoots, and 104 plants from 29 lines were transferred to the greenhouse. All transplanted plants were detected by GUS and PCR analysis, and 93％of which were confirmed as transformant. Most transgenes (AP1, NPTII and GUS) integrated into citrus genome with 1 to 2 copies by Southern blotting analysis. Different from non-transformed controls, transgenic plants had a dominant growth in axillary meristems, which resulted in 2 to 4 branches per plant. Real-time RT-PCR analysis in transgenic tissues showed various accumulation levels of AP1 RNA in different transgenic tissues, with the highest expression level in shoot, followed by root, and then callus and embryo. The expression levels of endogenous flowering genes like CiAP1, CiLFY, CiFT and CiTFL in transgenic tissues were up-regulated or down-regulated due to the different expression levels of AP1.3) AP1 transgenic plants were also acheived from calluses of other citrus genotypes, including Valencia and Succari sweet orange, Chazhigan mandarin, and the hybrid of Gailiangcheng orange×Weizhang Satsuma mandarin.3. Transformation of epicotyl segments were performed in four genotypes. Fourty-two GUS positive lines of from 3 genotypes were obtained, 21 lines of which were regenerated into whole plants and transferred to the greenhouse. 18 lines were confirmed by PCR analysis. Among transgenic plants, one ’Meiwa’ kumquat plant transformed with AP1 exhibited early-flowering. Over-expression of AP1 or LFY affected the expression of endogenous flowering genes, like CiLFY, CiFT, CiTFL, and CiAP1, as revealed by real-time RT-PCR. These results were elucidated as follows.1) Transformation of AP1 in ’Meiwa’ kumquat(Fortunella crossifolia Swing.): Nineteen GUS positive shoots were achieved from 1, 021 explants, with a transformation efficiency of 1.86％. Eight GUS positive plants were transferred to the greenhouse, 6 of which were confirmed as transformants by PCR analysis. Southern blotting analysis indicated that the transgenes integrated into citrus genome with 1 to 5 copies. No differences were observed on the growth between transgenic plants and non-transformed controls. One transgenic plant(J3) flowered 11 months after transfer to the greenhouse, while it needs 3 years for wild type to bolssom, this indicated that ectopic expression of AP1 promoted early-flowering in ’Meiwa’ kumquat. Expression levels of AP1 in transgenic lines were positively correlated with its copy number revealed by real-time RT-PCR analysis, while was irrelative to the precocity. The promotion of flowering by over-expression of AP1 was due to the up-regulation of CiLFY and CiFT and down-regulation of CiTFL, independent of CiAP1.2) Transformation of LFY in Bingtang sweet orange(Citrus sinensis Osbeck.): The ultimate transformation efficiency of this genotype was 8.88％. One GUS positive plant transformed with AP1 and 12 GUS positive plants transformed with LFY were achieved. All GUS positive plants, except one with transformed with LFY, were positive by PCR analysis. One LFY transgenic plant B1 was further confirmed by Southern blotting analysis with single copy of transgene integration. Real-time RT-PCR analysis showed that various accumulation levels of LFY RNA in different transgenic lines, and the expression level was higher in the shoot than in the root. The expression of endogenous CLAP1, CiLFY and CiFT was up-regulated or suppressed in different transgenic lines, while CiTFL was suppressed in all transgenic lines. These results indicated that LFY affected the expression of endogenous flowering genes.3) Transformation of AP1 in Hongkang kumquat: Eight resistant shoots regenerated from 50 explants, 3 of which was GUS positive, but all of them showed weak growth and failed to regenerate into whole plant in the end.4. Aspects favorite for improving the efficiency of Agrobacterium-mediated transformation in citrus, the copy number of T-DNA integrated into citrus genome, the necessity of construction of transgenic plant populations, and the potential usage of flowering genes in shortening juvenility were also discussed.更多还原