【作者】 仝铸；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2008， 博士

【摘要】 柑橘是世界上最重要的水果之一,果实品质一直是柑橘育种追求的主要目标。长期以来,童期长、珠心胚、性器官败育以及遗传上的高度杂合等因素一直严重影响着柑橘育种工作的开展。基因工程的发展为柑橘的育种工作开辟了一条新的途径,从而使利用单个基因改造柑橘的品质成为可能。本研究利用童期较短的早实枳作为转化材料,建立并优化了其高效再生和遗传转化的体系,以期使其成为柑橘基因功能验证和研究的平台。利用该转化体系和RNA干涉技术,获得了一批与柑橘类胡萝卜素代谢相关基因的遗传转化材料,并对这些材料进行了分子鉴定和基因表达的初步分析,本研究主要取得了以下结果:1.建立了早实枳（Poncirus trifoliata[L.]Raf.）和Cocktail葡萄柚（C.paradisiMacf.×C.reticulata Blanco.）的高效再生及遗传转化体系,对若干影响再生和转化效率的因素,如外植体的绿化、共培养条件,激素的组合和用量,筛选的方法等进行了优化。对黄化的早实枳茎段或Cocktail上胚轴进行4-5d的光照处理,可以显著提高外植体的再生能力和转化率;共培养过程中,0.4 mg L^-1的2,4-D可显著提高外植体的转化率,而过高的2,4-D浓度(0.6-0.8 mg L^-1)则抑制不定芽的再生,降低转化率。培养基G(MT+1 mg L^-1 BA+0.1 mg L^-1 NAA)对正常芽的再生最有利,过高的BA浓度(大于1.5 mg L^-1)虽然能提高芽的数量,但也造成大量畸形芽的出现。实验中还发现,潮霉素对早实枳茎段的再生有强烈的抑制作用。在早实枳再生试验中,通过嫁接再生的早实枳有36.4%（8/22）只需要9个月就能开花,而通过生根再生的植株在第12个月时有32%（8/25）的植株开花,第20个月时,这些再生植株有87.2%（41/47）开花。这表明早实枳早花早果的特性可以经过组织培养和再生过程稳定地遗传给后代,可以用于柑橘功能基因的研究。2.构建了BCH基因片段的反义表达载体pCABCH,并用于转化伏令夏橙（C.sinensis Osb.cv.Valencia）的胚性愈伤组织,得到了6个独立的转化株系,并测定了抗性愈伤组织中类胡萝卜素含量的变化,结果表明,大部分转化愈伤中叶黄素和α-胡萝卜素的含量有所增加。50-60 mg L^-1的潮霉素对伏令夏橙愈伤组织的再生影响较大,强烈抑制再生植株的生长。3.构建了CRTISO基因片段的反义表达载体pBICI,分别转化了早实枳和山金柑,得到了23株早实枳再生植株,其中12株PCR检测呈阳性;得到转化山金柑4株。4.分别以LCYB的基因序列片段,和LCYE基因的5’和3’端片段,构建了pLCYB,pLCYE1和pLCYE2的RNAi表达载体,并用于转化早实枳和Cocktail葡萄柚。Real-time PCR结果表明,在LCYE基因被抑制的的早实枳植株中,PDS基因的表达量显著增加,LCYB和ZEP基因的表达量下降。HPLC分析表明,发生LCYE基因抑制的植株叶片中,α-胡萝卜素含量的下降,β-胡萝卜素的含量和紫黄质的含量增加,而叶黄素的含量没有显著的变化。但β-胡萝卜素和紫黄质的含量却与其基因的表达水平呈反比!据此我们推测,对LCYE基因的干涉可能通过某种机制激活了上游PDS基因的活性。而β-胡萝卜素和紫黄质的含量增加可能与代谢物质的反馈调节有关:随着PDS基因表达量增加,上游ζ-类胡萝卜素和番茄红素的含量增加,导致下游的代谢产物β-胡萝卜素和紫黄质的含量显著增加,而这些代谢产物对其本身的合成基因LCYB和ZEP又产生了反馈抑制作用。在抑制LCYB表达的早实枳植株中,PSY,ZDS,CRTISO和ZEP基因的表达量呈下降趋势。同时,β-胡萝卜素和下游紫黄质的含量在转基因植株叶片中的含量下降,α-胡萝卜素和叶黄素的含量没有显著的变化。β-胡萝卜素的含量和LCYB基因的表达量呈正相关,即LCYB基因的表达量越高,叶片中β-胡萝卜素的含量也越高。而这一过程可能会导致番茄红素的积累。更多还原

【Abstract】 Citrus is one of the most important fruits all over the world and the fruit quality is always the main goal for citrus breeding. Citrus breeding has been hindered seriously by several factors, such as long juvenility, micellar embryo, female/male abortion and high hetetozygosis. Genetic engineering opens a new avenue for citrus breeding and makes it possible for using a single gene to improve the fruit quality in citrus. In this study, the regeneration and transformation conditions of the precocious trifoliate orange have been built and optimized to establish a short juvenility genetic transformation system for gene functional verification related to citrus fruits. On the basis of the transformation system, a group of regenerated plantlets containing citrus carotenoids metabolic genes were obtained. Molecular identification and gene expression analysis were also conducted. The main results of this study are as follows:1. High efficient Agrobacterium-mediated transformation and regeneration sytems for precocious trifoliate orange （Poncirus trifoliata [L.] Raf.） and Cocktail （C. paradisi Macf.×C. reticulata Blanco.） were established. Some factors which affect regeneration and transformation were optimized, such as, the illumination of etiolated explants, the coculture conditions, the selection media and the mode of selection etc. Higher regeneration frequencies and transformation frequencies were achieved in all 4-day-illuminated precocious trifoliate orange stem segments and 5-day-illuminated Cocktail epicotyl segment compared to etiolated controls. The highest transformation frequency obtained in coculture medium, which is MT basal medium supplemented with 0.4 mg L^-1 2, 4-D. However, increasing concentrations of 2, 4-D from 0.6 to 0.8 mg L^-1 suppressed this effect. It was proper for precocious trifoliate orange to produce normal shoots on medium G (MT + 1 mg L^-1 BA + 0.1 mg L^-1 NAA). For precocious trifoliate orange, when over 1.5 mg L^-1 BA was added to SRM, although more visible shoots were produced, most of these shoots were abnormal. Regeneration of precocious trifoliate orange was completely suppressed when hygromycin B was used as selection agent. In recovery shoots, 9 months after grafting, 36.4% of the plantlets flowered （8/22）. For the rooting lines （8/25）, the flower process was delayed for 12 months. 20 months after being grafted or transferred to greenhouse, 87.2% （41/47） of the transformants flowered. The results implies the in vitro regeneration plants could reserved the precocious traits of precocious trifoliate orange and the transformation system could be used for gene functional verification related to citrus fruits. 2. The anti-sense expression vector pCABCH containing BCH gene fragment was constructed and used to transform the embryonic callus of Valencia （C. sinensis Osb. cv. Valencia）. 6 independent transgenic callus lines were obtained. The carotenoid contents were detected by HPLC. The result revealed that lutein andα-carotene content increased in most of transgenic callus lines. And the growth of Valencia transgenic calluses was greatly suppressed by 50-60 mg L^-1 Hyg B.3. The anti-sense expression vector pBICI containing CRTISO gene fragment was constructed, and used to transform precocious trifoliate orange and Hongkong kumquat （Fortunella japonica [champ.] Swing.）. 4 independent transgenic Hongkong kumquat lines and 23 independent transgenic precocious trifoliate orange lines were obtained respectively.4. The RNAi expression vector pLCYB containing LCYB gene fragment, pLCYE1 and pLCYE2 containing LCYE gene 5’-end and 3’-end fragment were constructed respectively, and the three vectors was used in precocious trifoliate orange and Cocktail tansformation experiments. Results of Real-time PCR revealed that the expression of PDS gene markedly increased in LCFE-suppressed transgenic lines. The expression of LCYB and ZEP decreased. HPLC analysis revealed that in LCYE-suppressed transgenic plants, the content ofα-carotene decreased andβ-carotene and Violaxanthin contents increased, there were no significant changes in respect of lutein content. Unexpectedly, both ofβ-carotene and Violaxanthin increases were inversely proportional to the expression of LCYB and ZEP. According to these findings, we hypothesized that PDS gene was activated by some mechanisms in LCYE-suppressed transgenic plants. And the feedback effects should be responsible for the improvement ofβ-carotene and Violaxanthin:ζ-carotene and lycopene contents upstream improved with the increased expression of PDS gene, and this led to the dramatical increase of metabolic products such asβ-carotene and Violaxanthin downstream. These synthesis genes LCYB and ZEP were suppressed by their metabolic products conversely.In LCYB suppressed transgenic precocious trifoliate orange lines, the expression of genes PSY, ZDS , CRTISO and ZEP presented a downward tendency. At the same time, the content ofβ-carotene and Violaxanthin downstream decreased in the transgenic leaves and there were no significant changes in respect ofα-carotene and lutein contents. The results showed positive correlation between the conten of LCYB expression and β-carotene: the content ofβ-carotene elevated with the increasing of LCYB expression. This process may lead to the accumulation of lycopene.更多还原