【作者】 丁健；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2009， 博士

【摘要】 柑橘体细胞融合技术有效地克服了柑橘有性杂交过程中遇到的珠心胚干扰、雌雄败育等生殖障碍,近二十年来已创造了超过250例的不同种间、属间体细胞杂种材料。由于柑橘体细胞融合实际上是一个半筛选体系,理论上再生植株包括三类:四倍体体细胞杂种,二倍体胞质杂种以及悬浮亲本再生植株。而原生质体再生植株很容易产生体细胞无性系变异,因而能创造出一些特别的种质资源。本研究以由‘朋娜脐橙愈伤原生质体’+‘红橘叶肉原生质体’通过PEG融合后再生的6棵具有果实早期粒化症状的变异体植株为材料,首先从遗传背景上分析其来源,而后以朋娜脐橙(Citrus sinensis[L.]Osbeck)作为对照分析了两者果实在发育过程中细胞壁代谢的变化规律和相关基因的表达,并采用SSH结合反向Northern技术分析了两者差异表达的基因。主要研究结果如下:1.流式细胞仪的倍性测定显示变异植株均为二倍体,而几种分子标记,即核SSR,RAPD,cpSSR,mtCAPS和mtRFLP的结果表明,它们的DNA均来源于朋娜脐橙而缺少红橘的遗传信息,但在线粒体基因组上单株之间以及与朋娜脐橙都存在着一定程度的变异。而叶型指数与朋娜没有显著差异而与红橘差异显著,其中单株之间有差异;枝条与朋娜脐橙相似但其中单株1-3无刺而单株4-6有刺;其花期与朋娜脐橙一致而早于红橘,花的形态也与亲本不同;果实形态及生理分析表明其与两亲本有明显差异,果实表面粗糙,无种子,果实无脐,汁胞粒化现象严重,果实着色略早,可固、可滴定酸、Vc含量均低于朋娜。由此我们推测其可能是由朋娜脐橙悬浮细胞再生形成的体细胞无性系变异体。2.测定了不同发育时期粒化突变体与朋娜脐橙果实汁胞和果皮中果胶类物质和纤维素类物质的组成与含量变化以及相关水解酶类的活性变化。结果表明两者果皮中果胶类物质以及纤维素类物质没有显著差异,但在突变体果实汁胞中原果胶,纤维素,半纤维素,木质素含量显著高于朋娜脐橙,这些均是细胞壁结构的重要组成物质。其代谢途径的三个关键酶,果胶甲酯酶,多聚半乳糖醛酸酶以及纤维素酶活性在果实发育过程均明显低于朋娜脐橙同时期的酶活性,特别是成熟后期。3.利用Real-time PCR测定粒化突变体与朋娜脐橙果实中细胞壁代谢途径关键酶基因(果胶甲酯酶,多聚半乳糖醛酸酶以及纤维素酶)以及多聚半乳糖醛酸酶抑制蛋白在几个不同采样时期的表达。结果与酶活测定结果趋势一致,表明汁胞粒化与果实中这几种酶的活动密切相关。综合以上研究结果,认为粒化可能是由于纤维素代谢相关的基因以及抑制蛋白的表达发生变化,一方面导致纤维素酶活性变低,使纤维素、半纤维素分解速度减小,另一方面纤维素合成增多,分解速度小于合成速度,导致粒化发生,同时由于果胶酶水解原果胶生成脱甲酯果胶质,其中一部分被钙桥、酯键固定,果胶被凝胶化,进一步加重粒化。4.为了获得与粒化突变体果实特异性状相关的基因,我们构建了其与朋娜脐橙果实的抑制性差减文库(SSH文库),然后采用反向Northern技术从文库中筛选在果实发育过程中差异表达的基因。在对差异表达的基因进行测序以及序列分析后,我们总共得到357条非重复性的基因,64.4%的非冗余序列(207条单一序列与23条contig)。其中与细胞代谢、初生代谢、定位以及大分子代谢等相关的基因数目最多。这些基因所属的代谢途径包括丙酮酸代谢途径、淀粉和蔗糖代谢途径、三羧酸代谢途径及糖酵解代谢途径等。此外,还对发现的12个与粒化性状可能相关的酶和蛋白候选基因,即果胶甲酯酶抑制蛋白(PMEIP)、果胶酸盐水解酶(PL)、几丁质酶(Chitinase)、葡聚糖酶(Glucanase)、β-半乳糖苷酶(Gal)、扩展蛋白(Exp)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)、赤霉素调节蛋白(GRP)、反转录转座子(Retrotransposon)和钙离子结合蛋白(CaBP),对它们在6个不同发育时期的基因表达活性变化进行了Real-time PCR检测,发现它们也与粒化有一定的关联。本研究通过对从果实生长发育的整个阶段进行研究,从细胞壁结构物质含量的变化到相关合成及水解酶类的动态变化,然后结合抑制性差减杂交技术(SSH)、反向Northern杂交技术,后续的生物信息学分析以及荧光实时定量PCR技术,首次深入到基因表达的层面研究了粒化所涉及到的一系列变化,揭示了调控粒化形成的可能分子途径,为将来解决这一问题奠定了一定的基础。更多还原

【Abstract】 Somatic hybridization via protoplast fusion is a new alternative for circumventing some reproductive barriers in citrus traditional breeding, such as sexual incompatibility, female and/or male sterility and polyembryony. Progress in somatic hybridization in citrus over the past 20 years has produced > 250 different inter-generic and inter-specific somatic hybrids. Protoplasts from embryogenic calli have the capacity to undergo embryogenesis and to develop into mature plants, while mesophyll protoplasts themselves are not totipotent. Thus, the regenerated plants from such a fusion event are primarily composed of hybrids, cybrids, and embryogenic calli parent regenerates. And somaclonal variation is commonly observed in protoplast-derived and protoplast fusion-regenerated plants, which will bring some special germ plasm. In this study, we describe six plants with an early juice sac granulation trait derived from the inter-specific protoplast fusions between embryogenic calli of ’Bonanza’ navel orange {Citrus sinensis [L.] Osbeck) and mesophyll protoplasts of ’Dahongpao’ Red tangerine （C. reticulata Blanco.）; analyzed their genetic background; studied the relationship between cell wall metabolism and granulation, the texture, hydrolases activity and gene expressions in juice sacs in order to studying the mechanism of granulation in juice sacs. To identify potential important or novel genes involved in somaclonal variation and juice sac granulation, suppression subtractive hybridization （SSH） and reverse Northern were performed to decipher this variation during fruit development.Six plants were analyzed by flow cytometry and using molecular markers including simple sequence repeats （SSR） and restriction fragment length polymorphism （RFLP）. The results indicated that all the six plants are diploids and inherited their nuclear DNA from the embryogenic calli parent ’Bonanza’ navel orange. However, analysis of morphological and fruit characteristics and measurements of the components of the cell walls in the juice sacs showed that they are not true-to-type for ’Bonanza’ navel orange, especially for fruit traits such as juice sac granulation and navel structure. We found that the leaves and fruits of these plants were different from their parents. For example, their fruit rinds were rougher, easier to peel than navel orange, and did not have a navel structure. The fruits were seedless, with an orange colour instead of being seedy with a deep red skin of ’Dahongpao Red’ tangerine. In addition, they exhibited serious juice sac granulation before maturityThese results confirmed that these plants were not hybrids and are more likely to be somaclonal variants arising during the regeneration of navel orange protoplasts.The texture, hydrolases activity in juice sacs of six somaclonal variation plants （BH） and the control （B） Skagg’s Bonanza navel orange （Citrus sinensis [L.] Osbeck） were characterized at six developmental stages. Results showed that the contents of water soluble pectin （WSP）, propectin, total pectin （TP）, cellulose （CEL）, hemicellulose （HC）, lignin, and water between granulated juice sacs in BH and the normal sacs in Bonanza navel orange were significantly different. High and positive correlation coefficients were observed between polygalacturonase （PG） activity and content of WSP, while the contents of CEL, HC, and lignin were negatively correlated with levels of cellulase （Cx）.Moreover the expression of PG and Cx genes was the same as enzyme activities. They behaved a high level in B and much low in BH fruit juice sacs. The pectinmethylesterase （PME） showed little different expression in two samples. In addition, the expression of polygalacruronase inhibiting protein （PGIP） gene was much higher in BH versus in its control B. These results suggested that the granulation of juice sacs during fruit development might partially be explained by the hydrolases activity and the transcriptional level of genes directing the cell wall metabolism pathway.After sequencing of the differentially expressed clones, a total of 357 non-redundant transcripts were obtained and 64.4% （207 single sequences and 23 contigs） of them shared homology (E-value≤1×10^-10) with known gene products. These genes were involved in many metabolic pathways such as pyruvate metabolism, starch and sucrose metabolism, citrate cycle and glycolysis pathways. Moreover, 12 genes: Pectinmethylesterase inhibiting protein （PMEIP）, Pectate lyase （PL）, Chitinase, Endo-beta-1, 4-glucanase,β-galactosidase （Gal）, Expansins （Exp）, Peroxidase （POD）, Superoxide dismutase （SOD）, Glutathione peroxidase （GPX）, Gibberellin regulated protein （GRP）, retrotransposon and Ca²⁺-binding protein（CaBP） transcripts were studied by real time PCR in six different sample times of BH fruits and B fruits. The results showed that they may associate with granulation.Through this research, the content of cell wall structure materials, the dynamic changes of relevant synthesis and hydrolases during the different development times were studied, and then combined with suppression subtractive hybridization （SSH）, reverse Northern hybridization, the follow-up of bio-informatics analysis and real-time quantitative fluorescence PCR technology, for the first time analysis the level of geneexpression which may involved in changes of granulation. The gene expression,regulation and control has revealed the possible molecular mechanism of the formation ofgranulation, therefore make a firm foundation to solve this problem for the future incitrus.更多还原