【作者】 王家保；

【导师】 金志强；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 荔枝是一种亚热带至热带果树,在华南农业经济中占有重要地位。然而,采后衰老过程中果皮发生的一些变化如褐变及病菌侵染等严重降低了荔枝的商品价值,缩短了荔枝的货架期。深入研究荔枝采后生物学问题可为开发荔枝保鲜技术提供基础与依据。虽然已有学者对若干果皮衰老相关的若干问题进行了大量研究,但目前对采后荔枝果皮衰老的机制依然不甚明了,有关采后荔枝果皮衰老分子生物学的研究报道更少。为了探讨采后荔枝果皮的衰老机制,为进一步荔枝采后生物学研究提供基础,本研究以妃子笑荔枝果实为试材,研究了室温无包装条件下每隔8h的果皮生理变化。通过cDNA微阵列杂交筛选,获得了一批在采后不同阶段果皮及不同荔枝组织中差异表达的基因,进一步研究了这些基因的表达规律。主要结果如下:妃子笑荔枝果实在实验条件下72h内完全褐变。在褐变过程中,荔枝果实与果皮的失水量持续上升,果实失水主要由果皮失水引起。采后48h内,果皮主要散失自由水,在48h以后,束缚水下降较快。果皮的总酚含量在采后0-72h内持续下降,类黄酮、花色素苷含量在采后40h后下降。衰老过程中,果皮pH值缓慢上升。果皮相对电导率持续升高,至采后40h时达到最高;丙二醛含量则缓慢升高,至采后64h时达到最高。多酚氧化酶活性在32h后快速下降至一稳定水平。过氧化物酶活性在32h内上升,此后亦下降至一稳定水平。LOX活性在32h时有一高峰,此后剧烈下降至一稳定水平。果皮抗坏血酸含量在24h内保持稳定,此后持续下降。谷胱甘肽含量在采后24h内保持稳定,32h时升至最高,高峰保持16h后下降。抗坏血酸过氧化物酶活性总体上呈下降趋势,而谷光甘肽还原酶活性则呈单峰曲线,峰值维持在采后24h-32h。通过对获得的各项数据进行相关分析、逐步回归分析及通径分析后发现:果皮含水量与果皮失水量是影响果皮褐变的主要因素。聚类分析表明,以32h-40h之间为界,妃子笑荔枝果皮的衰老过程可分为2个阶段。前期,即阶段Ⅰ的果皮褐变指数变化较慢,主要散失自由水,可能主要发生非酶促褐变;阶段Ⅱ的果皮自由水与束缚水均散失,褐变指数在48h以后剧烈上升,可能主要发生酶促褐变。在生理变化研究基础上,我们进一步研究了果皮衰老过程中的基因差异表达。首先建立了快速从果皮中提取总RNA的方法,然后构建了采后0h果皮的cDNA文库,并分别以0h果皮总RNA和32h果皮总RNA为TESTER,构建了正向和反向抑制差减杂交(SSH)文库。之后,从cDNA文库中随机挑取了11636克隆,从正向SSH文库和反向SSH文库中各挑取了284个克隆及78个克隆, PCR扩增了这些克隆的插入片段,以回收的PCR产物作探针,制做了含12000探针的cDNA微阵列。以采后0h果皮样品为共同对照,设计了11组杂交组合,用以筛选在采后不同时间果皮中及荔枝不同组织中发生差异表达的基因。通过杂交分析,分别从采收0h果皮cDNA文库、正向SSH文库及反向SSH文库中选择了2444个、49个、17个差异表达克隆。对这些克隆进行测序,各获得了2136、45、17条有效EST序列,分别拼接成为836、20、16个基因簇(Clusters)。在来自0hcDNA文库的836个clusters中,含287个重叠群,549条单一序列。依据序列分析结果进一步测序获得了200余条含完整开放读码框的cDNA序列。Blastx结果表明,共403个Clusters获得了基因功能注释,这些基因中有221个在Gene Ontology数据库中进行了功能分类,KEGG注释将150余条基因填入了不同的代谢途径。共获得采后0-48h内差异表达基因709个,其中,从0h果皮cDNA文库中得到693个,正向SSH文库中得到20个,反向SSH文库中得到16个。经BLASTx比对有功能注释的基因405条,未知功能基因131条,无匹配基因172条。聚类分析表明,这些基因的表达规律可分为4种类型。随机选取若干基因进行RT-PCR检测基因表达的结果与cDNA微阵列杂交结果基本一致。众多基因发生了差异表达说明果皮的衰老是一个多基因调控的复杂过程。功能已知的差异表达基因中有大量基础代谢相关基因、次生代谢相关基因、细胞壁代谢相关基因、转录因子、胁迫响应蛋白、激素应答元件、细胞信号传导、细胞防御相关基因等。由于参与淀粉、蔗糖、脂肪酸、磷脂、甘油脂、蛋白质等分解代谢的基因多数上调表达,而参与其合成代谢的相关基因多呈下调表达,因此在果皮衰老过程中果皮的分解代谢受到促进,合成代谢受到抑制。另外,依据基因的表达规律推测,果皮衰老过程中类黄酮类色素合成降低,木质素合成则可能上升,且细胞壁的降解加快。通过综合分析各类基因表达规律后发现,编码某些热击蛋白、WRKY类转录因子、锌指蛋白类转录因子等的基因和淀粉与蔗糖代谢、细胞壁代谢、半乳糖代谢、类黄酮合成、叶绿体能量代谢等代谢途径可能在调节采后果皮衰老中具有重要的作用。依据cDNA微阵列杂交结果,我们还获得了分别在果肉、果皮、种子、叶片、花和根中特异表达的基因39、22、20、11、6和8个,其中,有功能注释的基因分别为27、18、16、6、5和5个。RT-PCR检测结果与cDNA微阵列检测结果基本一致。这些基因的获得为进一步克隆组织特异型启动子提供了基础。总之,本研究发现了影响果皮衰老的主要因素和果皮衰老的进程,获得了一批在果皮衰老过程中差异表达的基因及组织特异性表达基因,发现有大量差异表达基因参与了多种代谢途径,说明荔枝果皮衰老是一个多因素影响的复杂过程,其中一些基因的表达变化可能在调节荔枝果皮衰老过程中起重要作用。这些结果为进一步的荔枝采后生物学研究提供了基础与依据。更多还原

【Abstract】 Litchi(Litchi chinensis Sonn.) is a subtropical to tropical fruit and plays an important role in agriculture economy in southern China. However, some changes of pericarp during its senescence, such as browning or/and diseases-infecting, reduce the commercial value and shelf-life of postharvest litchi. Insights into the mechanism of pericarp senescence will provide basis for innovations in preservation technology of litchi. Though many researches have focused on some specific issues of pericarp senescence, such as pericarp browning, the mechanism of pericarp senescence has not been well understood, especially of its molecular biology.To get some knowledge on pericarp senescence, some physiological changes in pericarp of unpacked Feizixiao litchi fruit were investigated at an 8-hour interval in this study. A number of genes differentially expressed during pericarp senescence of postharvest litchi and in different litchi tissues were identified by cDNA microarray hybridization, and their express patterns were further analyzed. The main results were as follows:Litchi fruit turned fully brown within 72 hours after harvest (HAH). During the process of skin browning, both the whole fruit and pericarp lost water continuously. The water loss of whole fruit was mainly resulted from water loss of pericarp. Within 48 HAH, free water in pericarp decreased more quickly than the binding water and the binding water in pericarp lost rapidly after 48 HAH. The falvonoid and anthocyanin content in pericarp decreased after 40 HAH and the total phenols content decreased throughout the experiment time. The pH value in pericarp increased slowly along with the senescence. Relative electronic conductivity of pericap increased continuously within 40 HAH and reached the maximum at 40 HAH. Malondiadlehyde(MDA) content increased slowly and reached the maximum at 64HAH. In the first 32HAH, there was little change in PPO activity, a slight increase in POD activity and an activity plateau in LOX. After 32HAH, activities of the three enzymes decreased quickly to a stabled level. Ascorbate acid and reduced glutathione content showed no changes within the first 24 HAH. After that time, Vc content decreased rapidly and reduced glutathione content increased to a plateau. After 48HAH, reduced glutathione content decreased. Activity of ascorbate acid peroxidase showed a decreasing tendency and activity of glutathione reductase showed a plateau in 24-32HAH.Correlation analysis, progressively regression analysis, path analysis indicated that water content and water loss of pericarp were the key factors related with pericarp browning. Senescence were differentiated into two stages according to the cluster analysis results. On stageⅠ, which was from 0 HAH to 32 HAH, pericarp lost mainly free water and could suffer a non-enzymatic browning. While on stageⅡ, which was from 40 HAH to 72 HAH, pericarp lost both free and binding water and its browning index increased sharply after 48HAH.The differential gene expression was examined based upon the results above. A method for rapidly extracting total RNA from litchi pericarp was established firstly. Then a cDNA library of pericarp at 0 HAH(0 h cDNA library) was constructed. Two subtraction suppression (SSH) libraries were also constructed by using RNA from pericarp at 0 HAH and at 32 HAH as testers for forward and reverse SSH library, respectively. Insert fragments of 12000 recombinant clones,among which 11636 were randomly picked from 0 h cDNA library, 78 were picked from reverse SSH library and 286 were picked from the forward SSH library, were amplified. Then the PCR products were purified and used as probes for constructing a set of cDNA microarrays. Eleven hybridization combinations were designed with the sample from pericarp at 0 HAH as common reference to identify differentially expressed genes in pericarp during 0-48HAH and in different litchi tissues. According to the hybridization results, 2444,49,17 clones which showed differential expression in different hybridization combinations were identified from 0 h cDNA library, forward and reverse SSH library respectively, and 2316, 45, 17 clones of them were sequenced successfully. Then 836, 20 and 16 clusters were assembled respectively. There were 287 contigs and 549 singletons in the 836 clusters obtained from 0 h cDNA library. 403 clusters were functionally annotated with BLASTx, in which 221 had functional classification according to Gene Ontology database. More than 150 genes were put into different metabolism pathways in KEGG database.709 genes differentially expressed in pericarp during 0 to 48HAH were screened out of 12000 probes, among which 693, 20, 16 genes were identified from 0h cDNA library, forward and reverse SSH library, respectively. Of the 709 genes, 405 were annotated as functional genes by BLASTx, 131 were annotated as unknown function and 172 had no homologues in the database. The expression patterns of above genes were classified into 4 groups according to the fluorescent ratio obtained by cDNA microarray hybridization. RT-PCR examining results of randomly selected genes showed similar expression patterns to the results from cDNA microarry hybridization.Most of genes mentioned above were isolated from litchi pericarp for the first time. Some of them had homologues involved in primary metabolisms, secondary metabolisms, cell wall metabolism and so on. And others of them had homologues which coded transcription factors, stress-responsible proteins, hormone responsible elements, signal transduction proteins, cell defensive associated proteins et al. Because expression of most genes involved in catabolism of starch and sucrose, lipid acid, phospholipid, glycerolipid, protein and so on were up-regulated and genes involved in their biosynthesis were down-regulated during pericarp senescence of postharvest litchi, catabolism was enhanced and biosynthesis was somehow inhibited. The patterns of gene expression also indicated the reduced biosynthesis of flavonoids, enhanced biosynthesis of lignin and accelerated degradation of cell wall. Expression of genes coding some heat shock proteins, WRKY transcription factors, zinc finger transcription factors and the metabolism pathways of starch and sucrose metabolism, cell wall metabolism, galactose metabolism, biosynthesis of flavonoids as well as energy metabolism of chloroplast should play important roles in regulating the pericarp senescence of postharvest litchi.In addition, 39, 22, 20, 11, 6 and 8 genes specifically expressed in pulp, pericarp, seeds, leaves, flower and roots of litchi respectively were identified by cDNA microarray analysis, among which only 27, 18, 16, 6, 5, and 5 genes were functionally annotated by bioinformatics analysis. The express patterns of the genes revealed by cDNA microarray hybridization were confirmed by RT-PCR analysis.In conclusion, the senescence stages of pericarp and the key factors influencing pericarp browning were concluded based on the physiological changes of posthavest litchi in this study. A number of genes differentially expressed both during pericarp senescence and in different litchi tissues were identified for the first time. Their involving in various physiological and molecular events implied that many complicated molecular processes had occured during pericarp senescence. Expression patterns of these genes provided some new clues to further research on postharvest biology of litchi. All the above results should provide basis for further studying on the postharvest biology of litchi.更多还原