Analysis of Physiological Changes and Differential Gene Expression during Pericarp Senescence of Postharvest Litch

Author: WangJiaBao Tutor: JinZhiQiang School: South China University of Tropical Agriculture Course: Crop Genetics and Breeding Keywords: Litchi Pericarp Senescence Water Content Subtraction Suppression Hybridization cDNA Library cDNA Microarray Gene Expression CLC: S667.1 Type: PhD thesis Year: 2007 Dissertation ID:1637993 Read: Download Dissertation http://www.dissertationtopic.net/doc/1637993

Executive Summary
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 stage, which was from 0 HAH to 32 HAH, pericarp lost mainly free water and could suffer a nonenzymatic browning. While on stage, 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.

Full-text Catalog
Chinese Abstract 3-6 Abstract 6-13 1 Introduction 13-52 1.1 lychee industry overview 13-14 1.2 Postharvest Biology Overview 14-38 1.2.1 litchi fruit an overview 14-15 1.2.2 litchi fruit respiration and ethylene production of 15-16 1.2.3 postharvest litchi pericarp browning mechanism 16-27 1.2.4 postharvest litchi pulp quality 27-28 1.2.5 Postharvest preservation storage technology 28-37 Problems and Prospects of the 1.2.6 Postharvest biological research 37-38 1.3 Postharvest molecular biological research 38-44 1.3.1 tissue culture regeneration system of progress 38-41 1.3.2 lychee The important function of gene cloning and expression analysis 41-42 1.3.3 litchi genetic transformation of 42-43 1.3.4 Prospect of the study 43-44 1.4 suppression subtractive hybridization in plant research applications 44-48 1.4.1 suppression subtractive hybridization principle 44-46 1.4.2 suppression subtractive hybridization technology applications 46-48

1.5 cDNA microarray technology and its application in plant research 48-50 1.5.1 cDNA microarray technology and its features 48 1.5.2 cDNA micro-array technology applications 48-50 1.6 of this study purpose, significance and technical route 50-52 2 postharvest litchi pericarp aging process in a number of physiological changes 52-68 2.1 Materials and Methods 52-56 2.1.1 Materials 52 2.1.2 Determination of indicators and methods 52-56 2.2 Results and analysis 56-65 2.2.1 browning index with good fruit rate changes 56 2.2.2 peel moisture content changes 56-57 2.2.3 peel pigment and total phenolic content changes 57-58 2.2.4 peel The pH changes 58 2.2.5 Peel malondialdehyde content and relative conductivity changes 58 2.2.6 peel PPO and POD activity in 58-59 2.2.7 peel The LOX activity changes 59 2.2.8 peel Vc and GSH content changes 59 2.2.9 peel The APX and the CAT, GR and SOD activity changes 59-60 2.2.10 browning correlation analysis of the relevant indicators 60-64 2.2.11 browning-related factors, stepwise regression analysis and path analysis of 64 the 2.2.12 peel aging stage is cluster analysis 64-65 2.3 discussion 65-67 2.4 Summary 67-68 3 mature litchi fruit peel total RNA extraction and cDNA library construction 68-81 3.1 Materials and Methods 68-75 3.1.1 Materials 68 3.1.2 Reagents 68 3.1.3 experimental methods 68-75 3.2 Results and analysis 75-78 3.2.1 RNA extraction 75-77 3.2.2 double strand cDNA synthesis of the optimal number of cycles to determine the dscDNA segment recovery 77 3.2.3 cDNA library titer 77-78 3.2.4 cDNA library insert size of 78 3.3 discussion 78-80 3.4 Summary 80-81 4 cDNA microarray technology detection of postharvest litchi pericarp aging process differentially expressed genes and lychee tissue-specific expression of gene 81-130 4.1 Materials and Methods 81-91 4.1.1 Materials 81 4.1.2 method 81-91 4.2 Results and discussion 91-128 4.2.1 cDNA microarray hybridization signal and data quality analysis 91-93 4.2.2 chip hybridization analysis 93 4.2.3 of differentially expressed genes in EST sequence analysis 93-95

4.2.4 litchi pericarp of postharvest aging process differentially expressed genes 95-127 4.2.5 with cDNA microarray screening tissue-specific expression of genes 127-128 4.3 Summary 128-130 with suppression subtractive hybridization cloning of postharvest litchi pericarp senescence-associated genes 130-145 5.1 Materials and Methods 130-137 5.1.1 Materials 130 5.1.2 Reagents 130 5.1.3 method 130-137 5.2 Results and analysis 137-143 5.2.1 RNA quality 137-138 5.2.2 LD-PCR cycle number of determine 138 5.2.3 suppression subtractive hybridization PCR results 138-139 5.2.4 Construction of the inserted fragment suppression subtractive library size 139 5.2.5 sequence processing and splicing 139 5.2.6 cDNA microarray screening suppression subtractive hybridization library of differentially expressed genes 139-140 5.2.7 suppression subtractive hybridization library of differentially expressed genes and their expression 140-142 5.2.8 suppression subtractive hybridization library screening results and cDNA library screening results compare 142-143 5.3 discussion 143 5.4 Summary 143-145 6 general discussion 145-147 6.1 litchi pericarp in the postharvest aging process 145-146 6.2 on litchi litchi pericarp senescence important genes and metabolic pathways explore 146 6.3 next step of the research program 146-147 7 Conclusion 147-148 References 148-161 Appendix primer results 161-202 Schedule 1 RT-PCR detection of gene expression primers 161-162 Blastx results Schedule 2 in litchi peel aging process differentially expressed genes of known function and its expression 162-192 Schedule 3 by RT-PCR detection of gene expression of the SSH library primer sequences 192-193 0h and 32h of Schedule 4 of postharvest litchi pericarp suppression subtractive hybridization library sequencing to obtain the gene 193-202 Appendix gene fragment of the cDNA sequence 202-205 Appendix III during the PhD commitment with this research-related topics and published papers 205-206 Acknowledgements 206

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