An Analysis On Physiological Changes And Gene Differential Expressions In The Process Of Pummelo Fruit [Citrus Grandis (L.) Osbeck Cv. Guanxi-miyou] Juice Sac Granulation

'Guanxi-miyou'pummelo [Citrus grandis (L.) Osbeck cv. Guanxi-miyou], as one kind of subtropical evergreen fruit trees, is a primary cultivar in China. Juice sac granulation is one kind of wide-spread physiological diseases in pummelo category, so as in citrus category, which seriously hinders eating quality and commodity value of Guanxi-miyou'pummelo fruit. Thus, the effective study on juice sac granulation is crucial to improve Guanxi-miyou'pummelo fruit production. In this article, by the research on interrelation of juice sac granulation with active oxygen metabolism, cell structure, mineral, endogenous hormone, the establishment of the experimental system of'Guanxi-miyou'pummelo juice sac mRNA differential display and acquirement of differential fragments, the cloning of relative genes in the process of'Guanxi-miyou'pummelo juice sac granulation, the physiological and biochemical mechanism of juice sac granulation during the fruit maturation of'Guanxi-miyou'pummelo fruits was presented. By RACE techniques, the genes cDNA full length related to the normal-developed juice sac (normal growth and granulation) was cloned, which would lay a foundation for exploring orange juice sacs granulation mechanism in molecular level. The results were as follows:1. Fruits of pummelo from old-age trees with higher ratio of juicy sac granulation fruits, compared with normal fruits, and proper-age trees with lower ratio of granulation fruits, compared with normal fruits were used to explore the relationship between granulation and active oxygen metabolism of juicy sac in pummelo [ Citrus grandis (L.) Osbeck cv. Guanxi-miyou] fruit during maturation. The changes in content of MDA, H2O2, and lignin,activities of reactive-oxygen-scavenging enzymes(POD,SOD,CAT), contents of endogenous antioxidants( AsA,GSH) in process of juicy sac granulation were studied. The index of juicy sac granulation and contents of H2O2 and MDA rose while contents of ASA and GSH decreased, whereas SOD activity first declined and then increased and CAT activity increased but POD activity rose more significantly in the fruits from the old-age trees in contrast to proper-age trees. The results suggest that the disorder of active oxygen metabolism of juicy sac of pummelo fruit during maturation result in the accumulation of active oxygen(H2O2)and the increase in POD activity, which promotes the synthesis of lignin, and finally causes juicy sac granulation.2. By observation on paraffin sections of'Guanxi-miyou'pummelo fruit ( October 8) granulation and no-granulated juice sacs, it was found that the juice sacs were overstaffed, cystic wall horniness hyperplasia occurred, cell layer increased, cytoderm incrassated, and the lignification of second-growth infiller and complex ornament of cytoderm increased, as a result, the juice sac granulation emerged.3. In the process of'Guanxi-miyou'pummelo fruit growth, the contents of N, K maintained a relatively high level. Compared with no-granulated juice sacs, the granulated juice sacs were higher in contents of N, P, Zn, Mg, Ni and Cu, but lower in content of Ca.4. Due to the self-incompatibility, the natural parthenocarpy of'Guanxi-miyou'pummelo fruits is non-nuclear fruits. The imbalanced endogenesis hormone distribution in the process of granulation, particularly at the beginning and exceeding phrases, concentration of IAA, GA3, and ZR in juice sacs was quite low, but that of ABA was high. The endogenesis hormone imbalance caused by changes in concentration ratio among GA3, ZR and ABA is one reason of granulation.5.'Guanxi-miyou'pummelo fruit old-age tree (seed tree) juice sacs in different phrases was applied as experimental materials to establish mRNA differential display technique system, including juice sacs main RNA extraction and purification methods, DD-RT-PCR system optimization, and reversed northern-blot application to remove false positive rate of cDNA differential fragments. By the improved CTAB method in'Guanxi-miyou'juice sacs RNA extraction, the obtained RNA was of high quality. By comparing key factors in DDRT-PCR, the'Guanxi-miyou'juice sacs granulation gene DDRT-PCR system was established. The optimizing reaction system contained 2μL cDNA, 40μM dNTP mix, 0.4μmol.L-1 single primer, and 0.2U Taq DNA polymerase dosage. Through electrophoresis detection, these reaction products could get cDNA fragments in suitable quantity, clear bands and good reproducibility.6. Nine anchoring primer and 20 random primers made up 180 primer pairs. The reverse transcription pcr amplification was tested on'Guanxi-miyou'sample normal and granulated juice sacs. By application of mRNA differential display techniques,'Guanxi-miyou'normal and granulated juice sacs gene expressions were studied, and cDNA differential fragments were obtained. cDNA differential fragments were recycled and compared and analyzed by cloning, sequencing and homology comparison. 7 cDNA fragments concerning granulation gene differential expressions and 9 cDNA fragments related to un-granulation gene differential expressions were acquired. The accession number of GenBank database of Miyou1,2,7,8,17, these 5 fragments were FJ866625, FJ866626, FJ866629, FJ866627 and FJ866628. Through reverse northern blot from extracting differential fragments, 2 positive fragments were obtained.7. By RACE technique, cDNA full length related to genes of juice sacs growth (normal growth and granulation) was cloned. Based on Miyou22 cDNA fragments related to granulation, the primers were designed, conservative region clone was carried out, and'Guanxi-miyou'pummelo fruit juice sacs granulation process 5'end sequence was cloned by 5'RACE. Because 3'end sequence was anchoring primer, and it was jointed with 5'end sequence, a cDNA full length related to granulation was obtained and its The accession number of GenBank database was FJ866630. The sequence analysis indicated the full length of cDNA was 1602 bp, among which the size of 5'UTR was 142 bp, that of 3'UTR was 204 bp, and 3'UTR region included typical tail signals AATAA and poly (A) tail. Open reading frame (ORF) was made up of 1254 base, encoding 418 amino acid. The Blast showed this cDNA and nucleotide sequence had homology with 1-γmRNA full length of tobacco and rice translation elongation factor, 81.3% and 79% respectively. Translation elongation factor is a key factor in the process of protein synthesis, which was displayed in granulated juice sac of'Guanxi-miyou'. So it was speculated that its protein synthesis was vigorous8. According to Miyou1 cDNA fragments designed primers of juice sacs normal gene differential cDNA fragments, 5'full-length sequence was cloned by RACE technique and jointed with Miyou1 fragment, and then cDNA full length with the size of 1152 bp was gained which included a open reading frame of 819 bp and encoding 272 amino acid. Its accession number of GenBank database was FJ866624. Thereamong the size of 5'UTR was 77bp,that of 3'UTR was 244 bp and 3'UTR region contained poly(A) tail. The further blast analysis showed, this cDNA had high sequence identity withβ5 subunit of 20s proteasome. It had the highest homology with that of chickpea (84.9%), and next with that of rice (76.1%) and with that of wheat (74.6%). Proteasome is a kind of proteolytic enzyme compounds exiting in cell cytoplasm and nucleus and plays a important role in degrading the intracellular protein cell protein and maintaining cell normal metabolism, therefore 20s proteasome plays a role in sac growth of'Guanxi-miyou'fruit...